Probes for INS

Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin (5-hydroxytryptamine, 5-HT) synthesis, but abundant for vesicular glutamate transporter 3 (Vglut3) encoding a synaptic-vesicle associated glutamate transporter. Genetic fate maps show these non-classical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment - hindbrain rhombomere 2 (r2) - as serotonergic TPH2+ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1Tph2-high versus r2-Pet1Vglut3-high Using male and female mice, we found r2-Pet1 axonal boutons segregated anatomically largely by serotonin+ versus VGLUT3+ identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2-Pet1Tph2-high and r2-Pet1Vglut3-high neurons likely regulate distinct brain regions and behaviors. Some r2-Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3+ or VGLUT3+/5-HT+ identity; this suggests that some r2-Pet1Vglut3-high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations.Significance statementOur findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1+ precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2-Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2-Pet1 boutons-those that are VGLUT3+ or VGLUT3+/5-HT+ co-positive-comprise "baskets" encasing interneurons, suggesting they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.

We describe a 53-year-old man with HIV-1 who received allogeneic CCR5Δ32/Δ32 hematopoietic stem cell transplantation (HSCT) in 2013 to treat acute myeloid leukemia. Four years after analytic treatment interruption (ATI), the absence of viral rebound and the lack of immunological correlates of HIV-1 antigen persistence provide convincing evidence for HIV-1 cure.

Cytokines are regulators of the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the contribution of cytokine-secreting CD4+ and CD8+ memory T cells to the SARS-CoV-2-specific humoral immune response in immunocompromised kidney patients is unknown. Here, we profiled 12 cytokines after stimulation of whole blood obtained 28 days post second 100 μg mRNA-1273 vaccination with peptides covering the SARS-CoV-2 spike (S)-protein from patients with chronic kidney disease (CKD) stage 4/5, on dialysis, kidney transplant recipients (KTR), and healthy controls. Unsupervised hierarchical clustering analysis revealed two distinct vaccine-induced cytokine profiles. The first profile was characterized by high levels of T-helper (Th)1 (IL-2, TNF-α, and IFN-γ) and Th2 (IL-4, IL-5, IL-13) cytokines, and low levels of Th17 (IL-17A, IL-22) and Th9 (IL-9) cytokines. This cluster was dominated by patients with CKD, on dialysis, and healthy controls. In contrast, the second cytokine profile contained predominantly KTRs producing mainly Th1 cytokines upon re-stimulation, with lower levels or absence of Th2, Th17, and Th9 cytokines. Multivariate analyses indicated that a balanced memory T cell response with the production of Th1 and Th2 cytokines was associated with high levels of S1-specific binding and neutralizing antibodies mainly at 6 months after second vaccination. In conclusion, seroconversion is associated with the balanced production of cytokines by memory T cells. This emphasizes the importance of measuring multiple T cell cytokines to understand their influence on seroconversion and potentially gain more information about the protection induced by vaccine-induced memory T cells.

The mechanisms underlying liver fibrosis are multifaceted and remain elusive with no approved antifibrotic treatments available. The adult zebrafish has been an underutilized tool to study liver fibrosis. We aimed to characterize the single-cell transcriptome of the adult zebrafish liver to determine its utility as a model for studying liver fibrosis. We used single-cell RNA sequencing (scRNA-seq) of adult zebrafish liver to study the molecular and cellular dynamics at a single-cell level. We performed a comparative analysis to scRNA-seq of human liver with a focus on hepatic stellate cells (HSCs), the driver cells in liver fibrosis. scRNA-seq reveals transcriptionally unique populations of hepatic cell types that comprise the zebrafish liver. Joint clustering with human liver scRNA-seq data demonstrates high conservation of transcriptional profiles and human marker genes in zebrafish. Human and zebrafish HSCs show conservation of transcriptional profiles, and we uncover collectin subfamily member 11 (colec11) as a novel, conserved marker for zebrafish HSCs. To demonstrate the power of scRNA-seq to study liver fibrosis using zebrafish, we performed scRNA-seq on our zebrafish model of a pediatric liver disease with mutation in mannose phosphate isomerase (MPI) and characteristic early liver fibrosis. We found fibrosis signaling pathways and upstream regulators conserved across MPI-depleted zebrafish and human HSCs. CellPhoneDB analysis of zebrafish transcriptome identified neuropilin 1 as a potential driver of liver fibrosis. Conclusion: This study establishes the first scRNA-seq atlas of the adult zebrafish liver, highlights the high degree of similarity to human liver, and strengthens its value as a model to study liver fibrosis.

Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous cancer type with limited treatment options. Identifying drivers of tumor heterogeneity may lead to better therapeutic options and favorable patient outcomes. Here, we aimed to investigate whether apoptotic cell death and its spatial architecture is linked to tumor molecular heterogeneity using single-cell in situ hybridization analysis.We analyzed 254 tumor samples from two HCC cohorts using tissue microarrays. We developed a mathematical model to quantify cellular diversity among HCC samples using two tumor markers, CDKN3 and PRC1 as surrogates for heterogeneity and CASP3 as an apoptotic cell death marker. We further explored the impact of potential dying-cell hubs on tumor cell diversity and patient outcome by density contour mapping and spatial proximity analysis. We also developed a selectively controlled in vitro model of cell death using CRISPR/Cas9 to determine therapy response and growth under hypoxic conditions. We found that increasing levels of CASP3+ tumor cells are associated with higher tumor diversity. Interestingly, we discovered regions of densely populated CASP3+ , that we refer to as CASP3+ cell islands, in which the nearby cellular heterogeneity was found to be the greatest compared to cells further away from these islands and that this phenomenon was associated with survival. Additionally, cell culture experiments revealed higher levels of cell death, accompanied by increased CASP3 expression, led to greater therapy resistance and growth under hypoxia.These results are consistent with the hypothesis that increased apoptotic cell death may lead to greater tumor heterogeneity and thus worse patient outcomes.This article is protected by

Skeletal muscle repair is driven by the coordinated self-renewal and fusion of myogenic stem and progenitor cells. Single-cell gene expression analyses of myogenesis have been hampered by the poor sampling of rare and transient cell states that are critical for muscle repair, and do not inform the spatial context that is important for myogenic differentiation. Here, we demonstrate how large-scale integration of single-cell and spatial transcriptomic data can overcome these limitations. We created a single-cell transcriptomic dataset of mouse skeletal muscle by integration, consensus annotation, and analysis of 23 newly collected scRNAseq datasets and 88 publicly available single-cell (scRNAseq) and single-nucleus (snRNAseq) RNA-sequencing datasets. The resulting dataset includes more than 365,000 cells and spans a wide range of ages, injury, and repair conditions. Together, these data enabled identification of the predominant cell types in skeletal muscle, and resolved cell subtypes, including endothelial subtypes distinguished by vessel-type of origin, fibro-adipogenic progenitors defined by functional roles, and many distinct immune populations. The representation of different experimental conditions and the depth of transcriptome coverage enabled robust profiling of sparsely expressed genes. We built a densely sampled transcriptomic model of myogenesis, from stem cell quiescence to myofiber maturation, and identified rare, transitional states of progenitor commitment and fusion that are poorly represented in individual datasets. We performed spatial RNA sequencing of mouse muscle at three time points after injury and used the integrated dataset as a reference to achieve a high-resolution, local deconvolution of cell subtypes. We also used the integrated dataset to explore ligand-receptor co-expression patterns and identify dynamic cell-cell interactions in muscle injury response. We provide a public web tool to enable interactive exploration and visualization of the data. Our work supports the utility of large-scale integration of single-cell transcriptomic data as a tool for biological discovery.

Squamous cell carcinoma metastasis of the head and neck with unknown primary tumor (CUP) comprises a diagnostic challenge. Human papillomavirus (HPV) testing on cytologic specimens is gaining increasing focus as this may facilitate an early diagnosis of HPV-induced oropharyngeal carcinoma. This study aimed to prospectively assess PCR-based HPV-DNA testing on FNA smears in a clinical setting.Patients referred to a tertiary Head and Neck Cancer Center with suspected CUP were included from November 2016 to November 2018. Scraped cell material from FNA smears was analyzed for HPV-DNA with PCR using general primers (GP5 + /GP6 +) and correlated with the origin and histology of the primary tumor (oropharynx vs. outside oropharynx or benign tumor). The turn-around time reflecting the workflow for HPV-DNA testing by PCR was also calculated.A total of 93 patients were enrolled in the study. The sensitivity and specificity were 86.7% [95% CI 75.4-94.1%] and 92.0% [95% CI 74.0-99.0%], and the positive and negative predictive values were 96.3% [95% CI 87.3-99.0%] and 74.2% [95% CI 59.9-84.7%], respectively. The turn-around time for HPV testing was a mean four calendar days.HPV-DNA testing on FNA smears can be performed within a reasonable timeframe and can guide for the detection of an HPV-positive oropharyngeal primary tumor in the clinical setting for patients presenting with CUP of the head and neck.

