Probes for INS

tRF-28-QSZ34KRQ590K demonstrated a significant diagnostic value for AD in the ROC curve analysis. There were 28 target genes of tRF-28-QSZ34KRQ590K, including tachykinin receptor 1, RANBP2-like and GRIP domain-containing 1, non-SMC condensin I complex subunit H (NCAPH), ectodysplasin-A receptor, contactin-associated protein family member 5, cytochrome P450 family (CYP)27 subfamily C member 1, WD repeat domain 33, POTE ankyrin domain family member F, insulin receptor substrate 1, thyroid hormone receptor interactor 12, S-antigen visual arrestin, ATPase plasma membrane Ca2+ transporting 2, neurexophilin and PC-esterase domain family member 3, Rho GTPase activating protein 31, p21 (RAC1)-activated kinase 2, midnolin, unc-13 homolog A, potassium channel tetramerization domain containing 15, endogenous retrovirus group V member 1 envelope, zinc finger and SCAN domain containing 18, KIAA0930 (also known as C22orf9, LSC3), SET binding factor 1, forkhead box K1, AVL9 cell migration-associated, FKBP prolyl isomerase family member 6, X-ray repair cross complementing 2, phosphatase and actin regulator 1 and mitochondrial calcium uniporter regulator 1. A previous study reported on a pediatric patient with AD, mental retardation, autistic features, epilepsy, developmental delay and abnormal immunological results, who carried a 7.9 Mb de novo deletion of chromosome 22q13.2/qter, a region containing the NCAPH2, SH3 and multiple ankyrin repeat domains 3 and CYP2D6 genes (26). These genes are associated with the T-cell immune response and the inflammatory response has also been suggested to have roles in the pathogenesis of AD. Thus, tRF-28-QSZ34KRQ590K may be involved in the pathogenesis of AD by affecting the inflammatory response. Further studies are required to uncover the role of tRF-28-QSZ34KRQ590K in the pathogenesis of AD. At present, it remains undetermined whether tRF-28-QSZ34KRQ590K is specifically expressed in AD, although it has not been reported to be expressed in any other diseases, to the best of our knowledge. However, tRF-28-QSZ34KRQ590K exhibited a significant diagnostic value in the ROC curve analysis, which suggested that tRF-28-QSZ34KRQ590K may be a potential biomarker for pediatric patients with AD, although further studies are required to confirm the role of this tRF in AD.

One of the most striking aspects of the sensory epithelium of the mammalian cochlea, the organ of Corti, is the presence of precise boundaries between sensory and non-sensory cells at its medial and lateral edges. A particular example of this precision is the single row of inner hair cells and associated supporting cells along the medial (neural) boundary. Despite the regularity of this boundary, the developmental processes and genetic factors that contribute to its specification are poorly understood. In this study we demonstrate that Leucine Rich Repeat Neuronal 1 (Lrrn1), which codes for a single-pass, transmembrane protein, is expressed prior to the development of the mouse organ of Corti in the row of cells that will form its medial border. Deletion of Lrrn1 in mice of mixed sex leads to disruptions in boundary formation that manifest as ectopic inner hair cells and supporting cells. Genetic and pharmacological manipulations demonstrate that Lrrn1 interacts with the Notch signaling pathway and strongly suggest that Lrrn1 normally acts to enhance Notch signaling across the medial boundary. This interaction is required to promote formation of the row of inner hair cells and suppress the conversion of adjacent non-sensory cells into hair cells and supporting cells. These results identify Lrrn1 as an important regulator of boundary formation and cellular patterning during development of the organ of Corti.SIGNIFICANCE STATEMENT:Patterning of the developing mammalian cochlea into distinct sensory and non-sensory regions and the specification of multiple different cell fates within those regions are critical for proper auditory function. Here, we report that the transmembrane protein LRRN1 is expressed along the sharp medial boundary between the single row of mechanosensory inner hair cells and adjacent non-sensory cells. Formation of this boundary is mediated in part by Notch signaling, and loss of Lrrn1 leads to disruptions in boundary formation similar to those caused by a reduction in Notch activity, suggesting that LRRN1 likely acts to enhance Notch signaling. Greater understanding of sensory/non-sensory cell fate decisions in the cochlea will help inform the development of regenerative strategies aimed at restoring auditory function.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion - critical processes underlying neuronal health and function, and in turn memory and cognition.

