Probes for INS

The homeoboxB9 (HOXB9) gene is necessary for specification of the anterior-posterior body axis during embryonic development and expressed in various types of cancer. Here we show that the Wilms tumor transcription factor WT1 regulates the HOXB9 gene in a bidirectional manner. Silencing of WT1 activates HOXB9 in Wt1 expressing renal cell adenocarcinoma-derived 786-0 cells, mesonephric M15 cells and ex vivo cultured murine embryonic kidneys. In contrast, HOXB9 expression in U2OS osteosarcoma and human embryonic kidney (HEK) 293 cells, which lack endogenous WT1, is enhanced by overexpression of WT1. Consistently, Hoxb9 promoter activity is stimulated by WT1 in transiently transfected U2OS and HEK293 cells, but inhibited in M15 cells with CRISPR/Cas9-mediated Wt1 deletion. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrate binding of WT1 to the HOXB9 promoter in WT1-overexpressing U2OS cells and M15 cells. BASP1, a transcriptional co-repressor of WT1, is associated with the HOXB9 promoter in the chromatin of these cell lines. Co-transfection of U2OS and HEK293 cells with BASP1 plus WT1 prevents the stimulatory effect of WT1 on the HOXB9 promoter. Our findings identify HOXB9 as a novel downstream target gene of WT1. Depending on the endogenous expression of WT1, forced changes in WT1 can either stimulate or repress HOXB9, and the inhibitory effect of WT1 on transcription of HOXB9 involves BASP1. Consistent with inhibition of Hoxb9 expression by WT1, both transcripts are distributed in an almost non-overlapping pattern in embryonic mouse kidneys. Regulation of HOXB9 expression by WT1 might become relevant during kidney development and cancer progression.

Effective control of ipomoviruses that cause cassava brown streak disease (CBSD) in Africa has remained problematic despite eight remarkable decades (1930-2021) of research efforts. Molecular mechanisms underlying resistance breakdown in genetically improved cassava are still unknown. The vast genetic diversity of cassava brown streak viruses, which is crucial for the improvement of routine reverse transcription polymerase chain reaction (RT-qPCR) assays in CBSD-endemic regions of Africa, is controversial and underrepresented. From a molecular epidemiology viewpoint, this review discusses the reasons for why permanent control of CBSD is difficult in the modern era, even with the presence of diverse in silico and omics tools, recombinant DNA, and high throughput next-generation sequencing technologies. Following an extensive nucleotide data search in the National Centre for Biotechnology Information (NCBI) database and a literature review in PubMed and Scopus, we report that genomic data of 87.62% (474/541) strains of cassava brown streak virus are missing due to poor sequencing capacity in Africa. The evolution dynamics of viral virulence and pathogenicity has not yet been fully explored from the available 67 (12.38%) genomic sequences, owing to poor bioinformatics capacity. Tanzania and Zambia have the highest and lowest disease inoculum pressure, correspondingly. Knowledge gaps in molecular biology and the overall molecular pathogenesis of CBSD viruses impede effective disease control in Africa. Recommendations for possible solutions to the research questions, controversies, and hypotheses raised in this study serve as a roadmap for the invention of more effective CBSD control methods.

Background Major depressive disorder (MDD) is a pervasive and debilitating syndrome characterized by mood disturbances, anhedonia, and alterations in cognition. While the prevalence of MDD is twice as high for women compared to men, little is known about the molecular mechanisms that drive sex differences in depression susceptibility. Methods We discovered that Slit Guidance Ligand 1 (SLIT1), a secreted protein essential for axonal navigation and molecular guidance during development, is downregulated in the adult ventromedial prefrontal cortex (vmPFC) of depressed women compared to healthy controls, but not depressed men. This sex-specific downregulation of Slit1 was also observed in vmPFC of mice exposed to chronic variable stress. To identify a causal, sex-specific role for SLIT1 in depression-related behavioral abnormalities, we performed knockdown (KD) of Slit1 expression in vmPFC of male and female mice. Results When combined with stress exposure, vmPFC Slit1 KD reflected the human condition by inducing a sex-specific increase in anxiety- and depression-related behaviors. Further, we found that vmPFC Slit1 KD decreased the dendritic arborization of vmPFC pyramidal neurons, and decreased the excitability of the neurons, in female mice, effects not observed in males. RNA-sequencing analysis of vmPFC after Slit1 KD in female mice revealed an augmented transcriptional stress signature. Conclusions Together, our findings establish a crucial role for SLIT1 in regulating neurophysiological and transcriptional responses to stress within the female vmPFC, and provide mechanistic insight into novel signaling pathways and molecular factors influencing sex differences in depression susceptibility.

