Skeletal muscle myofibers are large and elongated cells with multiple and evenly distributed nuclei. Nuclear distribution suggests that each nucleus influences a specific compartment within the myofiber and implies a functional role for nuclear positioning. Compartmentalization of specific mRNAs and proteins has been reported at the neuromuscular and myotendinous junctions, but mRNA distribution in non-specialized regions of the myofibers remains largely unexplored. We report that the bulk of mRNAs is enriched around the nucleus of origin and that this perinuclear accumulation depends on recently transcribed mRNAs. Surprisingly, mRNAs encoding large proteins - giant mRNAs - are spread throughout the cell and do not exhibit perinuclear accumulation. Furthermore, by expressing exogenous transcripts with different sizes we found that size contributes to mRNA spreading independently of mRNA sequence. Both these mRNA distribution patterns depend on microtubules and are independent of nuclear dispersion, mRNA expression level and stability, and the characteristics of the encoded protein. Thus, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure cellular compartmentalization and simultaneous long-range distribution of giant mRNAs.

Despite notable genetic influences, obesity mainly results from the overconsumption of food, which arises from the interplay of physiological, cognitive and environmental factors. In patients with obesity, eating is determined more by external cues than by internal physiological needs. However, how environmental context drives non-homeostatic feeding is elusive. Here, we identify a population of somatostatin (TNSST) neurons in the mouse hypothalamic tuberal nucleus that are preferentially activated by palatable food. Activation of TNSST neurons enabled a context to drive non-homeostatic feeding in sated mice and required inputs from the subiculum. Pairing a context with palatable food greatly potentiated synaptic transmission between the subiculum and TNSST neurons and drove non-homeostatic feeding that could be selectively suppressed by inhibiting TNSST neurons or the subiculum but not other major orexigenic neurons. These results reveal how palatable food, through a specific hypothalamic circuit, empowers environmental context to drive non-homeostatic feeding.

Benign Prostatic Hyperplasia / Lower Urinary Tract Dysfunction (BPH/LUTD) is a classic disease of aging which affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging existing paradigms about when this disease process begins. We delivered a single bolus dose of a widespread environmental contaminant, present in the serum of most Americans (2,3,7,8 tetrachlorodibenzo-p-dioxin, TCDD, 1 µg/kg), and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation does not resolve, but rather persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency, a bothersome symptom in men. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.

To determine the substrate specificity of Abcb4 and Abcb5, we transfected HEK293 cells with empty vector (Vector) or with vectors encoding human _ABCB1_ (MDR-19), zebrafish _abcb4_ (ZF Abcb4), or zebrafish _abcb5_ (ZF Abcb5). We then performed cytotoxicity assays with the known human P-gp substrates etoposide, doxorubicin, and mitoxantrone. As shown in Figure 3A, zebrafish Abcb4, zebrafish Abcb5, and P-gp conferred comparable levels of resistance to etoposide, while zebrafish Abcb5 conferred much less resistance to doxorubicin and mitoxantrone, suggesting that the substrate specificity profile of zebrafish Abcb5 might be narrower compared to zebrafish Abcb4 and P-gp. Further evidence was garnered from flow cytometry assays with fluorescent substrates [Figure 3B]. When we incubated MDR-19, ZF Abcb4, and ZF Abcb5 with the fluorescent substrate BODIPY-ethylenediamine (EDA) in the presence or absence of the P-gp inhibitor valspodar, we noted decreased intracellular fluorescence of BODIPY-EDA in the MDR-19 and ZF Abcb4 cells that was reversed by the addition of valspodar. However, transport was not observed in the ZF Abcb5 cells. Since these results pointed to a difference in substrate specificity between the zebrafish homologs, we performed a high-throughput screen on the transfected cells using 90 substrates of P-gp[81]. By comparing the drug response area under the curve for cells transfected with empty vector and the different transporters, we found that the substrate specificity profile of zebrafish Abcb4 is similar to that of human P-gp[82]. Additionally, we found that the efficacy of human P-gp inhibitors varied for zebrafish Abcb4 and Abcb5 and depended on the substrate examined. Using RNAScope _in situ_ probes, we noted that zebrafish _abcb4_ colocalized with C219 staining in the zebrafish brain vasculature. Vessels in the zebrafish brain that were claudin-5 positive also stained positively with _abcb4_ probes[82]. These results support the hypothesis that zebrafish Abcb4 is the functional homolog of human P-gp at the zebrafish BBB.

