ALBERT

Description: Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20–40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini-review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. This article is protected by copyright. All rights reserved

Description: ABSTRACT Atrogin-1, an E3 ligase present in skeletal, cardiac and smooth muscle, down-regulates myocardin protein during skeletal muscle differentiation. Myocardin, the master regulator of smooth muscle cell (SMC) differentiation, induces expression of smooth muscle marker genes through its association with serum response factor (SRF), which binds to the CArG box in the promoter. Myocardin undergoes ubiquitylation and proteasomal degradation. Evidence suggests that proteasomal degradation of myocardin is critical for myocardin to exert its transcriptional activity, but there is no report about the E3 ligase responsible for myocardin ubiquitylation and subsequent transactivation. Here, we showed that overexpression of atrogin-1 increased contractility of cultured SMCs and mouse aortic tissues in organ culture. Overexpression of dominant-negative myocardin attenuated the increase in SMC contractility induced by atrogin-1. Atrogin-1 overexpression increased expression of the SM contractile markers while downregulated expression of myocardin protein but not mRNA. Atrogin-1 also ubiquitylated myocardin for proteasomal degradation in vascular SMCs. Deletion studies showed that atrogin-1 directly interacted with myocardin through its amino acids 284-345. Immunostaining studies showed nuclear localization of atrogin-1, myocardin and the Rpt6 subunit of the 26S proteasome. Atrogin-1 overexpression not only resulted in degradation of myocardin but also increased recruitment of RNA Polymerase II onto the promoters of myocardin target genes. In summary, our results have revealed the roles for atrogin-1 in the regulation of smooth muscle contractility through enhancement of myocardin ubiquitylation/degradation and its transcriptional activity. This article is protected by copyright. All rights reserved

Description: Cholesterol is an important component for cell physiology. It regulates the fluidity of cell membranes and determines the physical and biochemical properties of proteins. In the central nervous system, cholesterol controls synapse formation and function and supports the saltatory conduction of action potential. In recent years, the role of cholesterol in the brain has caught the attention of several research groups since a breakdown of cholesterol metabolism has been associated with different neurodevelopmental and neurodegenerative diseases, and interestingly also with psychiatric conditions. The aim of this review is to summarize the current knowledge about the connection between cholesterol dysregulation and various neurologic and psychiatric disorders based on clinical and preclinical studies. This article is protected by copyright. All rights reserved

Description: ABSTRACT Nestin (+) -cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin (+) cells that differentiated to a vascular phenotype in the peri-infarct/infarct region of post-MI mice albeit the transgene was not detected in nestin (+) -cardiomyocytes. α-MHC-driven expression of the reporter mCherry was detected in troponin-T (+) - and nestin (+) -cardiomyocytes in the peri-infarct/infarct region of post-MI mice. However, the cell cycle re-entry of nestin/mCherry (+) -cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12, 13-dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co-treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin was detected in cycling troponin-T (+) -cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re-entry. Thus, nestin expressed by pre-existing cardiomyocytes following ischemic damage recapitulated in part an embryonic phenotype and may provide the requisite phenotype to initiate cell cycle re-entry. However, the overt activation of the p38 MAPK pathway post-MI may in part limit the appearance and inhibit the cell cycle re-entry of nestin (+) -cardiomyocytes. This article is protected by copyright. All rights reserved

Description: Alkaptonuria (AKU) is an ultra-rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2-dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called “ochronosis”. The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co-localization of SAA with three cytoskeletal proteins: actin, vimentin and β-tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4-HNE presence has been evaluated by means of immunofluorescence. This article is protected by copyright. All rights reserved

Description: In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. This article is protected by copyright. All rights reserved

Description: In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium-vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging-induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2-photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT-PCR and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over-expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. This article is protected by copyright. All rights reserved

Description: Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). In the past ten years, the clinical efficacy of this therapeutic approach in several spontaneous models of tumors in animals has been shown. Moreover, some of the molecular and cellular mechanisms responsible for this phenomenon have been elucidated. Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols and their translation to humans. This article is protected by copyright. All rights reserved

Description: Submarine groundwater discharge (SGD) is a widely recognized process that carries considerable amounts of groundwater and dissolved chemicals to the coastal ocean. Despite its importance, a lack of suitable tools to assess SGD's spatial and temporal variability has hampered a complete understanding of the process. Here we report, for the first time, use of an unmanned aerial vehicle (UAV or “drone”) to assess SGD variations. An octocopter UAV platform equipped with a thermal infrared (TIR) system was flown along a coastline on Jeju Island, Korea. The UAV clearly captured thermal signatures of SGD plumes and their dynamic temporal fluctuations modulated by tidal variations. Based on a plume area-SGD flux relation we developed by combining aerial and field data, we estimated that the SGD flux of the study site ranged from 33,000 to 54,000 m 3 d −1 . The drone approach enabled acquisition of time series plume imagery with easy control of spatial resolution, flexible field operations, and remote sensing of SGD at low cost compared to conventional aerial surveys. Combining the UAV-TIR images with on-site sampling enables one to determine fluxes of nutrients and other dissolved species. UAV-TIR mapping can thus serve as a powerful tool for study of SGD and other coastal processes.

Description: Quantification of chlorophyll a (Chl a ) is essential to the study of aquatic ecosystems, yet differences in methodology may introduce significant errors to its determination in ethanol extracts. Insufficient acidification slows the conversion of Chl a to pheophytin a leading to an underestimate of Chl a concentration. Furthermore, slight differences in the postacidification reaction time can introduce greater errors in calculated Chl a and impede our ability to make cross-study comparisons. We used known concentrations of pure Chl a from the blue-green algae Anacystis nidulans dissolved in 95% ethanol to evaluate the effect of acid strength and postacidification reaction time on the spectrophotometric determination of Chl a . Increasing acid strength resulted in more rapid stabilization of calculated Chl a concentration. At reaction times less than 120 s estimates of Chl a deviated from known concentrations by as much as 84.8%. The magnitude of error in the calculated Chl a values were dependent on acid strength and reaction time, which allowed us to develop predictive equations to correct Chl a measurements that were insufficiently acidified or read prior to reaction completion. We validated our predictive equations using benthic periphyton samples from northern Alaska and northwestern Vermont, U.S.A. Our results indicate that under-acidified samples with known reaction times can be easily corrected so results from different methods can be standardized. For future analyses we recommend acidifying ethanol-extracted algal samples to 0.008 mol HCl L −1 and allowing samples to react for 30–60 min to ensure accurate and consistent results.

Description: The increasing availability and use of high-frequency water quality sensors has enabled unprecedented observations of temporal variability in water chemistry in aquatic ecosystems. However, we remain limited by the prohibitive costs of these probes to explore spatial variability in natural systems. To overcome this challenge, we have developed a novel auto-sampler system that sequentially pumps water from up to 12 different sites located within a 12 m radius to a single water quality probe. This system is able to generate high temporal frequency in situ water chemistry data from multiple replicated units during experiments as well as multiple sites and depths within natural aquatic ecosystems. Thus, with one water quality probe, we are able to observe rapid changes in water chemistry concentrations over time and space. Here, we describe the coupled multiplexer-probe system and its performance in two case studies: a mesocosm experiment examining the effects of water current velocity on nitrogen dynamics in constructed wetland sediment cores and a whole-ecosystem manipulation of redox conditions in a reservoir. In both lotic and lentic case studies, we observed minute-scale changes in nutrient concentrations, which provide new insight on the variability of biogeochemical processes. Moreover, in the reservoir, we were able to measure rapid changes in metal concentrations, in addition to those of nutrients, in response to changes in redox. Consequently, we believe that this coupled system holds great promise for measuring biogeochemical fluxes in a diverse suite of aquatic ecosystems and experiments.

Description: ABSTRACT Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 µl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 µl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2 and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of type I and IIb muscle fibres). This article is protected by copyright. All rights reserved

Description: Diabetic macular edema (DME), characterized by an increase of thickness in the eye macular area, is due to breakdown of the blood-retinal barrier (BRB). Hypoxia plays a key role in the progression of this pathology by activating the hypoxia-inducible factors. In the last years, various studies have put their attention on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in retinal dysfunction. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia on outer BRB. Therefore, in the present study, we have analyzed the role of these peptides on permeability, restoration of tight junctions expression and inhibition of hyperglycemia/hypoxia-induced apoptosis, in an experimental in vitro model of outer BRB. Our results have demonstrated that the peptides' treatment have restored the integrity of outer BRB induced by cell exposure to hyperglycemia/hypoxia. Their effect is mediated through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling pathways. In conclusion, our study further clarifies the mechanism through which PACAP and VIP perform the beneficial effect on retinal damage induced by hyperglycemic/hypoxic insult, responsible of DME progression. This article is protected by copyright. All rights reserved

Description: Meiofaunal biomass, despite its ecological importance, is rarely assessed in marine ecological studies due to time-consuming and costly indirect procedures or inaccurate direct methods. Here, we present a semi-automated image analysis method for obtaining the widths and lengths of photographed nematodes that employs the Leica LAS Image Analysis module and is used to estimate Nematoda (a dominant marine meiofaunal taxon) biomass. Samples of 100 individual nematodes from 10 replicate samples representing two different environments (muddy fjord bottom and seagrass-vegetated sands) were used for the analyses, which were performed manually and semi-automatically. Manual measurements were taken by three independent analysts to check for individual differences. The automated measurements were collected almost two times faster than the manual ones. The three analysts and the computer did not differ in their assessments of nematode length, but significant differences existed among the measurements of maximum width by the three analysts (due to different designations of the point of nematode maximum width required by the manual method). For estimating individual nematode biomass, a formula using the equation for the volume of cylinder (one of the three tested in the study) is recommended. The total nematode biomass values obtained with this method were comparable to those reported from similar habitats. This semi-automated method seems to be less biased and more time and cost effective compared with standard manual protocols. The wider application of this method should facilitate the more frequent inclusion of meiofaunal biomass assessments in marine benthic surveys.

