ALBERT

Description: Dynamic interactions with DNA allow replication protein A to direct single-stranded DNA-intermediates into different pathways for synthesis or repair. On pages 1156–1161 , Chen and Wold review recent discoveries that show that replication protein A (RPA), the major eukaryotic single-stranded DNA-binding protein, binds DNA dynamically and that this is important for correct processing of DNA intermediates. The cover shows a model of human RPA interacting with ssDNA based on the known structures of the domains of human RPA and the structure of Ustalago RPA bound to DNA. The three subunits of RPA are shown in blue (RPA1), red (RPA2), and green (RPA3) with ssDNA shown in black.

Description: Ramathal et al. have employed an elegant xenotransplantation technique to study the fate of human induced pluripotent stem cells (hiPSCs) from fertile males and from males carrying Y chromosome deletions of the azoospermia factor ( AZF ) region. When placed in a mouse testis niche, hiPSCs from fertile males differentiate into germ cell-like cells (GCLCs). Highlighting the crucial role of cell autonomous factors in male sterility, hiPSCs derived from azoospermic males prove to be less successful under similar circumstances. Their studies argue that the agametic “Sertoli cell only” phenotype of two of the AZF deletions likely arises from a defect in the maintenance of germline stem cells (GSCs) rather than from a defect in their specification. These observations underscore the importance of the dialogue between the somatic niche and its inhabitant stem cells, and open up interesting questions concerning the functioning of the somatic niche and how it communicates to the GSCs. In Ramathal et al., human induced pluripotent stem cells (hiPSCs) from wild type and azoospermic men adopt germ cell fate in vitro. Upon xenotransplantation into mouse seminiferous tubules, these hiPSCs acquired germ cell-like (GCL) fate. Remarkably, samples from azoospermic males were less efficient in both contexts.

Description: Gene transcription is strictly controlled by the interplay of regulatory events at gene promoters and gene-distal regulatory elements called enhancers. Despite extensive studies of enhancers, we still have a very limited understanding of their mechanisms of action and their restricted spatio-temporal activities. A better understanding would ultimately lead to fundamental insights into the control of gene transcription and the action of regulatory genetic variants involved in disease. Here, I review and discuss pros and cons of state-of-the-art genomics methods to localize and infer the activity of enhancers. Among the different approaches, profiling of enhancer RNAs yields the highest specificity and may be superior in detecting in vivo activity. I discuss their apparent similarities to promoters, which challenge the established view of enhancers and promoters as distinct entities, and present a unifying model of regulatory elements in transcriptional regulation, in which activity, transcriptional output and regulatory function is context specific. Profiling of enhancer RNAs (eRNAs) likely represents a paradigm shift in enhancer genomics, yielding high specificity in the localization of active enhancers. The similarities between enhancers and promoters are discussed and their established distinctions are deconstructed. Based on this, a unified model of regulatory elements in transcriptional regulation is presented.

Description: Comparing structural and evolutionary dynamics of proteins . On pages 209–218 Marsh and Teichmann discuss parallels between protein dynamics and evolution, and how we can gain insights into function from these studies. Buried residues, for example, are usually more evolutionarily conserved and evolve less quickly than exposed residues. These residues generally form more intramolecular contacts, and mutating them would therefore more likely lead to a destabilization of the protein. Having more intramolecular contacts on the other hand also means that these residues are less flexible to move around. In contrast, dynamic parts of a protein in real time are also dynamic over evolutionary time and evolve more quickly. The cover shows the buried (blue) and surface (red) residues for the receiver domain of sensor histidine kinase CKI1 from Arabidopsis .

Description: ABSTRACT We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning the sample with a high-frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca 2+ in cultured neurons. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Gonadotropin releasing hormone (GnRH) is a peptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the distribution pattern of two isoforms of GnRH-like peptides in the neural ganglia and testis of reproductively mature male abalone , H. asinina , by immunohistochemistry and whole mount immunofluorescence. We found octopus (oct) GnRH and tunicate-I (t) GnRH-I immunoreactivities (ir) in type 1 neurosecretory cells (NS1) and they were expressed mostly within the ventral horn of the cerebral ganglion, whereas in pleuropedal ganglia they were localized primarily in the dorsal horn. Furthermore, tGnRH-I-ir were strongly detected in fibers at the caudal part of the cerebral ganglia and both ventral and dorsal horns of the pleuropedal ganglia. In the testis, only octGnRH-ir was found primarily in the granulated cell and central capillaries within the trabeculae. These results suggest that multiple GnRH-like peptides are present in the neural ganglia which could be the principal source of their production, whereas GnRH may also be synthesized locally in the testis and act as the paracrine control of testicular maturation. Microsc. Res. Tech. 77:110–119, 2014 . © 2014 Wiley Periodicals, Inc.

Description: Gestational factors play a role in the development of several neuropsychiatric disorders including schizophrenia and autism. In utero conditions influence future mental health through epigenetic mechanisms, which alter gene expression without affecting DNA coding sequence. Environmental factors account for at least 60% of the risk for developing major depression, and earlier onset of depressive illness has been observed over the past decades. I speculate that gestational factors may play a greater role in programing depression than previously recognized. Here, I examine recent evidence for a role for gestational factors in programing mood disorders, and how epigenetic mechanisms mediate this effect. How does the gestational environment transduce vulnerability to depression to the fetus? The in utero environment alters gene expression through epigenetic mechanisms, which mediate long-term effects on physiology and behavior without changing DNA sequence. I examine recent work suggesting that gestational environment programs depression in adult offspring via epigenetics.

Description: Recent findings have provided evidence for the existence of non-vertebrate acquired immunity. We survey these findings and propose that all living organisms must express both innate and acquired immunity. This is opposed to the paradigm that only vertebrates manifest the two forms of immune mechanism; other species are thought to use innate immunity alone. We suggest new definitions of innate and acquired immunity, based on whether immune recognition molecules are encoded in the inherited genome or are generated through somatic processes. We reason that both forms of immunity are similarly ancient, and have co-evolved in response to lifestyle, cost-benefit tradeoffs and symbiosis versus parasitism. However, different species have evolved different immune solutions that are not necessarily genetically related, but serve a similar general function – allowing individuals to learn from their own immune experience; survival of species is contingent on the acquired immune experience of its individuals. We propose that all organisms express both innate and adaptive/acquired immunity. Acquired immune manifestations vary between species, but all use recognition molecules not directly encoded in the inherited genome, and all serve a similar function – allowing individuals to learn from their own immune experience, thus promoting species survival.

Description: The standard representation of the Central Dogma (CD) of Molecular Biology conspicuously ignores metabolism. However, both the metabolites and the biochemical fluxes behind any biological phenomenon are encrypted in the DNA sequence. Metabolism constrains and even changes the information flow when the DNA-encoded instructions conflict with the homeostasis of the biochemical network. Inspection of adaptive virulence programs and emergence of xenobiotic-biodegradation pathways in environmental bacteria suggest that their main evolutionary drive is the expansion of their metabolic networks towards new chemical landscapes rather than perpetuation and spreading of their DNA sequences. Faulty enzymatic reactions on suboptimal substrates often produce reactive oxygen species (ROS), a process that fosters DNA diversification and ultimately couples catabolism of the new chemicals to growth. All this calls for a revision of the CD in which metabolism (rather than DNA) has the leading role. The central dogma of Molecular Biology has shortcomings that need revision: (i) metabolism is the next step of the information flow, (ii) metabolism has the predominant role (thereby the hierarchy of actors should be flipped upside down) and (iii) metabolism feedbacks on DNA by means of reactive oxygen species.

Description: If validated, diet-derived foreign microRNA absorption and function in consuming vertebrates would drastically alter our understanding of nutrition and ecology. RNA interference (RNAi) mechanisms of Caenorhabditis elegans are enhanced by uptake of environmental RNA and amplification and systemic distribution of RNAi effectors. Therapeutic exploitation of RNAi in treating human disease is difficult because these accessory processes are absent or diminished in most animals. A recent report challenged multiple paradigms, suggesting that ingested microRNAs (miRNAs) are transferred to blood, accumulate in tissues, and exert canonical regulation of endogenous transcripts. Independent replication of these findings has been elusive, and multiple disconfirmatory findings have been published. In the face of mounting negative results, any additional positive reports must provide the proverbial “extraordinary proof” to support such claims. In this article, we review the evidence for and against a significant role for dietary miRNAs in influencing gene expression, and make recommendations for future studies. A report that foreign microRNAs from the diet rival endogenous vertebrate miRNAs in abundance and function has been refuted by multiple independent studies, although low-level uptake may occur. We present the current evidence for and against the paradigm-challenging but uncorroborated dietary xenomiR hypothesis, and discuss remaining questions in the field.

Description: Literature on maternal exposures and the risk of epigenetic changes or diseases in the offspring is growing. Paternal contributions are often not considered. However, some animal and epidemiologic studies on various contaminants, nutrition, and lifestyle-related conditions suggest a paternal influence on the offspring's future health. The phenotypic outcomes may have been attributed to DNA damage or mutations, but increasing evidence shows that the inheritance of environmentally induced functional changes of the genome, and related disorders, are (also) driven by epigenetic components. In this essay we suggest the existence of epigenetic windows of susceptibility to environmental insults during sperm development. Changes in DNA methylation, histone modification, and non-coding RNAs are viable mechanistic candidates for a non-genetic transfer of paternal environmental information, from maturing germ cell to zygote. Inclusion of paternal factors in future research will ultimately improve the understanding of transgenerational epigenetic plasticity and health-related effects in future generations. Animal and epidemiologic studies on various environmental exposures suggest that transgenerational epigenetic changes can be induced through the paternal germ line, ultimately affecting health status of the offspring. This essay suggests the existence of epigenetic windows of susceptibility to environmental insults during spermatogenesis or other early developmental processes.