Study question To elucidate whether AZF screening is indicated in men with proven non-obstructive azoospermia (NOA) or severe oligozoospermia (<5x106 sperm/mL) who concomitantly harbor chromosomal abnormalities. Summary answer Some chromosomal aberrations are concomitant with AZF microdeletions, and hence Y chromosome microdeletion (YCM) screening is necessary for these patients. What is known already In the era of assisted reproduction, finding cost-minimization strategies in infertility clinics without affecting the quality of diagnosis and treatment is becoming one of the top-priority topics for future research. Formulating definitive guidelines concerning the indications for paraclinical testing could be one of the strategies. Accordingly, definitive guidelines for AZF screening do not exist especially in men who also have chromosomal abnormalities. The current clinical practice is to decide per case whether to pursue further molecular testing, with the accompanying added psychological and socio-economic burden. Study design, size, duration An observational retrospective cohort study of 10,388 consecutive cases from a tertiary infertility referral center (Royan institute, Tehran, Iran) over the last 12 years. A comprehensive literature review was also performed to summarize scientific evidence. Participants/materials, setting, methods The study recruited the largest cohort of men with primary infertility (NOA or severe oligozoospermia) ever presented who underwent screening for cytogenetic abnormalities and YCMs using sequence-tagged sites-polymerase chain reaction (STS-PCR) with a primer set covering the AZF regions according to the EAA/EMQN guidelines, as part of the infertility workup prior to ART. Main results and the role of chance In total, one-third of men with YCMs concomitantly had an abnormal karyotype (excluding those with heteromorphisms) (169/565; 29.9%, 95% CI: 26.3-33.8). In respect to the frequency of YCMs, our findings suggest that the cases diagnosed with 46,X,add(X) with incidence of 1.8% (3/169; 95% CI: 0.6-5.1); 45,X and mosaic forms: 45,X/46,X,inv(Y)(p11.2q12); 45,X/46,X,del(Y); 45,X,der(Y;Autosome); 45,X/46,X,idic(Y)(p11.2); 45,X/46,XY,r(Y); and 45,X/46,X,idic(Y)(q11.2) (19/169; 11.2%, 95% CI: 7.3-16.9); and inv(Y)(p11.2q12) (2/169; 1.2%, 95% CI: 0.3-4.2) should also be referred for AZF deletion screening, as data suggests they are accompanied with YCMs. Limitations, reasons for caution The extension of the outcomes beyond the described population could introduce concerns on appropriate medical management. Confirmatory studies in other geographic/ethnic groups are still necessary to reach a consensus regarding the outcomes. Wider implications of the findings It has been recommended that all men with NOA who have chromosomal abnormalities, except those with 46,XY/45,X karyotype, do not need AZF testing. The results reflect a crucial need for reconsidering whether YCM testing is indicated in the population of men with certain karyotypic abnormalities. Trial registration number Not Applicable

Purpose : The formation of retinal ganglion cells (RGCs) is a stepwise process subject to tight genetic control. Our purpose in this study is to use single cell ATAC-seq (scATAC-seq) to investigate how the epigenetic landscape changes along the RGC developmental trajectory. Methods : We used two knock-in mouse lines, Atoh7-zaGreen and Pou4f2-tdTomato, to enrich different cell populations by FACS. scATAC-seq and scRNA-seq libraries were then generated using the 10X Chromium platform and sequenced on an Illumina sequencer. The sequence data were then analyzed using Cell Ranger, Seurat, and archR. We also used CRISPR and RNAscope in situ hybridization to investigate the function of one candidate enhancer of Pou4f2, a key gene for the RGC lineage. Results : We first performed UMAP clustering and gene score calculation of the scATAC-seq data and were able to cluster the cells into the same groups as achieved by scRNA-seq. We next identified enriched accessible peaks in individual clusters, which represented potential enhancers for each developmental stage/cell types. We further performed DNA motif enrichment and footprinting analysis which revealed stage/cell type-specific motif binding by key transcription factors. Further, we performed peak-to-gene linkage analysis by integrating scATAC-seq data with the scRNA-seq data and assigned the stage/cell type-specific enhancers to the genes they likely regulate, thus generating a global enhancer-gene linkage map for the developing retina at E14.5 and E17.5. We also compared the wild-type and Atoh7-null scATAC-seq data and identified peaks (enhancers) that were dependent on Atoh7. Finally, as a pilot validation, we used CRISPR to delete one of the candidate enhancers of Pou4f2, a key gene involved in RGC genesis, and confirmed by RNAscope in situ hybridization that the enhancer indeed played a critical role in the expression of Pou4f2. Conclusions : Using scATAC-seq, we identified stage/cell-specific enhancers in the developing retina, assigned them to genes they likely regulate, and identified DNA-motifs in them likely bound by key transcription factors . Our results shed significant new light on the shifting epigenetic landscape along the developmental trajectory of the RGC lineage.

