Imaging leukocyte migration through afferent lymphatics.

Afferent lymphatics mediate the transport of antigen and leukocytes, especially of dendritic cells (DCs) and T cells, from peripheral tissues to draining lymph nodes (dLNs). As such they play important roles in the induction and regulation of adaptive immunity. Over the past 15 years, great advances in our understanding of leukocyte trafficking through afferent lymphatics have been made through time-lapse imaging studies performed in tissue explants and in vivo, allowing to visualize this process with cellular resolution. Intravital imaging has revealed that intralymphatic leukocytes continue to actively migrate once they have entered into lymphatic capillaries, as a consequence of the low flow conditions present in this compartment. In fact, leukocytes spend considerable time migrating, patrolling and interacting with the lymphatic endothelium or with other intralymphatic leukocytes within lymphatic capillaries. Cells typically only start to detach once they arrive in downstream-located collecting vessels, where vessel contractions contribute to enhanced lymph flow. In this review, we will introduce the biology of afferent lymphatic vessels and report on the presumed significance of DC and T cell migration via this route. We will specifically highlight how time-lapse imaging has contributed to the current model of lymphatic trafficking and the emerging notion that - besides transport - lymphatic capillaries exert additional roles in immune modulation.

Profiling of syngeneic mouse HCC tumor models as a framework to understand anti-PD-1 sensitive tumor microenvironments.

BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. APPROACH AND RESULTS: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. CONCLUSIONS: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

In Silico Analysis of Genes Associated with the Pathogenesis of Odontogenic Keratocyst.

Odontogenic keratocyst (OK) is a benign intraosseous cystic lesion characterized by a parakeratinized stratified squamous epithelial lining with palisade basal cells. It represents 10-12% of odontogenic cysts. The changes in its classification as a tumor or cyst have increased interest in its pathogenesis. OBJECTIVE: Identify key genes in the pathogenesis of sporadic OK through in silico analysis. MATERIALS AND METHODS: The GSE38494 technical sheet on OK was analyzed using GEOR2. Their functional and canonical signaling pathways were enriched in the NIH-DAVID bioinformatic platform. The protein-protein interaction network was constructed by STRING and analyzed with Cytoscape-MCODE software v 3.8.2 (score > 4). Post-enrichment analysis was performed by Cytoscape-ClueGO. RESULTS: A total of 768 differentially expressed genes (DEG) with a fold change (FC) greater than 2 and 469 DEG with an FC less than 2 were identified. In the post-enrichment analysis of upregulated genes, significance was observed in criteria related to the organization of the extracellular matrix, collagen fibers, and endodermal differentiation, while the downregulated genes were related to defensive response mechanisms against viruses and interferon-gamma activation. CONCLUSIONS: Our in silico analysis showed a significant relationship with mechanisms of extracellular matrix organization, interferon-gamma activation, and response to viral infections, which must be validated through molecular assays.

Comprehensive Evaluation of the Impact of Sociodemographic Inequalities on Adverse Outcomes and Excess Mortality During the Coronavirus Disease 2019 (COVID-19) Pandemic in Mexico City.

BACKGROUND: The impact of the coronavirus disease 2019 (COVID-19) pandemic in Mexico City has been sharp, as several social inequalities at all levels coexist. Here we conducted an in-depth evaluation of the impact of individual and municipal-level social inequalities on the COVID-19 pandemic in Mexico City. METHODS: We analyzed suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases, from the Mexico City Epidemiological Surveillance System from 24 February 2020 to 31 March 2021. COVID-19 outcomes included rates of hospitalization, severe COVID-19, invasive mechanical ventilation, and mortality. We evaluated socioeconomic occupation as an individual risk, and social lag, which captures municipal-level social vulnerability, and urban population density as proxies of structural risk factors. Impact of reductions in vehicular mobility on COVID-19 rates and the influence of risk factors were also assessed. Finally, we assessed discrepancies in COVID-19 and non-COVID-19 excess mortality using death certificates from the general civil registry. RESULTS: We detected vulnerable groups who belonged to economically unfavored sectors and experienced increased risk of COVID-19 outcomes. Cases living in marginalized municipalities with high population density experienced greater risk for COVID-19 outcomes. Additionally, policies to reduce vehicular mobility had differential impacts modified by social lag and urban population density. Finally, we report an under-registry of COVID-19 deaths along with an excess mortality closely related to marginalized and densely populated communities in an ambulatory setting. This could be attributable to a negative impact of modified hospital admission criteria during the pandemic. CONCLUSIONS: Socioeconomic occupation and municipality-wide factors played a significant role in shaping the course of the COVID-19 pandemic in Mexico City.