Several neurodevelopmental disabilities are strongly associated with alterations in GABAergic transmission, and therapies to stimulate its normal development are lacking. Erythropoietin (EPO) is clinically used in neonatology to mitigate acute brain injury, and to stimulate neuronal maturation. Yet it remains unclear whether EPO can stimulate maturation of the GABAergic system. Here, with the use of a transgenic mouse line that constitutively overexpresses neuronal EPO (Tg21), we show that EPO stimulates postnatal GABAergic maturation in the hippocampus. We show an increase in hippocampal GABA-immunoreactive neurons, and postnatal elevation of interneurons expressing parvalbumin (PV), somatostatin (SST) and neuropeptide Y (NPY). Analysis of perineuronal net formation and innervation of glutamatergic terminals onto PV+ cells, shows to be enhanced early in postnatal development. Additionally, an increase in GABAAergic synapse density and inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells from Tg21 mice is observed. Detection of erythropoietin receptor (EPOR) mRNA was observed to be restricted to glutamatergic pyramidal cells and increased in Tg21 mice at postnatal day 7, along with reduced apoptosis. Our findings show that EPO can stimulate postnatal GABAergic maturation in the hippocampus, by increasing neuronal survival, modulating critical plasticity periods, and increasing synaptic transmission. Our data supports EPO's clinical use to balance GABAergic dysfunction.Significance Statement Using a mouse model that overexpresses recombinant human EPO in the CNS, we observed stimulation of the postnatal maturation of GABAergic transmission in the hippocampus, notably accelerated maturation of PV+ interneurons, enhanced glutamatergic inputs onto these interneurons, and enhanced inhibitory postsynaptic currents (IPSCs) onto pyramidal cells. We show that EPORs are expressed on pyramidal cells, therefore the impact of EPO on GABAergic maturation is likely to be indirect. Our data show that EPO can modulate hippocampal network maturation and support ongoing trials of the use of EPO in clinical neonatology to stimulate neuronal maturation after perinatal brain injury.

Cancer stem cells (CSCs) are believed to be responsible for tumor growth, invasion, and metastasis. Submucosal invasion, which greatly enhances metastasis risk, is a critical step in gastric cancer (GC) progression. To identify stem cell-related markers associated with submucosal invasion and lymph node (LN) metastasis in GCs, we investigated the expression of candidate CSC markers (CD133, CD44, and ALDH1A) and intestinal stem cell (ISC) markers (EPHB2, OLFM4, and LGR5) in early GCs that manifested submucosal invasion. We discovered that EPHB2 and LGR5 expression was frequently confined to the basal area of the lamina propria (basal pattern) in mucosal cancer, and the proportion of stem cell marker-positive cells substantially increased during submucosal invasion. CD44 expression showed a focal pattern, ALDH1A was predominantly expressed diffusely, and there was no expansion of CD44 or ALDH1A expression in the submucosal cancer cells. Unexpectedly, no CSC markers showed any associations with LN metastasis, and only loss of EPHB2 expression was associated with increased LN metastasis. Treatment of RSPO2, a niche factor, along with Wnt 3a, to GC cells led to increased EPHB2 and LGR5 mRNA levels. RNA in situ hybridization confirmed specific RSPO2 expression in the smooth muscle cells of the muscularis mucosa, suggesting that RSPO2 is responsible for the increased expression of ISC markers in GC cells at the basal areas. In summary, no stem cell markers were associated with increased LN metastasis in early GCs. Conversely, isolated EPHB2 expression was associated with lower LN metastasis. EPHB2 and LGR5 showed a basal distribution pattern along with enhanced expression in submucosal invading cells in early GCs, which was induced by a niche factor, RSPO2, from the muscularis mucosa.