Description: ABSTRACT Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt + /Vim + cell number and ΔNp63α expression when cells reached confluence and formed a 4-5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt + /Vim - cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim + /Krt + /ΔNp63α + /Pax-6 low /α6 integrin + phenotype. This article is protected by copyright. All rights reserved

Description: DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full -scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. This article is protected by copyright. All rights reserved

Description: Angiogenesis is associated with changes in endothelial cell (EC) proliferation and tube formation, controlled by extracellular receptor-activated kinase (ERK)/mitogen activated protein kinase (MAPK) and Akt signaling. Important regulators of these systems include hormones acting on G-protein-coupled receptors, such as beta 2-adrenoceptors (β2-ARs). In central nervous system (CNS) trauma, the importance of β2-AR modulation has been highlighted, although the effects on revascularization remain unclear. Vascular protection and revascularization are, however, key to support regeneration. We have investigated the angiogenic capacity of the specific β2-AR agonist terbutaline on ECs derived from the CNS, namely bEnd.3-cells. As angiogenesis is a multistep process involving increased proliferation and tube formation of ECs, we investigated the effects of terbutaline on these processes. We show that terbutaline significantly induced bEnd.3 tube formation in a matrigel in vitro assay. Moreover, administration of specific inhibitors of ERK and Akt signaling both inhibited terbutaline-induced tube formation. The proliferation rate of the ECs was not affected. In order to investigate the general effects of terbutaline in an organotypic system, we have used the chick chorioallantoic membrane (CAM)-assay. Most importantly, terbutaline increased the number of blood vessels in this in ovo setting. Although we observed a positive trend, the systemic administration of terbutaline did not significantly improve the functional outcome, nor did it affect revascularization in our spinal cord injury model. In conclusion, these data indicate that terbutaline is promising to stimulate blood vessel formation, underscoring the importance of further research into the angiotherapeutic relevance of terbutaline and β2-AR signaling after CNS-trauma. This article is protected by copyright. All rights reserved

Description: Extracellular ATP and other nucleotides induce autocrine and/or paracrine purinergic signalling via activation of the P2 receptors on the cell surface, which represents one of the most common signalling mechanisms. Mesenchymal stem cells (MSC) are a type of multipotent adult stem cells that have many promising applications in regenerative medicine. There is increasing evidence to show that extracellular nucleotides regulate MSC functions and P2 receptor-mediated purinergic signalling plays an important role in such functional regulation. P2 receptors comprise ligand-gated ion channel P2X receptors and G-protein-coupled P2Y receptors. In this review, we provide an overview of the current understanding with respect to expression of the P2X and P2Y receptors in MSC and their roles in mediating extracellular nucleotide regulation of MSC proliferation, migration and differentiation. This article is protected by copyright. All rights reserved

Description: Coring is one of several standard procedures to extract sediments and their faunas from open marine, estuarine, and limnic environments. Achieving sufficiently deep penetration, obtaining large sediment volumes in single deployments, and avoiding sediment loss upon retrieval remain problematic. We developed a piston corer with a diameter of 16 cm that enables penetration down to 1.5 m in a broad range of soft bottom types, yields sufficient material for multiple analyses, and prevents sediment loss due to a specially designed hydraulic core catcher. A novel extrusion system enables very precise slicing and preserves the original sediment stratification by keeping the liners upright. The corer has moderate purchase costs and a robust and simple design that allows for a deployment from relatively small vessels as available at most marine science institutions. It can easily be operated by two to three researchers rather than by specially trained technicians. In the northern Adriatic Sea, the corer successfully extracted more than 50 cores from a range of fine mud to coarse sand, at water depths from three to 45 m. The initial evaluation of the cores demonstrated their usefulness for fauna sequences along with heavy metal, nutrient and pollutant analyses. Their length is particularly suited for historical ecological work requiring sedimentary and faunal sequences to reconstruct benthic communities over the last millennia.

Description: Endometrioid adenocarcinomas represent 80% of endometrial carcinomas 1 . Molecular features, including microsatellite instability, mutations of the PTEN, PIK3CA, K-Ras and β-catenin genes 1 and dysregulations in sncRNAs (small non coding RNAs) are described for this disease. However, mechanisms and molecules that determine cell survival and response/resistance to therapy in different subtypes of this tumour are not fully clarified 1 . This article is protected by copyright. All rights reserved

Description: With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty L d (eL d ) molecules, an open form of murine MHC-I allele, in fibroblast-like cells. Pulse labeling of cell surface eL d with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC-I conformers. Kinetic modeling, using in-house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eL d demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eL d distributed into pre-degradative multivesicular bodies (MVBs), these LE subsets were not a source for eL d recycling. The LE recycling of eL d did not require Rab7 membrane domains, as demonstrated by Rab7-silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eL d in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. This article is protected by copyright. All rights reserved

Description: Neuronatin (NNAT) was first identified as a brain-specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post-developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca 2+ signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. This article is protected by copyright. All rights reserved

Description: Due to the major role played by diatoms in the marine biological pump, opal/biogenic silica (bSi) has a strong potential as a proxy for paleoproduction reconstructions. Here, we present a detailed evaluation of the independent Fourier transform infrared spectroscopy (FTIRS) calibration introduced by Meyer-Jacob et al. (2014 a ), which is based on synthetic sediment mixtures with known concentrations of bSi and the application of partial least squares (PLS) regression, for the determination of bSi in marine sediments. To test the robustness of the FTIRS calibration for marine samples we selected 199 globally distributed samples, with available wet chemically inferred bSi concentrations and compared the results of both methods. We observe that the spectral characteristics in marine samples are comparable to those reported for a synthetic FTIR spectrum composed solely of spectral features in quartz, biogenic silica, and calcite. FTIRS-inferred and wet-chemically measured bSi concentrations for the set of 199 marine sediment samples are strongly correlated ( R 2  = 0.87; RMSE = 5.27%). Together with the high reproducibility of bSi values by the independent calibration our results corroborate the applicability of the independent FTIRS bSi calibration for quantification in marine samples. This together with the succinct sample preparation procedure, rapid analysis, reduced consumable and machine maintenance costs makes FTIRS a powerful and robust alternative for the quantitative analysis of bSi in marine sediments.

Description: MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post-transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid-lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs and 821 pre-miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR-145) had significant differences in expression over the lactation cycle. Further studies revealed that miR-145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR-145. Overexpression of miR-145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR-145 impaired fatty acid synthesis. Inhibition of miR-145 increased methylation levels of fatty acid synthase ( FASN ), stearoyl-CoA desaturase 1 ( SCD1 ), peroxisome proliferator-activated receptor gamma ( PPARG ), and sterol regulatory element binding transcription factor 1 ( SREBF1 ). Luciferase reporter assays confirmed that insulin induced gene 1 ( INSIG1 ) is a direct target of miR-145. These findings underscore the need for further studies to evaluate the potential for targeting miR-145 for improving beneficial milk components in ruminant milk. This article is protected by copyright. All rights reserved

Description: Thrombin initiates proinflammatory signaling responses through activation of protease-activated receptors (PARs) in in vitro and in vivo systems. Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. Thrombin as a potent signaling molecule is strongly implicated in a number of proinflammatory disorders including severe sepsis, cancer, cardiovascular disease, and of special interest in this review neurodegenerative disorders. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases. This article is protected by copyright. All rights reserved

Description: Brain cancers account for less than 1,5% of all new cancer cases reported in the United States each year. Due to their invasive and heterogeneous nature, in addition to their resistance to multimodal treatments, these tumors are usually fatal. Gliomas, and in particular high-grade astrocytomas such as glioblastoma multiforme (GBM), are the most common and lethal primary tumors of the central nervous system. The median survival of most patients is less than one year after application of multimodal therapies. The question is why are these cancers so injurious? And above all, how is it possible for a so carefully orchestrated area like the brain to develop such tumors? This brings us to the study of glioma stem cells, their specialized niches (perivascular and hypoxic), and the neurogenic phenomena that takes place within the adult ventricular-subventricular zone: a structure that lies at the intersection between brain development and gliomagenesis. This article is protected by copyright. All rights reserved

Description: ABSTRACT Diabetic retinopathy (DR) is a serious complication of diabetes mellitus affecting about one third of diabetic adults. Despite its prevalence, treatment options are limited and often implemented only in the later stages of the disease. To date, the pathogenesis of DR has been extensively characterized in the context of elevated glucose, insulin, and VEGF signaling, although a growing number of other growth factors and molecules, including transforming growth factor-β (TGF-β) are being recognized as important contributors and/or therapeutic targets. Here, we review the complex roles of TGF-β signaling in DR pathogenesis and progression. This article is protected by copyright. All rights reserved

Description: Long-term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). We previously showed that CS destabilizes the networked morphology of mitochondria in human ASM by regulating the expression of mitochondrial fission and fusion proteins via multiple signaling mechanisms. Emerging data link regulation of mitochondrial morphology to cellular structure and function. We hypothesized that CS-induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling. Here, ASM cells were exposed to 1% cigarette smoke extract (CSE) for 48 hours to alter mitochondrial fission/fusion, or by inhibiting the fission protein Drp1 or the fusion protein Mfn2. Mitochondrial function was assessed via changes in bioenergetics or altered rates of proliferation and apoptosis. Our results indicate that both exposure to CS and inhibition of mitochondrial fission/ fusion proteins affect mitochondrial function (i.e., energy metabolism, proliferation and apoptosis) in ASM cells. In vivo , the airways in mice chronically exposed to CS are thickened and fibrotic, and the expression of proteins involved in mitochondrial function is dramatically altered in the ASM of these mice. We conclude that CS-induced changes in mitochondrial morphology (fission/ fusion balance) correlate with mitochondrial function, and thus may control ASM proliferation, which plays a central role in airway health. This article is protected by copyright. All rights reserved

Description: There is still a considerable debate concerning whether uric acid is neuroprotective or neurotoxic agent. To clarify this topic we tested the effects of uric acid on neuronal cells biology by using differentiated SHSY5Y neuroblastoma cells incubated with amyloid ß to reproduce an in vitro model of Alzheimer's disease. The incubation of cells with uric acid at the dose of 40 µM or higher significantly reduced cell viability and potentiated the proapoptotic effect of amyloid ß. Finally, uric acid enhanced the generation of 4-hydroxynonenal and the expression of PPARβ/δ promoted by amyloid β, indicating a prooxidant effects. In conclusion, uric acid could exert a detrimental influence on neuronal cell biology being this influence further potentiated by concomitant exposure to neurotoxic stimuli. This effect is evident for uric acid concentrations close to those achievable in cerebrospinal fluid in presence of mild hyperuricemia thus suggesting a potential role of uric acid in pathophysiology of cognitive dysfunction. These effects are influenced by the concentrations of uric acid and by the presence of favouring conditions that commonly occur in neurodegenerative disorders and well as in the aging brain, including increased oxidative stress and exposure to amyloid β. This article is protected by copyright. All rights reserved

