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Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single‐cell RNA‐sequencing (scRNA‐seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets – fibroblast progenitors and stem cells – indicative of an impairment of normal tissue self‐renewal. Tissue‐specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue‐remodeling. In degenerated AF, a novel disease‐associated subset is identified, which expresses disease‐promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFβ/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue‐specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment. Intervertebral (IVD) cells maintain tissue structure and biomechanical function while their depletion and dysfunction are key mechanisms in IVD degeneration and back pain. Using scRNA‐seq, cell type and compartment‐specific are resloved and shared mechanisms and their transcriptional regulators that are dysregulated in disease. These findings define novel genes and gene regulatory networks that enable more precise therapeutic targeting for the prevention and treatment of intervertebral disc degeneration.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival. Aberrant glycosylation patterns on cancer cells promote several pro-tumorigenic functions, including enhancing tumor cell proliferation. Here the authors provide data that show melanoma cells downregulate GCNT2 with consequent loss of I-branched glycans; this leads to the formation of extended i-linear glycans and enhances melanoma growth via increases, in part, by IGF-1- and extracellular matrix-induced signaling.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

ObjectivesSingle-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees.MethodsSingle-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy (n=6, n=7) and OA (n=6, n=6) knees. Expression of genes of interest was validated using immunohistochemistry and RNA-seq and function was analysed by gene overexpression and depletion.ResultsscRNA-seq analyses of human knee articular cartilage (70 972 cells) and meniscus (78 017 cells) identified a pathogenic subset that is shared between both tissues. This cell population is expanded in OA and has strong OA and senescence gene signatures. Further, this subset has critical roles in extracellular matrix (ECM) and tenascin signalling and is the dominant sender of signals to all other cartilage and meniscus clusters and a receiver of TGFβ signalling. Fibroblast activating protein (FAP) is also a dysregulated gene in this cluster and promotes ECM degradation. Regulons that are controlled by transcription factor ZEB1 are shared between the pathogenic subset in articular cartilage and meniscus. In meniscus and cartilage cells, FAP and ZEB1 promote expression of genes that contribute to OA pathogenesis, including senescence.ConclusionsThese single-cell studies identified a senescent pathogenic cell cluster that is present in cartilage and meniscus and has FAP and ZEB1 as main regulators which are novel and promising therapeutic targets for OA-associated pathways in both tissues.

Alzheimer's disease (AD) is the leading cause of dementia worldwide. Nevertheless, its cellular and molecular mechanisms remain incompletely understood, partially due to inadequate disease models. To illuminate early changes in AD, we developed a cerebrocortical organoid (CO) model with improved methodology. Our COs produce excitatory and inhibitory neurons alongside glia, utilizing established isogenic wild‐type and diseased human induced pluripotent stem cells (hiPSCs) carrying heterozygous familial AD mutations in PSEN1ΔE9/WT, PSEN1M146V/WT, or APPSwe/WT. In addition to amyloid‐beta (Aβ) accumulation, the AD COs display time‐progressive loss of monomeric Tau, and accumulation of aggregated high‐molecular‐weight (HMW) phospho(p)‐Tau species (pT181 and pT217). They also exhibit neuronal hyperexcitability reminiscent of early electroencephalography (EEG) clinical findings and synapse loss in AD patient brains. Single‐cell RNA‐sequencing analyses of AD and WT control COs reveal significant divergent molecular abnormalities in excitatory vs. inhibitory neurons, with several pathways being upregulated in one while downregulated in the other, providing insight into AD phenotypes. Finally, we show that chronic dosing with autophagy activators, including a novel mTOR inhibitor‐independent drug candidate, prevents pathologic Aβ and HMW p‐Tau accumulation, normalizes hyperexcitability, and rescues synaptic loss in AD COs. Collectively, our results demonstrate this CO model as a useful platform for assessing early features of familial AD pathogenesis and for testing small‐molecule candidate therapeutics. A new cerebrocortical organoid model using isogenic hiPSCs with familial Alzheimer's mutations recapitulates key AD features, including amyloid‐beta and phospho‐Tau aggregation, neuronal hyperexcitability, and synapse loss. Single‐cell RNA‐seq reveals aberrant pathways in excitatory and inhibitory neurons. Known and novel autophagy activators prevent pathological and synaptic deficits, supporting the model's utility for AD research and drug screening in the setting of early disease.