Description: Several general anesthetics produce their sedative effect by activating endogenous sleep pathways. We propose that general anesthesia is a two-step process targeting sleep circuits at low doses, and synaptic release mechanisms across the entire brain at the higher doses required for surgery. Our hypothesis synthesizes data from a variety of model systems, some which require sleep (e.g. rodents and adult flies) and others that probably do not sleep (e.g. adult nematodes and cultured cell lines). Non-sleeping systems can be made insensitive (or hypersensitive) to some anesthetics by modifying a single pre-synaptic protein, syntaxin1A. This suggests that the synaptic release machinery, centered on the highly conserved SNARE complex, is an important target of general anesthetics in all animals. A careful consideration of SNARE architecture uncovers a potential mechanism for general anesthesia, which may be the primary target in animals that do not sleep, but a secondary target in animals that sleep. Volatile general anesthetics such as isoflurane produce loss of behavioral responsiveness in all animals. We propose that this can be explained as a two-step process: activation of endogenous sleep pathways at low doses followed by attenuation of synaptic release at the drug concentrations required for surgery.

Description: ABSTRACT Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2-Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2-methoxyestradiol analog, (8R, 13S, 14S, 17S)-2-Ethyl-13-methyl-7, 8, 9, 11, 12,13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrane-3, 17-diyl bis(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor-positive breast adenocarcinoma cells line (MCF-7), estrogen receptor-negative highly metastatic breast cell line (MDA-MB-231) and a non-tumorigenic breast epithelial cell line (MCF-12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 μM after 24 h of exposure. Light microscopy and polarization-optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS-exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non-tumorigenic breast cell lines. Findings warrant in-depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The sperm ultrastructure of the short-faced scorpionfly Panorpodes kuandianensis Zhong et al. was investigated using transmission electron microscopy. The spermatozoon is composed of a short head containing a bilayered acrosome and an elongate nucleus, a neck transition region, and a long flagellum. The acrosome consists of an acrosomal vesicle and a central perforatorium. The nucleus has two deep, U-shaped lateral grooves and its chromatin contains a series of parallel, regularly arrayed nuclear fibers. The barrel-shaped centriolar adjunct occupies the most volume of the neck region. The flagellum is helical for its whole length and is formed by an axoneme, two mitochondrial derivatives, a pair of accessory bodies, and peculiar accessory structures. The axoneme has nine accessory microtubules at the anterior flagellum region, displaying a 9 + 9 + 2 microtubular pattern, but the accessory microtubules are short and disappear quickly. The two mitochondrial derivatives differ in length and diameter. In the more posterior region, the remaining anchor-shaped mitochondrial derivative has a circular crystalline region, differing from other mecopteran species. The filiform materials are peculiar and lie beside the nucleus and in the flagellum region. Sperm ultrastructural comparison of P. kuandianensis with other families supports a close affinity of Panorpodidae with Panorpidae. In addition, the occurrence of the nine accessory microtubules in the sperm axoneme of Panorpodes indicates that the 9 + 9 + 2 axonemal pattern might be a symplesiomorphy of the Mecoptera. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: The discovery of traces of a blood meal in the abdomen of a 50-million-year-old mosquito reminds us of the insights that the chemistry of fossils can provide. Ancient DNA is the best known fossil molecule. It is less well known that new fossil targets and a growing database of ancient gene sequences are paralleled by discoveries on other classes of organic molecules. New analytical tools, such as the synchrotron, reveal traces of the original composition of arthropod cuticles that are more than 400 my old. Pigments such as melanin are readily fossilized, surviving virtually unaltered for ∼200 my. Other biomarkers provide evidence of microbial processes in ancient sediments, and have been used to reveal the presence of demosponges, for example, more than 635 mya, long before their spicules appear in the fossil record. Ancient biomolecules are a powerful complement to fossil remains in revealing the history of life. Ancient biomolecules range from hundreds of thousand-year-old DNA to lipids and structural macromolecules that survive for billions of years. Extracted from fossils and sedimentary rocks, they reveal organism relationships, past environments, and the origin of fossil fuels. In the oldest rocks they may be the only evidence of particular life forms.

Description: ABSTRACT The wing of a dragonfly is thin and light, but can bear high frequent alternating stress and present excellent antifatigue properties. The surface morphology and microstructure of the wings of dragonfly Pantala flavescens were observed using SEM in this study. Based on the biological analysis method, the configuration, morphology, and structure of the vein were studied, and the antifatigue properties of the wings were investigated. The analytical results indicated that the longitudinal veins, cross veins, and membrane of dragonfly wing form a optimized network morphology and spacially truss-like structure which can restrain the formation and propagation of the fatigue cracks. The veins with multilayer structure present high strength, flexibility, and toughness, which are beneficial to bear alternating load during the flight of dragonfly. Through tensile-tensile fatigue failure tests, the results were verified and indicate that the wings of dragonfly P. flavescens have excellent antifatigue properties which are the results of the biological coupling and synergistic effect of morphological and structural factors. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: The encounter of amateur science with synthetic biology has led to the formation of several amateur/do-it-yourself biology (DIYBio) groups worldwide. Although media outlets covered DIYBio events, most seemed only to highlight the hope, hype, and horror of what DIYBio would do in the future. Here, we analyze the European amateur biology movement to find out who they are, what they aim for and how they differ from US groups. We found that all groups are driven by a core leadership of (semi-)professional people who struggle with finding lab space and equipment. Regulations on genetic modification limit what groups can do. Differences between Europe and the US are found in the distinct regulatory environments and the European emphasis on bio-art. We conclude that DIYBio Europe has so far been a responsible and transparent citizen science movement with a solid user base that will continue to grow irrespective of media attention. Here, we analyzed the European do-it-yourself biology (DIYBio) community consisting of enthusiastic biotechnologists, artists, and designers. We conclude that DIYBio Europe has so far been a responsible and transparent citizen science movement with a solid user base that will continue to grow irrespective of media attention.

Description: HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity , Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. Cellular proteins including cyclophilin A (CypA) interact with the capsid (CA) of HIV-1. Recent work shows that wild-type CA prevents the induction of an interferon (IFN) response. Increased CypA binding to mutated CA results in abortive infection and activation of the innate immune sensor cGAS that detects exposed cDNA.

Description: We recently described a novel form of stress-associated bidirectional plasticity at GABA synapses onto hypothalamic parvocellular neuroendocrine cells (PNCs), the apex of the hypothalamus-pituitary-adrenal axis. This plasticity may contribute to neuroendocrine adaptation. However, this GABA synapse plasticity likely does not translate into a simple more and less of inhibition because the ionic driving force for Cl − , the primary charge carrier for GABA A receptors, is dynamic. Specifically, stress impairs a Cl − extrusion mechanism in PNCs. This not only renders the steady-state GABA response less hyperpolarizing but also makes PNCs susceptible to the activity-dependent accumulation of Cl − . Accordingly, GABA synapse plasticity impacts both the robustness of GABA voltage response and dynamic Cl − loading, imposing nonlinear influences on PNC excitability during circuit activities. This theoretical consideration predicts roles for GABA transmission far more versatile than canonical inhibition. We propose potential impacts of GABA synapse plasticity on the experience-dependent fine-tuning of neuroendocrine stress responses. Hypothalamic parvovellular neuroendocrine cells (PNCs) form the apex of the hypothalamus-pituitary-adrenal axis, controlling the corticosteroid (CORT) response to stress. GABAergic inputs onto PNCs undergo multiple forms of plasticity following stress. We propose a hypothesis that GABA synapse plasticity impacts Cl − homeostasis, generates excitatory GABA responses, and tunes neuroendocrine response.

Description: Replication of the main chromosome in the halophilic archaeon Haloferax volcanii was recently reported to continue despite deletion of all active replication origins. Equally surprising, the deletion strain grew faster than the parent strain. It was proposed that origin-less H. volcanii duplicate their chromosomes via recombination-dependent replication. Here, we recall our present knowledge of this mode of chromosome replication in different organisms. We consider the likelihood that it accounts for the viability of H. volcanii deleted for its main specific replication origins, as well as possible alternative interpretations of the results. The selective advantages of having defined chromosome replication origins are discussed from a functional and evolutionary perspective. Hawkins et al. (Nature 503:544–7, 2013) reported the surprising finding that deleting all three replication origins from the archaea Haloferax volcanii main chromosome does not impair viability and rather increases growth rate. We discuss the authors' proposal that replication is then initiated from recombination intermediates, and present alternative hypotheses.

Description: The purinergic signalling system, which utilises ATP, related nucleotides and adenosine as transmitter molecules, appeared very early in evolution: release mechanisms and ATP-degrading enzymes are operative in bacteria, and the first specific receptors are present in single cell eukaryotic protozoa and algae. Further evolution of the purinergic signalling system resulted in the development of multiple classes of purinoceptors, several pathways for release of nucleotides and adenosine, and a system of ectonucleotidases controlling extracellular levels of purinergic transmitters. The purinergic signalling system is expressed in virtually all types of tissues and cells, where it mediates numerous physiological reactions and contributes to pathological responses in a variety of diseases. Purinergic chemical signalling appears early in evolution in species without a nervous system followed by purinergic neurotransmission in animals that developed a nervous system. Widespread purinoceptor expression on most mammalian cell types suggests ATP and adenosine have been retained as successful, compared to more restricted distributions of other chemical messengers.