Description: Choline is a quaternary ammonium base that represents an essential component of phospholipids and cell membranes. Malignant transformation is associated with an abnormal choline metabolism at a higher levels with respect to those exclusively due to cell multiplication. The use of Positron Emission Tomography/Computed Tomography (PET/CT) with radiocholine (RCH), labeled with 11 C or 18 F, is widely diffuse in oncology, with main reference to restaging of patients with prostate cancer. The enhanced concentration in neoplasm is based not only on the increasing growing rate, but also on more specific issues, such as the augmented uptake in malignant cells due to the up-regulation of choline kinase. Furthermore the role of hypoxia in decreasing choline's uptake determine an in vivo concentration only in well oxygenated tumors, with a lower uptake when malignancy increases, i.e. in tumors positive at 18 F-Fluoro-deoxyglucose. In this paper we have analyzed the most important issues related to the possible utilization of RCH in diagnostic imaging of human cancer. This article is protected by copyright. All rights reserved

Description: Bivalve larvae are small (50–400 μ m) and difficult to identify using standard microscopy, thus limiting inferences from samples collected in the field. With the advent of ShellBi, an image analysis technique, accurate identification of bivalve larvae is now possible but rapid image acquisition and processing remains a challenge. The objectives of this research were to (1) develop a benchtop automated image acquisition system for use with ShellBi, (2) evaluate the system, and (3) create a protocol that would maintain high classification accuracies for larvae of the eastern oyster, Crassostrea virginica . The automated system decreased image acquisition time from 2–13 h to 46 min per slide and resulted in the highest classification accuracies at the lowest tested magnification (7X) and shortest image acquisition time (46 min). Quality control tests indicated that classification accuracies were sensitive to camera and light source settings and that measuring changes in light source and color channel intensities over time was an important part of quality control during routine operations. Validation experiments indicated that under proper settings, automated image acquisition coupled with ShellBi could rapidly classify C. virginica larvae with high accuracies (80–93%). Results suggest that this automated image acquisition system coupled with ShellBi can be used to rapidly image plankton samples and classify C. virginica larvae allowing for expanded capability to understand bivalve larval ecology in the field. Additionally, the automated system has application for rapidly imaging other planktonic organisms at high magnification.

Description: Recently, Sack et al . presented an interesting, novel data set in JCP examining the effect of substrate stiffness on VEGF processing and signaling. The data represents a clear contribution to the field. However, the authors' conclusion that “extracellular matrix binding is essential for VEGF internalization” conflicts with other knowledge in the field, and is not supported by their data. Instead, their data demonstrates the effect of heparin addition and changing ECM stiffness on both VEGF binding to fibronectin and VEGF binding to endothelial receptors. This is consistent with other work showing that matrix- binding reduces VEGF-VEGFR internalization, shifting downstream signaling. This article is protected by copyright. All rights reserved

Description: PC3/Tis21 is a transcriptional cofactor that inhibits proliferation in several cell types, including neural progenitors. Here we report that PC3/Tis21 associates with HDAC1, HDAC4 and HDAC9 in vivo , in fibroblast cells. Furthermore, when HDAC1, HDAC4 or HDAC9 are silenced in fibroblasts or in a line of cerebellar progenitor cells, the ability of PC3/Tis21 to inhibit proliferation is significantly reduced. Overexpression of HDAC1, HDAC4 or HDAC9 in fibroblasts and in cerebellar precursor cells synergizes with PC3/Tis21 in inhibiting the expression of cyclin D1, a cyclin selectively inhibited by PC3/Tis21. Conversely, the depletion of HDAC1 or HDAC4 (but not HDAC9) in fibroblasts and in cerebellar precursor cells significantly impairs the ability of PC3/Tis21 to inhibit cyclin D1 expression. An analysis of HDAC4 deletion mutants shows that both the amino- terminal moiety and the catalytic domain of HDAC4 associate to PC3/Tis21, but neither alone is sufficient to potentiate the inhibition of cyclin D1 by PC3/Tis21. As a whole, our findings indicate that PC3/Tis21 inhibits cell proliferation in a way dependent on the presence of HDACs, in fibroblasts as well as in neural cells. Considering that several reports have demonstrated that HDACs can act as transcriptional corepressors on the cyclin D1 promoter, our data suggest that the association of PC3/Tis21 to HDACs is functional to recruit them to target genes, such as cyclin D1, for repression of their expression. This article is protected by copyright. All rights reserved

Description: Epithelial–mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis in HCC. Growing evidence indicates that upregulation of Snail, the major EMT inducer, significantly correlates with the metastasis and poor prognosis of HCC. Here, we investigate the underlying mechanism of miR-30b in suppressing metastasis of hepatoma cells by targeting Snail. In this study, we found that miR-30b was significantly downregulated and negatively associated with Snai1 production in HCC cell lines with higher metastatic potentials. Gain- and loss-of-function studies revealed that miR-30b could dramatically inhibit in vitro HCC cell migration and invasion. In vivo orthotopic liver xenograft model further demonstrated that stable over-expression of miR-30b significantly repressed the local invasion and lung metastasis of hepatoma cells. Meanwhile, the restoration of miR-30b expression suppressed the distant colonization of hepatoma cells. Both gain- and loss-of-function studies showed that miR-30b suppressed the EMT of hepatoma cells as indicated by the morphology changes and deregulation of epithelial and mesenchymal markers. Using RNAi, we further investigated the role of Snai1 in HCC cell EMT and demonstrated that knockdown of Snai1 significantly inhibited the EMT and cancer cell metastasis. Additionally, miR-30b exhibited inhibitory effects on HCC cell proliferation in vitro and in vivo . In conclusion, our findings highlight the significance of miR-30b downregulation in HCC tumor metastasis and invasiveness, and implicate a new potential therapeutic target for HCC metastasis. This article is protected by copyright. All rights reserved

Description: The molecular and cellular mechanisms underlying vascular remodeling are currently investigated several experimental strategies which aim to mimic the complex environmental conditions found in vivo . Some of them focus on the tubulogenic activity of dispersed endothelial cell populations, while others evaluate vascular sprouting. Here we propose a new method to assess matrigel invasion starting from confluent or subconfluent monolayers of human microvascular ECs (HMVEC) seeded on different substrates. The experimental setting is also validated by an improved hybrid multiscale mathematical approach, which integrates a mesoscopic grid-based cellular Potts model, that describes HMVEC phenomenology, with a continuous one, accounting for the kinetics of diffusing growth factors. Both experimental and theoretical approaches show that the endothelial potential to invade, migrate and organize in tubule structures is a function of selected environmental parameters. The present methodology is intended to be simple to use, standardized for rapid screening and suitable for mechanistic studies. This article is protected by copyright. All rights reserved

Description: A lack of fine-scale methods for sampling planktonic microbial populations hinders advancement in understanding the responses of these communities to environmental conditions. Current methods provide resolution at scales of centimeters to meters, but not at the millimeter-scale required to understand highly stratified communities. To address this we developed two cryogenic sampling tools to collect spatially-precise samples from aquatic environments while simultaneously preserving the microbial communities. The application of these samplers was examined over a 5.5 h period using a cyanobacterial scum ( Microcystis ) formed in experimental mesocosms. A cryogenic “surface snatcher” collected a discrete layer (ca. 1 mm) of surface water. Compared to conventional surface sampling methods, the surface snatcher samples contained up to 22-times more microcystin, indicating that less underlying water was incorporated into the sample. A cryogenic “cold finger” sampler was used to collect vertical profiles of the upper 40 mm of the water column. This profiler provided new insights into the fine-scale structure of Microcystis scums, demonstrating that more microcystin-producing Microcystis was contained in the surface 5 mm than the 35 mm below. The results also showed that upregulation of microcystin production was highly localized in the top 2.5 mm of the Microcystis scum. Our results demonstrate that extreme changes in cyanobacterial communities can occur over small distances, and indicate that sampling resolution is of great importance for improving knowledge on cyanobacterial blooms and toxin production. While this study focused on microcystin-producing Microcystis , the cryogenic sampling tools described here could be applied to any planktonic microbial community.

Description: A “StingerCam” camera system provided high temporal resolution image data on the presence of large cubozoan jellyfish over nearly five years on the tropical coast of northeastern Australia. There was strong seasonality in the occurrence of Chironex fleckeri and an unnamed species of the family Carybdeidae ( Morbakka spp.). Jellyfish of both species were only found between December and May of each year; primarily in the wet season. It was estimated that jellyfish were released from polyps from September. From a sample of 〉1000 C. fleckeri and 493 Morbakka we determined the temperature and salinity range in which these taxa were detected being between 21.7°C–31.6°C and 25.2–34.9 PSU for C. fleckeri , and 20.2–30.2°C and 25.4–35.4 PSU for Morbakka sp. Daily wind speed influenced detection rates with less jellyfish observed in winds that exceeded 28 km h −1 . Data reduction software greatly improved processing time by identifying images without jellyfish with an accuracy of 93–98% in the two case studies used here, an image series from a night when jellyfish were highly active and in winter when jellyfish were absent. The StingerCam not only provided strong ecological data and information of high utility to reduce the risk of envenomation to the public, but also detected boney fishes, sharks and marine reptiles. We conclude that StingerCams are an effective way of collecting data to determine the range of physical conditions that jellyfish can tolerate and this information can be used in predictive models; especially given a global focus on climate change.