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.

The clinical outcome of porcine circovirus 3 (PCV3) infection is still controversial. Herein, a novel PCV3 isolate (PCV3-China/DB-1/2017) with the molecular characterization of 24A and 27K in the Cap protein was used to inoculate three-week-old cesarean-derived, colostrum-deprived piglets. The nine PCV3 DB-1 inoculated piglets exhibited no obvious clinical symptoms or macroscopic lesions. PCV3 displayed a broad histotropism, including the heart, liver, spleen, lung, kidney, brain, lymph nodes, and tonsil, and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs. From 7 days after PCV3 DB-1 inoculation, the piglets showed obvious IgG antibody responses against PCV3 rCap-VLPs. The cumulative results demonstrated that PCV3 trend to low pathogenicity.

Rationale: Extraembryonic tissues, including the yolk sac and placenta, and the heart within the embryo, work to provide crucial nutrients to the embryo. The association of congenital heart defects (CHDs) with extraembryonic tissue defects further supports the potential developmental relationship between the heart and extraembryonic tissues. Although the development of early cardiac lineages has been well-studied, the developmental relationship between cardiac lineages, including epicardium, and extraembryonic mesoderm remains to be defined. Objective: To explore the developmental relationships between cardiac and extraembryonic lineages. Methods and Results: Through high-resolution single cell and genetic lineage/clonal analyses, we show an unsuspected clonal relationship between extraembryonic mesoderm and cardiac lineages. Single-cell transcriptomics and trajectory analyses uncovered two mesodermal progenitor sources contributing to left ventricle cardiomyocytes, one embryonic and the other with an extraembryonic gene expression signature. Additional lineage-tracing studies revealed that the extraembryonic-related progenitors reside at the embryonic-extraembryonic interface in gastrulating embryos, and produce distinct cell types forming the pericardium, septum transversum, epicardium, dorsolateral regions of the left ventricle and atrioventricular canal myocardium, and extraembryonic mesoderm. Clonal analyses demonstrated that these progenitors are multipotent, giving rise to not only cardiomyocytes and serosal mesothelial cell types but also, remarkably, extraembryonic mesoderm. Conclusions: Overall, our results reveal the location of previously unknown multipotent cardiovascular progenitors at the embryonic-extraembryonic interface, and define the earliest embryonic origins of serosal mesothelial lineages, including the epicardium, which contributes fibroblasts and vascular support cells to the heart. The shared lineage relationship between embryonic cardiovascular lineages and extraembryonic mesoderm revealed by our studies underscores an underappreciated blurring of boundaries between embryonic and extraembryonic mesoderm. Our findings suggest unexpected underpinnings of the association between congenital heart disease and placental insufficiency anomalies, and the potential utility of extraembryonic cells for generating cardiovascular cell types for heart repair.

The mammalian cerebral cortex has an unparalleled diversity of cell types, which are generated during development through a series of temporally orchestrated events that are under tight evolutionary constraint and are critical for proper cortical assembly and function1,2. However, the molecular logic that governs the establishment and organization of cortical cell types remains unknown, largely due to the large number of cell classes that undergo dynamic cell-state transitions over extended developmental timelines. Here we generate a comprehensive atlas of the developing mouse neocortex, using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing. We sampled the neocortex every day throughout embryonic corticogenesis and at early postnatal ages, and complemented the sequencing data with a spatial transcriptomics time course. We computationally reconstruct developmental trajectories across the diversity of cortical cell classes, and infer their spatial organization and the gene regulatory programs that accompany their lineage bifurcation decisions and differentiation trajectories. Finally, we demonstrate how this developmental map pinpoints the origin of lineage-specific developmental abnormalities that are linked to aberrant corticogenesis in mutant mice. The data provide a global picture of the regulatory mechanisms that govern cellular diversification in the neocortex.