Despite significant improvements in life expectancy of kidney transplant patients due to advances in surgery and immunosuppression, Chronic Allograft Nephropathy (CAN) remains a daunting problem. A complex network of cellular mechanisms in both graft and peripheral immune compartments complicates the non-invasive diagnosis of CAN, which still requires biopsy histology. This is compounded by non-immunological factors contributing to graft injury. There is a pressing need to identify and validate minimally invasive biomarkers for CAN to serve as early predictors of graft loss and as metrics for managing long-term immunosuppression. We used DNA microarrays, tandem mass spectroscopy proteomics and bioinformatics to identify genomic and proteomic markers of mild and moderate/severe CAN in peripheral blood of two distinct cohorts (n = 77 total) of kidney transplant patients with biopsy-documented histology. Gene expression profiles reveal over 2400 genes for mild CAN, and over 700 for moderate/severe CAN. A consensus analysis reveals 393 (mild) and 63 (moderate/severe) final candidates as CAN markers with predictive accuracy of 80% (mild) and 92% (moderate/severe). Proteomic profiles show over 500 candidates each, for both stages of CAN including 302 proteins unique to mild and 509 unique to moderate/severe CAN. This study identifies several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAN, the most common cause of late allograft failure. These biomarkers are the necessary first step to a proteogenomic classification of CAN based on peripheral blood profiling and will be the targets of a prospective clinical validation study.

Background Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit. Results From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r 2 values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r 2 values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2. Conclusions Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.

Alzheimer's disease (AD) is the most common form of dementia worldwide. Despite extensive progress, the cellular and molecular mechanisms of AD remain incompletely understood, partially due to inadequate disease models. To illuminate the earliest changes in hereditary (familial) Alzheimer's disease, we developed an isogenic AD cerebrocortical organoid (CO) model. Our refined methodology produces COs containing excitatory and inhibitory neurons alongside glial cells, utilizing established isogenic wild-type and diseased human induced pluripotent stem cells (hiPSCs) carrying heterozygous familial AD mutations, namely PSEN1 , PSEN1 , or APP . Our CO model reveals time-progressive accumulation of amyloid beta (Aβ) species, loss of monomeric Tau, and accumulation of aggregated high-molecular-weight (HMW) phospho(p)-Tau species. This is accompanied by neuronal hyperexcitability, as observed in early human AD cases on electroencephalography (EEG), and synapse loss. Single-cell RNA-sequencing analyses reveal significant differences in molecular abnormalities in excitatory vs. inhibitory neurons, helping explain AD clinical phenotypes. Finally, we show that chronic dosing with autophagy activators, including a novel CNS-penetrant mTOR inhibitor-independent drug candidate, normalizes pathologic accumulation of Aβ and HMW p-Tau, normalizes hyperexcitability, and rescues synaptic loss in COs. Collectively, our results demonstrate these COs are a useful human AD model suitable for assessing early features of familial AD etiology and for testing drug candidates that ameliorate or prevent molecular AD phenotypes.

Chronic hepatitis C (CHC) disrupts host humoral immune response by impairing the timely generation of neutralizing antibody (nAb) and durable immune memory. However, the underlying mechanisms and their reversibility after viral clearance remain poorly defined. Here, through integrated single-cell transcriptomics and antibody repertoire characterization, we show that B cells from CHC patients retain transcriptional dysregulation even after successful antiviral therapy. Sustained TNF-α signaling emerged as a central driver of chronic B cell hyperactivation, persistent dysregulation and unresolved inflammation following cure. Furthermore, a CD86hi memory B cell subset, responsible for an IGHV1-69-encoded multi-donor class recall nAb response, declined rapidly following viral clearance, compromising immune memory against reinfection. Together, these findings reveal how CHC imprints lasting B cell dysregulation, impairs nAb memory, and sustains inflammation in the B cell compartment, after viral clearance. The insights underscore the need for strategies aimed at restoring B cell homeostasis to achieve durable immune protection.

B cells in CHC patients retained dysregulated transcriptional profiles despite successful DAA treatment B cell dysregulation is marked by global B cell hyperactivation and antigen-specific atypical MBC expansion Sustained upregulation of TNF-α signaling via NF-κB (TNF-α/NF-κB) is a central driver of persistent B cell dysregulation and chronic inflammation Viral clearance leads to restoration of IFN responses and IFN-stimulated gene (ISG) signatures in B cells but not TNF-α/NF-κB HCV IGHV1-69 bnAb-producing B cells are enriched within the CD86hi IgG MBC subset before treatment CD86hi IgG MBC subset contracts rapidly post-cure in parallel with the rapid decline of cross-nAb responses and loss of protective immune memory against HCV reinfection Chronic hepatitis C (CHC) disrupts host humoral immune response by impairing the timely generation of neutralizing antibody (nAb) and durable immune memory. However, the underlying mechanisms and their reversibility after viral clearance remain poorly defined. Here, through integrated single-cell transcriptomics and antibody repertoire characterization, we show that B cells from CHC patients retain transcriptional dysregulation even after successful antiviral therapy. Sustained TNF-α signaling emerged as a central driver of chronic B cell hyperactivation, persistent dysregulation and unresolved inflammation following cure. Furthermore, a CD86hi memory B cell subset, responsible for an IGHV1-69-encoded multi-donor class recall nAb response, declined rapidly following viral clearance, compromising immune memory against reinfection. Together, these findings reveal how CHC imprints lasting B cell dysregulation, impairs nAb memory, and sustains inflammation in the B cell compartment, after viral clearance. The insights underscore the need for strategies aimed at restoring B cell homeostasis to achieve durable immune protection.