Description: Clusterin (CLU) is a chaperone-like protein and plays a protective role against renal ischemia-reperfusion injury (IRI); however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU-mediating pathways in kidney cells by using bioinformatics analysis. CLU null renal tubular epithelial cells (TECs) expressing human CLU cDNA (TEC-CLU hCLU ) or empty vector (TEC-CLU -/- ) were exposed to normoxia or hypoxia (1% O 2 ). Transcriptome profiling with a significant 2-fold change was performed using SurePrint G3 Mouse Gene Expression 8 × 60K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis. Here, we showed that compared to CLU null controls, ectopic expression of human CLU in CLU null kidney cells promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU expression affected expression of 3864 transcripts (1893 up-regulated) in normoxia and 3670 transcripts (1925 up-regulated) in hypoxia. CLU functions in normoxia were associated mostly with AKT2/PPP2R2B-dependent PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling and as well with GSK3B-mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU-enhanced cell survival in hypoxia was also associated with PIK3CD/MAPK1-dependent PI3K/AKT, HIF-α, PTEN, VEGF and ERK/MAPK signaling. In conclusion, our data showed that CLU functions in kidney cells were mainly mediated in a cascade manner by PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney. This article is protected by copyright. All rights reserved

Description: Glioblastoma, the most common brain tumor, is characterized by high proliferation rate, invasion, angiogenesis and chemo- and radio-resistance. One of most remarkable feature of glioblastoma is the switch toward a glycolytic energetic metabolism that leads to high glucose uptake and consumption and a strong production of lactate. Activation of several oncogene pathways like Akt, c-myc and ras, induces glycolysis and angiogenesis and acts to assure glycolysis prosecution, tumor proliferation and resistance to therapy. Therefore, the high glycolytic flux depends on the overexpression of glycolysis-related genes resulting in an overproduction of pyruvate and lactate. Metabolism of glioblastoma thus represents a key issue for cancer research. Flavopiridol is a synthetic flavonoid that inhibits a wide range of Cyclin-dependent kinase, that has been demonstrate to inactivate glycogen phosphorylase, decreasing glucose availability for glycolysis. In this work is reported the study of glucose metabolism upon flavopiridol treatment in the two different glioblastoma cell lines. The results obtained point towards an effect of flavopiridol in glycolytic cells, thus suggesting a possible new use of this compound or flavopiridol-derived formulations in combination with anti-proliferative agents in glioblastoma patients. This article is protected by copyright. All rights reserved

Description: Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi-lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1 , Scl/tal1 , and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down-regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi-lineage hematopoiesis in the early yolk sac of developing embryos. This article is protected by copyright. All rights reserved

Description: The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell-matrix mechanical crosstalk and disease-induced tissue stiffness alterations. This article is protected by copyright. All rights reserved

Description: Immune function, inflammation and atherosclerosis display sex differences and are influenced by 17β-estradiol through estrogen receptors subtypes ERα and ERβ. Male tissues express active ERs, but their possible involvement in inflammation in males has never been assessed. Macrophages express both ERα and ERβ and offer the opportunity to evaluate the role of ER levels and activation in inflammation. We assessed the ability of lipopolysaccharide (LPS) to modulate, in a sex-specific way, the expression and the activation status of ERα and ERβ in blood monocytes-derived macrophages (MDMs) from men and women. MDMs were incubated with 100 ng/ml LPS for 24 h and used to evaluate ERα, ERβ, P- ERα, p38 and P-p38 expression by Western Blotting. In basal conditions, ERα and ERβ were significantly higher in female MDMs than in male MDMs. LPS up-regulated ERα and ERα phosphorylation in both sexes, with a significantly higher effect observed in male MDMs, and down-regulated ERβ level only in female MDMs. p38 and P-p38 proteins, indicative of ERβ activity, did not show sex differences both in basal conditions and after LPS treatment. Finally, ERα/ERβ and P-ERα/ERα ratios were significantly higher in male MDMs than in female ones. Our data indicate, for the first time, that LPS affects ERα but not ERβ activation status. We identify a significant role of ERα in LPS-mediated inflammatory responses in MDMs, which represents an initial step in understanding the influence of sex in the relationship between LPS and ERα. This article is protected by copyright. All rights reserved

Description: The cardiac Z-line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z-line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z-line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z-line proteins and the pathways leading from mutations in Z-line proteins to cardiomyopathy, although genotype-phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z-line and Z-line associated proteins involved in human cardiomyopathies with special emphasis on knock-in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z-line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. This article is protected by copyright. All rights reserved

Description: Chrysophanol is an anthraquinone compound, mainly isolated from rhubarb, with anti-cancer effects on some types of cancer cells. However, effects of chrysophanol on human choriocarcinoma cells are not known. Therefore, the objective of this study was to determine effects of chrysophanol on choriocarcinoma cells (JAR and JEG-3) and identify signal transduction cascades activated by chrysophanol. Results of present study showed that chrysophanol decreased cell viability and induced apoptosis of JEG-3, but not JAR cells, in a dose-dependent manner. Chrysophanol also increased oxidative stress in JEG-3 cells by inducing ROS generation followed by mitochondrial dysfunction including depolarization of mitochondrial inner membrane potential. Western blot analysis revealed that ERK1/2, P90RSK, AKT and P70S6K were increased significantly in JEG-3 cells by chrysophanol. Next, we investigated chrysophanol-mediated effects on proliferation of JEG-3 cells using pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126). Inhibition of AKT and ERK1/2 prevented chrysophanol-induced stimulation of proliferation of JEG-3 cells. In addition, the phosphorylation of AKT and ERK1/2 was suppressed by LY294002 and U0126 in JEG-3 cells treated with chrysophanol, whereas the AKT protein was activated by pre-treatment of JEG-3 cells with U0126. Furthermore, we compared therapeutic effects of chrysophanol with cisplatin and paclitaxel which are conventional salvage regimens for choriocarcinoma. Our results verified that chrysophanol has synergistic effects with traditional therapy to increase apoptosis of JEG-3 cells. Collectively, these results indicate that chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy and minimizing side effects of conventional treatment regimens. This article is protected by copyright. All rights reserved

Description: Molecular size and dissolved organic carbon (DOC) distributions of dissolved organic matter (DOM) in seawater were determined qualitatively and quantitatively by high-performance size exclusion chromatography (HPSEC). To estimate the relationship between molecular size distribution and the DOC concentration of seawater DOM, seawater was desalinated with an electric dialyzer. The recent technological advancements of desalination make us possible to recover a high percentage of seawater DOM. Surface coastal seawater samples were collected from three locations in Japan: Tokyo Bay, Kashima Port, and Cape Inubo. Samples were evaluated by a chromatography system coupled with ultraviolet (UV) absorbance, fluorescence, and non-dispersive infrared total organic carbon detectors in series. Two peaks, 1 (1.24 × 10 5 −1.54 × 10 5 Da) and 2 (9.48 × 10 2 −1.81 × 10 3 Da), were detected in all sample chromatograms. Peak 1 showed little fluorescence and UV absorption in all samples. It has been shown that carbohydrates, one of the main components of seawater DOM, exhibit almost no fluorescence or UV absorption; thus, Peak 1 could mainly consist of carbohydrates which are usually labile DOM. Total DOC concentrations of the Tokyo Bay, Kashima Port, and Cape Inubo were 1.38, 0.92, and 0.80 mg C L −1 , respectively. Peak 2 accounted for 75–80% of the total DOC and showed a substantial humic-like fluorescence and UV absorption in all samples. These results indicate that the photochemical characteristics of seawater DOM significantly differ depending on its molecular size. Thus, analysis of molecular size with our HPSEC provides a new approach for quantitative and qualitative characterization of seawater DOM.

Description: The 15 N isotope pairing technique is widely used to quantify anammox, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) in sediments. However, the effects of DNRA on anammox and denitrification in slurry incubations and on genuine N 2 production in intact core incubations have not been fully explored. We developed a mathematical model describing these effects of DNRA, and tested this model in field and computational studies. Our model calculations revealed that for slurry incubations the presence of DNRA tends to under -and overestimate the actual anammox and denitrification rates, respectively, when calculated according to Thamdrup and Dalsgaard (2002). We found this underestimate of anammox to be proportional to the 15 mole fraction ( F A ), and the overestimate of denitrification to be related to both F A and the relative contribution of anammox to the total N 2 production ( ra ). We propose three alternative procedures to better quantify anammox and denitrification rates in slurry incubations and two procedures for intact core incubations. Our model calculations also revealed that for intact core incubations, the presence of DNRA leads to an overestimate of the genuine N 2 production rate when calculated according to Risgaard-Petersen et al. (2003). This overestimate depends on the ra and the mole fractions of 15 and 15 in intact core incubations. The results of our field experiments and numerical modeling indicated that the overestimate of the genuine N 2 production was less than 1% at two sites in the East China Sea.

Description: Conventional sensors for the quantification of O 2 availability in aquatic environments typically have limits of detection (LOD) of 〉 1 μmol L −1 and do not have sufficient resolution to reliably measure concentrations in strongly O 2 depleted environments. We present a novel trace optical sensor based on the palladium(II)-benzoporphyrin luminophore, immobilized in a perfluorinated matrix with high O 2 permeability. The trace sensor has a detection limit of ∼5 nmol L −1 with a dynamic range extending up to ∼2 μmol L −1 . The sensor demonstrates a response time 〈 30 s and a small, predictable, and fully reversible response to hydrostatic pressure and temperature. The sensor showed excellent stability for continuously measurements during depth profiling in Oxygen Minimum Zones (OMZ). The novel sensor was deployed in situ using a Trace Oxygen Profiler instrument (TOP) equipped with two additional O 2 optical sensors, with higher dynamic range, allowing, when combined, measurements of O 2 concentration from ∼5 nmol L −1 to 1000 μmol L −1 with a single instrument. The TOP instrument was deployed in the OMZ regions of the Eastern Tropical North Pacific (ETNP) and Bay of Bengal (BoB). The measurements demonstrated that O 2 concentrations in the ETNP generally were below the LOD of the trace sensor, but that large sub-micromolar O 2 intrusions, spanning 60–80 m with maximum O 2 concentrations above 50 nmol L −1 , could be observed in the OMZ core. The O 2 concentrations in the BoB were high compared to the ETNP and rarely decreased below 50 nmol L −1 , but demonstrated tremendous small-scale variability.