Lymph node metastasis (LNM) is an important reference indicator for the prognosis of endometrial cancer (EC). Even in patients with early low-risk EC, many people still have LNM. The purpose of this study was to investigate the related factors influencing LNM in early-stage EC and determine the optimal positive threshold of immunohistochemical parameter Ki67 for predicting LNM, providing auxiliary reference indicators for clinical diagnosis and treatment.The clinicopathological data of 651 patients with "apparent" early-stage EC who underwent standard surgical treatment were included. Univariate and multivariate logistics regression were used to analyze the correlation between each clinicopathological factor and LNM. Receiver operating characteristic curve (ROC curve) and Youden index were used to determine the optimal positive threshold of Ki67 for predicting LNM. Finally, correlation between Ki67 and various clinicopathological factors was analyzed, and the predictive value of each prognostic factor was compared.Multivariate analysis found that histologic grade (P=0.023), lymphatic vessel space invasion (LVSI) (P < 0.001), serological index Ca125 (P=0.002), immunohistochemical parameter Ki67 (P < 0.001), ER (P < 0.001) and P53 (P=0.001) were independent prognostic factors of LNM. ROC curve and Youden index showed that the optimal positive thresholds of Ki67 to predict LNM were 40%. Based on this, ROC curve showed that the area under the curve (AUC) of Ki67 (AUC=0.714) was larger than other single predictors, and Ki67 combined with other predictors can significantly increase the AUC value (AUC= 0.847 and 0.868, respectively).Ki67 was an important predictor for predicting the LNM in early-stage EC and taking a positive percentage of about 40% can be used as the positive threshold of the immunohistochemical parameter Ki67. On this basis, Ki67 combined with other predictive indicators can significantly improve prediction performance and can be used for segmentally predicting LNM of early-stage EC.

The human immunodeficiency virus (HIV) enters the central nervous system (CNS) within a few days after primary infection, establishing viral reservoirs that persist even with combined antiretroviral therapy (cART). We show that monocytes from people living with HIV (PLWH) on suppressive cART harboring integrated HIV, viral mRNA, and/or viral proteins preferentially transmigrate across the blood-brain barrier (BBB) to CCL2 and are significantly enriched post-transmigration, and even more highly enriched posttransmigration than T cells with similar properties. Using HIV-infected ART-treated mature monocytes cultured in vitro, we recapitulate these findings and demonstrate that HIV+ CD14+ CD16+ ART-treated monocytes also preferentially transmigrate. Cenicriviroc and anti-JAM-A and anti-ALCAM antibodies significantly and preferentially reduce/block transmigration of HIV+ CD14+ CD16+ ART-treated monocytes. These findings highlight the importance of monocytes in CNS HIV reservoirs and suggest targets to eliminate their formation and reseeding.IMPORTANCE We characterized mechanisms of CNS viral reservoir establishment/replenishment using peripheral blood mononuclear cells (PBMC) of PLWH on cART and propose therapeutic targets to reduce/block selective entry of cells harboring HIV (HIV+) into the CNS. Using DNA/RNAscope, we show that CD14+ CD16+ monocytes with integrated HIV, transcriptionally active, and/or with active viral replication from PBMC of PLWH prescribed cART and virally suppressed, selectively transmigrate across a human BBB model. This is the first study to our knowledge demonstrating that monocytes from PLWH with HIV disease for approximately 22 years and with long-term documented suppression can still carry virus into the CNS that has potential to be reactivated and infectious. This selective entry into the CNS-and likely other tissues-indicates a mechanism of reservoir formation/reseeding in the cART era. Using blocking studies, we propose CCR2, JAM-A, and ALCAM as targets on HIV+ CD14+ CD16+ monocytes to reduce and/or prevent CNS reservoir replenishment and to treat HAND and other HIV-associated comorbidities.