Description: ABSTRACT Muscle fiber images play an important role in the medical diagnosis and treatment of many muscular diseases. The number of nuclei in skeletal muscle fiber images is a key bio-marker of the diagnosis of muscular dystrophy. In nuclei segmentation one primary challenge is to correctly separate the clustered nuclei. In this article, we developed an image processing pipeline to automatically detect, segment, and analyze nuclei in microscopic image of muscle fibers. The pipeline consists of image pre-processing, identification of isolated nuclei, identification and segmentation of clustered nuclei, and quantitative analysis. Nuclei are initially extracted from background by using local Otsu's threshold. Based on analysis of morphological features of the isolated nuclei, including their areas, compactness, and major axis lengths, a Bayesian network is trained and applied to identify isolated nuclei from clustered nuclei and artifacts in all the images. Then a two-step refined watershed algorithm is applied to segment clustered nuclei. After segmentation, the nuclei can be quantified for statistical analysis. Comparing the segmented results with those of manual analysis and an existing technique, we find that our proposed image processing pipeline achieves good performance with high accuracy and precision. The presented image processing pipeline can therefore help biologists increase their throughput and objectivity in analyzing large numbers of nuclei in muscle fiber images. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Cystoisospora belli is an opportunistic protozoan that causes human cystoisosporiasis, an infection characterized by diarrhea, steatorrhea, abdominal pain, fever, and weight loss. The lack of animal models susceptible to C. belli , and the difficulty in obtaining clinical samples with fair amounts of oocysts have limited the research pertaining to the basic biology of this parasite. This study aimed to describe the ultrastructure of endogenous stages of C. belli in Monkey Rhesus Kidney Cells (MK2) and Human Ileocecal Adenocarcinoma cells (HCT-8). Zoites of C. belli exhibited typical morphological features of coccidia, which included a trilaminar pellicle, an apical complex formed by a conoid, polar rings, rhoptries, and micronemes, in addition to dense granules and the endoplasmic reticulum. No crystalloid body was observed but various lipid and amylopectin granules were usually present in the cytoplasm of zoites. We observed a tendency of the endoplasmic reticulum of the host cell to be located near the parasitophorous vacuole membrane. Merozoites were formed by endodyogeny and during replication, the apical complex of the mother cell remained intact. The formation of gametes or oocysts was not observed. The ultrastructural findings of C. belli are further evidence of its proximity to Sarcocystidae family members and corroborate their reclassification as Cystoisospora spp. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Arge pullata Zadd is an important phytophagous pest that damages red birch Betula albo-sinensis in Hubei Province, South China. Massive ecological and economic losses have been caused by this species, which threatens the ecological security of the Shennongjia Nature Reserve. To investigate the mechanoreception, chemoreception, and oviposition processes of A. pullata , scanning electron microscopy and optical confocal microscopy were used to reveal the typology, morphology, and distribution of ovipositor and antennal sensilla. The results show that A. pullata has clavate antennae and eight types of sensilla in total, including sensilla chaetica, sensilla trichodea (types 1–3), sensilla basiconica, sensilla coeloconica (types 1 and 2), and Böhm's bristles. Sensilla trichodea type 1 distributed only on male antennae; the densities of sensilla trichodea type 2 and sensilla basiconica differed between the sexes. The binding pattern of ovipositor valvulae was discovered, and one type of sensilla chaetica, two types of sensory pits, and tooth-like cones as well as two types of microtrichia were found in the ovipositor. Based on morphological evidence and research on Hymenoptera, putative functions are suggested to increase our understanding of the mechanisms by which this species finds hosts and mates, and how oviposition takes place. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The longicorn beetle Xylotrechus grayii (White, 1855) has been spreading rapidly in China, causing mass mortality of honeysuckle which is economically and medicinally important. In order to elucidate the mechanisms of mate and host location and to advance efficient control methods, antennal sensilla features were investigated in both sexes of X. grayii using scanning electron microscopy (SEM). The filiform antennae of both sexes consist of scape, pedicel, and nine flagellomeres (f1–9). Five types of sensilla were observed: sensilla chaetica (5 subtypes, SC1–5), sensilla basiconica (4 subtypes, SB1–4), Böhm bristles (Bm), grooved peg sensilla (Gp), and sensilla campaniformia (Ca). SC were most common on the antennae, followed by SB and Bm. No significant sexual differences in the type, amounts, and distribution of antennal sensilla were found except for the distribution of SB clusters and Ca. SB clusters and Ca occurred on f1–8 of male antennae but were absent on those segments in females, suggesting a potential function as receptors for female sex pheromones. The putative functions of other sensilla are discussed based on their characteristics in related species. This study provides an important foundation for further research on sensory mechanisms and control measures of X. grayii . Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT In mammals, the cerebral cortex microvasculature (CCM) of the neopallium plays important roles in the physiological and pathological processes of the brain. The aim of the present work is to analyze the CCM by use of the SEM-vascular corrosion cast technique, and to examine the immunocytochemical characteristics of the CCM in adult domestic ruminants (cattle, buffalo, and sheep) by using the SEM-immunogold technique. The CCM originated from the very small, finger-like terminal branches of the macrovasculature of the brain. The superficial cortical arterioles were more numerous than the deep straight arterioles which proceeded toward the white matter. The surface casts of the arterioles and capillaries of the cerebral cortex showed ring-shaped formations in the arterioles and at the origin of the capillaries. All capillaries down-stream from these ring-shaped formations were flaccid. Casts of the capillaries showed wrinkles due to the presence of endothelial folds, which is characteristic of varying blood pressure. Formations having intense anti-GIFAP immunoreactivity were frequently evident along the course of the blood capillaries in the cerebral cortex. These formations were probably astrocytes that might regulate the cerebral microcirculation based on physiological and pathological stimuli, such as neuronal activation. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The effects of anti-angiogenic therapies in guiding tumor angioarchitecture prompted us to examine the modifications in the vascular network of the oral squamous cell carcinoma (SCC) produced by the multitargeted tyrosine kinase inhibitor sunitinib malate. Twelve Syrian hamsters had their right buccal pouches submitted to tumor induction with dimethylbenzanthracene and carbamide peroxide for 55 days. The animals were then divided into two groups of six animals each; group I was treated with sunitinib malate and group II (control) was remained untreated. After 4 weeks, the hamsters had their vascular networks casted by Mercox ® resin and analyzed by scanning electron microscopy. The qualitative study of the vascular network of the control tumor-bearing pouches showed images of intussusception and sprouting angiogenesis, flattened blood vessels, abrupt variations in their diameter, and a tortuous course. The samples treated with sunitinib exhibited a qualitative reduction of the signs of vascular proliferation. In addition, these casts presented an attenuation of the morphological features observed in the untreated tumor-bearing pouches. Quantitative analysis demonstrated that the pouches treated with sunitinib did not show a decrease ( P 〉 0.05) in the vascular diameter and intervessel distances when compared with the control group. The results of the present study suggest that sunitinib may act on the vascular network of oral SCC, normalizing the blood vessels. However, further experiments should be performed in order to determine a judicious dose of this anti-angiogenic therapy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Genomes are inherently unstable because of the need for DNA sequence variation as a substrate for evolution through natural selection. However, most multicellular organisms have postmitotic tissues, with limited opportunity for selective removal of cells harboring persistent damage and deleterious mutations, which can therefore contribute to functional decline, disease, and death. Key in this process is the role of genome maintenance, the network of protein products that repair DNA damage and signal DNA damage response pathways. Genome maintenance is beneficial early in life by swiftly eliminating DNA damage or damaged cells, facilitating rapid cell proliferation. However, at later ages accumulation of unrepaired damage and mutations, as well as ongoing cell depletion, promotes cancer, atrophy, and other deleterious effects associated with aging. As such, genome maintenance and its phenotypic sequelae provide yet another example of antagonistic pleiotropy in aging and longevity. DNA damage has since long been suspected to be a major cause of aging. Here, I will discuss the dual role of genome maintenance systems in providing DNA sequence variation in the germline as the substrate for evolution and mediating DNA damage-induced aging phenotypes in the soma.

Description: Disruption of Nodal in the lateral plate mesoderm (LPM) usually leads to left-right (LR) patterning defects in multiple organs. However, whether the LR patterning of organs is always regulated in a coupled way has largely not yet been elucidated. In addition, whether other crucial regulators exist in the LPM that coordinate with Nodal in regulating organ LR patterning is also undetermined. In this paper, after briefly summarizing the common process of LR patterning, the most puzzling question regarding the initiation of asymmetry is considered and the divergent mechanisms underlying the uncoupled LR patterning in different organs are discussed. On the basis of cases in which different organ LR patterning is determined in an uncoupled way via an independent mechanism or at a different time, we propose that there are other critical factors in the LPM that coordinate with Nodal to regulate heart LR asymmetry patterning during early LR patterning. Also watch the Video Abstract . Based on the fact that distinct mechanisms determine different organ left-right (LR) patterning in an uncoupled way, we hypothesize that other critical factors in the lateral plate mesoderm (LPM) may coordinate with Nodal to regulate heart LR asymmetry patterning during LR patterning, and this can be evaluated by our proposed model.