Description: Proteasome-mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well-documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. 62 of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP-PIPs were associated with protein biosynthesis and, unlike CP-PIPs, were under represented by chaperon/ATP-binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. This article is protected by copyright. All rights reserved

Description: ABSTRACT The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein-protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. This article is protected by copyright. All rights reserved

Description: Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow-induced vascular remodeling by increasing MMP activity. TIMP1 deficient ( Timp1 −/− ) and wild-type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha-1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1 −/− mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1 −/− mice. Timp1 −/− mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1 −/− mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF-A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1 −/− mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. This article is protected by copyright. All rights reserved

Description: Coumestrol is the one of the major phytoestrogens which is abundant in soybeans, legumes, brussel sprouts and spinach. The beneficial effects of coumestrol are well known in various biological processes including; neuroprotective effects on the nervous system, function of the female reproductive system, anti-bacterial properties and anti-cancer effects. Although the anti-tumor activity of coumestrol has been demonstrated for ovarian, breast, lung and cervical cancers, little is known of its effects on prostate cancer. Therefore, in the present study, we investigated the chemotherapeutic effects of coumestrol on two prostate cancer cell lines, PC3 and LNCaP. Our results showed that coumestrol decreased proliferation and migration and induced apoptosis in both PC3 and LNCaP cells. Moreover, effects of coumestrol on cell signaling pathways were investigated and it increased phosphorylation of ERK1/2, JNK, P90RSK and P53 proteins in a dose- and time-dependent manner whereas phosphorylation of AKT was reduced by coumestrol under the same conditions for culture of PC3 and LNCaP cells. In addition, mitochondrial dysfunction was induced by coumestrol as evidenced by a significant loss of mitochondrial membrane potential. Furthermore, cleavage of caspase-3 and caspase-9, the apoptotic proteins associated with mitochondria, also changed in response to coumestrol. Coumestrol also caused mitochondrial dysfunction resulting in an increase in ROS production in PC3 and LNCaP cells. These results suggest that coumestrol can inhibit progression of prostate cancer and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signaling pathways. This article is protected by copyright. All rights reserved

Description: A sensitive and specific ultra-high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC–MS/MS) method was developed for the purpose of decreasing the detection limit of quantification (LOQ) and reducing sample analysis time. In total, 14 marker pigments were determined and validated using an internal standard (IS) method with trans-8′-Apo-beta-carotenal as the IS. Electrospray ionization in positive mode was applied for the detection of pigments. The 14 types of pigments were separated in less than 15 min and presented good linearity, with the coefficients ranging from 0.9915 to 0.9991 using a UHPLC C8 column. The LOQ attained for 14 pigments were from 0.005 ng mL −1 to 0.500 ng mL −1 , which enabled quantification of the pigments in seawater at ppt level. Both intra-day and inter-day precisions were less than 15%. The method was validated using seawater samples and the recovery of the analytes ranged from 62.8% to 120.2% at two spiked concentrations. Compared with previous methods, UHPLC–MS/MS method improved the efficiency of qualitative analysis and significantly decreased the detection LOQ for analyzing marker phytoplankton pigments at trace/ultra-trace levels in complex matrices.

Description: Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)-dependent pathway. We previously reported that 3-methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3-MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt-mediated mechanism. The mechanism underlying the effects of 3-MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3-MC modified heat shock protein 90 (HSP90), caveolin-1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA-dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3-MC reduced eNOS phosphorylation through the AhR/RhoA-mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3-MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin-1. Immunofluorescence and western blot analysis revealed that 3-MC reduced the amount of membrane-bound activated eNOS, and a modified Griess assay revealed that 3-MC concomitantly reduced NO production. However, simvastatin reduced 3-MC–mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3-MC. This article is protected by copyright. All rights reserved

Description: Measuring the quantity and composition of sinking particulate matter is key to understanding biogeochemical processes in the ocean. This has been done in the past with sequencing sediment traps, which collect and store particulate matter in sample bottles for subsequent laboratory analysis. Having a limited number of bottles, these traditional traps force the user to choose between finer time resolution and longer deployment duration. We have built a new sediment trap that analyzes the collected material in situ, eliminating the need to preserve and store the samples. This new instrument, the Sedimentation Event Sensor (SES), captures macro images of the sample with front and back lighting, and takes fluorometric measurements in two bands as proxies for the presence of chlorophyll a and accessory pigments. The SES can process 6200 samples during a single deployment, which is the equivalent of 15 samples per day for more than a year. Here, we describe the design of the SES and present the results of its first three deployments at 3910 m depth. Images and fluorometry data revealed high variability of sinking particulate matter composition on the order of hours. Sedimentation patterns detected by the SES largely agreed with mass flux patterns measured from traditional traps deployed concurrently nearby. Given the functional differences, the SES is best used to complement rather than replace traditional sediment traps.

Description: Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. This article is protected by copyright. All rights reserved

Description: An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH) 2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH) 2 D 3 and 25(OH)D 3 affect myoblast proliferation, differentiation and myotube size and whether these cells are able to metabolize 25(OH)D 3 and 1,25(OH) 2 D 3 . We showed that myoblasts not only respond to 1,25(OH) 2 D 3 , but also to the precursor 25(OH)D 3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH) 2 D 3 as well as 25(OH)D 3 stimulate VDR mRNA expression and in myotubes 1,25(OH) 2 D 3 also stimulates MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes express CYP27B1 and CYP24 mRNA which are required for vitamin D 3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH) 2 D 3 or 25(OH)D 3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D 3 to 24R,25(OH) 2 D 3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D 3 metabolites, but is also able to metabolize 25(OH)D 3 and 1,25(OH) 2 D 3 . This article is protected by copyright. All rights reserved

Description: It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale . POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum . Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from Day 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. This article is protected by copyright. All rights reserved

Description: Inhibition of RUNX2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)-induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2 dox cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2-driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT-qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 ( Wif1 ), and Wif1 protein was readily detectable in medium conditioned by cultures co-treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox-driven alkaline phosphatase (ALP) activity in control ST2/Rx2 dox cells, but not in cells in which Wif1 was silenced. Unlike its anti-mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S-phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2-driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2 dox cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1 . This article is protected by copyright. All rights reserved

Description: ABSTRACT The last decade has seen significant progress in understanding the organisation of regenerative cells in the adult lung. Cell lineage tracing and in vitro clonogenic assays have enabled the identification and characterisation of endogenous lung epithelial stem and progenitor cells. Selective lung injury models, and genetically engineered mice have revealed highly conserved gene networks, factors, signalling pathways, and cellular interactions important in maintaining lung homeostasis and regulating lung regeneration and repair following injury. This review describes the current models of lung epithelial stem and progenitor cell organisation in adult mice, and the impediments encountered in translational studies aiming to identify and characterise their human homologs. This article is protected by copyright. All rights reserved

Description: A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt-villus axis. One important check-point in this process occurs in the transit amplifying zone of the intestinal crypts where different signalling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development and tissue renewal. This article is protected by copyright. All rights reserved

Description: ABSTRACT DICER is the central enzyme that cleaves precursor microRNAs (miRNAs) into 21-25 nucleotide duplex in cell lineage differentiation, identity and survival. In the current study, we characterized the specific bone metabolism genes and corresponding miRNAs and found that DICER and Runt-related transcription factor 2 (Runx2) expressions increased simultaneously during osteogenic differentiation. Luciferase assay showed that Runx2 significantly increased the expression levels of DICER luciferase promoter reporter. Our analysis also revealed weaker DICER expression in embryos of Runx2 knock out mice (Runx2 -/-) compared with that of Runx2 +/− and Runx2 +/+ mice. We further established the calvarial bone critical-size defect (CSD) mouse model. The bone marrow stromal cells (BMSCs) transfected with siRNA targeting DICER were combined with silk scaffolds and transplanted into calvarial bone CSDs. Five weeks post-surgery, micro-CT analysis revealed impaired bone formation and repairing in calvarial defects with the siRNA targeting DICER group. In conclusion, our results suggest that DICER is specifically regulated by osteogenic master gene Runx2 that binds to the DICER promoter. Consequently, DICER cleaves precursors of miR-335-5p and miR-17-92 cluster to form mature miRNAs, which target and decrease the Dickkopf-related protein 1 (DKK1) and proapoptotic factor BIM levels, respectively, leading to an enhanced Wnt/β-catenin signaling pathway. These intriguing results reveal a central mechanism underlying lineage-specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. Our study also suggests a potential application of modulating DICER expression for bone tissue repair and regeneration. This article is protected by copyright. All rights reserved

Description: Genetic experiments have positioned the fgfr1 gene at the top of the gene hierarchy that governs gastrulation, as well as the subsequent development of the major body axes, nervous system, muscles, and bones, by affecting downstream genes that control the cell cycle, pluripotency and differentiation, as well as microRNAs. Studies show that this regulation is executed by a single protein, the nuclear isoform of FGFR1 (nFGFR1), which integrates signals from development-initiating factors such as Retinoic Acid (RA), and operates at the interface of genomic and epigenomic information. nFGFR1 cooperates with a multitude of Transcriptional Factors (TFs), and targets thousands of genes encoding for mRNAs, as well as miRNAs in top ontogenic networks. nFGFR1 binds to the promoters of ancient proto-oncogenes and tumor suppressor genes, in addition to binding to metazoan morphogens that delineate body axes, and construct the nervous system, as well as mesodermal and endodermal tissues. The discovery of pan-ontogenic gene programming by Integrative Nuclear FGFR1 Signaling (INFS) impacts our understanding of ontogeny, as well as developmental pathologies, and holds new promise for reconstructive medicine, and cancer therapy. This article is protected by copyright. All rights reserved

Description: A microwave-preservation method was developed to quantify total dimethylsulfoniopropionate (DMSP T ), dimethylsulfoxide (DMSO T ), and acrylate (acrylate T ) concentrations in unfiltered samples to alleviate problems associated with the acidification method when applied to samples containing Phaeocystis . Microwave- and acid-preservation methods were compared using batch cultures of Phaeocystis antarctica and 11 other marine phytoplankton species for DMSP T , batch P. antarctica cultures for DMSO T and acrylate T , and unfiltered Delaware Estuary water samples for DMSP T to demonstrate the general applicability of this method. Acidification of P. antarctica culture samples resulted in the underestimation of DMSP T (42–69%) and overestimation of dimethylsulfide (DMS) (2156–3819%), DMSO T (9–101%), and acrylate T (71–249%). By comparison, DMSP T concentrations in microwaved samples agreed with non-microwaved, non-acidified controls. In contrast to P. antarctica results, the microwave- and acid-preservation methods yielded DMSP T concentrations that were statistically indistinguishable for 11 other marine phytoplankton species and Delaware Estuary samples. Unfiltered samples stored frozen following microwave treatment or stored at room temperature if acidified after the microwaving step, resulted in no change in DMSP T or acrylate T ; DMSO T concentrations increased slightly (∼ 15%) when they were not sparged to remove DMS prior to acidification and room temperature storage. Based on these findings, we propose microwaving small sample volumes (≤ 7 mL) of unfiltered seawater or culture samples as a general approach to preserve samples for subsequent DMSP T , DMSO T , and acrylate T analyses, especially when the phytoplankton composition of the samples is unknown.

