Probes for INS

Porcine circovirus 3 (PCV3) was first discovered in 2016, almost concomitantly by two groups of researchers in the United States. The novel case was reported in a group of sows with chronic reproductive problems with clinical presentation alike porcine dermatitis and nephropathy syndrome (PDNS), where metagenomic sequencing revealed a genetically divergent porcine circovirus designated PCV3. The discovery of PCV3 in a PDNS case, which used to be considered as part of PCVAD attributed to PCV2 (porcine circovirus 2), has garnered attention and effort in further research of the novel virus. Just when an infectious molecular DNA clone of PCV3 has been developed and successfully used in an in vivo pathogenicity study, yet another novel PCV strain surfaced, designated PCV4 (porcine circovirus 4). So far, PCV3 has been reported in domestic swine population globally at low to moderate prevalence, from almost all sample types including organ tissues, faecal, semen and colostrum samples. PCV3 has been associated with a myriad of clinical presentations, from PDNS to porcine respiratory disease complex (PRDC). This review paper summarizes the studies on PCV3 to date, with focus on diagnosis.

Peroxisome Proliferator-Activated Receptor Gamma (PPARG) is one of the three members of the PPAR family of transcription factors. Besides its roles in adipocyte differentiation and lipid metabolism, we recently demonstrated an association between PPARG and metastasis in prostate cancer. In this study a functional effect of PPARG on AKT serine/threonine kinase 3 (AKT3), which ultimately results in a more aggressive disease phenotype was identified. AKT3 has previously been shown to regulate PPARG co-activator 1 alpha (PGC1α) localisation and function through its action on chromosome maintenance region 1 (CRM1). AKT3 promotes PGC1α localisation to the nucleus through its inhibitory effects on CRM1, a known nuclear export protein. Collectively our results demonstrate how PPARG over-expression drives an increase in AKT3 levels, which in turn has the downstream effect of increasing PGC1α localisation within the nucleus, driving mitochondrial biogenesis. Furthermore, this increase in mitochondrial mass provides higher energetic output in the form of elevated ATP levels which may fuel the progression of the tumour cell through epithelial to mesenchymal transition (EMT) and ultimately metastasis.

Aging results in gray and white matter degeneration, but the specific microglial responses are unknown. Using single-cell RNA sequencing from white and gray matter separately, we identified white matter-associated microglia (WAMs), which share parts of the disease-associated microglia (DAM) gene signature and are characterized by activation of genes implicated in phagocytic activity and lipid metabolism. WAMs depend on triggering receptor expressed on myeloid cells 2 (TREM2) signaling and are aging dependent. In the aged brain, WAMs form independent of apolipoprotein E (APOE), in contrast to mouse models of Alzheimer's disease, in which microglia with the WAM gene signature are generated prematurely and in an APOE-dependent pathway similar to DAMs. Within the white matter, microglia frequently cluster in nodules, where they are engaged in clearing degenerated myelin. Thus, WAMs may represent a potentially protective response required to clear degenerated myelin accumulating during white matter aging and disease.

Rodent primary somatosensory cortex (S1) is organized in defined layers, where layer IV serves as the main target for thalamocortical projections. Serotoninergic signaling is important for the organization of thalamocortical projections and consequently proper barrel field development in rodents, and the vesicular monoamine transporter 2 (VMAT2) can be detected locally in layer IV S1 cortical neurons in mice as old as P10, but the identity of the Vmat2-expressing neurons is unknown. We here show that Vmat2 mRNA and also Vmat2-Cre recombinase are still expressed in adult mice in a sub-population of the S1 cortical neurons in the barrel field. The Vmat2-Cre cells showed a homogenous intrinsically bursting firing pattern determined by whole-cell patch-clamp, localized radial densely spinous basal dendritic trees and almost exclusively lack of apical dendrite, indicative of layer IV spiny stellate cells. Single cell mRNA sequencing analysis showed that S1 cortical Vmat2-Cre;tdTomato cells express the layer IV marker Rorb and mainly cluster with layer IV neurons, and RNAscope analysis revealed that adult Vmat2-Cre neurons express Vmat2 and vesicular glutamate transporter 1 (Vglut1) and Vglut2 mRNA to a high extent. In conclusion, our analysis shows that cortical Vmat2 expression is mainly confined to layer IV neurons with morphological, electrophysiological and transcriptional characteristics indicative of spiny stellate cells.