Description: ABSTRACT Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera . Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC 50 (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Autofocusing technology is indispensable for routine use of microscopes on a large scale in biological field. The autofocusing method using the angle of Hilbert space is brought forward to measure whether the image is focused or not. The angle of Hillbert space can be used to evaluate accurately the similarity degree of two images. The experiment results show that the autofocusing method can decrease the computational cost and get accuracy for real-time biological and biomedical images with noise robustness. The focus curves are smooth and possess the unimodality, the monotonicity and the symmetry. Compared with other classic and optimum focus method, the Hilbert method demonstrates its robustness to noise and can improve the focus speed. The experiments showed that the proposed method can increase the overall performance of an autofocus system and has strong applicability in various autofocusing algorithms. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The structure of ovary in a representative of the scale insect family Matsucoccidae, Matsucoccus pini , is described at the ultrastructural level. The paired ovaries of M. pini are composed of about 50 ovarioles of telotrophic type that develop asynchronously. An individual ovariole consists of an anterior tropharium (trophic chamber) and posterior vitellarium. The tropharium encloses trophocytes (nurse cells) and early previtellogenic oocytes termed arrested oocytes. In the vitellarium from 1 to 6, linearly arranged oocytes may develop. Analysis of serial sections has shown that each ovariole contains 32 germ cells (trophocytes, arrested oocytes, and developing oocytes). In the cytoplasm of all these cells, small rod-shaped bacteria are present. In the early vitellogenic oocytes, accessory nuclei arise. As vitellogenesis progresses, these nuclei migrate toward the cortical ooplasm. The obtained results are discussed in a phylogenetic context. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Gene retrocopies are generated by reverse transcription and genomic integration of mRNA. As such, retrocopies present an important exception to the central dogma of molecular biology, and have substantially impacted the functional landscape of the metazoan genome. While an estimated 8,000–17,000 retrocopies exist in the human genome reference sequence, the extent of variation between individuals in terms of retrocopy content has remained largely unexplored. Three recent studies by Abyzov et al., Ewing et al. and Schrider et al. have exploited 1,000 Genomes Project Consortium data, as well as other sources of whole-genome sequencing data, to uncover novel gene retrocopies. Here, we compare the methods and results of these three studies, highlight the impact of retrocopies in human diversity and genome evolution, and speculate on the potential for somatic gene retrocopies to impact cancer etiology and genetic diversity among individual neurons in the mammalian brain. Three recent studies have employed whole genome sequencing data to identify novel gene retrocopies in humans, by exploiting distinguishing retrocopy hallmarks including exon-exon junctions and genomic locations distal to parent genes. Gene retrocopies contribute to human genetic diversity.

Description: The environment can have a long-lasting influence on an individual's physiology and behavior. While some environmental conditions can be beneficial and result in adaptive responses, others can lead to pathological behaviors. Many studies have demonstrated that changes induced by the environment are expressed not only by the individuals directly exposed, but also by the offspring sometimes across multiple generations. Epigenetic alterations have been proposed as underlying mechanisms for such transmissible effects. Here, we review the most relevant literature on these changes and the developmental stages they affect the most. We discuss current evidence for transgenerational effects of prenatal and postnatal factors on bodily functions and behavioral responses, and the potential epigenetic mechanisms involved. We also discuss the need for a careful evaluation of the evolutionary importance with respect to health and disease, and possible directions for future research in the field. Evidence for epigenetic inheritance of acquired traits in mammals is growing. Transgenerational transmission is based on mechanisms involving DNA-methylation, histone post-translational modifications, and non-coding small RNAs in the germline. These epigenetic modifications constitute potential vehicles for effects of early life stress and nutrition on brain and behavior across generations.

Description: Understanding in vivo regeneration of complex structures offers a fascinating perspective for translation into medical applications. Unfortunately, mammals in general lack large-scale regenerative capacity, whereas planarians, newts or Hydra can regenerate complete body parts. Such organisms are, however, poorly annotated because of the lack of sequence information. This leads to limited access for molecular biological investigations. In the last decade, high throughput technologies and new methods enabling the effective generation of transgenic animals have rapidly evolved. These developments have allowed the extensive use of niche model organisms as part of a trend towards the accessibility of a greater panel of model species for scientific research. The case study that follows provides an insight into the impact of high throughput techniques on the landscape of models of regeneration. The cases presented here give evidence of alternative stem cell maintenance pathways, the identification of new protein families and new stem cell markers. Regeneration of complex structures is a capability mastered by a small number of niche model organisms. Although such organisms are poorly annotated, current technologies allow the extensive use of such organisms. Representative studies give evidence of alternative stem cell maintenance pathways, new protein families, and new stem cell markers.

Description: ABSTRACT Many species that belong to Artiodactyls order show an interdigital sinus (IS), as it occurs in sheep, in all four extremities. These are considered to be scent glands responsible for sexual communication having strong attractiveness to mature males at the peak of the breeding season. The aim of this study was to evaluate, in IS in cyclic ewes, the microscopic and ultrastructure (scanning and transmission electron microscopy) anatomy, secretion composition, and mRNA and protein expression of estrogen receptors α and β and progesterone receptors. Glandular sebaceous structures occupy a superficial area of the pouch. The other glands present in the IS show a coiled tubular structure and tall and polyhedral secretory cells with irregular luminal surface resulting from the secretory process. Protein and mRNA gene transcription studies were performed to determine the presence of ER (α and β) and P4r in IS. At the follicular phase, IS cell populations analyzed using flow cytometry expressed higher levels of ERβ compared with ERα ( P  〈 0.05), whereas no difference was observed between them in the luteal phase. The IS amount of secretion was the highest in the follicular phase compared with luteal phase ( P  〈 0.05) or pregnancy ( P  〈 0.001).To the best of our knowledge, for the first time, the presence of ER (α and β) within the IS was demonstrated. As estrogen action is mediated by specific receptors in target cells, the presence of these receptors in IS might be needed to trigger signaling pathways involved in conspecific chemical (sexual) communication attributed to this area. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Development, life cycle evolution and immunity were among the topics discussed at a recent meeting in Tutzing dedicated to the biology of the ‘basal’ metazoan taxa Porifera, Ctenophora, Placozoa and Cnidaria.

Description: ABSTRACT Light microscopy has undergone a revolution with the advent of super-resolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: The gain of a selective advantage in cancer as well as the establishment of complex traits during evolution require multiple genetic alterations, but how these mutations accumulate over time is currently unclear. There is increasing evidence that a mutator phenotype perpetuates the development of many human cancers. While in some cases the increased mutation rate is the result of a genetic disruption of DNA repair and replication or environmental exposures, other evidence suggests that endogenous DNA damage induced by AID/APOBEC cytidine deaminases can result in transient localized hypermutation generating simultaneous, closely spaced (i.e. “clustered”) multiple mutations. Here, we discuss mechanisms that lead to mutation cluster formation, the biological consequences of their formation in cancer and evidence suggesting that APOBEC mutagenesis can also occur genome-wide. This raises the possibility that dysregulation of these enzymes may enable rapid malignant transformation by increasing mutation rates without the loss of fitness associated with permanent mutators. Some environmental agents and APOBEC cytidine deaminases result in transient hypermutation by causing lesions in ssDNA regions within double strand break repair intermediates and at replication forks. In human cancers such multiple lesions produce mutation clusters, also termed kataegis. Hypermutation of multiple simultaneously formed ssDNA regions may accelerate cancer development.

Description: Homologous recombination (HR) is required to protect and restart stressed replication forks. Paradoxically, the Mrc1 branch of the S phase checkpoints, which is activated by replicative stress, prevents HR repair at breaks and arrested forks. Indeed, the mechanisms underlying HR can threaten genome integrity if not properly regulated. Thus, understanding how cells avoid genetic instability associated with replicative stress, a hallmark of cancer, is still a challenge. Here I discuss recent results that support a model by which HR responds to replication stress through replicative and repair activities that operate at different stages of the cell cycle (S and G2, respectively) and in distinct subnuclear structures. Remarkably, the replication checkpoint appears to control this scenario by inhibiting the assembly of HR repair centers at stressed forks during S phase, thereby avoiding genetic instability. Homologous recombination proteins Rad52/BRCA2 and Rad51 escort and help stressed replication forks. I propose that the Mrc1-dependent checkpoint restricts recombinational repair to G2 by inhibiting the assembly of repair centers at stressed forks, a scenario that would interfere with proper replication and might promote fork restart by detrimental recombinogenic events.