Description: Increasing human activities cause local to global changes in sea surface temperatures, ocean acidity, eutrophication, and rising sea levels. Many laboratory experiments investigate the effects of these regime shifts on single species and single stressors, showing variable responses within and among species, while different combinations of stressors can have synergistic, additive or antagonistic effects. Large-scale multi-species and multi-stressor experiments can more reliably predict future ecosystem changes. A unique mesocosm facility was developed and set up at the AWI Wadden Sea Station – Sylt, Northern Germany to investigate the particular effects of future climate changes on predominant marine intertidal communities. Each of 12 benthic mesocosms serves as an independent experimental unit with novel techniques of tide and current simulations as well as multi parameter measurement systems to simulate multi-factorial climate change scenarios including the combination of warming, acidification, nutrient enrichment, and sea level rise. Temperature, pH, oxygen, and salinity can be continuously monitored and logged, while discretely collected samples of total alkalinity, light availability, chlorophyll a (Chl a ), nutrients and seston supplement these online datasets. Herein we demonstrate the functionality of the new benthic mesocosm system including first experimental results on the responses of Fucus vesiculosus forma mytili , and its associated community to the combination of warming, ocean acidification, and increased nutrient enrichment.

Description: The phenotype of primary cells in culture varies according to the donor environmental condition. We recently showed that the time of the day imposes a molecular program linked to the inflammatory response that is heritable in culture. Here we investigated whether microRNAs (miRNAs) would show differential expression according to the time when cells were obtained, namely daytime or nighttime. Cells obtained from explants of cremaster muscle and cultivated until confluence (∼20 days) presented high CD133 expression. Global miRNA expression analysis was performed through deep sequencing in order to compare both cultured cells. A total of 504 mature miRNAs were identified, with a specific miRNA signature being associated to the light versus dark phase of a circadian cycle. miR-1249 and miR-129-2-3p were highly expressed in daytime cells, while miR-182, miR-96-5p, miR-146a-3p, miR-146a-5p and miR-223-3p were highly expressed in nighttime cells. Nighttime cells are regulated for programs involved in cell processes and development, as well as in the inflammation, cell differentiation and maturation; while daytime cells express miRNAs that control stemness and cytoskeleton remodeling. In summary, the time of the day imposes a differential profile regarding to miRNA signature on CD133 + cells in culture. Understanding this daily profile in the phenotype of cultured cells is highly relevant for clinical outputs, including cellular therapy approaches. This article is protected by copyright. All rights reserved

Description: Mechanisms behind the emergence of brown adipocyte-like (brite or beige) adipocytes within white adipose tissue (WAT) are of interest. Retinoblastoma protein gene (Rb) haploinsufficiency associates in mice with improved metabolic regulation linked to a greater capacity for fatty acid oxidation and thermogenesis in WAT. We aimed to explain a feasible mechanism of WAT-to-BAT remodeling in this model. Differentiated primary adipocytes and Sca1-positive preadipocytes derived from adipose depots of Rb +/− mice and wild-type siblings were compared. Primary white Rb +/− adipocytes displayed under basal conditions increased glucose uptake and an enhanced expression of brown adipocyte–related genes (Pparg, Ppargc1a, Ppargc1b, Prdm16, Cpt1b), but not of purported beige/brite transcriptional markers (Cd137, Tmem26, Tbx1, Slc27a1, Hoxc9, Shox2). Lack of induction of beige markers phenocopied results in WAT of adult Rb +/− mice. Flow cytometry analysis evidenced an increased number of preadipocytes in WAT depots of Rb +/− mice. Sca1 + preadipocytes from WAT of Rb +/− mice displayed increased gene expression of several transcription factors common to the brown and beige adipogenic programs (Prdm16, Pparg, Ppargc1a) and of receptors of bone morphogenetic proteins (BMPs); however, amongst the recently proposed beige markers, only Tbx1 was upregulated. Adult Rb +/− mice had increased circulating levels of BMP7. These results indicate that preadipose cells resident in WAT depots of Rb +/− mice retain an increased capacity for brown-like adipogenesis that appears to be different from beige adipogenesis, and suggest that the contribution of these precursors to the Rb +/− adipose phenotype is driven, at least in part, by interaction with BMP7 pathways. This article is protected by copyright. All rights reserved

Description: Acoustic surveys of the distribution and abundance of freshwater zooplankton were conducted in Lake Giles, an oligotrophic freshwater lake. Volume backscatter data from a 710 kHz scientific echosounder were converted to high-resolution spatial and temporal numerical density estimates of small zooplankton. Vertical net tows of a 153 μm mesh closing bongo net at multiple depth intervals provided both identification of the types and sizes (0.5–1.5 mm length) of crustacean zooplankton present in the lake as well as an independent measurement of zooplankton numerical density. Net and acoustic estimates of zooplankton abundance, biovolume, and distribution were very similar. The improved resolution of the high-frequency acoustic sampling provides insight into several aspects of freshwater zooplankton ecology including: separation of migrating and non-migrating zooplankton, high resolution measurements of in situ zooplankton biovolume, calculation of in situ vertical velocities of migrating zooplankton, and fine-scale (sub-meter) horizontal and vertical zooplankton distribution during daytime, nighttime, and vertical migration events. These methods allow for more detailed and accurate estimates of zooplankton distribution than traditional net sampling methods can provide, including determining the total abundance of organisms within a specific habitat. They also provide higher resolution data in both space and time of smaller zooplankton taxa than have been measured previously in freshwater ecosystems.

Description: Regulation of cytosolic Ca 2+ is critical for pancreatic acinar cell function. Disruptions in normal Ca 2+ concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca 2+ homeostasis by promoting rapid Ca 2+ movement. Determining how expression of Ca 2+ modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory pathway Ca 2+ ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last 4 exons of the Atp2c2 gene ( Atp2c2c ). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2' s 24 th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca 2+ . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca 2+ levels. Combined, these results suggest SPCA2C affects Ca 2+ homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca 2+ ATPases. This article is protected by copyright. All rights reserved

Description: Exosomes are released by cells as self-contained vesicles with an intact lipid bilayer that encapsulates a small portion of the parent cell. Exosomes have been studied widely as information-rich sources of potential biomarkers that can reveal cellular physiology. We suggest that quantification is essential to understand basic biological relationships between exosomes and their parent cells and hence the underlying interpretation of exosome signals. The number of methods for quantifying exosomes has expanded as interest in exosomes has increased. However, a consensus on proper quantification has not developed, making each study difficult to compare to another. Overcoming this ad hoc approach will require widely available standards that have been adequately characterized, and multiple comparative studies across platforms. We outline the current status of these technical approaches and our view of how they can become more coherent. This article is protected by copyright. All rights reserved

Description: Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up-regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide-induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for four days (D5-9) with 300 µM hydrogen peroxide resulted in fewer alkaline phosphatase-positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression ( p  〈 0.05). This suppression was partially blocked when cells were pretreated 12h with 20 µM QRC. Hydrogen peroxide also produced sustained up-regulation of heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC), which was partially blocked in hydrogen peroxide-treated cells that first received QRC pretreatment. The alterations in the antioxidant stress response coincided with alterations in phosphorylated ERK1/2, but not Nrf2. These results suggest that QRC suppresses hydrogen peroxide-induced activation of the antioxidant response, and partially preserves osteoblast phenotypic development. This article is protected by copyright. All rights reserved

Description: Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation and survival. Leptin receptor is integrated inside cholesterol-rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl-β-cyclodextrin (β-MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/mL). Unlike β-MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. This article is protected by copyright. All rights reserved

Description: Monocyte chemoattractant protein-1 (MCP-1) has been reported to induce the expression of monocyte chemotactic protein-induced protein 1 (MCPIP1), which undergoes ubiquitination degradation. Therefore, we predict that in vascular smooth muscle (VSMCs), MCPIP1 may be induced by MCP-1 and undergo degradation, which can be inhibited by the proteasome inhibitor, MG132. Our results showed that treatment of human VSMCs with MCP-1 did not increase the expression of MCPIP1. Treatment with MG132, however, elevated MCPIP1 protein levels through stimulation of the gene transcription, but not thorugh increasing protein stability. MCPIP1 expression induced by MG132 was inhibited by α-amanitin inhibition of gene transcription or cycloheximide inhibition of protein synthesis. Our further studies showed that MCPIP1 expression induced by MG132 was inhibited by the inhibitors of AKT and p38 kinase, suggesting a role of the AKT-p38 pathway in MG132 effects. We also found that treatment with MG132 induces apoptosis, but overexpression of MCPIP1 inhibited bromodeoxyuridine (BrdU) incorporation of human VSMCs without induction of significant apoptosis. In summary, MCPIP1 expression is induced by MG132 likely through activation of the AKT-p38 pathway. MCPIP1 inhibits SMC proliferation without induction of apoptosis. This article is protected by copyright. All rights reserved

Description: ABSTRACT Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. This article is protected by copyright. All rights reserved

Description: ABSTRACT Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, representing the second most common primary malignant bone tumor in children and adolescents. Although the development of a multimodal therapy, including both local control (surgery and/or radiation) and systemic multidrug chemotherapy, has determined a significant improvement in survival, patients with metastatic and recurrent disease still face a poor prognosis. Moreover, considering that ES primarily affects young patients, there are concerns about long-term adverse effects of the therapy. Therefore, more rational strategies, targeting specific molecular alterations underlying ES, are required. Recent studies suggest that SRC family kinases (SFKs), which are aberrantly activated in most cancer types, could represent key therapeutic targets also for ES. Here, we challenged ES cell lines with a recently developed selective SFK inhibitor (a pyrazolo[3,4- d ]pyrimidine derivative, called SI221), which was previously shown to be a valuable proapoptotic agent in other tumor types while not affecting normal cells. We observed that SI221 significantly reduced ES cell viability and proved to be more effective than the well-known SFK inhibitor PP2. SI221 was able to induce apoptosis in ES cells and also reduced ES cell clonogenic potential. Furthermore, SI221 was also able to reduce ES cell migration. At the molecular level, our data suggest that SFK inhibition through SI221 could reduce ES cell viability at least in part by hindering an SFK-NOTCH1 receptor-p38 mitogen-activated protein kinase (MAPK) axis. Overall, our study suggests a potential application of specific SFK inhibition in ES therapy. This article is protected by copyright. All rights reserved

