Probes for INS

The estrogen receptor beta (ERβ) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERβ, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERβ (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERβ. Herein, we established an immunohistochemical detection protocol for ERβ protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERβ. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERβ. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERβ, is also suitable for reliable detection of murine ERβ. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERβ in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

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.

The polyamines putrescine, spermidine and spermine are organic polycations that regulate many cell functions including proliferation and differentiation. It is known that certain genes of the polyamine system are dysregulated after kidney ischemia reperfusion injury. Here we examined the hypothesis that different forms of acute and chronic kidney injury lead to similar changes in the expression patterns of the polyamine system. In different models of acute and chronic kidney injury expression of genes involved in polyamine homeostasis were analyzed by RT-qPCR and RNAScope. In these models, expression of catabolic enzymes (Aoc1 and Sat1) was upregulated, and the anabolic enzymes (Odc1, Sms) were downregulated. The putrescine-degrading enzyme AOC1 exhibits the most striking changes. Interestingly, it can act together with ODC1 as gatekeepers of the polyamine system. The detected increase of Aoc1 takes place in the injured but regenerating proximal tubules. As a screening for stimuli of increased Aoc1 expression, we used mouse embryonic kidney explants. Here we observed changes of Aoc1expression under hypoxia and hyperosmotic conditions. These changes were further examined in mouse models of hypoxia. However, in vivo, hypoxia did not lead to changes of Aoc1 expression. Hyperosmolarity was confirmed as a stimulus by using the kidney cell lines M15 and 209/MDCT as well as cultured primary proximal tubules. Using reporter gene and RNA-stability assays, we could show that the increase in Aoc1 expression is based on mRNA-stabilization and transcriptional activation of one certain isoform. The activated isoform contains an additional set of 22 amino acids N-terminally that lead to an altered subcellular localization. In conclusion, different models of kidney injury exhibit a similar pattern of dysregulation of the polyamine system with the most striking change being the upregulation of Aoc1 in proximal tubules. Using hyperosmolarity as a stimulus, we provide first insights into the regulation of Aoc1 under harmful conditions.

Although anesthesia is commonly used in the fields of medicine and scientific research, the neural mechanisms and circuits through which it produces analgesia is still unclear. Utilizing c-fos labeling of neuronal activity, this project aimed to investigate the brain regions of C57BL/6 mice, which become activated subsequent to isoflurane anesthesia. RNAscope in situ hybridization was used to examine c-fos mRNA activation in the brain. Confocal microscopy was utilized to locate and characterize brain regions displaying c-Fos activation. Finally, manual quantification of c-fos activation in identified brain regions was conducted through Fiji software. The brain regions identified resemble brain areas that have been associated with pain regulation in literature, including the central nucleus of amygdala (CeA), paraventricular nucleus of the hypothalamus (PVN), centrally-projecting Edinger-Westphal nucleus (EWcp), piriform cortex (PC), and para-supraoptic nucleus (ParaSON). Furthermore, the CeA displayed the greatest average number of positive cells and the densest activation, supporting its importance in pain and analgesia. The identified brain regions validate the prominent findings of prior studies, which also found c-Fos activation subsequent to isoflurane anesthesia in the CeA, PVN, and ParaSON (Hua et al., Nat Neurosci, 2020). New regions of c-fos activation, including the EWcp and PC, found in this study are in need of further exploration. PC activation may also be caused by smell from isoflurane. The connections and coordination which the identified brain regions have in producing analgesia is also an area for future investigation. This study is supported by Duke University Anesthesiology Fund and NIH grant R01-DE29342. This study is supported by Duke University Anesthesiology Fund and NIH grant R01-DE29342.