Background Corticotropin releasing factor (CRF) neural systems are important stress mechanisms in central amygdala (CeA), bed nucleus of stria terminalis (BNST), nucleus accumbens (NAc) and related structures. CRF-containing neural systems are traditionally posited to generate aversive distress states that motivate over-consumption of rewards and relapse in addiction. However, CRF-containing systems may alternatively promote incentive motivation to increase reward pursuit and consumption, without requiring aversive states. Methods We optogenetically stimulated CRF-expressing neurons in CeA, BNST or NAc, using Crh-Cre+ rats (n=37 female, n=34 male) to investigate roles in incentive motivation versus aversive motivation. We paired CRF-expressing neuronal stimulations with earning sucrose rewards in two-choice and progressive ratio tasks and investigated recruitment of distributed limbic circuitry. We further assessed valence with CRF-containing neuron laser self-stimulation tasks. Results Channelrhodopsin excitation of CRF-containing neurons in CeA and NAc amplified and focused incentive motivation and recruited activation of mesocorticolimbic reward circuitry. CRF systems in both CeA and NAc supported laser self-stimulation, amplified incentive motivation for sucrose in a breakpoint test, and focused ‘wanting’ on laser-paired sucrose over a sucrose alternative in a two-choice test. Conversely, stimulation of CRF-containing neurons in BNST produced negative-valence or aversive effects and recruited distress-related circuitry, as stimulation was avoided and suppressed motivation for sucrose. Conclusions CRF-containing systems in NAc and CeA can promote reward consumption by increasing incentive motivation, without involving aversion. By contrast, stimulation of CRF-containing systems in BNST is aversive but suppresses sucrose reward pursuit and consumption, rather than increase as predicted by traditional hedonic self-medication hypotheses.

Common steps in analysis of microRNA expression levels between different tissues, developmental stages, or disease states is to study microRNA expression levels by several methods as: NGS, microarray analysis, real-time PCR, Northern blots, in situ hybridization, and solution hybridization. Of these techniques, quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR) is one the most sensitive and accurate method. For qRT-PCR applications, the tools include: a) Effective method of microRNA isolation from samples; b) RT-qPCR reagents optimized for microRNA detection; c) Assays specific to the microRNAs of interest, and d) Real-time analytical instruments and reagents validated for microRNA detection. MicroRNAs have also been employed diagnostically, using liquid biopsies. Growing interest and utility of Circulating Cell-Free DNA [cfDNA] and interest in their role in oncology re-search is continue to grow in importance, in order to exploit their role as biomarkers for detecting premalignant and early stage cancers. The field of microRNA-based cancer research has witnessed a remarkable evolution over the last two decades, is the role of microRNAs as disease prognostic biomarkers, as well as recent attempts to exploit their role as therapeutic targets, as their small size and their stability in a variety of body fluids make them attractive substrates for employment as biomarkers. Current approaches for detecting microRNAs in blood and other body fluids is inadequate. The advantage of using microRNA approach is based on concurrently tar-geting multiple effectors of pathways involved in cell differentiation, proliferation, as well as in cell survival. In this review, we have employed regulatory small microRNAs as unifying molecules, which have shown a strong correlation with induction and progression of many human cancers, as they progress from the non- to the invasive stages of various types of human cancers, as detailed in this review below.

The interest and utility of high-plex spatial profiling of RNA and protein biomarkers has increased over the last few years. The implementation of high-plex analyte spatial platforms, such as GeoMx^® Digital Spatial Profiler (DSP), is increasing within discovery and development of biomarkers associated with clinical outcome. The surge in spatial platforms comes with an increased adoption of digital pathology in translational and clinical research studies. The integration of these two workflows has the potential to benefit diagnostic and therapeutic development. This study aims to facilitate the implementation of DSP in tissue analysis workflows helping researchers involved in drug discovery and development efforts to (1) assess platform feasibility for their research, (2) design effective DSP experiments, and (3) enable generation of high-quality, analyzable spatial data from large cohortstudies. The GeoMx DSP Biopharma and CRO Consortium has developed consensus-based best practices incorporating expertise of members from biopharma and contract research organizations (CROs). Best practices guidelines for spatial profiling of tissue biopsies in drug discovery and development using GeoMx stands to advance current standard practices in tissue analysis. These recommendations encompass every step in the implementation of DSP for standard tissue analysis workflows, emphasizing the importance of multidisciplinary stakeholder involvement, defining experimental conditions and testing these prior to execution of large-scale studies, and considerations in assessing assay performance. This document offers a practical reference for optimal implementation of GeoMx DSP in exploratory sample analysis for use in research supporting drug discovery and development. Here we present a practical reference for the optimal implementation of GeoMx DSP in exploratory analysis for drug discovery and development studies. Best practices insights for the application of this technology to breast cancer research have been previously published (1) and should also be taken in consideration when designing relevantspatial studies.