Distinguishing different contexts is thought to involve a form of pattern separation that minimizes overlap between neural ensembles representing similar experiences. Theoretical models suggest that the dentate gyrus (DG) segregates cortical input patterns before relaying its discriminated output patterns to the CA3 hippocampal field. This suggests that the evaluation of neural ensembles in DG and CA3 could be an important means to investigate the process of pattern separation. In the past, measurement of entorhinal cortex (EC), DG, and CA3 ensembles was largely dependent upon in vivo electrophysiological recording, which is technically difficult. This protocol provides a method to instead measure pattern separation by a molecular method that provides direct spatial resolution at the cellular level.

Highly multiplexed protein and RNA in situ detection on a single tissue section concurrently is highly desirable for both basic and applied biomedical research. CO-detection by inDEXing (CODEX) is a new and powerful platform to visualize up to 60 protein biomarkers in situ, and RNAscope in situ hybridization (RNAscope) is a novel RNA detection system with high sensitivity and unprecedent specificity at a single-cell level. Nevertheless, to our knowledge, the combination of CODEX and RNAscope remained unreported until this study. Here, we report a simple and reproducible combination of CODEX and RNAscope. We also determined the cross-reactivities of CODEX anti-human antibodies to rhesus macaques, a widely used animal model of human disease.

Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Neurons, astrocytes and oligodendrocytes locally regulate protein translation within distal processes. Here, we tested whether there is regulated local translation within peripheral microglial processes (PeMPs) from mouse brain. We show that PeMPs contain ribosomes that engage in de novo protein synthesis, and these are associated with transcripts involved in pathogen defense, motility and phagocytosis. Using a live slice preparation, we further show that acute translation blockade impairs the formation of PeMP phagocytic cups, the localization of lysosomal proteins within them, and phagocytosis of apoptotic cells and pathogen-like particles. Finally, PeMPs severed from their somata exhibit and require de novo local protein synthesis to effectively surround pathogen-like particles. Collectively, these data argue for regulated local translation in PeMPs and indicate a need for new translation to support dynamic microglial functions.

Fluorescence in situ hybridization (FISH) is a useful tool for analyzing RNA expression, but difficulties arise with low-abundance RNA and in tissues that are formalin-fixed paraffin-embedded (FFPE) because reagents can be expensive. In this protocol, we adapt a previously designed FISH amplification protocol (SABER [signal amplification by exchange reaction]) for adult mouse FFPE lung sections by using probes that are extended and branched to amplify the signal. We combine FISH and immunostaining to identify cell-specific RNA. For complete details on the use and execution of this protocol, please refer to Kishi et al.1 and Lyu et al.2.

Multicellular organisms typically develop from a single fertilized egg and therefore consist of clonal cells. We report an extraordinary reproductive system in the yellow crazy ant. Males are chimeras of haploid cells from two divergent lineages: R and W. R cells are overrepresented in the males' somatic tissues, whereas W cells are overrepresented in their sperm. Chimerism occurs when parental nuclei bypass syngamy and divide separately within the same egg. When syngamy takes place, the diploid offspring either develops into a queen when the oocyte is fertilized by an R sperm or into a worker when fertilized by a W sperm. This study reveals a mode of reproduction that may be associated with a conflict between lineages to preferentially enter the germ line.