Animal‐borne video systems provide insight into the reproductive behavior of the Asian black bear

Previous studies on the mating system of the Asian black bear (Ursus thibetanus) have been limited to observations of captive populations and estimations of multiple paternities. Hence, the mating system of wild bears remains poorly understood. Animal‐borne camera systems (i.e., cameras mounted on animals) provide novel tools to study the behavior of elusive animals. Here, we used an animal‐borne video system to record the activities of wild bears during the mating season. Video camera collars were attached to four adult Asian black bears (male “A” and “B,” and female “A” and “B”) captured in Tokyo, central Japan, in May and June 2018. The collars were retrieved in July 2018, after which the video data were downloaded and analyzed in terms of bear activity and mating behavior. All the bears were found to interact with other uniquely identifiable bears for some of the time (range 9–22 days) during the deployment period (range 36–45 days), and multiple mating in males was documented. Both males and females exhibited different behaviors on social days (i.e., days when the bear interacted with conspecifics) compared with solitary days (i.e., days with no observed interactions with conspecifics). Compared with solitary days, the bears spent a lower proportion of time on foraging activities and higher proportion of time on resting activities on social days. Our results suggest that Asian black bears have a polygamous mating system, as both sexes consort and potentially mate with multiple partners during a given mating season. Furthermore, bears appeared to reduce their foraging activities on social days and engaged more in social interactions.     

Stromal Liver Kinase B1 [STK11] Signaling Loss Induces Oviductal Adenomas and Endometrial Cancer by Activating Mammalian Target of Rapamycin Complex 1

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.     

Corticotropin-releasing hormone receptor 1 gene variants in irritable bowel syndrome

Background

Corticotropin-releasing hormone (CRH) acts mainly via the CRH receptor 1 (CRH-R1) and plays a crucial role in the stress-induced pathophysiology of irritable bowel syndrome (IBS). Several studies have demonstrated that variants of the CRH-R1 gene carry a potential risk for depression, but evidence for an association between CRH-R1 genotypes and IBS is lacking. We tested the hypothesis that genetic polymorphisms and haplotypes of CRH-R1 moderate the IBS phenotype and negative emotion in IBS patients.

Methods

A total of 103 patients with IBS and 142 healthy controls participated in the study. Three single-nucleotide polymorphisms of the CRH-R1 gene (rs7209436, rs242924, and rs110402) were genotyped. Subjects'' emotional states were evaluated using the Perceived-Stress Scale, the State-Trait Anxiety Inventory, and the Self-rating Depression Scale.

Results

The TT genotype of rs7209436 (P = 0.01) and rs242924 (P = 0.02) was significantly more common in patients with IBS than in controls. Total sample analysis showed significant association between bowel pattern (normal, diarrhea, constipation, or mixed symptoms) and the T allele of rs7209436 (P = 0.008), T allele of rs242924 (P = 0.019), A allele of rs110402 (P = 0.047), and TAT haplocopies (P = 0.048). Negative emotion was not associated with the examined CRH-R1 SNPs.

Conclusion

These findings suggest that genetic polymorphisms and the CRH-R1 haplotypes moderate IBS and related bowel patterns. There was no clear association between CRH-R1 genotypes and negative emotion accompanying IBS. Further studies on the CRH system are therefore warranted.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.     

RBMX: a regulator for maintenance and centromeric protection of sister chromatid cohesion

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.     

A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.     

Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).     

Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method

Background

Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.

Methods/Principal Findings

A new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion. MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation.

Conclusions/Significance

We demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.     

Combination treatment of rosuvastatin or atorvastatin, with regular exercise improves arterial wall stiffness in patients with coronary artery disease

Objective

Statin- and exercise-therapy are both clinically beneficial by preventing cardiovascular events in patients with coronary artery disease (CAD). However, there is no information on the vascular effects of the combination of statins and exercise on arterial wall stiffness in CAD patients.

Methods

The present study is a sub-analysis of PRESET study that determined the effects of 20-week treatment with statins (rosuvastatin, n = 14, atorvastatin, n = 14) combined with regular exercise on arterial wall stiffness assessed by measurement of brachial and ankle pulse wave velocity (baPWV) in CAD patients.

Results

The combination of statins and regular exercise significantly improved exercise capacity, lipid profile, including low- and high-density lipoprotein cholesterol, and high-sensitivity C-reactive protein (hs-CRP), baPWV (baseline: 1747±355, at 20 weeks of treatment: 1627±271 cm/s, p = 0.008), and basophil count (baseline: 42±32, 20 weeks: 26±15 cells/µL, p = 0.007), but had no effect on blood pressure (baseline: 125±22, 20 weeks: 121±16 mmHg). Changes in baPWV correlated significantly with changes in basophil count (r = 0.488, p = 0.008), but not with age, lipids profile, exercise capacity, or hs-CRP.

Conclusion

In CAD patients, the combination treatment with statins and exercise resulted in significant amelioration of arterial wall stiffness, at least in part, through reduction of circulating basophils.