Probes for INS

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with schizophrenia risk. Integration of RNA-sequencing data from postmortem human brains with these risk SNPs identified transcripts associated with increased schizophrenia susceptibility, including a class of exon 9-spliced isoforms of Sorting nexin-19 (SNX19d9) and an isoform of Arsenic methyltransferase (AS3MT) splicing out exons 2 and 3 (AS3MTd2d3). However, the biological function of these transcript variants is unclear. Defining the cell types where these risk transcripts are dominantly expressed is an important step to understand function, in prioritizing specific cell types and/or neural pathways in subsequent studies. To identify the cell type-specific localization of SNX19 and AS3MT in the human dorsolateral prefrontal cortex (DLPFC), we used single-molecule in situ hybridization techniques combined with automated quantification and machine learning approaches to analyze 10 postmortem brains of neurotypical individuals. These analyses revealed that both pan-SNX19 and pan-AS3MT were more highly expressed in neurons than non-neurons in layers II/III and VI of DLPFC. Furthermore, pan-SNX19 was preferentially expressed in glutamatergic neurons, while pan-AS3MT was preferentially expressed in GABAergic neurons. Finally, we utilized duplex BaseScope technology, to delineate the localization of SNX19d9 and AS3MTd2d3 splice variants, revealing consistent trends in spatial gene expression among pan-transcripts and schizophrenia risk-related transcript variants. These findings demonstrate that schizophrenia risk transcripts have distinct localization patterns in the healthy human brains, and suggest that SNX19 transcripts might disrupt the normal function of glutamatergic neurons, while AS3MT may lead to disturbances in the GABAergic system in the pathophysiology of schizophrenia.

Central oxytocin receptor (OTR) expression is extremely sensitive to circulating steroid hormones and OTRs influence many of the neurobehavioural adaptations associated with female reproduction (e.g., postpartum caregiving, aggression, cognition, affective responses). Changes in central OTR expression across female reproduction have often been studied, but almost all of such research has focused on the forebrain, ignoring hormone-sensitive midbrain sites such as the serotonergic dorsal raphe (DR) that are also critical for postpartum behaviours. To investigate the effects of female reproductive state on OTRs in the DR, we first used autoradiography to examine OTR binding across four female reproductive states in laboratory rats: dioestrous virgin, pregnancy day 10, the day of parturition and postpartum day 7. OTR binding in the rostral DR (but not other DR subregions) was approximately 250% higher in parturient rats compared to dioestrous virgins and dropped back down to virgin levels by postpartum day 7. Given the chemical heterogeneity of the DR, we then examined OTR expression in the three most abundant neuronal phenotypes of the DR (i.e., serotonin, GABA and dopamine) in dioestrous virgins and recently parturient females. Using dual-label immunohistochemistry and in situ hybridisation, we found that twice as many dopaminergic cells in the parturient rostral DR contained OTR immunoreactivity compared to that found in virgins. On the other hand, mothers had fewer rostral DR GABAergic cells expressing OTRs than did virgins. OTR expression in serotonin cells did not differ between the two groups. Overall, these results suggest that the rostral subregion of the midbrain DR is uniquely sensitive to oxytocin around the time of parturition, with subpopulations of cells that become more sensitive (i.e., dopamine), less sensitive (i.e., GABA) and show no change (i.e., serotonin) to this neuropeptide. This dynamic OTR signalling in the female DR may help drive the numerous behavioural changes across female reproduction that are necessary for successful motherhood.

Hitherto, the recognition of the microbiota role in tumorigenesis and clinical studies mostly focused on the intestinal flora. In contrast to the gut microbiome, microorganisms resident in tumor tissue are in close contact with cancer cells and therefore have the potential to have similar or even different functional patterns to the gut flora. Some investigations have shown intratumoral bacteria, which might come from commensal microbiota in mucosal areas including the gastrointestinal tract and oral cavity, or from nearby normal tissues. The existence, origin, and interactions of intratumoral bacteria with the tumor microenvironment all contribute to intratumoral microorganism heterogeneity. Intratumoral bacteria have a significant role in tumor formation. They can contribute to cancer at the genetic level by secreting poisons that directly damage DNA and also intimately related to immune system response at the systemic level. Intratumoral bacteria have an impact on chemotherapy and immunotherapy in cancer. Importantly, various properties of bacteria such as targeting and ease of modification make them powerful candidates for precision therapy, and combining microbial therapies with other therapies is expected to improve the effectiveness of cancer treatment. In this review, we mainly described the heterogeneity and potential sources of intratumoral bacteria, overviewed the important mechanisms by which they were involved in tumor progression, and summarized their potential value in oncology therapy. At last, we highlight the problems of research in this field, and look forward to a new wave of studies using the various applications of intratumoral microorganisms in cancer therapy.