Description: The small intestine is the main organ involved in the digestion and absorption of nutrients, i.e., it is in an ideal position to sense the availability of energy in the lumen in addition to its absorptive function. Consumption of a high-fat diet (HFD) influences the metabolic characteristics of the small intestine. Therefore, to better understand the metabolic features of the small intestine and their changes in response to dietary fat, we characterized the metabolism of duodenal, jejunal and hepatic cell lines and assessed the metabolic changes in the enterocytes and the liver after short-term (3 days) or medium-term (14 days) HFD feeding in mice. Experiments with immortalized enterocytes indicated a higher glycolytic capacity in the duodenal cell line compared to the other two cell lines, whereas the jejunal cell line exhibited a high oxidative metabolism. Short-term HFD feeding induced changes in the expression of glucose and lipid metabolism-related genes in the duodenum and the jejunum of mice, but not in the liver. When focusing on fatty acid oxidation both, short- and medium-term HFD feeding induced an upregulation of 3-hydroxy-3-methylglutaryl-coenzyme A, the key enzyme of ketogenesis, at the protein level in the intestinal epithelial cells, but not in the liver. These results suggest that HFD feeding induces an early adaptation of the small intestine rather than the liver in response to a substantial fat load. This highlights the importance of the small intestine in the adaptation of the body to the metabolic changes induced by HFD exposure. This article is protected by copyright. All rights reserved

Description: A systematic meta-analysis of the available gene expression profiling datasets for the whole normal human heart generated a quantitative transcriptome reference map of this organ. TRAM (Transcriptome Mapper) software integrated 32 gene expression profile datasets from different sources returning a reference value of expression for each of the 43,360 known, mapped transcripts assayed by any of the experimental platforms used in this regard. Main findings include the visualization at the gene and chromosomal levels of the classical description of the basic histology and physiology of the heart, the identification of suitable housekeeping reference genes, the analysis of stoichiometry of gene products and the focusing on chromosome 21 genes, which are present in one excess copy in Down syndrome subjects, presenting cardiovascular defects in 30-40% of cases. Independent in vitro validation showed an excellent correlation coefficient (r = 0.98) with the in silico data. Remarkably, heart/non-cardiac tissues expression ratio may also be used to anticipate that effects of mutations will most probably affect or not the heart. The quantitative reference global portrait of gene expression in the whole normal human heart illustrates the structural and functional aspects of the whole organ and is a general model to understand the mechanisms underlying heart pathophysiology. This article is protected by copyright. All rights reserved

Description: Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterise various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterise the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. This article is protected by copyright. All rights reserved

Description: Organic matter is the dominant pool of reduced carbon in marine and freshwater systems. Mineralization of organic matter is largely attributed to complex and diverse microbial communities that mediate degradation and ultimately yield the terminal respiratory product, carbon dioxide (CO 2 ). The factors that constrain the lability and degradation of organic matter remain unclear, but they involve a complex interplay between structural and chemical properties of the compounds, physical properties of the matrices, and the functional potential of the microorganisms that are present. To investigate these relationships, we developed a novel bioreactor system—called Isotopic Carbon Respirometer-Bioreactor (IsoCaRB)—that permits real-time monitoring of microbial CO 2 production rates and collects sequential samples of this CO 2 for off-line isotopic analyses ( 13 C, 14 C). Application of this system to organic-rich sediments from Salt Pond, MA reveals that organic matter is oxidized both abiotically and microbially, and that the pattern of microbial respiration by the native sediment community is complex, accessing different carbon substrates over the course of incubation. Isotopic measurements show modern organic matter of progressively older ages (≤ ca. 50 yr) is consumed, and this material has variable origins (salt marsh grasses, terrestrial, and marine organic matter). Collectively, the IsoCaRB system provides coupled insights into the sources, ages, and inherent biological and abiotic reactivity of natural organic matter.

Description: ABSTRACT Adipogenesis is a physiological process required for fat-tissue development, mainly involved in regulating the organism energetic-state. Abnormal distribution-changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal-cells (MSC) induced and not induced to differentiate into adipocytes by next (second)-generation sequencing. A total of 2,866 differentially-expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane-organelle biosynthesis and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation and differentiation. Analyses of mRNA-miRNA interaction showed that repressed miRNA-encoding genes can act downregulating PPARG-related genes; mostly the PPARG activator ( PPARGC1A ). Induced miRNA-encoding genes regulate downregulated genes related to TGFB1 . These results shed new light to understand adipose-tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type-2 diabetes and osteoporosis. This article is protected by copyright. All rights reserved

Description: The 18 O technique is considered the most direct in vitro method for measuring gross primary production (GPP) in aquatic ecosystems. This method measures the 18 O enrichment of the dissolved O 2 pool through photosynthesis after spiking a water sample with a tracer amount of 18 O-labeled water ( 18 O-H 2 O) and incubating it under natural light conditions. Despite its advantages, the 18 O technique has only scarcely been used to measure GPP in the ocean. The lack of 18 O-based primary productivity measurements is most likely due to the technical difficulty associated with sample collection, handling, and processing, and to the need of an isotope ratio mass spectrometer (IRMS) for sample analysis, which is not available for the majority of research groups. The current procedure also precludes at sea measurements. In this manuscript, we demonstrate that the biological 18 O enrichment of dissolved O 2 , after incubation of seawater enriched with 18 O-H 2 O, can be precisely measured by shipboard or laboratory-based membrane inlet mass spectrometry (MIMS). The method was validated in the low-productivity oligotrophic North Pacific Subtropical Gyre, where the measured GPP ranged from 0.2 to 1.1 μmol O 2 L −1 d −1 , with an approximate precision for surface waters of ± 0.02 μmol O 2 L −1 d −1 . This new approach has the advantages of simple water sample handling and analysis, accurate dissolved gas measurements, capability of analysis on board of a ship, and use of relatively inexpensive instrumentation, and therefore has the potential to improve our understanding of primary production in the ocean and other aquatic environments.

Description: ABSTRACT Obesity is characterized by a disruption in energy balance regulation that results in an excess accumulation of body fat. Its increasing prevalence poses a major public health concern because it is a risk factor for a host of additional chronic conditions, including type 2 diabetes, hypertension, and cardiovascular disease. Obesity is increasingly recognized as a growing cause of cancer risk. In particular excessive adipose expansion during obesity causes adipose dysfunction and inflammation that can regulate tumor growth. In obesity, dysregulated systemic metabolism and inflammation induce hyperinsulinemia, hyperglycemia, dyslipidemia and enhance sex hormone production with increased secretion of proinflammatory adipokine that impact breast cancer development and progression. This review describes how adipose inflammation that characterizes obesity is responsible of microenvironment to promote cancer, and discuss how steroid hormones, that are essential for the maintenance of the normal development, growth and differentiation of the cells, influence the induction and progression of breast cancer. This article is protected by copyright. All rights reserved

Description: This study compared horizontal black disk visibility (BDV), a measure of visual water clarity, to turbidity and total suspended solids (TSS) concentrations in five rivers of the southwestern Ozarks of Arkansas, U.S. to assess its usefulness in evaluating optical water quality. We investigated correlative relationships between BDV vs. turbidity and BDV vs. TSS, and compared them to those reported in similar studies. Our results indicated that replicate measures of horizontal BDV were subject to less error than replicate turbidity (coefficients of variation were 5.0% and 6.4%, respectively), and BDV was more strongly correlated with TSS concentrations than was turbidity (correlations coefficients were −0.97 and 0.92, respectively). We also assessed the validity of the black disk method as a surrogate for TSS concentrations and derived a predictive model for each river in the study. The benefits of the horizontal black disk method are both scientific and practical. Horizontal BDV provides a means of quantifying the beam attenuation coefficient. And, the low cost, ease of use, and usefulness in shallow rivers make the black disk method a versatile tool in water quality assessment. We ultimately conclude that the horizontal black disk method should be added to routine monitoring programs in the U.S., and that it be adopted by public groups involved in volunteer monitoring and water quality communication that may be limited by equipment availability or budget.

Description: ABSTRACT Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates expression of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. This article is protected by copyright. All rights reserved

Description: ABSTRACT In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling, however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro . In addition, Wnt3A induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. This article is protected by copyright. All rights reserved

Description: Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc-finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF-кB transcriptional activity in Egr3-inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V + cell fraction and apoptotic protein activity including caspase-3 and caspase-7, and Poly-ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan-caspase inhibitor Z-VAD-FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. This article is protected by copyright. All rights reserved

Description: ABSTRACT Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen-activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH-related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase-1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS-induced apoptosis by targeting MKP1. In osteoblastic MC3T3-E1 and MG-63 cells, H 2 O 2 triggered p38, JNK, ERK and p66 Shc phosphorylation and cell apoptosis. Meanwhile, PTHrP (1-37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK or p66 Shc activation. H 2 O 2 -induced p38 and ERK phosphorylation and apoptosis were both decreased by pre-treatment with specific kinase inhibitors or PTHrP (1-37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1-37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1-37) promptly enhanced MKP1 protein and gene expression and MKP1-dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1-37) adsorbed in an implanted hydroxyapatite-based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1-induced dephosphorylation of MAPKs in osteoblasts. This article is protected by copyright. All rights reserved

Description: Many zooplankton species produce dormant stages to bridge episodes that are unfavorable for juveniles and adults. Although a variety of methods has been used to study the morphology and internal structure of these propagules, surprisingly little is known about links between propagule traits and life history characteristics such as dormancy, hatching behavior and fitness of emerging larvae. A major constraint is that most available methods are lethal to the embryo and processing can generate visual artefacts. Here, we investigate the potential of High Resolution X-ray Computed Tomography (HRXCT or µCT scanning) to compile whole mount 3D reconstructions of the propagules of three zooplankton species that differ in size (∼200–1200 μm) and shape (spherical eggs vs. semi-circular ephippia). Our results show that µCT scanning is a suitable technique for whole mount reconstruction of propagules. In addition, by combining the scanning procedure with a laboratory hatching experiment we showed that while eggs that were exposed to µCT scanning had significantly lower hatching fractions and lower motility of hatchlings than control eggs, some of the scanned eggs or larvae could still be used in subsequent life table experiments. Overall, µCT scanning represents a valuable, non-invasive technique for internal and external characterization of zooplankton propagules and to study associations between propagule structure and life history traits.

