Probes for INS

While toll-like receptors (TLRs), which mediate innate immunity, are known to play an important role in host defense, recent work suggest their involvement in some integrated behaviors, including anxiety, depressive and cognitive functions. Here, we investigated the potential involvement of the flagellin receptor, TLR5, in anxiety, depression and cognitive behaviors using male TLR5 knock-out (KO) mice. We aobserved a specific low level of basal anxiety in TLR5 KO mice with an alteration of the hypothalamo-pituitary axis (HPA) response to acute restraint stress, illustrated by a decrease of both plasma corticosterone level and c-fos expression in the hypothalamic paraventricular nucleus where TLR5 was expressed, compared to WT littermates. However, depression and cognitive-related behaviors were not different between TLR5 KO and WT mice. Nor there were significant changes in the expression of some cytokines (IL-6, IL-10 and TNF-α) and other TLRs (TLR2, TLR3 and TLR4) in the prefrontal cortex, amygdala and hippocampus of TLR5 KO mice compared to WT mice. Moreover, mRNA expression of BDNF and glucocorticoid receptors in the hippocampus and amygdala, respectively, was not different. Finally, acute intracerebroventricular administration of flagellin, a specific TLR5 agonist, or chronic neomycin treatment did not exhibit a significant main effect, only a significant main effect of genotype was observed between TLR5 KO and WT mice. Together, those findings suggest a previously undescribed and specific role of TLR5 in anxiety and open original prospects in our understanding of the brain-gut axis function.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Obesity is a disease that affects 1/3 of the US population that could lead to several comorbidities. Obesity induces cognitive and behavioral impairment. Low plasma vitamin D levels are associated with increased body mass index (BMI). Therefore, vitamin D supplementation has shown benefits in BMI, glucose homeostasis and cognitive/behavioral function; however, contradictory data exists in animal models and remains poorly studied. Obesity in males increases 5.7-fold the risk for severe complications and death due to SARSCoV-2. Brain RAS activity, especially activation of the Angiotensin II receptor (AT1R), has been implicated in cognitive impairment and may participate in persistent neurological symptoms seen in 1 in 3 severe COVID-19 cases, i.e., disorientation, inattention and ataxia. Supplementation with Vitamin D (VitD), known to ameliorate respiratory infections and thromboembolism, may protect against COVID symptoms by increasing ACE2 and Masr expression. To generate a diet-induced obesity (DIO) model, C57Bl6/J male mice (7 wks of age) were randomly assigned to 3 groups (n=10/group) and were fed ad libitum with a) chow diet (CD), b) lard-based high-fat diet (HFD, 60% fat; D12492; Research diet, New Brunswick, NJ), or c) HFD supplemented with vitamin D (60% fat plus 15,000 IU/kg diet, HVitD). Behavioral and metabolic endpoints were measured starting at 7 wks on diet. The HFD diet was effective in generating DIO vs CD despite equivalent food intake consumption on a kcal-basis. Body composition analysis (EchoMRI) showed increased fat mass in HFD and HVitD when compared to CD. Contrary to our hypothesis, ip glucose (0.25 mg/kg) caused greater glucose intolerance in HVitD relative to HFD at 15 (P

Study question: Can our newly developed testicular organoid (TO) growth platform advance the robustness of murine TOs? Summary answer: The platform resulted in more consistent TO histology. Moreover, improved germ cell survival was observed after a two-week culture with numbers comparable to fresh samples. What is known already: Organ cultures have traditionally been used for invitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Organoids made from testicular cell suspensions offer these features. Although TO cultures can result in organoids with compartmentalized testicular architecture, histological heterogeneity between individual TOs limits reproducibility of the results, offering unreliable readouts. Moreover, germ cell loss is characteristic during the reorganization phase. Study design, size, duration: Here, we tested a new TO growth platform. Firstly, the focus was put on improving germ cell survival in TOs during tubulogenesis in the first two weeks of culture. For this, four different growth media (A-D), supplemented with other combinations or concentrations of growth factors, were compared. Next, five cell seeding densities (I-V) were tested for their ability to recreate the testicular architecture in TOs in the selected culture media. Participants/materials, setting, methods: Testicular cells from 5 days old C57BL/6J mice were grown in our TO platform with alpha-MEM-based medium, previously found to support TO generation in mice (medium A). Three additional conditions were tested in their ability to improve germ cell survival during tubulogenesis (B-D). Finally, the ideal cell density (I-V) was determined based on histological resemblance to native tissue: one tubule-like structure and surrounding interstitium. Cellular reorganization and germ cell maintenance were characterized by (immuno)histochemistry. Main results and the role of chance: During short-term cultures of 2 weeks, testicular cells self-assembled and compacted into organoids in our platform. Interestingly, media B and D resulted in the highest amount of germ cells (p < 0.05), comparable to the fresh control. Particularly TOs cultured in medium D also exhibited the largest surface area, indicative for better in-vitro growth. Finally, TOs that were cultured in condition D had the best histology when grown at cell density IV and V (p < 0.05). Limitations, reasons for caution: Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells typically observed in organ culture, but also in TOs. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings: . The opportunities TOs offer to manipulate cells (genetic modification, inclusion and exclusion) are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity and drug discovery studies.