GPR37 is an orphan GPCR and expressed in different brain regions. However, its biological function in pain regulation remains poorly understood. Recently, we identified Neuroprotectin D1 (NPD1) as a novel ligand of GPR37. NPD1 is a specialized pro-resolving mediator (SPM) and bio-synthesized from fish oil DHA (docosahexaenoic acid) . Here we reported a protective role of GRP37/NPD1 signaling in traumatic brain injury (TBI)-induced neuropathic pain. Mild TBI was induced by closed-head impact and the neuropathic pain was assessed by periorbital and cutaneous mechanical allodynia. In contrast to DHA (300 μg) showing no effects, peri-surgical treatment of NPD1 via intravenous injection (300 ng) effectively prevented TBI-induced locomotor deficiency and mechanical hypersensitivity in mice. Intraperitoneal post-treatment of NPD1 also significantly reduced established neuropathic pain in TBI mice. We also found that NPD1 treatment could inhibit TBI-induced neuroinflammation, characterized by microglia and astrocyte activation in the cortex and hippocampus. Furthermore, demyelination occurred after TBI, which was prevented by peri-surgical treatment of NPD1. RNAscope in situ hybridization revealed high Gpr37 mRNA expression in oligodendrocytes of the motor cortex and hippocampus . TBI resulted in a significant decrease in GPR37 expression, which could be restored by NPD1 treatment. GPR37 is protective and mice lacking Gpr37 (Gpr37-/-) exhibited prolonged pain symptoms after TBI. Furthermore, the protective effect of NPD1 was abolished in Gpr37-/- mice. All these findings suggest that activation of NPD1/GPR37 signaling pathway is a promising therapeutic strategy for preventing and treating neuropathic pain and its comorbidities after TBI. Funding: DoD grant W81XWH2110885.

Adult hippocampal neurogenesis (AHN) is a well-studied phenomenon that involves the derivation of new neurons from neural progenitor cells in the dentate gyrus region of the hippocampus, an area responsible for cognitive functions such as learning and memory storage. Moreover, the hippocampus is known to be implicated in neurological conditions such as Alzheimer's disease. Although AHN has been extensively observed in animal models for twenty years, its existence and persistence in humans have been widely debated in academia, heavily based on post-mortem immunohistochemical markers. Using the search engines PubMed and Google Scholar for “Adult Human Neurogenesis,” 143 articles that were most relevant to the history of AHN discovery, detection in rodents, immunohistochemical studies on post-mortem human sections, and therapeutic development targeting AHN were reviewed. This review article highlights the current understanding of AHN in rodents and humans, its implications in neurodegenerative diseases and therapeutics, and the inconsistencies and methodological variabilities encountered in studying AHN in humans. Furthermore, the correlation between AHN and diseases such as mood disorders and Alzheimer's disease is still not well established, with conflicting findings reported. Standardization of transcriptomic methodologies and increased availability of post-mortem human brain samples are crucial in advancing AHN research. This review article attempts to discover the fascinating and controversial world of adult human neurogenesis and its potential implications in treating neurological disorders. Apart from the discussion on AHN existence, tackling devastating diseases with this supplemental knowledge can lead to therapeutic advancements which greatly rely on understanding not only the presence of AHN but the mechanisms mediating its availability.

SELENON-related congenital myopathy is characterized by proximal weakness starting in infancy, early respiratory insufficiency, and early development of severe scoliosis. While changes in the SELENON gene, which encodes the protein SelN, are known to cause this disease the mechanisms through which loss of SelN lead to myopathy are not well understood. Previous studies suggest that SelN may have multiple roles in muscle, including regulating development of Type II muscle fibers, modulating excitation-contraction coupling through interactions with RYR1 and other muscle calcium channels, and possibly supporting satellite cell activation and proliferation following muscle injury. One particular challenge to understanding the role of SelN in skeletal muscle has been the inability to directly visualize SelN expression within muscle fibers and supporting cells due to a lack of robust antibodies for immunohistochemistry. Studies of mRNA expression and Western blot analysis of protein expression suggest significant post-transcriptional regulation of protein expression with an overall pattern of high expression in developing muscle and other developing tissues and low-level ubitquitous expression in mature tissues but evaluation of SelN expression in more limited sub-populations of cells has not been possible. Experiments in mouse suggest that loss of SelN expression results in decreased satellite cell proliferation following muscle injury. Here, I use a newly developed zebrafish model with mNeonGreen-tagged SelN to directly visualize SelN expression in satellite cells following muscle injury and show that SelN expression increases in activated satellite cells following mechanical muscle injury. This provides support SelN playing a role in satellite cell activation and proliferation during muscle repair following injury.

Although best known for their involvement in modulating nociception, Neuropeptide FF (NPFF) group peptides have been suggested to fulfil a variety of biological functions such as feeding, anxiety behaviors and thermogenesis. However, evidence supporting these functions of NPFF is mostly pharmacological, leaving the physiological relevance unaddressed. Here we examined the physiological impact of lack of NPFF signalling in both genders using a Npff-/- mouse model. NPFF expression in the mouse is restricted to the spinal cord and brainstem while its cognate receptor NPFFR2 has wider distribution throughout the brain. Both male and female Npff-/- mice showed reduced repetitive behaviors evidenced in the marble burying test and self-grooming test. A decrease in anxiety-related behaviors in the Npff-/- mice was also observe in the open field test and to a lesser degree in an elevated plus maze test. Moreover, both male and female Npff-/- mice exhibited increased water intake resulting from increases in drinking size, rather than number of drinking events. During a fasting-refeeding challenge, Npff-/- mice of both genders displayed alterations in reparatory exchange ratio that reflect a greater fuel type flexibility. Npff-/- mice were otherwise wild-type-like regarding body weight, body composition, feeding behaviors, locomotion or energy expenditure. Together, these findings reveal the important physiological roles of NPFF signalling in the regulation of anxiety-related and repetitive behaviors, fluid homeostasis and oxidative fuel selection, highlighting the therapeutical potential of the NPFF system in a number of behavioral and metabolic disorders.

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.