Recent advances in sequencing technologies have uncovered the existence of thousands of long noncoding RNAs (lncRNAs) with dysregulated expression in cancer. As a result, there is burgeoning interest in understanding their function and biological significance in both homeostasis and disease. RNA interference (RNAi) enables sequence-specific gene silencing and can, in principle, be employed to silence virtually any gene. However, when applied to lncRNAs, it is important to consider current limitations in their annotation and current principles regarding lncRNA regulation and function when assessing their phenotype in cancer cell lines. In this chapter we describe the analysis of lncRNA splicing variant expression, including subcellular localization, transfection of siRNAs in cancer cell lines, and validation of gene silencing by quantitative PCR and single molecule in situ hybridization. All protocols can be performed in a laboratory with essential equipment for cell culture, molecular biology, and imaging.

Pruritus (itch) is a debilitating symptom in liver diseases with cholestasis, which severely affects patients' quality of life. Limited treatment options are available for cholestatic itch, largely due to the incomplete understanding of the underlying molecular mechanisms. Several factors have been proposed as pruritogens for cholestatic itch, such as bile acids, bilirubin, lysophosphatidic acid, and endogenous opioids. Recently, two research groups independently identified Mas-related G protein-coupled receptor X4 (MRGPRX4) as a receptor for bile acids and bilirubin and demonstrated its likely role in cholestatic itch. This discovery not only opens new avenues for understanding the molecular mechanisms in cholestatic itch but provides a promising target for developing novel anti-itch treatments. In this review, we summarize the current theories and knowledge of cholestatic itch, emphasizing MRGPRX4 as a bile acid and bilirubin receptor mediating cholestatic itch in humans. We also discuss some future perspectives in cholestatic itch research.Thieme. All rights reserved.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Human papillomavirus (HPV) is a well-established mucosotropic carcinogen, but its impact on urothelial neoplasm is unclear. We aimed to clarify the clinical and pathological features of HPV-related urothelial carcinoma (UC).Tissue samples of 228 cases of UC were obtained from the bladder, upper and lower urinary tract, and metastatic sites to construct a tissue microarray. The samples were analyzed for the presence of HPV by a highly sensitive and specific mRNA in situ hybridization (RISH) technique (RNAscope) with a probe that can detect 18 varieties of high-risk HPV. We also conducted immunohistochemistry (IHC) for a major HPV capsid antibody and DNA-PCR.The HPV detection rates varied among the methods; probably due to low HPV copy numbers in UC tissues and the insufficient specificity and sensitivity of the IHC and PCR assays. The RISH method had the highest accuracy and identified HPV infection in 12 (5.2%) of the cases. The histopathological analysis of the HPV-positive UC showed six cases of usual type UC, five cases of UC with squamous differentiation (UC_SqD), and one case of micropapillary UC. The HPV detection rate was six-fold higher in the cases of UC_SqD than in the other variants of UC (odds ratio [OR] =8.9, p = 0.002). In addition, HPV infection showed a significant association with tumor grade (OR =9.8, p = 0.03) and stage (OR =4.7, p = 0.03) of UC. Moreover, the metastatic rate was higher in HPV-positive than in negative UC (OR =3.4).These data indicate that although the incidence of HPV infection in UC is low, it is significantly associated with squamous differentiation and poor prognosis. Furthermore, our observations show that RNAscope is an ideal method for HPV detection in UC compared with the other standard approaches such as IHC and PCR assays.