A meta-analysis study was conducted to measure the consequence of diabetic foot ulcers (DFUs) and other risk factors (RFs) on the prevalence of lower extremity amputation (LEA). A comprehensive literature inspection till February 2023 was applied and 2765 interrelated studies were reviewed. Of the 32 chosen studies enclosed, 9934 subjects were in the chosen studies' starting point, and 2906 of them were with LEA. Odds ratio (OR) in addition to 95% confidence intervals (CIs) were used to compute the value of the effect of DFUs and other RFs on the prevalence of LEA by the continuous and dichotomous approaches and a fixed or random effect model. Male gender (OR, 1.30; 95% CI, 1.17-1.44, P < .001), smoking (OR, 1.24; 95% CI, 1.01-1.53, P = .04), previous foot ulcer (OR, 2.69; 95% CI, 1.93-3.74, P < .001), osteomyelitis (OR, 3.87; 95% CI, 2.28-6.57, P < .001), gangrene (OR, 14.45; 95% CI, 7.03-29.72, P < .001), hypertension (OR, 1.17; 95% CI, 1.03-1.33, P = .01), and white blood cells count (WBCC) (MD, 2.05; 95% CI, 1.37-2.74, P < .001) were significantly shown to be an RF in LEA in subjects with DFUs. Age (MD, 0.81; 95% CI, -0.75 to 2.37, P = .31), body mass index (MD, -0.55; 95% CI, -1.15 to 0.05, P = .07), diabetes mellitus type (OR, 0.99; 95% CI, 0.63-1.56, P = .96), and glycated haemoglobin (MD, 0.33; 95% CI, -0.15 to 0.81, P = .17) were not shown to be an RF in LEA in subjects with DFUs. Male gender, smoking, previous foot ulcer, osteomyelitis, gangrene, hypertension, and WBCC were significantly shown to be an RF in LEA in subjects with DFUs. However, age and diabetes mellitus type were not shown to be RF in LEA in subjects with DFUs. However, caused of the small sample sizes of several chosen studies for this meta-analysis, care must be exercised when dealing with its values.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent muscular dystrophies, ranging from 1 in 8,333 to 1 in 20,000. Currently no treatment exists that alters the course of FSHD, and therapy development remains an unmet need in the field. Abnormal reactivation of the DUX4 gene in skeletal muscle has emerged as an underlying cause of muscle weakness and wasting in FSHD. We propose that DUX4 silencing is the most direct route to FSHD therapy. Toward this goal, we developed an AAV6-CRISPR-Cas13 strategy to silence DUX4 mRNA. Cas13 targets and cleaves RNA instead of DNA, and avoids potential risks of permanent off-target genome editing that could arise with DNA-targeting systems. Intramuscular delivery of an AAV6 vector encoding a PspCas13b enzyme and DUX4-targeting guide RNAs reduced DUX4 mRNA by >50% and improved histopathological outcomes in FSHD mice. To investigate possible off-target effects, we performed RNA-seq of treated versus control or untreated human myoblasts and also examined potential collateral RNA cleavage activity using a dual reporter system. Although we did not detect collateral cleavage, our RNA-sequencing results suggested some guide RNAs could induce potential off-target gene expression changes. We are currently exploring mechanisms to explain these differential off-target effects. To address whether PspCas13b can activate a mammalian host immune response, we injected wild-type mice with AAV-Cas13b and investigated immune cell infiltration and pro-inflammatory cytokine profiles. We find evidence of an immune response against PspCas13b in injected mouse muscles. Importantly, transient immunosuppression reduced immune responses to Cas13b in treated animals. In conclusion, our data support that Cas13b can target and reduce DUX4 expression in FSHD muscles, but minimizing cellular immune response may be necessary to translate AAV-Cas13b therapy.

Rationale DNA damage accumulation is a hallmark of vascular smooth muscle cell (VSMC) ageing. Importantly, VSMC DNA damage accumulation and ageing has been implicated in the progression of cardiovascular disease (CVD), including atherosclerosis and vascular calcification. Chemotherapy drugs used in the treatment of many cancers are known to induce DNA damage in cardiovascular cells and accelerate CVD. Histone deacetylase (HDAC) inhibitors are drugs being investigated for novel treatments of many cancers. HDACs perform many vital functions in cells; HDAC6 is known to deacetylate alpha-tubulin to regulate microtubule stability and flexibility. We have recently shown that microtubule stability regulates both VSMC morphology and contractility. Therefore, in this study we investigate the impact of HDAC6 inhibition upon VSMC function. Methodology We use polyacrylamide hydrogels (PAHs)

Hv1 is the only voltage-gated proton-selective channel in mammalian cells. It contains a conserved voltage-sensor domain, shared by a large class of voltage-gated ion channels, but lacks a pore domain. Its primary role is to extrude protons from the cytoplasm upon pH reduction and membrane depolarization. The best-known function of Hv1 is the regulation of cytosolic pH and the nicotinamide adenine dinucleotide phosphate oxidase-dependent production of reactive oxygen species. Accumulating evidence indicates that Hv1 is expressed in nervous systems, in addition to immune cells and others. Here, we summarize the molecular properties, distribution, and physiological functions of Hv1 in the peripheral and central nervous systems. We describe the recently discovered functions of Hv1 in various neurological diseases, including brain or spinal cord injury, ischemic stroke, demyelinating diseases, and pain. We also summarize the current advances in the discovery and application of Hv1-targeted small molecules in neurological diseases. Finally, we discuss the current limitations of our understanding of Hv1 and suggest future research directions.

