Probes for INS

Many people with epilepsy remain drug-resistant, despite continuous efforts and advances in research and treatment. It is mandatory to understand the epilepsy's underlying etiology, whether it is structural, genetic, infectious, metabolic, immune or (currently) unknown, as it contains major information about the clinical phenotype, cognitive comorbidities, (new) drug targets and also help to predict postsurgical outcome. A multimodal approach, including digital slides and multichannel immunofluorescence labelling can increase the diagnostic yield of subtle pathologies, while DNA methylation arrays could helps in the diagnosis of difficult-to-classify lesions. Such techniques are not always available, however, in low-income countries. Even without access to expensive molecular techniques, automated analysis scripts and machine learning algorithms can be developed by Latin American researchers to improve our diagnostic yield from routine Hematoxylin & Eosin stained tissue sections. The pathology community of Latin America contributed substantially to our current knowledge of etiologies related to human epilepsies and experimental epilepsy models. To further boost the impact of Latin American research, local centers should adhere to modern, multimodal neuropathology techniques, integrate different levels of knowledge, and strengthen their scientific collaborations. Dedicated teaching courses in Epileptology, such as the Latin American Summer Schools of Epilepsy (LASSE) or International Summer School for Neuropathology and Epilepsy Surgery (INES) addressing young researcher and neurologists, are most successful to promote this endeavor. In this review, we will describe the state of neuropathology at the 21st century and also highlight Latin American researchers' contributions to the current knowledge in neuropathology of epilepsy.

Introduction: The role of eosinophils in intestinal inflammation and fibrosis in inflammatory bowel disease (IBD) is largely unknown. Aim: Therefore, we assessed the functional role of eosinophils in a chronic murine model of colitis and associated fibrosis via anti-CCR3 mediated eosinophil depletion. Methods: 6-8-week-old C57BL/6 RAG-/- mice received three cycles of dextran sodium sulphate (DSS) (1.75% - 2.25% - 2.25%) each interspersed with 14 days of recovery. Twice weekly, anti-CCR3 antibody (n=8), isotype (n=8) or saline injections (n=8) were given intraperitoneally. At the same timepoints, the disease activity index (DAI; mouse weight, stool consistency and presence of blood) was determined. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation). Colonic single cells were isolated and stained for flow cytometry, where eosinophils were characterized as CD45+ CD11b+ Siglec-F+ CD117- cells. Intestinal fibrosis was scored via colon weight/length, collagen deposition, using a colorimetric hydroxyproline assay and Martius Scarlet Blue staining (MSB), and COL1A1 expression by PCR.Results: Anti-CCR3 mediated eosinophil depletion resulted in decreased disease activity compared to the other DSS treated groups injected with saline or isotype, determined by the area under the curve of the DAI (74.6±18.4 vs. 127.5±42.9 and 136.9±33.6, p=0.01 and p=0.0008 respectively). The macroscopic damage score also suggested eosinophil depleted mice to be partially protected from colonic inflammation compared to the saline and isotype injected mice that received DSS (1.1±1.0 vs. 2.1±1.2 and 3.0±0.7, p=0.09 and p=0.001 respectively). Colon weight/length and hydroxyproline assay showed a trend towards increased fibrosis in the anti-CCR3 injected group compared to saline (p=0.03 and 0.07, respectively) but not isotype (p=0.3 and 0.1, respectively) injected groups. However, COL1A1 expression levels were significantly increased in the eosinophil depleted mice compared to the saline and isotype injected mice receiving DSS (43.2±11.4 vs. 23.3±8.7 and 30.1±11.0, p=0.002 and 0.04 respectively), indicating increased collagen expression. Moreover, MSB staining showed increased collagen deposition in the anti-CCR3 treated group compared to the isotype (p=0.0008), but not the saline (p=0.09) injected group exposed to DSS. Conclusions: Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection against colonic inflammation, but was associated with increased collagen expression and deposition. Caution is therefore needed when designing therapeutic interventions targeting eosinophils

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.

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.