The developmental process of central nervous system (CNS) myelin sheath formation is characterized by well-coordinated cellular activities ultimately ensuring rapid and synchronized neural communication. During this process, myelinating CNS cells, namely oligodendrocytes (OLGs), undergo distinct steps of differentiation, whereby the progression of earlier maturation stages of OLGs represents a critical step toward the timely establishment of myelinated axonal circuits. Given the complexity of functional integration, it is not surprising that OLG maturation is controlled by a yet fully to be defined set of both negative and positive modulators. In this context, we provide here first evidence for a role of lysophosphatidic acid (LPA) signaling via the G protein-coupled receptor LPA6 as a negative modulatory regulator of myelination-associated gene expression in OLGs. More specifically, cell surface accessibility of LPA6 was found to be restricted to the earlier maturation stages of differentiating OLGs, and OLG maturation was found to occur precociously in Lpar6 knockout mice. To further substantiate these findings, a novel small molecule ligand with selectivity for preferentially LPA6 and LPA6 agonist characteristics was functionally characterized in vitro in primary cultures of rat OLGs and in vivo in the developing zebrafish. Utilizing this approach, a negative modulatory role of LPA6 signaling in OLG maturation could be corroborated. During development, such a functional role of LPA6 signaling likely serves to ensure timely coordination of circuit formation and myelination. Under pathological conditions as seen in the major human demyelinating disease multiple sclerosis (MS), however, persistent LPA6 expression and signaling in OLGs can be seen as an inhibitor of myelin repair. Thus, it is of interest that LPA6 protein levels appear elevated in MS brain samples, thereby suggesting that LPA6 signaling may represent a potential new druggable pathway suitable to promote myelin repair in MS.This article is protected by

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It's unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived from Nts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in the Gal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in the Nts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Dorsal root ganglion (DRG) neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of Gal-positive (Gal+) DRG neurons. However, the characteristics and functions of Zcchc12+ neurons are largely unknown. Here, we genetically labelled Zcchc12+ neurons in Zcchc12-CreERT2::Ai9 mice, and verified that Zcchc12 represented a new subpopulation of DRG neurons in both sexes. Zcchc12+ neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Besides, Zcchc12+ neurons also formed circumferential endings surround the hair follicles in hairy skin. Functionally, in vivo calcium imaging in DRGs revealed that Zcchc12+ neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation of Zcchc12+ DRG neurons reduced the sensitivity to noxious heat in mice. Taken together, we identify a new subpopulation of Zcchc12+ nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENT:Zcchc12 represents a new subpopulation of DRG neurons. The characteristics and functions of Zcchc12+ neurons are largely unknown. Here we genetically labelled Zcchc12 neurons, and showed that the fibers of Zcchc12+ DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally, Zcchc12+ DRG neurons responded to noxious mechanical and heat stimuli. Ablation of Zcchc12+ DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulatipn of DRG neurons required for noxious heat sensation.

Stromal-epithelial interactions are critical to the morphogenesis, differentiation, and homeostasis of the prostate, but the molecular identity and anatomy of discrete stromal cell types is poorly understood. Using single-cell RNA sequencing, we identified and validated the in situ localization of three smooth muscle subtypes (prostate smooth muscle, pericytes, and vascular smooth muscle) and two novel fibroblast subtypes in human prostate. Peri-epithelial fibroblasts (APOD+) wrap around epithelial structures, whereas interstitial fibroblasts (C7+) are interspersed in extracellular matrix. In contrast, the mouse displayed three fibroblast subtypes with distinct proximal-distal and lobe-specific distribution patterns. Statistical analysis of mouse and human fibroblasts showed transcriptional correlation between mouse prostate (C3+) and urethral (Lgr5+) fibroblasts and the human interstitial fibroblast subtype. Both urethral fibroblasts (Lgr5+) and ductal fibroblasts (Wnt2+) in the mouse contribute to a proximal Wnt/Tgfb signaling niche that is absent in human prostate. Instead, human peri-epithelial fibroblasts express secreted WNT inhibitors SFRPs and DKK1, which could serve as a buffer against stromal WNT ligands by creating a localized signaling niche around individual prostate glands. We also identified proximal-distal fibroblast density differences in human prostate that could amplify stromal signaling around proximal prostate ducts. In human benign prostatic hyperplasia, fibroblast subtypes upregulate critical immunoregulatory pathways and show distinct distributions in stromal and glandular phenotypes. A detailed taxonomy of leukocytes in benign prostatic hyperplasia reveals an influx of myeloid dendritic cells, T cells and B cells, resembling a mucosal inflammatory disorder. A receptor-ligand interaction analysis of all cell types revealed a central role for fibroblasts in growth factor, morphogen, and chemokine signaling to endothelia, epithelia, and leukocytes. These data are foundational to the development of new therapeutic targets in benign prostatic hyperplasia.