A novel trophic cascade between cougars and feral donkeys shapes desert wetlands.

Introduced large herbivores have partly filled ecological gaps formed in the late Pleistocene, when many of the Earth's megafauna were driven extinct. However, extant predators are generally considered incapable of exerting top-down influences on introduced megafauna, leading to unusually strong disturbance and herbivory relative to native herbivores. We report on the first documented predation of juvenile feral donkeys Equus africanus asinus by cougars Puma concolor in the Mojave and Sonoran Deserts of North America. We then investigated how cougar predation corresponds with differences in feral donkey behaviour and associated effects on desert wetlands. Focusing on a feral donkey population in the Death Valley National Park, we used camera traps and vegetation surveys to compare donkey activity patterns and impacts between wetlands with and without cougar predation. Donkeys were primarily diurnal at wetlands with cougar predation, thereby avoiding cougars. However, donkeys were active throughout the day and night at sites without predation. Donkeys were ~87% less active (measured as hours of activity a day) at wetlands with predation (p < 0.0001). Sites with predation had reduced donkey disturbance and herbivory, including ~46% fewer access trails, 43% less trampled bare ground and 192% more canopy cover (PERMANOVA, R2  = 0.22, p = 0.0003). Our study is the first to reveal a trophic cascade involving cougars, feral equids and vegetation. Cougar predation appears to rewire an ancient food web, with diverse implications for modern ecosystems. Our results suggest that protecting apex predators could have important implications for the ecological effects of introduced megafauna.

The P522R protective variant of PLCG2 promotes the expression of antigen presentation genes by human microglia in an Alzheimer's disease mouse model.

The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild-type mice were generated by transplanting PLCG2-P522R or isogenic wild-type human induced pluripotent stem cell microglia. At 7 months of age, single-cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2-P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune-intact AD mice further demonstrated that the PLCG2-P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2-P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.

Clinical significance of quantitative categorization of HER2 fluorescent in situ hybridization results in invasive breast cancer patients treated with HER2-targeted agents.

HER2 (ERBB2) gene status serves as a strong predictive marker of response to HER2-targeted agents in invasive breast cancers, albeit with heterogeneous response. Our aim was to determine the distribution and prognosis of HER2 groups by fluorescent in situ hybridization (FISH) using the updated 2018 American Society of Clinical Oncology-College of American Pathologist (ASCO-CAP) guidelines. We identified 226 cases of equivocal or positive HER2 FISH invasive breast cancer (interpreted by ASCO-CAP guidelines at the time of reporting) who received HER2-targeted agents from 2006 to 2017. We subcategorized Group 1 further into three subgroups: low amplified (HER2/CEP17 ratio ≥ 2.0-2.99, mean HER2/cell 4.0-5.9), amplified (HER2/CEP17 ratio ≥ 2.0-2.99, mean HER2/cell ≥ 6), and excessive amplification (HER2/CEP17 ratio ≥ 3.0, mean HER2/cell ≥ 4.0). Outcomes studied were recurrence, metastasis, second breast primary, disease-specific survival (DSS), and overall survival (OS). Univariate analysis showed that the five categories of HER2 FISH were significantly associated with OS (p < 0.01), specifically higher HER2 amplification was associated with fewer deaths. HER2 FISH status also statistically significantly relates to DFS (p < 0.01) and metastasis (p = 0.01) but not with recurrence or second breast primary in our study. Tumor type and HER2 ISH Groups are independent predictors for both OS and DFS in our cohort. The proposed Group 1 subcategories were significantly associated with OS (p < 0.01) and DFS (p < 0.01), excessive HER2 amplification was associated with longer median survival. The Cox regression models showed better survival outcomes for the excessive amplification subgroup than the low amplified subgroup, with OS (hazard ratio = 0.63, 95% CI 0.42-0.93) and DFS (HR = 0.55, 95% CI 0.37-0.83). We demonstrated that in HER2 FISH Group 1 patients, high HER2 amplification was significantly associated with longer OS and DFS; these patients seem to benefit more from HER2-targeted regimens. We recommend reporting these Group 1 subcategories when assessing HER2 FISH.

