Exon 2 duplications of the DMD gene, encoding the dystrophin protein, account for around 6-11% of all duplication mutations associated with X-linked Duchenne muscular dystrophy (DMD). As part of the preclinical development of a U7snRNA vector currently in a clinical trial (ClinicalTrials.gov NCT04240314), we have previously evaluated the therapeutic efficacy, absence of off-target splicing effects in AAV9.U7snRNA-mediated skipping of exon 2 in a murine Dmd model, and lack of toxicity in non-human primates. Here we report that 3-month-old Dup2 mice systemically injected with scAAV9.U7.ACCA vector, containing four copies of U7snRNA targeted to the exon 2 splice acceptor and splice donor sites, showed efficient exon 2 skipping, long-term dystrophin expression, and skeletal muscle function correction 18-months post vector administration. The RT-PCR data showed that a single vector injection (3E13 vg/kg) resulted in significant exon 2 skipping in tibialis anterior (TA), diaphragm (Dia) and heart tissues, showing an average of 46%, 32% and 73% total therapeutic transcripts, respectively. To determine the degree of functional rescue, in situ and in vitro physiology studies on TA and Dia muscles were performed. Both Dia and TA from 21-month-old control Dup2 mice exhibited a functional deficit with a significant reduction in specific force output (45-61%) compared with Bl6 mice. The significant force drop was also observed in those mice compared with Bl6 following a rigorous fatigue protocol. The single vector infusion resulted in a dramatic improvement in specific force output up to 64-76% in Dia and TA, and better protection of the TA muscle (up to 73%) from repeated fatigue. Overall, our results confirm that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in straight muscles from Dup2 mice and functional recovery of TA and Dia muscles 18-month post vector administration.

Puberty onset is a complex physiological process which enables the capacity for reproduction through increased gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus (ARC) are believed to govern the timing of puberty, the degree to which KNDy neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the ARC. Fluorescent in situ hybridization revealed that the majority (> 74%) of ARC neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer ARC cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of ARC cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and supports the idea that NKB and dynorphin play a role in puberty onset in the female pig.

Chronic kidney disease (CKD) is characterized by persistent abnormalities in kidney function, accompanied by structural changes. Interstitial fibrosis, characterized by the accumulation of extracellular matrix (ECM) proteins, is frequently detected during CKD development. Given the multiple underlying causes of CKD, numerous animal models have been developed to advance our understanding of human nephropathy. Herein, we compared two reliable toxin-induced mouse kidney fibrosis models in terms of fibrosis and inflammation. Administration of folic acid (250 mg/kg, intraperitoneal injection) or an adenine diet (0.25 % for three weeks) afforded similar effects on kidney function, as detected by increased serum nitrogen levels. In addition, the kidneys exhibited a similar extent of tubule dilation and kidney damage. The degree of fibrosis was compared using various biological methods. Although both models developed a significant fibrotic phenotype, the adenine diet-fed model showed a marginally higher increase in fibrosis than the folic acid model, as reflected by increased kidney ECM gene and protein levels. We further compared inflammatory responses in the kidneys. Interestingly, pro-inflammatory responses, including cytokine expression and immune cell infiltration, were significantly increased in adenine diet-fed kidneys. Furthermore, collagen expression was identified in the macrophage-infiltrated region, implying the importance of inflammation in fibrogenesis. Collectively, we observed that the adenine diet-fed kidney fibrosis model presented a higher inflammatory response with increased fibrosis when compared with the folic acid-induced kidney fibrosis model, indicating the importance of the inflammatory response in fibrosis development.

Glioblastoma is a glioma characterized by highly malignant features. Numerous studies conducted on the relationship between glioblastoma and the microenvironment have indicated the significance of tumor-associated macrophages/microglia (TAMs) in glioblastoma progression. Since interleukin (IL)-1β secreted by TAMs has been suggested to promote glioblastoma growth, we attempted to elucidate the detailed mechanisms of IL-1β in glioblastoma growth in this study. A phospho-receptor tyrosine kinase array and RNA-sequencing studies indicated that IL-1β induced the activation of signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. Glioblastoma cells stimulated by IL-1β induced the production of IL-6 and CXCL8, which synergistically promoted glioblastoma growth via signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. By immunohistochemistry, IL-1β expression was seen on TAMs, especially in perinecrotic areas. These results suggest that IL-1β might be a useful target molecule for anti-glioblastoma therapy.