Store-operated calcium entry (SOCE) is critical for T cell-mediated immunity, specifically, for T cell activation, clonal expansion, and differentiation. In T cells, SOCE is facilitated by the plasma membrane (PM) localized protein Orai1 and the endoplasmic reticulum (ER) membrane localized protein STIM1. Upon T cell receptor activation, ER calcium stores are depleted, leading to STIM1 activation, which undergoes a conformational change, allowing it to interact with Orai1 at ER/PM junctions. This interaction enables Orai1 gating, leading to calcium entry into the cell. Although the importance of Orai1 and STIM1 in SOCE has been established, it is still unclear what mechanisms regulate the formation of Orai1/STIM1 complexes at ER/PM junctions. We have found that, among several other regulatory T cell proteins, both Orai1 and STIM1 are S-acylated. S-acylation is the post-translational lipidation of a cysteine residue via a labile thioester bond. Using biochemical, electrophysiological, and advanced imaging approaches, we have found that S-acylation of Orai1 and STIM1 is critical for their function. Using acylbiotin exchange, we determined not only are Orai1 and STIM1 S-acylated, but also the S-acylation of Orai1 is rapid, transient, and calcium-dependent. To further study the importance of S-acylation on SOCE, we used whole cell recording to show that the S-acylation of both Orai1 and STIM1 is important for proper calcium channel currents. Lastly, after performing total internal reflection fluorescence, Fo¨ rster resonance energy transfer, and Fura-2 calcium imaging, we determined acylation-deficient Orai1 significantly decreases SOCE and does not efficiently interact with STIM1 to form functional calcium channels. This work describes a previously unknown signaling pathway that regulates SOCE in T cells, and contributes to T cell-mediated immunity.

Ovulation is an integral part of the women's menstrual cycle and fertility. Understanding the mechanisms of ovulation has broad implications for the treatment of anovulatory diseases and development of novel contraceptives. Now, few studies have developed effective models that both faithfully recapitulate the hallmarks of ovulation and possess scalability. We established a 3D encapsulated in vitro follicle growth (eIVFG) system that recapitulates folliculogenesis and produces follicles that undergo ovulation in a controlled manner. Here, we determined whether ex vivo ovulation preserves molecular signatures of ovulation and demonstrated its use in discovering novel ovulatory pathways and non-hormonal contraceptive candidates through a high-throughput ovulation screening. Mature murine follicles from eIVFG were induced to ovulate ex vivo using human chorionic gonadotropin and collected at 0, 1, 4, and 8 hours post-induction. Phenotypic analyses confirmed key ovulatory events, including cumulus expansion, oocyte maturation, follicle rupture, and luteinization. Single-follicle RNA-sequencing analysis revealed the preservation of ovulatory genes and dynamic transcriptomic profiles and signaling. Soft clustering identified distinct gene expression patterns and new pathways that may critically regulate ovulation. We further used this ex vivo ovulation system to screen 21 compounds targeting established and newly-identified ovulatory pathways. We discovered that proprotein convertases activate gelatinases to sustain follicle rupture and do not regulate luteinization and progesterone secretion. Together, our ex vivo ovulation system preserves molecular signatures of ovulation, presenting a new powerful tool for studying ovulation and anovulatory diseases as well as for establishing a high-throughput ovulation screening to identify novel non-hormonal contraceptives for women.

BACKGROUND: Pediatric craniopharyngioma is associated with long-term survival, but tumor- and therapy-related complications often negatively impact quality of life (QoL). Standard treatments include resection and radiation, but institutional practices vary and recurrence rates remain high. In this review, we utilized a cohort from the Children’s Brain Tumor Network (CBTN) to evaluate outcomes for craniopharyngioma. METHODS: CBTN provides clinical and genomic data for pediatric patients diagnosed with primary central nervous system tumors across 25+ institutions. We collected data for 124 patients, ages 0-21, diagnosed with craniopharyngioma between 2012-2020. Variables collected included treatment, recurrence/progression, and comorbidities. RESULTS: Excluding patients without confirmed pathologic diagnosis (n=10) or follow-up data (n=39), 75 patients remained. For initial treatment, most (n=46, 61%) received surgery alone (9 partial, 33 near-total resection). Twenty-six (35%) underwent both surgery and radiation, with 9 receiving both therapies upfront and 17 receiving radiation at progression/recurrence. Four (5%) patients received chemotherapy. Over half of the cohort (n=39, 52%) had at least one progression/recurrence, and four died (5%). Significantly higher rates of progression/recurrence (84% vs. 32%, p=4.0e-5) were identified in patients that had surgery and radiation, compared to surgery alone. Time to recurrence, progression, or death was shorter for the surgery and radiation group (HR=4.1, p&lt;1.0e-4), and for those that underwent partial versus near-total resection (HR=2.7, p=0.1.2e-2). Comorbidities were likely underreported, based on low rates of visual (32%), neuroendocrine (27%), and neurologic (28%) deficits at diagnosis, and 29 patients (39%) with unspecified medical history. CONCLUSIONS: CBTN provides a robust repository of information on treatment and survival of craniopharyngioma patients. However, we found a paucity of data on associated comorbidities and QoL outcomes. We advocate that future datasets and clinical trials routinely collect functional outcomes alongside therapy and survival data, particularly in craniopharyngioma where long-term survival is balanced with future QoL.