Somatic Mutations in Circulating Cell-Free DNA and Risk for Hepatocellular Carcinoma in Hispanics.

Hispanics are disproportionally affected by liver fibrosis and hepatocellular carcinoma (HCC). Advanced liver fibrosis is a major risk factor for HCC development. We aimed at identifying somatic mutations in plasma cell-free DNA (cfDNA) of Hispanics with HCC and Hispanics with advanced liver fibrosis but no HCC. Targeted sequencing of over 262 cancer-associated genes identified nonsynonymous mutations in 22 of the 27 HCC patients. Mutations were detected in known HCC-associated genes (e.g., CTNNB1, TP53, NFE2L2, and ARID1A). No difference in cfDNA concentrations was observed between patients with mutations and those without detectable mutations. HCC patients with higher cfDNA concentrations or higher number of mutations had a shorter overall survival (p < 0.001 and p = 0.045). Nonsynonymous mutations were also identified in 17 of the 51 subjects with advanced liver fibrosis. KMT2C was the most commonly mutated gene. Nine genes were mutated in both subjects with advanced fibrosis and HCC patients. Again, no significant difference in cfDNA concentrations was observed between subjects with mutations and those without detectable mutations. Furthermore, higher cfDNA concentrations and higher number of mutations correlated with a death outcome in subjects with advanced fibrosis. In conclusion, cfDNA features are promising non-invasive markers for HCC risk prediction and overall survival.

Sclerosing TSC1 mutated renal cell carcinoma: An unusual pattern mimicking MITF family translocation renal cell carcinoma.

The tuberous sclerosis genes and MTOR are increasingly being found to have important roles in novel subtypes of renal cancer, particularly emerging entities eosinophilic solid and cystic renal cell carcinoma (RCC) and high-grade oncocytic renal tumor (HOT)/RCC with eosinophilic and vacuolated cytoplasm. We report a unique renal neoplasm in a 66-year-old woman that initially mimicked MITF family translocation RCC due to mixed clear and eosinophilic cells, extensive stromal hyalinization, and psammoma bodies, yet which was negative for TFE3 and TFEB fluorescence in situ hybridization and a next generation sequencing (NGS) gene fusion assay. Cytoplasmic stippling triggered consideration of TSC-associated neoplasms, and a targeted NGS assay revealed a variant in exon 21 of TSC1 resulting in c.2626G>T p.(Glu876*) truncating mutation. This report adds to the morphologic spectrum of TSC-related renal neoplasms, including prominent stromal hyalinization as a potentially deceptive pattern. Due to the overlap in cytoplasmic stippling between eosinophilic solid and cystic RCC and HOT/RCC with eosinophilic and vacuolated cytoplasm, it is debatable which category this example would best fit. Further understanding of these entities and other renal neoplasms with alterations in the TSC genes will elucidate whether they should be considered a family of tumors.

Using 4-vinylcyclohexene diepoxide as a model of menopause for cardiovascular disease.

There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women.NEW & NOTEWORTHY Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.

Impacts of the biocide chlorothalonil on biomarkers of oxidative stress, genotoxicity, and sperm quality in guppy Poecilia vivipara.