There are few prostate cancer prognostic biomarkers. However, clinical difficulties in distinguishing between aggressive and non-aggressive tumors have been observed. CD73 is a 70-kDa glycosylphosphatidylinositol (GPI)-linked ecto-enzyme that reduces antitumor immunity in mouse models of tumor, particularly prostate cancer. It's believed to be a promising biomarker for predicting the clinical development and prognosis of certain tumor types. Its function in prostate cancer, however, is unknown. This study aims to investigate the hypothesis that CD73 may be used as a biomarker in prostate cancer diagnosis and/or prognosis. Nuclear and cytoplasmic CD73 staining has been evaluated by immunohistochemistry using benign (23) and malignant (75) prostate tissues. The immunohistochemical study showed nuclear and cytoplasmic CD73 staining in cancerous and non-cancerous prostate tissues. Increased CD73 staining was shown in prostate cancer tissues compared to benign prostate tissues. A negative association between CD73 expression and Gleason scores has been observed. However, increased cytoplasmic CD73 staining was significantly associated with increasing tumor size. This finding suggests that CD73 may have a role in cancer development or aggressiveness, indicating that more research is needed to better understand its function and determine whether it might be used as a diagnostic biomarker for prostate cancer.

We previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFβRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFβ signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

SR-17018 was identified as a highly G protein-biased mu opioid peptide (MOP) receptor agonist and lacked MOP agonist-associated adverse effects in mice. The aim of this study was to determine the functional profile of spinal and systemic administration of SR-17018 in non-human primates. In vivo effects of SR-17018 were compared with those of MOP agonists in different intrinsic efficacies, DAMGO, morphine, heroin, and buprenorphine, in behavioral assays established in rhesus monkeys (Macaca mutatta). Nociceptive, itch-scratching, and operant behaviors were measured by experimenters blinded to the dosing conditions. Following intrathecal delivery, SR-17018 (30-300 ug), buprenorphine (3-10 ug), morphine (10-30 ug), and DAMGO (1-3 ug), dose-dependently attenuated capsaicin-induced thermal allodynia (p < 0.05). However, unlike DAMGO and morphine eliciting robust scratching activities, intrathecal SR-17018 and buprenorphine only elicited mild scratching responses, indicating that SR-17018 has low efficacy for activating spinal MOP receptors. In the intravenous drug self-administration assay, heroin (0.3-10 ug/kg/infusion) produced a higher reinforcing strength (abuse liability) as compared to lower reinforcing strengths by SR-17018 (3-30 ug/kg/infusion) and buprenorphine (1-10 ug/kg/infusion) in primates under the progressive-ratio schedule of reinforcement (p < 0.05). The intrathecal opioid-induced itch and intravenous drug self-administration have been documented to distinguish MOP receptor agonists with different intrinsic efficacies. Our findings reveal that in vivo apparent low efficacy of SR-17018 is similar to that of a MOP partial agonist buprenorphine measured by the primate assays with translation relevance. Such a low intrinsic efficacy explains its improved side-effect profile of a highly G protein-biased MOP agonist, S

SHROOM4 is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy number variations (CNVs) in SHROOM4 have been associated with the neurodevelopmental disorder Stocco dos Santos syndrome, but not with congenital anomalies of the urinary tract and the visceral or the cardiovascular system.Here, exome sequencing and CNV analyses besides expression studies in zebrafish and mouse and knockdown (KD) experiments using a splice blocking morpholino in zebrafish were performed to study the role of SHROOM4 during embryonic development.In this study, we identified putative disease-causing SNVs and CNVs in SHROOM4 in six individuals from four families with congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems (CNS). Embryonic mouse and zebrafish expression studies showed Shroom4 expression in the upper and lower urinary tract, the developing cloaca, the heart and the cerebral CNS. KD studies in zebrafish larvae revealed pronephric cysts, anomalies of the cloaca and the heart, decreased eye-to-head ratio and higher mortality compared with controls. These phenotypes could be rescued by co-injection of human wild-type SHROOM4 mRNA and morpholino.The identified SNVs and CNVs in affected individuals with congenital anomalies of the urinary tract, the anorectal, the cardiovascular and the central nervous systems, and subsequent embryonic mouse and zebrafish studies suggest SHROOM4 as a developmental gene for different organ systems.