Description: ABSTRACT This study focused on test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different resin reinforced glass ionomer cements (RRGICs). Forty-eight male Wistar rats were used, distributed into four groups ( n  = 12), as follows: Group C (Control, polyethylene), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass), in subcutaneous tissue. The events of edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts, and collagen formation were analyzed at 7, 15, and 30 days. The degree of conversion was evaluated by the Fourier method. Biocompatibility and degree of conversion were assessed using the Kruskal–Wallis and Dunn tests, and ANOVA and Tukey's test, respectively ( P  〈 0.05). It was observed that, there was significant difference between Groups FOB and UBL for the presence of young fibroblasts at 15 days ( P  = 0.034) and between the Control and MCG Groups for the presence of multinucleated giant cells at 30 days ( P  = 0.009). Monomer conversion increased progressively until day 30, with significant difference between Group FOB and Groups UBL and MCG ( P  = 0.013) at 15 days. The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Several treatments have been developed aiming the prevention of bone loss. There are discussions about the best prophylactic and therapeutic procedures for osteoporosis. This study evaluated the effects of physical exercise associated with risedronate as a prophylactic and therapeutic procedure in osteopenic bones of rats submitted to ovariectomy. We used 48 Wistar rats divided into: ovariectomized or subjected to sham surgery. Ovariectomized rats were divided into the following sub-groups: OVX, 12 weeks sedentary; OVX-EX, treadmill training for 12 weeks; OVX-RA, 12 weeks with risedronate administration; and OVX-EX-RA, 12 weeks with risedronate administration and treadmill training. Rats subjected to sham surgery were divided into the following sub-groups: SH, 12 weeks sedentary; SH-EX, treadmill training for 12 weeks; SH-RA, 12 weeks with risedronate administration; and SH-EX-RA, 12 weeks with risedronate administration and training on the treadmill. The effectiveness of the treatment was evaluated in tibias using biomechanical, radiological, histomorphometric, and immunohistochemical analyses. Data were analyzed by statistical tests, with significance level of P  〈 0.05. Results of mechanical tests showed that the SH-RA group had lower values compared with OVX-RA group; densitometry showed no significant differences; according to histomorphometric methods, OVX group presented lower results than the SH-EX, OVX-RA, SH-EX-RA, and OVX-EX-RA groups, and SH-EX-RA and OVX-EX-RA groups showed values higher than SH-RA, SH, and OVX-EX groups. The SH-EX-RA and OVX-EX-RA groups had decreased immunostaining for tartrate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B ligand and increased osteoprotegerin immunostaining. In this experimental model, it was concluded that the physical training associated with use of risedronate exerted positive effects on biomechanical and microstructural properties in bones of ovariectomized rats. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Histone H3.3 turnover displays distinct dynamics at various genomic elements such as promoters, enhancers, gene bodies, and heterochromatic regions, suggesting that it is differentially regulated according to chromatin context. Incorporation of variant histones into chromatin provides a mechanism to modulate chromatin states in addition to histone modifications. The replication-independent deposition and replacement of histone variant H3.3, i.e. H3.3 turnover, is mainly associated with transcriptional activity. H3.3 or H3.3-like histone turnover has been studied in various organisms from yeast to mammals. Here, we review the recent progress on this topic. The diversified turnover profiles of H3.3, and their corresponding underlying mechanisms, may reflect distinct requirements for chromatin accessibility in different biological events. Histone H3.3 turnover displays distinct dynamics at various genomic elements such as promoters, enhancers, gene bodies, and heterochromatic regions. The diversified turnover profiles of H3.3, and their corresponding underlying mechanisms, may reflect distinct requirements for chromatin accessibility in different biological events.

Description: Artificial genetic constructs that direct the synthesis of self-replicating RNA molecules are used widely to induce gene silencing, for bioproduction, and for vaccination. Interestingly, one variant of the self-replicon has not been discussed in the literature: namely, transgenic organisms that synthesise alien replicons. For example, plant cells may be easily genetically modified to produce bacteriophages or insect viruses. Alien replicon-producing organisms (ARPOs) may serve as a unique tool for biocontrol or to selectively influence the characteristics of a target organism. The ARPO approach would have to meet strict biosafety criteria, and its practical applications are problematic. However, a discussion on ARPO applicability would be valuable to outline the full set of options available in the bioengineering toolbox. In this paper, RNA replicons for bioengineering are reviewed briefly, and the ARPO approach is discussed. Genetic construct produces two mRNAs: for viral replicon (1), and for corresponding coat protein (2). Coat protein (3) specifically packs the replicon mRNA into transmissible virus-like particles. Plants interact with a target organism susceptible for this virus and the replicon may spread over its population.

Description: The study presents an old icon painted in egg tempera on lime wood, with a poor conservation condition and clogged dirt deposits. The icon is attributed to an anonymous painter of XIXth century and to the neo-classical style of painting. The painting layer was done with only a hand full of pigments, earth colors that were often used in painting the icons from XVIIth to XIXth century in Eastern Europe, that have Byzantine influences. Taking into consideration the nature and the structure of the materials from the upper layers of the painting, affected by deposits of dirt over time, a series of cleaning recipes were studied, using the so called cleaning tests with compatible mixtures of different juices and infusion from indigenes plants, that were freshly done and odorless. A low alkaline 95% ethyl alcohol solution, combined with a few drops of ammoniac 25%, was used as a reference system, due to its compatibility with the greasy deposits found on the polychrome layer and on the wood. The cleaning capacity of the new systems used, in comparison with the standard solution, was analyzed through modern analytical methods of evaluating the degree of cleaning, more exactly by means of visible and UV reflectography, CIE L*a*b* colorimetry by reflection assisted by SEM-EDX and IR spectroscopy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Background : There is a discrepancy between the interest in joint-reconstructions and the current knowledge about the healing-processes involved. Major reconstructions are performed with osteosynthesized allografts and fresh allografts for cartilage. Objectives : The main question to be answered is: what do we know about metaphyseal and epiphyseal cancellous bone healing, contact healing of the subchondral bone and its influence on cartilage healing? Can we achieve healing of all four compartments in contact? Purpose : The purpose is to systematically investigate through animal testing the healing processes of metaphyseal and epiphyseal bone, including the subchondral bone and the healing of cartilage of press-fit-inserted grafts, considering nondemineralized high-resolution histology. Material and Methods : Primary cancellous-bone healing of osteosynthesized hemi-osteotomies was studied in 13 canine tibial heads, the contact healing was investigated in 7 dogs and 18 giant-rabbits comparing contact-healing of press-fit-inserted autologs cylindrical grafts with empty defects applying the wet-grinding diamond-technology. Bench-experiments on the epiphyseal bones of swine including pullout-tests of cylindrical grafts formed the basis for validation of that press-fit diamond technology. Results : Primary metaphyseal and epiphyseal contact healing, including hyaline cartilage, was found in all compartments of the meta-and epiphysis in the precisely performed experiments. The press-fit principle, which employs cylindrical grafts and diamond instrumentation featuring a difference of 15/100 mm between graft and recipient bed, achieved high loads between 73.48 and 178.95 N (mean value 118.16 and standard deviation 32.79) in the pullout tests. Conclusion : Autologous press-fit grafting with alignment of the bony baseplate using wet-grinding precision has attained promising histo-morphological results. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Due to its very low hardness and atomic number, pure lithium cannot be prepared by conventional methods prior to scanning electron microscopy analysis. Here, we report on the characterization of pure lithium metallic sheets used as base electrodes in the lithium-ion battery technology using electron backscatter diffraction (EBSD) and x-ray microanalysis using energy dispersive spectroscopy (EDS) after the sheet surface was polished by broad argon ion milling (IM). No grinding and polishing were necessary to achieve the sufficiently damage free necessary for surface analysis. Based on EDS results the impurities could be characterized and EBSD revealed the microsctructure and microtexture of this material with accuracy. The beam damage and oxidation/hydration resulting from the intensive use of IM and the transfer of the sample into the microscope chamber was estimated to be 〈50 nm. Despite the fact that the IM process generates an increase of temperature at the specimen surface, it was assumed that the milling parameters were sufficient to minimize the heating effect on the surface temperature. However, a cryo-stage should be used if available during milling to guaranty a heating artefact free surface after the milling process. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Over the past decade, high-throughput studies have identified many novel transcripts. While their existence is undisputed, their coding potential and functionality have remained controversial. Recent computational approaches guided by ribosome profiling have indicated that translation is far more pervasive than anticipated and takes place on many transcripts previously assumed to be non-coding. Some of these newly discovered translated transcripts encode short, functional proteins that had been missed in prior screens. Other transcripts are translated, but it might be the process of translation rather than the resulting peptides that serves a function. Here, we review annotation studies in zebrafish to discuss the challenges of placing RNAs onto the continuum that ranges from functional protein-encoding mRNAs to potentially non-functional peptide-producing RNAs to non-coding RNAs. As highlighted by the discovery of the novel signaling peptide Apela/ELABELA/Toddler, accurate annotations can give rise to exciting opportunities to identify the functions of previously uncharacterized transcripts. There is accumulating evidence that translation occurs at many sites outside of known protein-coding regions. This phenomenon of pervasive translation presents a challenge for gene annotations, but also gives rise to exciting opportunities to identify functions for uncharacterized genes.

Description: We propose for the first time to divide histone proteolysis into “histone degradation” and the epigenetically connoted “histone clipping”. Our initial observation is that these two different classes are very hard to distinguish both experimentally and biologically, because they can both be mediated by the same enzymes. Since the first report decades ago, proteolysis has been found in a broad spectrum of eukaryotic organisms. However, the authors often not clearly distinguish or determine whether degradation or clipping was studied. Given the importance of histone modifications in epigenetic regulation we further elaborate on the different ways in which histone proteolysis could play a role in epigenetics. Finally, unanticipated histone proteolysis has probably left a mark on many studies of histones in the past. In conclusion, we emphasize the significance of reviving the study of histone proteolysis both from a biological and an experimental perspective. Also watch the Video Abstract . By categorizing histone proteolysis into “Histone Clipping” and “Histone Degradation” we illustrate that these biologically diverse processes are mediated by the same enzymes, such as Cathepsin L and Neutrophil Elastase. Experimentally distinguishing both classes is a prerequisite to define the role of histone clipping in epigenetics in the future.

Description: Communication and common understanding between politicians, scientists, and the society can lead to evidence-based science policy, a core principle that guides high caliber research and open innovation for a sustainable future.