Chlorothalonil is a fungicide present in antifouling paints and other formulations used in agriculture, although studies have shown this chemical to be toxic to fish species. To clarify the deleterious effects of chlorothalonil for these non-target organisms, the present study evaluated the toxic effects of this biocide for the estuarine guppy Poecilia vivipara in terms of an acute mortality test (96 h) and the analysis of biomarkers of oxidative stress, genotoxicity, and sperm quality. The LC50 calculated for P. vivipara was 40.8 µg/L of chlorothalonil. For the analysis of biomarkers, fish were exposed (96 h) to 1 and 10 µg/L of chlorothalonil. It was observed that chlorothalonil alters the levels of pro- and antioxidants towards oxidative stress. In the gills, a negative effect on total antioxidant capacity (ACAP) was detected, while there was a reduction in the activity of glutathione S-transferase (GST) in the liver. However, levels of glutathione (GSH) and the activity and glutamate-cysteine-ligase (GCL) increased in both tissues, as a possible detoxification response. Following chlorothalonil exposure, oxidative damage measured by lipoperoxidation (LPO) significantly increased at the cellular level only (red blood cells (RBCs) and sperm cells). An increase in fluidity of membranes, reactive oxygen species concentration and micronuclei (MNs) incidence were also seen in RBCs. In sperm cells, LPO increased, while membrane and mitochondrial functionality as well as sperm motility decreased. Based on these results, chlorothalonil can be considered as a toxic compound for fish, causing genotoxicity and affecting the RBCs physiology and the fertility of males of P. vivipara.

Impacts of Heterogeneous Host Densities and Contact Rates on Pathogen Transmission in the Channel Island Fox (Urocyon littoralis).

Diseases threaten wildlife populations worldwide and have caused severe declines resulting in host species being listed as threatened or endangered. The risk of a widespread epidemic is especially high when pathogens are introduced to naive host populations, often leading to high morbidity and mortality. Prevention and control of these epidemics is based on knowledge of what drives pathogen transmission among hosts. Previous disease outbreaks suggest the spread of directly transmitted pathogens is determined by host contact rates and local host density. While theoretical models of disease spread typically assume a constant host density, most wildlife populations occur at a variety of densities across the landscape. We explored how spatial heterogeneity in host density influences pathogen spread by simulating the introduction and spread of rabies and canine distemper in a spatially heterogeneous population of Channel Island foxes (Urocyon littoralis), coupling fox density and contact rates with probabilities of viral transmission. For both diseases, the outcome of pathogen introductions varied widely among simulation iterations and depended on the density of hosts at the site of pathogen introduction. Introductions into areas of higher fox densities resulted in more rapid pathogen transmission and greater impact on the host population than if the pathogen was introduced at lower densities. Both pathogens were extirpated in a substantial fraction of iterations. Rabies was over five times more likely to go locally extinct when introduced at low host density sites than at high host-density sites, leaving an average of >99% of foxes uninfected. Canine distemper went extinct in >98% of iterations regardless of introduction site, but only after >90% of foxes had become infected. Our results highlight the difficulty in predicting the course of an epidemic, in part due to complex interactions between pathogen biology and host behavior, exacerbated by the spatial variation of most host populations.

Effects of Aqueous Extracts from Amazon Plants on Plutella xylostella (Lepidoptera: Plutellidae) and Brevicoryne brassicae (Homoptera: Aphididae) in Laboratory, Semifield, and field trials.