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumour that is resistant to existing therapeutics. Identifying signalling pathways deregulated in GBM that can be targeted therapeutically is critical to improve the present dismal prognosis for GBM patients. In this report, we have identified that the BRG1 (Brahma-Related Gene-1) catalytic subunit of the SWI/SNF chromatin remodelling complex promotes the malignant phenotype of GBM cells. We found that BRG1 is ubiquitously expressed in tumour tissue from GBM patients, and high BRG1 expression levels are localized to specific brain tumour regions. Knockout (KO) of BRG1 by CRISPR-Cas9 gene editing had minimal effects on GBM cell proliferation, but significantly inhibited GBM cell migration and invasion. BRG1-KO also sensitized GBM cells to the anti-proliferative effects of the anti-cancer agent temozolomide (TMZ), which is used to treat GBM patients in the clinic, and selectively altered STAT3 tyrosine phosphorylation and gene expression. These results demonstrate that BRG-1 promotes invasion and migration, and decreases chemotherapy sensitivity, indicating that it functions in an oncogenic manner in GBM cells. Taken together, our findings suggest that targeting BRG1 in GBM may have therapeutic benefit in the treatment of this deadly form of brain cancer.

Insufficient eradication capacity and dysfunction are common occurrences in T cells that characterize cancer immunotherapy failure. De novo DNA methylation promotes T cell exhaustion, whereas methylation inhibition enhances T cell rejuvenation in vivo. Decitabine, a DNA methyltransferase inhibitor approved for clinical use, may provide a means of modifying exhaustion-associated DNA methylation programmes. Herein, anti-tumour activities, cytokine production, and proliferation are enhanced in decitabine-treated chimeric antigen receptor T (dCAR T) cells both in vitro and in vivo. Additionally, dCAR T cells can eradicate bulky tumours at a low-dose and establish effective recall responses upon tumour rechallenge. Antigen-expressing tumour cells trigger higher expression levels of memory-, proliferation- and cytokine production-associated genes in dCAR T cells. Tumour-infiltrating dCAR T cells retain a relatively high expression of memory-related genes and low expression of exhaustion-related genes in vivo. In vitro administration of decitabine may represent an option for the generation of CAR T cells with improved anti-tumour properties.

Chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC) and is a serious health problem in China, East Asia, and North African countries. Effective treatment of HBV-related HCC is currently unavailable. This study evaluated the therapeutic potential of TIGIT blockade in HBV-related HCC.A mouse model of spontaneous HBV-related HCC was generated by replacing wild-type hepatocytes with HBsAg+ hepatocytes (namely HBs-HepR mice). The tumors in HBs-HepR mice were inflammation-associated HCC, similar to HBV-related HCC in patients, which was distinguished from other HCC mouse models, such as diethylnitrosamine (DEN)-induced HCC, Tak1-knockout-induced HCC, HCC in stelic animal model (STAM), or nonalcoholic steatohepatitis (NASH)-induced HCC. HCC in HBs-HepR mice was characterized by an increased number of CD8+ T cells whereas the production of IL-2, TNF-α, and IFN-γ by intrahepatic CD8+ T cells was decreased. Increased expression of TIGIT on CD8+ T cells was responsible for functional exhaustion. The therapeutic effect of TIGIT blockade was investigated at the early and middle stages of HCC progression in HBs-HepR mice. TIGIT blockade reinvigorated intrahepatic CD8+ T cells with increased TNF-α and IFN-γ production and an increased number of CD8+ T cells in tumors, thereby slowing the development of HCC in HBs-HepR mice. Blocking PD-L1 did not show direct therapeutic effects or synergize with TIGIT blockade.Blockade of TIGIT alone enhanced the anti-tumor activity of CD8+ T cells during the progression of HBV-related HCC in a spontaneous HCC mouse model.This article is protected by

A geroscience-informed approach to the increasing prevalence of bladder control problems in older adults requires understanding the impact of aging on dynamic mechanisms that ensure resilience in response to stressors challenging asymptomatic voluntary control over urine storage and voiding. Bladder control is predicated on sensory neural information about bladder volume. Modulation of volume-induced bladder wall tensions by autonomic and mucosal factors controls neural sensitivity to bladder volume. We hypothesized that HCN (hyperpolarization-activated cyclic nucleotide-gated) channels integrate these factors and thereby mediate adrenergic detrusor tension control. Furthermore, loss of HCN expression compromises that integration, and could result in loss of precision of detrusor control. Using a lifespan mouse model, RT-qPCR and pharmacologic studies in pre-tensioned intact and mucosa-denuded bladder strips were made. The dominant hcn1 expression declines with maturation and aging, however aging is also associated with increased variance around mean values. In strips from mature animals, isoproterenol had less effect in denuded muscle strips than in intact strips, and HCN blockade diminished isoproterenol responsiveness. With aging, variances about mean response values significantly increased, paralleling hcn1 expression. Our findings support a role for HCN in providing neuroendocrine/paracrine integration and suggest an association of increased heterogeneity of HCN expression in aging with reductions in response precision to neuroendocrine control. The functional implication is an increased risk of dysfunction of brainstem/bladder regulation of neuronal sensitivity to bladder volume. This supports the clinical model of the aging bladder phenotype as an expression of loss of resilience, and not as emerging bladder pathology with aging.