Description: The emphasis of systems and synthetic biology on quantitative understanding of biological objects and their eventual re-design has raised the question of whether description and construction standards that are commonplace in electric and mechanical engineering are applicable to live systems. The tuning of genetic devices to deliver a given activity is generally context-dependent, thereby undermining the re-usability of parts, and predictability of function, necessary for manufacturing new biological objects. Tolerance (acceptable limits within the unavoidable divergence of a nominal value) and allowance (deviation introduced on purpose for the sake of flexibility and hence modularity, i.e. fitting together with a variety of other components) are key aspects of standardization that need to be brought to biological design. These should endow functional building blocks with a pre-specified level of confidence for bespoke biosystems engineering. However, in the absence of more fundamental knowledge, fine-tuning necessarily relies on evolutionary/combinatorial gravitation toward a fixed objective. Assembly of functional objects within a pre-existing frame might happen through forced standardization of the boundaries (A), through joining interacting parts with flexible interfaces permitting allowance and tolerance (B) or random exploration of a solution space (C). We argue that this is true both for mechanical engineering and synthetic biology.

Description: One of the distinctive features of the primate genome is the Alu element, a repetitive short interspersed element, over a million highly similar copies of which account for 〉10% of the genome. A direct consequence of this feature is that primates' transcriptome is highly enriched in long stable dsRNA structures, the preferred target of adenosine deaminases acting on RNAs (ADARs), which are the enzymes catalyzing A-to-I RNA editing. Indeed, A-to-I editing by ADARs is extremely abundant in primates: over a hundred million editing sites exist in their genomes. However, there are few essential editing sites conserved across mammals that have maintained their editing level despite the radical change in ADAR target landscape. Here, we review and discuss the cost of having an unusual amount of dsRNA and editing in the transcriptome, as well as the opportunities it presents, which might have contributed to the accelerated evolution of the primates. Invasion of Alu repeats into the primate genome created a plethora of new A-to-I RNA editing targets. How can the editing machinery maintain regulation of editing levels in the few essential sites in face of the drastic change in the editome landscape? Has necessity to overcome this challenge led to innovative functional editing in the course of primate evolution?

Description: ABSTRACT Biological reactions between biomaterials and surrounding tissues, analyzed by histology, may be important predictors of clinical healing pattern and selection of slide preparation techniques requires a careful consideration regarding sample properties. In this study, we compared histology of bone specimens prepared with or without decalcification and performed histological and histomorphometrical assessments. For the histological evaluation, one-wall intrabony defects were created around the mandibular molars of six adult dogs, filled with biphasic calcium phosphate, synthetic bone graft material/recombinant human bone morphogenic protein-2, and healing pattern was histologically evaluated at 4 and 12 weeks. New bone formation in 5 × 4 × 4 mm defects and the length of new cementum, connective tissue attachments around the teeth and number of osteoclasts were measured by histomorphometric analysis. After decalcification, new cementum was easily observed and was significantly increased at week 4. In nondecalcified samples, significantly increased connective tissue attachments were seen at week 12. After 12 weeks, the number of countable multinucleated osteoclasts was significantly increased by 62% in nondecalcified versus calcified tissue sections ( P = 0.030). Histomorphometric results may be significantly affected by histological preparation method and therefore, selecting the most appropriate histological preparation method is essential for reliable diagnosis and evaluation of bony samples in studies analyzing tissue regeneration. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This article presents the experimental results of a research on six manuscripts (three of the XVIII century and three of XIX century) belonging collection of old religious books to the Moldovan Metropolitan Church of Romania. Non-invasive techniques (optical microscopy [OM], scanning electron microscopy/energy dispersive X-ray system, X-ray fluorescence analysis, shrinkage temperature, and Fourier transform infrared spectroscopy/attentuated total reflectance) provided information on the degree of degradation and identification of the leather bookbinding type. Moreover, visual assessment and OM revealed the extent of the surface degradation (wane, biological attack, change color, etc.). The degradation extent of the skin bindings was determined on the 12 samples. The insight on the mechanism of degradation was accomplished by analyzing the deterioration of collagen fibers in terms of shrinkage temperature and chemical modifications induced by oxidative and hydrolytic processes. Shrinkage temperature values were lower compared with the literature data for collagen, indicating that the leather bookbinding suffered intrinsic damage. Morphological analysis was accomplished by microscopy and allowed the identification of skin type and provided information about its processing technique. Mineral elements were identified for leather composition and contributed to the information regarding the origin and the extent of degradation of the leather bookbinding, of the studied manuscripts. The analyzed results were useful in determining the state of preservation and were able to provide an increased efficiency of further restoration. The correlation of the obtained data brought new contributions to the knowledge of the leather covers for the book technique in the XVIII and XIX centuries in monastic workshops of Eastern Europe. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: After more than a century of research on glycolysis, we have detailed descriptions of its molecular organization, but despite this wealth of knowledge, linking the enzyme properties to metabolic pathway behavior remains challenging. These challenges arise from multi-layered regulation and the context and time dependence of component functions. However, when viewed as a system that functions according to the principles of supply and demand, a simplifying theoretical framework can be applied to study its regulation logic and to assess the coherence of experimental interpretations. These principles are universally applicable, as they emphasize the common metabolic tasks of glycolysis: the provision of free-energy carriers, and precursors for biosynthesis and stress-related compounds. Here we will review the regulation of multi-tasking by glycolysis and consider how an understanding of this central metabolic pathway can be pursued using general principles, rather than focusing on the biochemical details of constituent components. An intuitive understanding of multi-tasking by glycolysis is complicated by many interactions and dynamic, multi-layered regulation. Viewed as a system that functions according to the principles of supply and demand, a simplifying theoretical framework can be applied to study its regulation logic and to assess the coherence of experimental interpretations.

Description: ABSTRACT Vascular remodeling in the brain occurs as a plastic change following neural over-activity. The auditory midbrain (or inferior colliculus, IC) is an ideal place to study sound-induced vascular changes because it is the brain's most vascularized structure and it is tonotopically organized. However, its micro-vascular pattern remains poorly understood. Since the IC is a sphere-like structure, the histological assessment of vasculature could depend on the angle of sectioning. Here, we studied the effects of cutting the IC at different angles on microvascular assessment, specifically: micro-vascular density and the shape of microvascular lumen. Photomicrographs were taken from 5 µm toluidine blue-stained histological sections obtained at two angles of sectioning: (a) the conventional coronal sectioning, and (b) a novel “tangential” sectioning (tangential to the dorso-medial surface of the IC). Results showed that the tangential sections, in comparison with the coronal sections, yielded (a) a higher count of micro-vascular density and (b) a higher proportion of round-shaped micro-vascular lumens. This discrepancy in results between two cut angles is likely related to the spatial pattern of blood vessels supplying the IC. We propose that the tangential sectioning should be adopted as standard for the accurate study of microvasculature in the IC. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The effect of different Ca contents on the microstructure and mechanical properties of Mg-5Al-1Bi-0.3Mn (AMB501) magnesium alloys was investigated by conventional melting and casting technique using different Ca contents (1.0, 2.0, and 3.0 wt %). Increasing the Ca content resulted in higher hardness and yield strength, but decreased elongation. The improved tensile properties of the AM50-1Bi- x Ca alloys were due to the changes in AMB501 alloy microstructure when the Ca content increased, as demonstrated by scanning electron microscope, energy dispersive spectrum, and X-ray diffractometer. The alloy microstructure indicated that the amount of β-Mg 17 Al 12 phase on grain boundaries decreased and the morphology of β-Mg 17 Al 12 phase on grain boundaries changed from quasicontinuous-net shape to dispersed particles. The Mg 17 Al 12 phase disappeared and a new secondary phase Al 2 Ca appeared after a 3.0 wt % Ca addition. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Transcription-coupled repair (TCR) is a phenomenon that exists in a wide variety of organisms from bacteria to humans. This mechanism allows cells to repair the actively transcribed DNA strand much faster than the non-transcribed one. At the sites of bulky DNA damage RNA polymerase stalls, initiating recruitment of the repair machinery. It is a commonly accepted paradigm that bacterial cells utilize a sole coupling factor, called Mfd to initiate TCR. According to that model, Mfd removes transcription complexes stalled at the lesion site and simultaneously recruits repair machinery. However, this model was recently put in doubt by various discrepancies between the proposed universal role of Mfd in the TCR and its biochemical and phenotypical properties. Here, I present a second pathway of bacterial TCR recently discovered in my laboratory, which does not involve Mfd but implicates a common repair factor, UvrD, in a central position in the process. Two pathways of transcription-coupled repair. Left: During acute stress RNAP stalled at a lesion is pushed back by UvrD without transcript loss and after repair by the NER machinery transcription resumes. Right: At steady-state level of DNA damage stalled RNAP is pushed forward by Mfd, which displaces RNA polymerase to allow the repair.

Description: The analysis of genetic and epigenetic mechanisms of the genotype–phenotypic connection has, so far, only been possible in a handful of genetic model systems. Recent technological advances, including next-generation sequencing methods such as RNA-seq, ChIP-seq and RAD-seq, and genome-editing approaches including CRISPR-Cas, now permit to address these fundamental questions of biology also in organisms that have been studied in their natural habitats. We provide an overview of the benefits and drawbacks of these novel techniques and experimental approaches that can now be applied to ecological and evolutionary vertebrate models such as sticklebacks and cichlid fish. We can anticipate that these new methods will increase the understanding of the genetic and epigenetic factors influencing adaptations and phenotypic variation in ecological settings. These new arrows in the methodological quiver of ecologist will drastically increase the understanding of the genetic basis of adaptive traits – leading to a further closing of the genotype–phenotype gap. Novel methods are currently revolutionizing the fields of ecology and evolutionary biology. They facilitate the investigation of genotype–phenotype-associations and permit the analysis of cis-regulatory and epigenetic underpinnings of adaptive traits. For the first time transgenesis and genome-editing tools allow the validation of targets in ecological model organisms.

