ALBERT

Description: Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock out ( ko/ko ) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4 ko/ko cells were verified by green immunofluorescence and PCR. BMP2/4 ko/ko osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4 ko/ko cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4 ko/ko osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. This article is protected by copyright. All rights reserved

Description: Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tβ4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio-protective effect of Tβ4 in angiotensin II (Ang II)-induced cardiomyocytes growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tβ4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, β-catenin and Wnt-induced secreted protein-1 (WISP-1) were evaluated by quantitative real-time PCR, Western blotting and fluorescent microscopy. Pre-treatment of Tβ4 resulted in reduction of cell size, hypertrophy marker genes and Wnt-associated gene expression and protein levels; induced by Ang II in cardiomyocytes. WISP-1 was overexpressed in NRVM and, the effect of Tβ4 in Ang II-induced cardiomyocytes growth was evaluated. WISP-1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tβ4. This is the first report which demonstrates that Tβ4 targets Wnt/WISP-1 to protect Ang II-induced cardiomyocyte growth. This article is protected by copyright. All rights reserved

Description: We have previously shown that retinoic acid (RA) has protective effects on high glucose (HG)-induced cardiomyocyte apoptosis. To further elucidate the molecular mechanisms of RA effects, we determined the interaction between nuclear factor (NF)-κB and RA signaling. HG induced a sustained phosphorylation of IKK/IκBα and transcriptional activation of NF-κB in cardiomyocytes. Activated NF-κB signaling has an important role in HG-induced cardiomyocyte apoptosis and gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α and monocyte chemoattractant protein-1 (MCP-1). All-trans RA (ATRA) and LGD1069, through activation of RAR/RXR-mediated signaling, inhibited the HG-mediated effects in cardiomyocytes. The inhibitory effect of RA on NF-κB activation was mediated through inhibition of IKK/IκBα phosphorylation. ATRA and LGD1069 treatment promoted protein phosphatase 2A (PP2A) activity, which was significantly suppressed by HG stimulation. The RA effects on IKK and IκBα were blocked by okadaic acid or silencing the expression of PP2Ac-subunit, indicating that the inhibitory effect of RA on NF-κB is regulated through activation of PP2A and subsequent dephosphorylation of IKK/IκBα. Moreover, ATRA and LGD1069 reversed the decreased PP2A activity and inhibited the activation of IKK/IκBα and gene expression of MCP-1, IL-6 and TNF-α in the hearts of Zucker diabetic fatty rats. In summary, our findings suggest that the suppressed activation of PP2A contributed to sustained activation of NF-κB in HG-stimulated cardiomyocytes; and that the protective effect of RA on hyperglycemia-induced cardiomyocyte apoptosis and inflammatory responses is partially regulated through activation of PP2A and suppression of NF-κB-mediated signaling and downstream targets. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSC) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass in early aging, the rate of loss was higher in males. Similar expression of bone- and adipocyte-related genes was seen in males and females at 3 and 9 months, while at 6 months, females exhibited a two-fold greater expression of these genes. Under osteogenic culture conditions, bone marrow MSCs from female 3- and 6-month-old mice expressed similar levels of bone-related genes, but significantly greater levels of adipocyterelated genes, than male MSCs. Female MSCs also responded to rosiglitazone-induced suppression of osteogenesis at a 5-fold lower (10 nM) concentration than male MSCs. Female MSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as MSCs grown in serum containing estrogen. MSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the MSCs came. These results also may explain the sensitivity of women to the deleterious effects of rosiglitazone on bone. © 2014 Wiley Periodicals, Inc.