Description: For almost three decades, flow cytometry has allowed researchers to investigate ocean planktonic communities using size and cell fluorescence properties. However, oceanographic applications must face two constraints. First, when dealing with marine microbes, instruments must be sensitive because these organisms are very small and with low fluorescence. Second, instruments must be portable to be used on board ships. We compared the performance of two instruments, the BD FACSCanto™ and BD Accuri™ C6. The former is an expensive laboratory-based instrument which has a very good sensitivity, whilst the latter is less sensitive but presents critical advantages for field studies (easy handling and transportation, relatively low cost). We have analyzed 102 samples from the South Atlantic Ocean from three transects off Brazil, within the euphotic zone. We compared cell abundance of heterotrophic bacteria, Prochlorococcus and Synechococcus , as well as photosynthetic pico- and nano-eukaryotes. Heterotrophic bacteria, pico- and nano-eukaryotes could be easily detected with both cytometers. Prochlorococcus and Synechococcus populations were severely under-estimated with the BD Accuri™ C6, particularly for samples from the well-lit layers of the water column. Correction of abundance data using previously suggested approaches was not sufficient to fully compensate for the low sensibility. Our data suggest that the BD Accuri™ C6 is suitable for counting marine bacteria and photosynthetic eukaryotes, but not Prochlorococcus and Synechococcus .

Description: Dedicator of cytokinesis 2 (DOCK2) is a CDM family protein containing DOCK homology region (DHR) -1 and DHR-2, Src-homology 3 (SH3) domain, and C-terminal polybasic amino acid cluster. The CDM family consists of 11 mammalian members and is classified into four subfamilies, the DOCK-A, -B, -C, and -D. DOCK2 is a member of DOCK-A subfamily and an atypical guanine exchange factor regulating the loading of GTP to activate Rac. It is primarily found in peripheral blood, spleen, and thymus and mainly expressed in lymphocytes and macrophages of various organs. DOCK2 is also expressed in microglial in brain and is induced in neointima smooth muscle following vascular injury. Functionally, DOCK2 is involved in cell motility, polarity, adhesion, proliferation, and apoptosis. It is essential for lymphocyte migration and activation as well as neutrophil chemotaxis. DOCK2 also regulates the differentiation of natural killer T cells, type 2 T helper cells, and plasmacytoid dendritic cells. In addition, it is important for the growth of B cell lymphoma and prostate cancer cells. Deletion of DOCK2 enables long-term cardiac allograft survival. Moreover, DOCK2 is associated with the Alzheimer Disease, HIV development, and the early-onset of invasive infections. Recently, we found that DOCK2 plays a critical role in SMC phenotypic modulation and vascular remodeling. In this review, we will briefly summarize recent advancement of DOCK2 function. This article is protected by copyright. All rights reserved

Description: Ca 2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal and amplitude features. Here we investigate the role of Store-Operated Ca 2+ Entry (SOCE) in regulating the temporal dynamics of Ca 2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca 2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca 2+ release is coupled to Ca 2+ store depletion, this triggers the activation of SOCE that translates into a low amplitude tonic Ca 2+ signal. SOCE has also been implicated in fueling Ca 2+ oscillations when activated at low levels. Here we show that sustained SOCE activation in the presence of IP 3 to gate IP 3 receptors (IP 3 R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca 2+ oscillations and produces a tonic Ca 2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca 2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca 2+ uptake into the ER, followed by release through open IP 3 Rs at distant sites, a process we refer to as ‘Ca 2+ teleporting’. Therefore, sustained SOCE activation in the presence of an IP 3 -dependent ‘leak’ pathway at the ER membrane results in a switch from oscillatory to tonic Ca 2+ signaling. This article is protected by copyright. All rights reserved

Description: The efficacy of chemotherapy is hindered by both tumor heterogeneity and acquired or intrinsic multi- drug resistance caused by the contribution of multidrug resistance proteins and stemness-associated prosurvival markers. Therefore, targeting multi-drug resistant cells would be much more effective against cancer. In this study, we characterized the chemoresistance properties of adherent (anchorage- dependent) lung H460 and breast MCF-7 cancer cells growing under prolonged periods of serum starvation (PPSS). We found that under PPSS, both cell lines were highly resistant to Paclitaxel, Colchicine, Hydroxyurea, Obatoclax, Wortmannin and LY294002. Levels of several proteins associated with increased stemness such as Sox2, MDR1, ABCG2 and Bcl-2 were found to be elevated in H460 cells but not in MCF-7 cells. While pharmacological inhibition of either MDR1, ABCG2, Bcl-2 with Verapamil, Sorafenib or Obatoclax respectively decreased the levels of their target proteins under routine culture conditions as expected, such inhibition did not reverse PX resistance in PPSS conditions. Paradoxically, treatment with inhibitors in serum-starved conditions produced an elevation of their respective target proteins. In addition, we found that Digitoxin, an FDA approved drug that decrease the viability of cancer cells growing under PPSS, downregulates the expression of Sox2, MDR1, phospho- AKT, Wnt5a/b and β-catenin. Our data suggests that PPSS-induced chemoresistance is the result of extensive rewiring of intracellular signaling networks and that multi-resistance can be effectively overcome by simultaneously targeting multiple targets of the rewired network. Furthermore, our PPSS model provides a simple and useful tool to screen drugs for their ability to target multiple pathways of cancer resistance. This article is protected by copyright. All rights reserved

Description: ABSTRACT Advances in our understanding of desmosomal diseases have provided a clear demonstration of the key role played by desmosomes in tissue and organ physiology, highlighting the importance of their dynamic and finely regulated structure. In this context, non-desmosomal regulatory molecules have acquired increasing relevance in the study of this organelle resulting in extending the desmosomal interactome, named the “desmo-adhesome”. Spatiotemporal changes in the expression and regulation of the desmo-adhesome underlie a number of genetic, infectious, autoimmune, and malignant conditions. The aim of the present article was to examine the structural and functional relationship of the desmosome, by providing a comprehensive, yet focused overview of the constituents targeted in human disease. The inclusion of the novel regulatory network in the desmo-adhesome pathophysiology opens new avenues to a deeper understanding of desmosomal diseases, potentially unveiling pathogenic mechanisms waiting to be explored. This article is protected by copyright. All rights reserved

Description: DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na + /H + exchanger 1 (NHE1). ROS formation in CD4 + T cells plays a decisive role in regulating inflammatory responses. In the present study we explored whether DJ-1 is expressed in CD4 + T cells and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript and protein levels were quantified by qRT-PCR and Western blotting respectively, intracellular pH (pHi) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' –dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4 + T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4 + T cells from DJ-1 deficient mice than in CD4 + T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4 + T cells, and blunted the difference between DJ-1 -/- and DJ-1 +/+ CD4 + T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1 -/- CD4 + T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4 + T cells. This article is protected by copyright. All rights reserved

Description: The importance of the submersion regime for the physiology, behavior and ecology of intertidal organisms, as well as limitations of available measurement methods, motivated us to develop a new remote sensing method. This new method is based on video monitoring of visual references installed at interest positions in the intertidal zone. Video imagery processing algorithms discriminate between states of submersion and emersion. The signal is based on the fluctuation in the color contrast between the visual reference and the adjacent rock. Our remote sensing method was validated favorably by comparing with direct field observations of submersion time series and comparison of different submersion metrics, including submersion percentage and maximum emersion duration. Since the remote sensing method captures and stores imagery remotely, obtaining long records of high temporal resolution is facilitated and the risk of discontinuity of time series is reduced in comparison to the use of sensors at the zone of wave impact. The high temporal resolution is critical for environments with high exposure to waves and allows for reliable estimates of a wide variety of relevant submersion metrics. This low cost remote sensing method should encourage quantification of submersion regimes of organisms inhabiting rocky intertidal zones, as well as the exploration of physiological, behavioral and ecological consequences of these regimes. Identifying the character of the forces underlying submersion regimes, together with critical environmental variables, such as temperature and solar radiation, will allow better hind-casts and forecasts of the ecological consequences of past and future climate change on these communities.

Description: ABSTRACT Basic, pre-clinical and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide - LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin ( Ocn ), bone sialoprotein ( Bsp ) and runt-related transcription factor 2 ( RunX2 ). In conclusion, LRAP uptake by cementoblast involves flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by flotillin-1. This article is protected by copyright. All rights reserved

Description: Lagrangian particle tracking and Lagrangian coherent structures (LCS) analysis tools aid in the studies of fluid flow and are especially helpful in understanding the role of transport in marine ecosystems. However, most existing particle tracking and analysis tools operate in conjunction with a specific model and/or require execution in multiple programming languages. The Particle Tracking and Analysis TOolbox (PaTATO) for Matlab aims to increase the availability of particle tracking and analysis techniques to a wider audience. PaTATO is compatible with many different types of velocity data and can compute forward and backward trajectories in two and/or three dimensions. PaTATO can compute standard LCS metrics, like Lyapunov exponents and relative dispersion, as well as the maximal extent of a trajectory (MET), a relatively new metric. Most importantly, PaTATO is computationally efficient and easy-to-use. We describe PaTATO, and present examples using the time-periodic double gyre, high frequency radar surface current observations, the Massachusetts Institute of Technology general circulation model (MITgcm), altimeter-derived geostrophic velocities (AVISO), and Nucleous for European Modelling of the Ocean (NEMO).

Description: The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture-expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand-receptor complex formation. We previously reported on an FGF2-interacting HS variant (termed HS2) isolated in microgram amounts from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here we detail the isolation of gram amounts of an FGF2 affinity-purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP-2 or VEGF 165 . This process used a peptide sequence derived from the heparin-binding domain of FGF2 as a substrate to affinity-isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF-BB, or VEGF 165 . Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long-term supplementation of cultures with HS8 increased both hMSC numbers and their colony-forming efficiency without adversely affecting the expression of hMSC-related surface antigens. This strategy further exemplifies the utility of affinity-purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture-expansion of hMSCs destined for cellular therapy. This article is protected by copyright. All rights reserved

Description: Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio-temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole-organism context. This article is protected by copyright. All rights reserved