Description: Egg and sperm have, understandably, been the “stars” of mammalian fertilization biology, particularly because artificial reproductive technologies allow for fertilization to occur outside of the female reproductive tract without other apparent contributions from either sex. Yet, recent research, including an exciting new paper, reveals unexpected and important contributions of seminal plasma to fertility. For example, seminal plasma proteins play critical roles in modulating female reproductive physiology, and a new study in mice demonstrates that effects of some of these proteins on the female can even affect the health of her progeny. Furthermore, although several actions of seminal plasma have been conserved across taxa, male accessory glands and their products are diverse – even among mammals. Taken together, these studies suggest that the actions of seminal plasma components are important to understand, and also to consider in future development of assisted reproductive technologies (ART) for humans, farm species and endangered species of mammals. Although eggs and sperm can combine to generate embryos (upper equation), studies in a range of organisms show that seminal plasma components (stars, in the lower equation) enhance fertility by modulating the physiology of the female and – based on a recent study – improving the health of her progeny. The sperm diagram is based on a photo of a human sperm in Smith et al. (2009, Cell Motil. Cytoskel . 66 : 220–36). The very happy emoticon in the lower equation is from: http://www.graphicsfuel.com/2012/09/happy-and-sad-emoticons-psd/ .

Description: Although music and other forms of art can develop in diverse directions, they are linked to the genetic profiles of populations. Hearing music is a strong environmental trigger that serves as an excellent model to study the crosstalk between genes and the environment. We propose that the ability to enjoy and practice music requires musical aptitude, which is a common and innate trait facilitating the enjoyment and practice of music. The innate drive for music can only have arisen by exposure to music, and it develops with motivation and training in musically rich environments. Recent genomic approaches have shown that the genes responsible for inner ear development, auditory pathways and neurocognitive processes may underlay musical aptitude. It is expected that genomic approaches can be applied to musical traits and will reveal new biological mechanisms that affect human evolution, brain function, and civilisation. Can genes explain why some people are more interested in music than others? Genomics approaches enable to study the biological background of musical aptitude in an unbiased, hypothesis-free fashion, without any prior knowledge about the biological background. Genome-wide analyses of musical aptitude identified candidate genes that affect the auditory pathway and neurocognitive functions.

Description: Environmental factors routinely influence an organism's biology. The inheritance or transmission of such influences to descendant generations would be an efficient mode of information transfer across generations. The developmental stage at which a specific environment is encountered by the ancestral generation, and the number of generations over which information about that environment is registered, determines an inter- vs. trans-generational effect of ancestral influence. This commentary will outline the distinction between these influences. While seductive in principle, inter- and trans-generational inheritance in mammals is a hotly debated area of research inquiry. We present constructive criticism of such inheritance, and suggest potential experimental avenues for reconciliation. Finally, epigenetic mechanisms present an avenue for gene regulation that is dynamic. We briefly discuss how such malleability affords the potential for a reversal of any detrimental environmental influences that might have adversely impacted ancestral or descendant generations. Influence of ancestral environments on descendant biology are accumulating. It is important to distinguish these influences as inter- or trans-generational inheritance. Questions remain about how gametes are marked by ancestral environmental factors, and how genetic loci that are marked by epigenetic mechanisms escape from epigenetic reprogramming.

Description: Microscopy has revealed tremendous diversity of bacterial and eukaryotic forms. Recent molecular analyses show discordance in estimates of biodiversity between morphological and molecular analyses. Moreover, phylogenetic analyses of the diversity of microbial forms reveal evidence of convergence at scales as deep as interdomain: morphologies shared between bacteria and eukaryotes. Here, we highlight examples of such discordance, focusing on exemplary lineages such as testate amoebae, ciliates, and cyanobacteria. These have long histories of morphological study, enabling deeper analyses on both the molecular and morphological sides. We discuss examples in two main categories: (i) morphologically identical (or highly similar) individuals that are genetically distinct and (ii) morphologically distinct individuals that are genetically the same. We argue that hypotheses about discordance can be tested using the concept of neutral morphologies, or more broadly neutral phenotypes, as a null hypothesis. Recent phylogenies of microbial lineages, combined with microscopical studies, reveal instances of discordance between molecular and morphological evolution. Here, we focus on exemplary microbial lineages with long histories of morphological study. We propose that instances of discordance can be tested using the concept of neutral morphologies as a null hypothesis.

Description: The acidic (leucine-rich) nuclear phosphoprotein 32 kDa (ANP32) family is composed of small, evolutionarily conserved proteins characterized by an N-terminal leucine-rich repeat domain and a C-terminal low-complexity acidic region. The mammalian family members (ANP32A, ANP32B, and ANP32E) are ascribed physiologically diverse functions including chromatin modification and remodelling, apoptotic caspase modulation, protein phosphatase inhibition, as well as regulation of intracellular transport. In addition to reviewing the widespread literature on the topic, we present a concept of the ANP32s as having a whip-like structure. We also present hypotheses that ANP32C and other intronless sequences should not currently be considered bona fide family members, that their disparate necessity in development may be due to compensatory mechanisms, that their contrasting roles in cancer are likely context-dependent, along with an underlying hypothesis that ANP32s represent an important node of physiological regulation by virtue of their diverse biochemical activities. This essay surveys our understanding of the ANP32 proteins, a family of multifunctional factors conserved in most eukaryotes. Based primarily on studies of mammalian ANP32A, ANP32B, and ANP32E, we hypothesize that these regulators of chromatin, caspases, phosphatases, and mRNA transport coordinate disparate pathways and potentially impact disease states.

Description: Lysine methylation has been traditionally associated with histones and epigenetics. Recently, lysine methyltransferases and demethylases – which are involved in methylation of non-histone substrates – have been frequently found deregulated in human tumours. In this realm, a new discovery has unveiled the methyltransferase SMYD3 as an enhancer of Ras-driven cancer. SMYD3 is up-regulated in different types of tumours. SMYD3-mediated methylation of MAP3K2 increases mutant K-Ras-induced activation of ERK1/2. Methylation of MAP3K2 prevents it from binding to the phosphatase PP2A, thereby impeding the impact of this negative regulator on Ras-ERK1/2 signals, leading to the formation of lung and pancreatic adenocarcinomas. Furthermore, depletion of SMYD3 synergises with a MEK inhibitor, currently in clinical trials, to block Ras-driven pancreatic neoplasia. These results underscore the importance of lysine methylation in the regulation of signalling pathways relevant for tumourigenesis and endorse the development of drugs targeting unregulated lysine methylation as therapeutic agents in the struggle against cancer. Lysine methylation is traditionally associated with histones and epigenetics. Recently, lysine methyltransferases involved in methylation of non-histone substrates have been frequently found deregulated in tumours. Now methyltransferase SMYD3 has been identified as an enhancer of Ras-driven cancer via methylation of MAP3K2, which prevents it from binding to the phosphatase PP2A, thereby mitigating its negative regulation of Ras-ERK1/2 signals, hence promoting tumourigenesis.

Description: Secondary lymphoid organs form in utero through an inherited and well-established developmental program. However, maternal non-heritable features can have a major impact on the gene expression of the embryo, hence influencing the future health of the offspring. Recently, maternal retinoids were shown to regulate the formation of immune structures, shedding light on the role of maternal nutrition in the genetic signature of emergent immune cells. Here we highlight evidence showing how the maternal diet influences the establishment of the immune system, and we also discuss how unbalanced maternal diets may set the response to infection and vaccination in the progeny. Maternal nutrition has a major impact on the developing immune system, hence influencing the future health of the progeny. Recent findings revealed that retinoic acid, derived from the maternal diet intake of vitamin A, is critical for the development of secondary lymphoid organs in the embryo, such as lymph nodes and Peyer's patches, which in turn pre-set the efficiency of adaptive immune responses throughout life.

Description: Oncogene activation leads to cellular transformation by deregulation of biological processes such as proliferation and metabolism. Paradoxically, this can also sensitize cells to nutrient deprivation, potentially representing an Achilles' heel in early stage tumors. The mechanisms underlying this phenotype include loss of energetic and redox homeostasis as a result of metabolic reprogramming, favoring synthesis of macromolecules. Moreover, an emerging mechanism involving the deregulation of mRNA translation elongation through inhibition of eukaryotic elongation factor 2 kinase (eEF2K) is presented. The potential consequences of eEF2K deregulation leading to cell death under nutrient depletion are discussed. Finally, the relevance of eEF2K as a master regulator of the response to nutrient deprivation in vivo, and its potential exploitation for therapeutic targeting of cancers, are elaborated. Overall, a better understanding of the adaptive mechanisms allowing tumors to circumvent oncogene-induced hypersensitivity to nutrient deprivation is a promising avenue for uncovering novel therapeutic targets in cancers. Oncogene activation leads to hypersensitivity to nutrient deprivation by deregulation of specific metabolic pathways. Novel mechanisms underlying this phenotype are emerging, such as altered control of mRNA translation elongation by inhibition of the eEF2 kinase (eEF2K). The adaptive mechanisms used to circumvent oncogene-mediated cell death under nutrient deprivation are described.