Many general anesthetics potentiate gamma-aminobutyric acid (GABA) A receptors but their neuroanatomic sites of action are less clear. GABAergic neurons in the rostromedial tegmental nucleus (RMTg) send inhibitory projections to multiple arousal-promoting nuclei, but the role of these neurons in modulating consciousness is unknown. In this study, designer receptors exclusively activated by designer drugs (DREADDs) were targeted to RMTg GABAergic neurons of Vgat-ires-Cre mice. DREADDs expression was found in the RMTg and other brainstem regions. Activation of these neurons decreased movement and exploratory behavior, impaired motor coordination, induced electroencephalogram (EEG) oscillations resembling nonrapid eye movement (NREM) sleep without loss of righting and reduced the dose requirement for sevoflurane-induced unconsciousness. These results suggest that GABAergic neurons in the RMTg and other brainstem regions promote sedation and facilitate sevoflurane-induced unconsciousness.

Genetic variant calling from DNA sequencing has enabled understanding of germline variation in hundreds of thousands of humans. Sequencing technologies and variant-calling methods have advanced rapidly, routinely providing reliable variant calls in most of the human genome. We describe how advances in long reads, deep learning, de novo assembly and pangenomes have expanded access to variant calls in increasingly challenging, repetitive genomic regions, including medically relevant regions, and how new benchmark sets and benchmarking methods illuminate their strengths and limitations. Finally, we explore the possible future of more complete characterization of human genome variation in light of the recent completion of a telomere-to-telomere human genome reference assembly and human pangenomes, and we consider the innovations needed to benchmark their newly accessible repetitive regions and complex variants.

Solid tumors have a dynamic ecosystem in which malignant and non-malignant (endothelial, stromal, and immune) cell types constantly interact. Importantly, the abundance, localization, and functional orientation of each cell component within the tumor microenvironment vary significantly over time and in response to treatment. Such intratumoral heterogeneity influences the tumor course and its sensitivity to treatments. Recently, high-dimensional imaging mass cytometry (IMC) has been developed to explore the tumor ecosystem at the single-cell level. In the last years, several studies demonstrated that IMC is a powerful tool to decipher the tumor complexity. In this review, we summarize the potential of this technology and how it may be useful for cancer research (from preclinical to clinical studies).

Inhibitors of fibroblast growth factor receptor (FGFR) signaling have been investigated in various human cancer diseases. Recently, the first compounds received FDA approval in biomarker-selected patient populations. Different approaches and technologies have been applied in clinical trials, ranging from protein (immunohistochemistry) to mRNA expression (e.g., RNA in situ hybridization) and to detection of various DNA alterations (e.g., copy number variations, mutations, gene fusions). We review, here, the advantages and limitations of the different technologies and discuss the importance of tissue and disease context in identifying the best predictive biomarker for FGFR targeting therapies.

Tumor cells present complex behaviors in their interactions with other cells. This intricate behavior is driving the need to develop new tools to understand these ecosystems. The surge of spatial technologies allows evaluation of the complexity of relationships between cells present in a tumor, giving insights about tumor heterogeneity and the tumor microenvironment while providing clinically relevant metrics for tumor classification. In this review, we describe key results obtained using spatial techniques, present recent advances in methods to uncover spatially relevant biological significance, and summarize their main characteristics. We expect spatial technologies to significantly broaden our understanding of tumor biology and to generate clinically relevant tools that will ultimately impact personalized medicine.

RESULTS : Uniform Manifold Approximation and Projection clustering identified distinct expression signatures from the ganglion cell layer(GCL), inner nuclear layer(INL), retinal pigment epithelium (RPE)/choroid/sclera, optic nerve, and ciliary body (Fig, 1) but not the outer nuclear layer(ONL) which was contaminated with expression from other layers. Our findings highlight Clu, C4b, Apoe, and C1qa genes (z-score 3.0, 2.4, 2.3, and 2.2) as potential markers of disease in the RPE. Gene Set Enrichment analysis between rd6 and WT eyes showed upregulation of glycolysis and carbon metabolism pathways in the GCL and Rap1, Hippo and lysosome pathways in the RPE/Choroid/sclera. The ribosomal pathway was downregulated in these layers. No significant pathways were found in the INL, ciliary body or optic nerve.

As we skillfully navigate through familiar places, neural computations of distances and coordinates escape our attention. However, we perceive clearly the division of space into socially meaningful territories. 'My space' versus 'your space' is a distinction familiar to all of us. Spatial frontiers are social in nature since they regulate individuals' access to utilities in space depending on hierarchy and affiliation. How does the brain integrate spatial geometry with social territory? We propose that the action of oxytocin (OT) in the entorhinal-hippocampal regions supports this process. Grounded on the functional role of the hypothalamic neuropeptide in the hippocampal system, we show how OT-induced plasticity may bias the geometrical coding of place and grid cells to represent social territories.

In the last decades, cannabinoid receptor 2 (CB2R) has continued to receive attention as a key therapeutic target in neuroprotection. Indeed, several findings highlight the neuroprotective effects of CB2R through suppression of both neuronal excitability and reactive microglia. Additionally, CB2R seems to be a more promising target than cannabinoid receptor 1 (CB1R) thanks to the lack of central side effects, its lower expression levels in the central nervous system (CNS), and its inducibility, since its expression enhances quickly in the brain following pathological conditions. This review aims to provide a thorough overview of the main natural and synthetic selective CB2R modulators, their chemical classification and their potential therapeutic usefulness in neuroprotection, a crucial aspect for the treatment of neurodegenerative diseases.