Targeting specific opioid receptor types in distinct sensory neurons could lead to safer and more effective treatments against pain. However, the extent to which different DRG neurons that express opioid receptors (MOR, DOR, KOR) innervate distinct organs, and what sensory information is encoded by these neurons, represent long-standing questions in the field. To fill this knowledge gap, we utilized novel knock-in mouse lines in which the DNA recombinases Cre and/or Flp are expressed in opioid receptor-positive DRG neurons. We injected adeno-associated viruses to express tdTomato and analyzed the organization of DRG axon terminals in peripheral tissues using tissue clearing and immunostaining protocols. In hairy skin, we observed circumferential nerve endings around hair follicles that are either MOR+ or DOR+. However, DOR+ circumferential endings were also NFH+ whereas MOR+ circumferential endings were not, suggesting that MOR is expressed by high-threshold mechanoreceptors, while DOR is expressed by low-threshold mechanoreceptors activated by stroking of the skin. In glabrous skin, we found a similar divergent organization, with MOR+ and DOR+ axon terminals co-expressing CRGP and NFH, respectively. In the colon, we observed innervation by both KOR+ and MOR+ axons whereas, in the muscle (soleus) and kidney, we found axons that are either MOR+, DOR+, or KOR+. Remarkably, these MOR+, DOR+, or KOR+ axons innervate different sub-regions within these organs and form distinct nerve-ending structures. Collectively, our findings show that MOR+, DOR+, and KOR+ DRG neurons are expressed in largely non-overlapping DRG neuron types that distinctly innervate tissues and presumably differently contribute to sensory perception. National Institutes of Health grant R01DA044481 New York Stem Cell Foundation.

Introduction: Hyperglycemia is a common finding in ACS patients in both diabetic and non-diabetic, it is considered a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury is not fully understood, whether the Sodium Glucose Co-Transporter 1(SGLT1) plays a role in increase injury, before and/or after reperfusion, remains to be elucidated. SGLT2 inhibitors clinical trials have shown significant improvements in cardiovascular outcomes in diabetic and non-diabetic, yet the mechanism is not fully understood and whether SGLT1 plays a role in infarct augmentation remains to be elucidated. Hypothesis: High glucose at reperfusion leads to excess myocardial injury and the increased injury is mediated through the activity of SGLT1. Methods: RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and Zucker diabetic rats. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose on myocardium at reperfusion. Canagliflozin a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 and SGLT2 transporter and 5nmol/L to block only the SGLT2 transposer) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results: Our data reveal that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model and it is abrogated through the administration of both mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, and through the administration of novel specific SGLT1 inhibitor, Mizagliflozin. Conclusions: We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

FSGS is the final common pathway to nephron loss in most forms of severe or progressive glomerular injury. Although podocyte injury initiates FSGS, parietal epithelial cells (PECs) are the main effectors. Because PDGF takes part in fibrotic processes, we hypothesized that the ligand PDGF-B and its receptor PDGFR-β participate in the origin and progression of FSGS.We challenged Thy1.1 transgenic mice, which express Thy1.1 in the podocytes, with anti-Thy1.1 antibody to study the progression of FSGS. We investigated the role of PDGF in FSGS using challenged Thy1.1 mice, 5/6 nephrectomized mice, Col4-/- (Alport) mice, patient kidney biopsies, and primary murine PECs, and challenged Thy1.1 mice treated with neutralizing anti-PDGF-B antibody therapy.The unchallenged Thy1.1 mice developed only mild spontaneous FSGS, whereas challenged mice developed progressive FSGS accompanied by a decline in kidney function. PEC activation, proliferation, and profibrotic phenotypic switch drove the FSGS. During disease, PDGF-B was upregulated in podocytes, whereas PDGFR-β was upregulated in PECs from both mice and patients with FSGS. Short- and long-term treatment with PDGF-B neutralizing antibody improved kidney function and reduced FSGS, PEC proliferation, and profibrotic activation. In vitro, stimulation of primary murine PECs with PDGF-B recapitulated in vivo findings with PEC activation and proliferation, which was inhibited by PDGF-B antibody or imatinib.PDGF-B-PDGFR-β molecular crosstalk between podocytes and PECs drives glomerulosclerosis and the progression of FSGS.

Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility.Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes.Three groups were compared: a. uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); b. subjects dying of COVID-19 (virus-negative in testes; n = 15); c. subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method.SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups.In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.