Description: ABSTRACT The cysts of nine Chinese populations of parthenogenetic Artemia were studied by scanning electron microscope. In the 270 cysts examined, 15 different morphological patterns were recognized with most of them not recorded in previous studies and the “tubercled shell surface” being the most common pattern. Results also displayed high intrapopulation variability, with the maximum of 11 patterns (in 30 cysts) recorded from the Barkol population. No positive correlation between the diversity of cyst shell patterns and ploidy compositions was found. Principal components analysis suggests higher similarity among coastal populations than among inland populations, which may be attributed to the identity of physicochemical conditions among coastal salterns and dissimilarity among inland saline lakes. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: The nomination of candidate genes underlying complex traits is often focused on genetic variations that alter mRNA abundance or result in non-conservative changes in amino acids. Although inconspicuous in complex trait analysis, genetic variants that affect splicing or RNA editing can also generate proteomic diversity and impact genetic traits. Indeed, it is known that splicing and RNA editing modulate several traits in humans and model organisms. Using high-throughput RNA sequencing (RNA-seq) analysis, it is now possible to integrate the genetics of transcript abundance, alternative splicing (AS) and editing with the analysis of complex traits. We recently demonstrated that both AS and mRNA editing are modulated by genetic and environmental factors, and potentially engender phenotypic diversity in a genetically segregating mouse population. Therefore, the analysis of splicing and RNA editing can expand not only the regulatory landscape of transcriptome and proteome complexity, but also the repertoire of candidate genes for complex traits. The complex molecular mechanisms that modulate the relationship between genetic variations and quantitative traits are often depicted in broad cellular transcriptional networks. Therefore, a comprehensive genetic analysis of the cellular transcript abundance, levels of alternative splicing, and RNA editing, will help to delineate the mechanisms that modulate the genotype-phenotype relationship.

Description: Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. Replication protein A (RPA) binds single-strand DNA intermediates in DNA replication, repair, and recombination. Recent studies have demonstrated that RPA-DNA interactions are highly dynamic and suggest that this dynamism contributes to the functions of RPA and is necessary for genome stability.

Description: Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools. A recent single-molecule pull-down method (SiMPull) has been described in detail, which enables determination of absolute copy number of protein complexes, the stoichiometry of each protein in the complex and their functional properties all in matter of minutes directly from whole cell or tissue extracts.

Description: ABSTRACT The results of microstructural characterization of mortars containing fly ash class C (High Calcium Fly Ash) from combustion of lignite are presented. The evaluation of the microstructure was performed using scanning electron microscope, optical, and confocal microscope. The tested beams were bent till the crack and microcracks opening, which were healed during the different curing time. The results showed that the replacement of cement with fly ash class C influenced the process of crack healing. The addition of HCFA, at both 30% and 60%, speeds up the self-healing process in cracks and particularly in micro-cracks. In the research, the completely filling up of the cracks by new phases has not been observed, only the beginning of such process has been noticed. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Here, we propose that the heterogeneity of mutational types in populations underpins alternative pathways of evolutionary adaptation. Point mutations, deletions, insertions, transpositions and duplications cause different biological effects and provide distinct adaptive possibilities. Experimental evidence for this notion comes from the mutational origins of adaptive radiations in large, clonal bacterial populations. Independent sympatric lineages with different phenotypes arise from distinct genetic events including gene duplication, different insertion sequence movements and several independent point mutations. The breadth of the mutational spectrum in the ancestral population should be viewed as a form of bet-hedging, reducing the risk of evolutionary dead ends and complementing the phenotypic and epigenetic heterogeneities that improve the survival capabilities of a population. Different mutational events arise from distinct cellular processes and are subject to separate environmental impacts, so the availability of any particular type of mutation may constrain or promote adaptive pathways in populations. Distinct types of mutation have different phenotypic effects. Sub-populations containing different mutations provide alternative starting points for adaptation and originate parallel lines of evolution, initiating sympatric divergence. The mutational heterogeneity can be viewed as a form of bet-hedging, initiating alternative evolutionary solutions that have different risks associated with them.

Description: Myelin is required for efficient nerve conduction, but not all axons are myelinated to the same extent. Here we review recent studies that have revealed distinct myelination patterns of different axonal paths, suggesting that myelination is not an all or none phenomenon and that its presence is finely regulated in central nervous system networks. Whereas powerful reductionist biology has led to important knowledge of how oligodendrocytes function by themselves, little is known about their role in neuronal networks. We still do not understand how oligodendrocytes integrate information from neurons to adapt their function to the need of the system. An intricate cross talk between neurons and glia is likely to exist and to determine how neuronal circuits operate as a whole. Dissecting these mechanisms by using integrative systems biology approaches is one of the major challenges ahead. Beyond the role of myelin in speeding up nerve conduction, its function in the fine-tuning of neuronal networks is just emerging. This new concept of myelin plasticity implies that oligodendrocytes are able to sense neuronal activity and adapt myelination, resulting in a wider range of consequences than previously thought.

Description: ABSTRACT We propose two-photon excitation-based light-sheet technique for nano-lithography. The system consists of 2 -configured cylindrical lens system with a common geometrical focus. Upon superposition, the phase-matched counter-propagating light-sheets result in the generation of identical and equi spaced nano-bump pattern. Study shows a feature size of as small as few tens of nanometers with a inter-bump distance of few hundred nanometers. This technique overcomes some of the limitations of existing nano-lithography techniques, thereby, may pave the way for mass-production of nano-structures. Potential applications can also be found in optical microscopy, plasmonics, and nano-electronics. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Recent studies uncovered critical roles of the adhesion protein E-cadherin in health and disease. Global inactivation of Cdh1 , the gene encoding E-cadherin in mice, results in early embryonic lethality due to an inability to form the trophectodermal epithelium. To unravel E-cadherin's functions beyond development, numerous mouse lines with tissue-specific disruption of Cdh1 have been generated. The consequences of E-cadherin loss showed great variability depending on the tissue in question, ranging from nearly undetectable changes to a complete loss of tissue structure and function. This review focuses on these studies and discusses how they provided important insights into E-cadherin's role in cell adhesion, proliferation and differentiation, and its consequences for biological processes as epithelial-to-mesenchymal transition, vascularization, and carcinogenesis. Lastly, we present some perspectives and possible approaches for future research. E-cadherin is a cell adhesion protein with critical roles in development, tissue homeostasis, and disease. We focus on mouse lines with organ-specific E-cadherin disruption and the insights they provided into E-cadherin's role in cell adhesion, proliferation and differentiation, epithelial-to-mesenchymal transition, vascularization, and carcinogenesis.

Description: Recent findings in several organ systems show that cytoneme-mediated signaling transports signaling proteins along cellular extensions and targets cell-to-cell exchanges to synaptic contacts. This mechanism of paracrine signaling may be a general one that is used by many (or all) cell types in many (or all) organs. We briefly review these findings in this perspective. We also describe the properties of several signaling systems that have previously been interpreted to support a passive diffusion mechanism of signaling protein dispersion, but can now be understood in the context of the cytoneme mechanism. Also watch the Video Abstract . The importance of paracrine signaling for animal development has been recognized for almost 100 years, but the discovery that epithelial cells transport signaling proteins over long distances with filopodia that make direct contact with target cells is recent. In this perspective we review the new findings and explore some of their many implications.

Description: ABSTRACT This study evaluated the bond strength (BS) and the adhesive interface of four endodontic sealers to root canal dentine, before, and after immersion in phosphate buffered saline (PBS) to simulate an in vivo environment. Eighty roots were instrumented using ProTaper rotatory files, under irrigation with 17% EDTA and 1% NaOCl. Posteriorly were divided into four groups ( n = 20) according to the sealer used: Endofill, AH Plus, Sealapex, and MTA Fillapex. Each group was divided into two subgroups ( n = 10) and stored at 37°C immersed in water for 7 days and in PBS for 60 days. From each subgroup, 1 mm thick sections were obtained. One section of each region (coronal, middle, and apical) was submitted to the push-out test and failures were observed. Twelve sections of each subgroup (four from each region) were evaluated under SEM. Three-way ANOVA evaluation for BS showed significant differences between groups and regions ( P 〈 0.0001), but not between subgroups ( P 〉 0.05). AH Plus had significantly higher BS than the others sealers, regardless of the analyzed subgroup (Tukey's test, P 〈 0.5). The most common failures were adhesive to dentine and cohesive of the sealer. The SEM evaluation (Kruskal–Wallis, Mann–Whitney) showed homogeneous adhesive interface formed and sealer tags in all groups with significant statistical differences with AH Plus, regardless of PBS immersion. AH Plus was superior to the other sealers for both BS and quality of interface formation. Immersion in PBS did not interfere on BS or adhesive interface of the sealers tested. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans. Also watch the Video Abstract . Understanding the diversity of genome-environment interactions requires integrative, multidisciplinary, and modeling-based approaches. Representative cnidarians and sponges provide novel opportunities to investigate how the environment interacts with developmental processes and, in turn, how developmental evolution affects the environment.

Description: Take it up . It was recently shown that horizontal gene transfer (HGT) not only takes place in the form of long DNA fragments, but that it may also occur with short and damaged DNA (down to 20 bp in length). The integration of these short fragments is achieved at replication forks, and this process opens up the possibility for genetic exchange across distinct species in both time and space. On pages 1005–1010 of this issue, Søren Overballe-Petersen and Eske Willerslev further elaborate on their findings and speculate on the potential evolutionary consequences of this phenomenon. Cover by Søren Overballe-Petersen and Eske Willerslev.