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Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment.

Two drugs, miconazole and clobetasol, have functions that modulate differentiation of oligodendrocyte progenitor cells directly, enhance remyelination, and significantly reduce disease severity in mouse models of multiple sclerosis. Remyelination in multiple sclerosis Multiple sclerosis is characterized by an autoimmune response and failure of remyelination in the brain due to defects in differentiation of myelin-producing cells from oligodendrocyte progenitor cells. Most current treatments target the immune system. Paul Tesar and colleagues screened for compounds that can enhance oligodendrocyte maturation from mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitors. They found two drugs — miconazole (an antifungal) and clobetasol (a steroid) — that enhance myelin production in vivo in mouse models of multiple sclerosis and enhanced the differentiation of human oligodendrocytes progenitors in vitro . Mechanistically, these compounds appear to target both the immune response and oligodendrocyte progenitor cells. Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes 1 . These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention 2 . To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells 3 , 4 , 5 . Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro . Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro . Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Brain tumor initiating cells (BTICs) utilize high-affinity glucose uptake, which is normally active in neurons to maintain energy demands and self-renew. Leveraging metabolomic and genomic analyses, Wang et al . report that de novo purine biosynthesis reprograms BTIC metabolism, revealing a potential point of fragility amenable to targeted cancer therapy. Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

Microenvironmental pressures in glioblastoma select for glioma stem cells (GSCs) subpopulations that are maintained through preferential activation of BMI1 and EZH2 in different niches. Given the high degree of intratumor heterogeneity, combined pharmacological inhibition of Polycomb repressive complexes targets proneural and mesenchynmal GSCs and expands lifespan in mice, warranting the therapeutic evaluation of this approach in patients with glioblastoma. Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile, and hypoxic regions showing a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress due to downregulation of the E3 ligase RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are more sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo , suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be effective in overcoming therapy resistance caused by intratumoral heterogeneity.

An in vivo RNA interference screening strategy in glioblastoma enabled the identification of a host of epigenetic targets required for glioblastoma cell survival that were not identified by parallel standard screening in cell culture, including the transcription pause–release factor JMJD6, and could be a powerful tool to uncover new therapeutic targets in cancer. In vivo screening of brain tumour transcription factors Most high-throughput target discovery screens for glioblastoma have been limited to in vitro models with uncertain physiological relevance. Here, Jeremy Rich and colleagues perform two parallel RNA interference screens for transcriptional regulators, comparing an in vitro screen in cell lines to an in vivo screen that recapitulates the tumour microenvironment. They find several transcriptional elongation factors that are specifically required for glioblastoma cell survival in vivo , particularly the transcriptional pause release factor JMJD6 which is highly expressed in gliomas. This type of in vivo functional screen has the potential to uncover novel therapeutic targets for cancer that have not been identified in previous in vitro approaches. Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months 1 . Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology 2 , 3 , 4 for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro . We identified transcription pause–release and elongation factors as one set of in vivo -specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause–release, promoting cell survival specifically in vivo . Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3′ single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis. Clonal dynamics are inferred from mitochondrial variants in primary human cells.

The elastic properties of renal glomeruli and their capillaries permit them to maintain structural integrity in the presence of variable hemodynamic forces. Measured by micro-indentation, glomeruli have an elastic modulus (E, Young's modulus) of 2.1 kPa, and estimates from glomerular perfusion studies suggest that the E of glomeruli is between 2 and 4 kPa. F-actin depolymerization by latrunculin, inhibition of acto-myosin contractility by blebbistatin, reduction in ATP synthesis, and reduction of the affinity of adhesion proteins by EDTA reduced the glomerular E to 1.26, 1.7, 1.5, and 1.43 kPa, respectively. Actin filament stabilization with jasplakinolide and increasing integrin affinity with Mg2+ increased E to 2.65 and 2.87 kPa, respectively. Alterations in glomerular E are reflected in commensurate changes in F/G actin ratios. Disruption of vimentin intermediate filaments by withaferin A reduced E to 0.92 kPa. The E of decellularized glomeruli was 0.74 kPa, indicating that cellular components of glomeruli have dominant effects on their elasticity. The E of glomerular basement membranes measured by magnetic bead displacement was 2.4 kPa. Podocytes and mesangial cells grown on substrates with E values between 3 and 5 kPa had actin fibers and focal adhesions resembling those of podocytes in vivo. Renal ischemia and ischemia-reperfusion reduced the E of glomeruli to 1.58 kPa. These results show that the E of glomeruli is between 2 and 4 kPa. E of the GBM, 2.4 kPa, is consistent with this value, and is supported by the behavior of podocytes and mesangial cells grown on variable stiffness matrices. The podocyte cytoskeleton contributes the major component to the overall E of glomeruli, and a normal E requires ATP synthesis. The reduction in glomerular E following ischemia and in other diseases indicates that reduced glomerular E is a common feature of many forms of glomerular injury and indicative of an abnormal podocyte cytoskeleton.

In this trial involving patients with CKD, type 2 diabetes, and hypertension, the use of a personalized algorithm and practice facilitators in primary care clinics did not reduce hospitalization at 1 year.

In two semester-long studies, we examined whether college students could improve their ability to accurately predict their own exam performance across multiple exams. We tested whether providing concrete feedback and incentives (i.e., extra credit) for accuracy would improve predictions by improving students’ metacognition, or awareness of their own knowledge. Students’ predictions were almost always higher than the grade they earned and this was particularly true for low-performing students. Experiment 1 demonstrated that providing incentives but minimal feedback failed to show improvement in students’ metacognition or performance. However, Experiment 2 showed that when feedback was made more concrete, metacognition improved for low performing students although exam scores did not improve across exams, suggesting that feedback and incentives influenced metacognitive monitoring but not control.