We evaluated the insecticide activities of aqueous extracts of five species of plants from the Ecuadorian Amazon (Deguelia utilis (ACSm.) AMGAZEVEDO (Leguminosae: Papilionoideae), Xanthosoma purpuratum K. Krause (Alismatales: Araceae), Clibadium sp. (Asteracea: Asterales), Witheringia solanacea L'Hér (Solanales: Solanaceae), and Dieffenbachia costata H. Karst. ex Schott (Alismatales: Araceae)) plus Cymbopogon citratus Stapf. (Poales: Poaceae) under laboratory, open-field conditions in Plutella xylostella L. (diamondback moth), and semifield conditions in Brevicoryne brassicae L. Tap water was used as a negative control, and synthetic insecticides were used as positive controls. In a laboratory bioassay, aqueous extracts of D. utilis resulted in P. xylostella larval mortality. In contrast to chlorpyrifos, all botanicals were oviposition deterrents. All extracts except Clibadium sp. decreased leaf consumption by P. xylostella larvae. In semifield experiments, D. utilis, Clibadium sp., D. costata, and X. purpuratum initially controlled the population of B. brassicae, but 7 d after application, all botanicals except the D. utilis lost their ability to control the pest. In field experiments on broccoli crops in both dry and rainy seasons, the extracts did not control the abundance of P. xylostella, where as a mixture of two insecticides (chlorpyrifos + lambda cyhalothrin) did. These results show some incongruences from laboratory to semifield and field conditions, indicating that more studies, including the identification of the chemicals responsible for the biological activity, its stability, and the effects of chemotypes on insecticidal activity, are needed to understand the potential of these plant species as botanical insecticides.

Pleomorphic fibroma of the skin with MDM2 immunoreactivity: A potential diagnostic pitfall.

Pleomorphic fibroma is a rare benign cutaneous neoplasm characterized by spindle-shaped cells and multinucleated giant cells scattered throughout collagenous stroma. These morphologic features can lead to diagnostic confusion, including atypical lipomatous tumor as one consideration. In contrast to atypical lipomatous tumor, previous studies have found pleomorphic fibroma to be negative for MDM2 immunohistochemical staining and MDM2 gene amplification. Here, we present a case of pleomorphic fibroma of skin with nuclear MDM2 immunoreactivity in the absence of MDM2 gene amplification, underscoring the superiority of fluorescence in situ hybridization as a diagnostic test in this differential diagnosis. The RB1 locus is also explored for differential diagnosis with pleomorphic/spindle cell lipoma and related entities.

ß-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

Lower Hospital Charges and Societal Costs for Catheter Device Closure of Atrial Septal Defects.

Atrial septal defects (ASD) are among the most common congenital heart defects. As more ASDs are corrected by interventional catheterization instead of surgery, it is critical to understand the associated clinical and societal costs. The goal of this study was to use a national U.S. database to describe hospital charges and societal costs for surgical and catheter-based (ASD) closure. Retrospective review of hospital discharge data from the Kids' Inpatient Database from January 2010 to December 2012. The database was queried for admissions for <21 years old with ICD-9 procedure codes for surgical (35.51 or 35.61) or catheter (35.52) ASD closure; those with other cardiac conditions and/or additional cardiac procedures were excluded. Age, length of stay (LOS), and hospital charges and lost parental wages (societal costs) were compared between groups using t test or Mann-Whitney U test, as appropriate. Four hundred and eighty-six surgical and 305 catheter ASD closures were identified. LOS, hospital charges, and total societal costs were higher in surgical ASD compared to catheter ASD admissions (3.6 vs. 1.3 days, p < 0.001, $87,465 vs. $64,109, p < 0.001, and $90,000 vs. $64,966, p < 0.001, respectively). In this review of a large national inpatient database, we found that hospital and societal costs for surgical ASD closure are significantly higher than catheter ASD closure in the United States in the current era. Factors that likely contribute to this include longer LOS and longer post-operative recovery. Using "real-world" data, this study demonstrates a substantial cost advantage for catheter ASD closure compared to surgical.

Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling.

The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

ANG II-induced hypertension in the VCD mouse model of menopause is prevented by estrogen replacement during perimenopause.