Description: Early timber inventories in dry forests of the western United States offer detailed data sets that might provide historical information to guide restoration and preparation for future forests, but inventories have errors, biases, and limitations. We reviewed early documentation of errors and estimated errors by comparing inventory estimates to nearby tree-ring and plot estimates. In a case study in the Greenhorn Mountains, southern Sierra Nevada, California, we studied how selection and use of evidence affects findings and compared timber-inventory, land-survey, and other early evidence about historical forests and fire. Early documents showed inventories were unreliable, often with large underestimation errors from poor visual estimates, requiring correction multipliers of 2.0–2.5. Comparing inventory estimates to tree-ring estimates, we found commonly used two-chain-wide inventories required correction multipliers of about 1.4–3.2, consistent with, but wider than the 2.0–2.5 range. These needed corrections were not applied in any study. The case study showed (1) tree-density estimates from narrower one-chain-wide inventories could be more accurate, (2) data are often available, but unused, that provide quantitative evidence about historical high-severity fires consistent with nearby historical reports, and (3) differences in forest structure between inventories and land surveys may be explained by tree growth, stand development, and especially a significant fire. Our review also documented biased placement of inventories in merchantable timber, often excluding younger forests, chaparral, and other indicators of preceding mixed/high-severity fires. We found added significant bias introduced by omitting areas burned in mixed/high-severity fires, or by missing evidence of these fires on parts of forms or associated archival materials. Use of early timber inventories could be improved by (1) avoiding use of unreliable two-chain-wide inventories or applying correction multipliers to inventory estimates, (2) completing an accuracy test of one-chain-wide inventories, (3) locating and using notes, maps, and other data about small trees and high-severity fires often available in inventory archives, or omitting conclusions about these, (4) deriving an envelope model of inference space for inventories, and (5) specifying a large area, then including all available inventory data within it, or using unbiased selection criteria. These improvements could help bring timber-inventory data into congruence with other historical sources.

Description: Predator space use influences ecosystem dynamics, and a fundamental goal assumed for a foraging predator is to maximize encounter rate with prey. This can be achieved by disproportionately utilizing areas of high prey density or, where prey are mobile and therefore spatially unpredictable, utilizing patches of their prey's preferred resources. A third, potentially complementary strategy is to increase mobility by using linear features like roads and/or frozen waterways. Here, we used novel population-level predator utilization distributions (termed “localized density distributions”) in a single-predator (wolf), two-prey (moose and caribou) system to evaluate these space-use hypotheses. The study was conducted in contrasting sections of a large boreal forest area in northern Ontario, Canada, with a spatial gradient of human disturbances and predator and prey densities. Our results indicated that wolves consistently used forest stands preferred by moose, their main prey species in this part of Ontario. Direct use of prey-rich areas was also significant but restricted to where there was a high local density of moose, whereas use of linear features was pronounced where local moose density was lower. These behaviors suggest that wolf foraging decisions, while consistently influenced by spatially anchored patches of prey forage resources, were also determined by local ecological conditions, specifically prey density. Wolves appeared to utilize prey-rich areas when regional preferred prey density exceeded a threshold that made this profitable, whereas they disproportionately used linear features that promoted mobility when low prey density made directly tracking prey distribution unprofitable.

Description: Given the widespread degradation of aquatic systems caused by land-use changes associated with palm oil production in South-East Asia, it is imperative to identify and study the remaining undisturbed rivers and streams. Stream macroinvertebrates are reliable indicators of environmental health. Linking the community structure of these organisms to natural hydraulic and geomorphic conditions (categorized as biotopes) is vital for the conservation and restoration of streams. This study characterizes the effects of biotopes on macroinvertebrate community structure in three streams within Ulu Temburong National Park in northern Borneo. Biotopes within these streams were categorized as either bedrock (waterfalls and cascades) or mixed substrate (riffles and pools). In total, 119 taxa were collected from all sampled biotopes, but not all taxa were collected from each stream. Biotopes were statistically distinct in terms of taxonomic richness, but not mean individual density or average community biomass. There were differences in community structure between waterfalls, cascades, pools, and riffles. The survey suggests that pool and riffle biotopes were more vulnerable to scouring flows and had similar community structure, while waterfalls and cascades likely experienced lower sheer stress during floods and had similar macroinvertebrate communities. This study has found that classification and mapping of macroinvertebrates with biotope theory in pristine, tropical streams is a useful framework for simplifying the many linkages between ecology, geomorphology, and hydrology. These natural patterns increase our understanding of tropical streams and can be used to assess the impacts of forest degradation.