Loss of kidney function is a common feature of COVID-19 infection, but serum creatinine (SCr) is not a sensitive or specific marker of kidney injury. We tested whether molecular biomarkers of tubular injury measured at hospital admission were associated with AKI in those with COVID-19 infection.This is a prospective cohort observational study consisting of 444 consecutive SARS-CoV-2 patients enrolled in the Columbia University Emergency Department at the peak of New York's pandemic (March-April 2020). Urine and blood were collected simultaneously at hospital admission (median time: day 0, IQR 0-2 days) and urine biomarkers analyzed by ELISA and by a novel dipstick. Kidney biopsies were probed for biomarker RNA and for histopathologic acute tubular injury (ATI) scores.Admission uNGAL was associated with AKI diagnosis (267±301 vs. 96±139 ng/mL, P < 0.0001) and staging; uNGAL levels >150ng/mL demonstrated 80% specificity and 75% sensitivity to diagnose AKI-stage 2-3. Admission uNGAL quantitatively associated with prolonged AKI, dialysis, shock, prolonged hospitalization, and in-hospital death, even when admission SCr was not elevated. The risk of dialysis increased almost 4-fold per standard deviation of uNGAL independently of baseline SCr, co-morbidities, and proteinuria [OR(95%CI): 3.59 (1.83-7.45), P < 0.001]. In COVID-19 kidneys, NGAL mRNA expression broadened in parallel with severe histopathological injury (ATI). Conversely, low uNGAL levels at admission ruled out stage 2-3 AKI (NPV 0.95, 95%CI: 0.92-0.97) and the need for dialysis (NPV: 0.98, 95%CI: 0.96-0.99)). While proteinuria and uKIM-1 implicated tubular injury, neither were diagnostic of AKI stages.In COVID-19 patients, uNGAL quantitatively associated with histopathological injury (ATI), the loss of kidney function (AKI), and the severity of patient outcomes.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.

P-glycoproteins from the ATP-binding cassette transporter family are responsible for drug evasion by bacterial pathogens and neoplastic cells. More recently, these multidrug resistance transporters have been investigated for contributions to drug resistance in nematode parasites. In this study, we cloned and characterized the P-glycoprotein Tca-Pgp-11.1 from Toxocara canis, the canine intestinal ascarid. Large numbers of Tca-Pgp-11 transcripts were observed in the intestine of adult male and female worms. Heterologous expression studies confirmed sensitivity to known P-glycoprotein inhibitors. Interestingly, the competitive inhibitor verapamil had lower IC50 values than newer generation inhibitors that are designed to allosterically modulate mammalian P-glycoprotein. Consistent with other nematode P-glycoproteins, Tca-Pgp-11.1 was sensitive to ivermectin and selamectin but not moxidectin. Taken together, our data suggests that T. canis P-glycoproteins represent nematode-specific drug targets that could be exploited to enhance efficacy of existing anthelmintics.