Premenopausal females are resistant to the development of hypertension, and this protection is lost after the onset of menopause, resulting in a sharp increase in disease onset and severity. However, it is unknown how a fluctuating ovarian hormone environment during the transition from perimenopause to menopause impacts the onset of hypertension, and whether interventions during perimenopause prevent disease onset after menopause. A gradual transition to menopause was induced by repeated daily injections of 4-vinylcyclohexene diepoxide (VCD). ANG II (800 ng·kg(-1)·min(-1)) was infused into perimenopausal and menopausal female mice for 14 days. A separate cohort of mice received 17ß-estradiol replacement during perimenopause. ANG II infusion produced significantly higher mean arterial pressure (MAP) in menopausal vs. cycling females, and 17ß-estradiol replacement prevented this increase. In contrast, MAP was not significantly different when ANG II was infused into perimenopausal and cycling females, suggesting that female resistance to ANG II-induced hypertension is intact during perimenopause. ANG II infusion caused a significant glomerular hypertrophy, and hypertrophy was not impacted by hormonal status. Expression levels of aquaporin-2 (AQP2), a collecting duct protein, have been suggested to reflect blood pressure. AQP2 protein expression was significantly downregulated in the renal cortex of the ANG II-infused menopause group, where blood pressure was increased. AQP2 expression levels were restored to control levels with 17ß-estradiol replacement. This study indicates that the changing hormonal environment in the VCD model of menopause impacts the severity of ANG II-induced hypertension. These data highlight the utility of the ovary-intact VCD model of menopause as a clinically relevant model to investigate the physiological mechanisms of hypertension that occur in women during the transition into menopause.

Creation and validation of a novel body condition scoring method for the magellanic penguin (Spheniscus magellanicus) in the zoo setting.

This research aims to validate a novel, visual body scoring system created for the Magellanic penguin (Spheniscus magellanicus) suitable for the zoo practitioner. Magellanics go through marked seasonal fluctuations in body mass gains and losses. A standardized multi-variable visual body condition guide may provide a more sensitive and objective assessment tool compared to the previously used single variable method. Accurate body condition scores paired with seasonal weight variation measurements give veterinary and keeper staff a clearer understanding of an individual's nutritional status. San Francisco Zoo staff previously used a nine-point body condition scale based on the classic bird standard of a single point of keel palpation with the bird restrained in hand, with no standard measure of reference assigned to each scoring category. We created a novel, visual body condition scoring system that does not require restraint to assesses subcutaneous fat and muscle at seven body landmarks using illustrations and descriptive terms. The scores range from one, the least robust or under-conditioned, to five, the most robust, or over-conditioned. The ratio of body weight to wing length was used as a "gold standard" index of body condition and compared to both the novel multi-variable and previously used single-variable body condition scores. The novel multi-variable scale showed improved agreement with weight:wing ratio compared to the single-variable scale, demonstrating greater accuracy, and reliability when a trained assessor uses the multi-variable body condition scoring system. Zoo staff may use this tool to manage both the colony and the individual to assist in seasonally appropriate Magellanic penguin nutrition assessment.

Array-CGH shows amplification of 8q including MYC as the sole aberration in a leiomyosarcoma of the female lower genital tract.

Leiomyosarcomas (LMS) are uncommon in the female genital tract, and the literature on LMS of the vagina consists mostly of case reports. We report the cytogenetic, FISH and array-CGH findings in a LMS of the vagina. Tumor microscopy showed a cellular spindle cell tumor composed of oval to spindly cells arranged in sheets or fascicles supported by a rich vascular network and stained positive for smooth muscle markers (SMA, HHF35). The majority of metaphase cells from the short-term cultures of tumor cells had 49 chromosomes including 3 copies of a derivative chromosome that consisted in most part of 8q material. The remaining cells had 4 copies of the derivative chromosome. FISH studies showed that each derivative chromosome consisted in its entirety of chromosome 8 material, had 2 copies of MYC and 8qter signals, lacked an 8pter signal, and was devoid of a centromeric alpha satellite or satellite III signal of any of the 24 chromosomes. Strong positive staining for MYC was demonstrated in tumor nuclei. Microarray-CGH study on DNA extracted from the tumor confirmed amplification of 8q12.1∼q22.2 and 8q24.13∼qter, and to a lesser extent 8q22.3∼q24.12, as the sole genetic abnormality in the tumor. The present report, to the best of our knowledge, is the first cytogenetically characterized primary LMS of the vagina, and our findings indicate that amplification of 8q including MYC is a primary genetic abnormality as well as a pathway of evolution in the tumor.