Description: The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (~750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with antizonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation. © 2014 Wiley Periodicals, Inc.

Description: The retinoid X receptors (RXR), peroxisome proliferator activated receptor gamma (PPARγ) and liver X receptors (LXR) all have been shown to regulate bone homeostasis. Tributyltin (TBT) is an environmental contaminant that is a dual RXRα/β and PPARγ agonist. TBT induces RXR, PPARγ and LXR-mediated gene transcription and suppresses osteoblast differentiation in vitro . Bone marrow multipotent mesenchymal stromal cells derived from female C57BL/6J mice were more sensitive to suppression of osteogenesis by TBT than those derived from male mice. In vivo , oral gavage of 12 week old female, C57Bl/6J mice with 10 mg/kg TBT for 10 weeks resulted in femurs with a smaller cross-sectional area and thinner cortex. Surprisingly, TBT induced significant increases in trabecular thickness, number, and bone volume fraction. TBT treatment did not change the Rankl : Opg RNA ratio in whole bone, and histological analyses showed that osteoclasts in the trabecular space were minimally reduced. In contrast, expression of cardiotrophin-1, an osteoblastogenic cytokine secreted by osteoclasts, increased. In primary bone marrow macrophage cultures, TBT marginally inhibited the number of osteoclasts that differentiated, in spite of significantly suppressing expression of osteoclast markers Nfatc1 , Acp5 and Ctsk and resorptive activity. TBT induced expression of RXR- and LXR-dependent genes in whole bone and in vitro osteoclast cultures. However, only an RXR antagonist, but not an LXR antagonist, significantly inhibited TBTs ability to suppress osteoclast differentiation. These results suggest that TBT has distinct effects on cortical versus trabecular bone, likely resulting from independent effects on osteoblast and osteoclast differentiation that are mediated through RXR. This article is protected by copyright. All rights reserved

Description: The rigorous program of monitoring humpback whales ( Megaptera novaeangliae ), implemented by Glacier Bay National Park and Preserve in 1985, augmented by additional data collected in southeastern Alaska since 1968, constitutes one of the longest studies of living whales in the world. This monitoring program, now a National Park Service (NPS) Inventory and Monitoring Program Vital Sign, employed consistent methods for summer surveys from 1985 to 2014 to document the number of whales and gather longitudinal records on individuals in Glacier Bay and Icy Strait. Survey effort averaged 355.4 h/yr (SD = 45.8), resulting in 9485 encounters with 662 individual whales, including 276 calves. The population increased at a rate of 5.1%/yr, from 41 individuals in 1985 to 239 individuals in 2013, primarily due to long-term site fidelity and local recruitment. We documented sighting histories of 〉30 yr in southeastern Alaska, for 54 whales, including one 45-yr sighting history. Almost half of the whales first identified as calves returned in subsequent years, at a mean age of 3.2 yr (SE = 0.28, range = 1–17 yr). Over 75% of females had their first calf by age 13. The maximum female reproductive span was 32 yr, and the maximum number of calves was 11. We estimated mean effective calving rate with a simple ratio and used logistic regression to estimate calving probability. Both methods resulted in similar maximal estimates that were somewhat lower than previously published values for this species: 0.324 (95% CL: 0.28–0.36) calves·female −1 ·yr −1 vs. calving probability of 0.319 (95% CL: 0.29–0.35). Minimal estimates, in which the first calf of each known-aged female was omitted, were 0.302 (95% CL: 0.27–0.34) calves·mature female −1 ·yr −1 vs. calving probability of 0.305 (95% CL: 0.27–0.34) calves·mature female −1 ·yr −1 . This monitoring program has guided Park management actions and documented this once critically endangered population's trajectory toward recovery, often through collaboration with other agencies and organizations, fostering the continued protection and understanding of this distinctive species. Our findings highlight the value of marine protected areas for migratory species with strong seasonal site fidelity and the role of long-term monitoring in interpreting population-level responses to changing marine ecosystems.