In 2020, the WHO published a new system for classifying invasive endocervical adenocarcinoma based on histological features and high-risk human papillomavirus (HPV) infection. However, immunophenotypes of each histological subtype require further investigation. We immunohistochemically analyzed 66 invasive endocervical adenocarcinomas using three cell-lineage-specific markers: claudin 18 (CLDN18) for gastric, cadherin 17 (CDH17) for intestinal, and PAX8 for Müllerian epithelial cells. We identified five immunophenotypes of endocervical adenocarcinoma: gastric (21%); intestinal (14%); gastrointestinal (11%); Müllerian (35%); and not otherwise specified (NOS) (20%). Adenocarcinomas with gastric immunophenotype, characterized by aging (p = 0.0050), infrequent HPV infection (p < 0.0001), concurrent lobular endocervical glandular hyperplasia (p = 0.0060), lymphovascular invasion (p = 0.0073), advanced clinical stage (p = 0.0001), and the poorest progression-free (p < 0.0001) and overall (p = 0.0023) survivals, were morphologically compatible with gastric-type adenocarcinoma of the WHO 2020 classification. Conversely, most adenocarcinomas with Müllerian (91%) and intestinal (89%) immunophenotypes were HPV associated and morphologically compatible with usual- or intestinal-type adenocarcinomas of the WHO 2020 classification. The morphology of adenocarcinomas with gastrointestinal immunophenotype was intermediate or mixed between those of gastric and intestinal immunophenotypes; 57% were HPV associated. Adenocarcinomas with NOS immunophenotype were mainly HPV associated (85%) and histologically poorly differentiated. Multivariate analysis revealed that gastric (p = 0.008), intestinal + gastrointestinal (p = 0.0103), and NOS (p = 0.009) immunophenotypes were independent predictors of progression-free survival. Immunophenotypes characterized by CLDN18, CDH17, and PAX8 exhibited clinicopathological relevance and may improve the diagnostic accuracy and prognostic value of conventional histological classification.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was up-regulated by heart and neural crest derivatives expressed 2 (HAND2) and down-regulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.

Emerging evidence indicates that long noncoding RNAs (lncRNAs) are important regulators of various biological processes, and their expression can be altered following certain pathological conditions, including central nervous system injury. Retinal ganglion cells (RGCs), whose axons form the optic nerve, are a heterogeneous population of neurons with more than 40 molecularly distinct subtypes in mouse. While most RGCs, including the ON-OFF direction-selective RGCs (ooDSGCs), are vulnerable to axonal injury, a small population of RGCs, including the intrinsically photosensitive RGCs (ipRGCs), are more resilient.By performing systematic analyses on RNA-sequencing data, here we identify lncRNAs that are expressed in ooDSGCs and ipRGCs with and without axonal injury. Our results reveal a repertoire of different classes of lncRNAs, including long intergenic noncoding RNAs and antisense ncRNAs that are differentially expressed between these RGC types. Strikingly, we also found dozens of lncRNAs whose expressions are altered markedly in response to axonal injury, some of which are expressed exclusively in either one of the types. Moreover, analyses into these lncRNAs unraveled their neighboring coding genes, many of which encode transcription factors and signaling molecules, suggesting that these lncRNAs may act in cis to regulate important biological processes in these neurons. Lastly, guilt-by-association analysis showed that lncRNAs are correlated with apoptosis associated genes, suggesting potential roles for these lncRNAs in RGC survival.Overall, the results of this study reveal RGC type-specific expression of lncRNAs and provide a foundation for future investigation of the function of lncRNAs in regulating neuronal type specification and survival.

The fate of neurons in the developing brain is largely determined by the combination of transcription factors they express. In particular, stem cells must follow different transcriptional cascades during differentiation in order to generate neurons with different neurotransmitter properties, such as glutamatergic and GABAergic neurons. In the mouse cerebral cortex, it has been shown that large Maf family proteins, MafA, MafB and c-Maf, regulate the development of specific types of GABAergic interneurons but are not expressed in glutamatergic neurons. In this study, we examined the expression of large Maf family proteins in the developing mouse olfactory bulb by immunohistochemistry and found that the cell populations expressing MafA and MafB are almost identical, and most of them express Tbr2. Since Tbr2 is expressed in glutamatergic neurons in the olfactory bulb, we further examined the expression of glutamatergic and GABAergic neuronal markers in MafA and MafB positive cells. The results showed that in the olfactory bulb, MafA and MafB are expressed exclusively in glutamatergic neurons, but not in GABAergic neurons. We also found that few cells express c-Maf in the olfactory bulb. These results indicate that, unlike the cerebral cortex, MafA and/or MafB may regulate the development of glutamatergic neurons in the developing olfactory bulb. This study advances our knowledge about the development of glutamatergic neurons in the olfactory bub, and also provides insight into the mechanism by which the cortex and olfactory bulb, although both generated from the telencephalon, generate projection and interneurons with different properties. This article is protected by