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Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.

N-methyl-D-aspartate receptors (NMDARs emerging from GRIN genes) are tetrameric receptors that form diverse channel compositions in neurons, typically consisting of two GluN1 subunits combined with two GluN2(A-D) subunits. During prenatal stages, the predominant channels are di-heteromers with two GluN1 and two GluN2B subunits due to the high abundance of GluN2B subunits. Postnatally, the expression of GluN2A subunits increases, giving rise to additional subtypes, including GluN2A-containing di-heteromers and tri-heteromers with GluN1, GluN2A, and GluN2B subunits. The latter  emerge as the major receptor subtype at mature synapses in the hippocampus. Despite extensive research on purely di-heteromeric receptors containing two identical GRIN variants, the impact of a single variant on the function of other channel forms, notably tri-heteromers, is lagging. In this study, we systematically investigated the effects of two de novo GRIN2B variants (G689C and G689S) in pure, mixed di- and tri-heteromers. Our findings reveal that incorporating a single variant in mixed di-heteromers or tri-heteromers exerts a dominant negative effect on glutamate potency, although ‘mixed’ channels show improved potency compared to pure variant-containing di-heteromers. We show that a single variant within a receptor complex does not impair the response of all receptor subtypes to the positive allosteric modulator pregnenolone-sulfate (PS), whereas spermine completely fails to potentiate tri-heteromers containing GluN2A and -2B-subunits. We examined PS on primary cultured hippocampal neurons transfected with the variants, and observed a positive impact over current amplitudes and synaptic activity. Together, our study supports previous observations showing that mixed di-heteromers exhibit improved glutamate potency and extend these findings towards the exploration of the effect of Loss-of-Function variants over tri-heteromers. Notably, we provide an initial and crucial demonstration of the beneficial effects of GRIN2B -relevant potentiators on tri-heteromers. Our results underscore the significance of studying how different variants affect distinct receptor subtypes, as these effects cannot be inferred solely from observations made on pure di-heteromers. Overall, this study contributes to ongoing efforts to understand the pathophysiology of GRINopathies and provides insights into potential treatment strategies.

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

Oligo‐astheno‐teratozoospermia (OAT) is a common cause of male infertility, but the genetic basis of most OAT cases is still unknown. Here, one homozygous loss‐of‐function (LOF) variant in TDRD6, c.G1825T/p.Gly609X, was identified in an infertile patient with severe OAT by whole‐exome sequencing (WES) and Sanger confirmation. Furthermore, Tdrd6‐mutant mice (p.Gly615X; equivalent to p.Gly609X in human TDRD6) were generated. Remarkably, the Tdrd6‐mutated mice mimicked the severe OAT symptoms of the patient. In addition, the architecture of chromatoid bodies (CBs) were disrupted in round spermatids from Tdrd6‐mutant mice, leading to blocked spermatogenesis in the round spermatids. The assembly of PIWIL1, TDRD1, TDRD7 and DDX25 in CBs was disturbed in the Tdrd6‐mutant mice. Applying immunoprecipitation‐mass spectrometry (IP‐MS), we identified some TDRD6‐interacting partners, including CB proteins TDRD7, MAEL and PCBP1. Moreover, we described the assisted reproductive technology (ART) outcomes of the infertile patient and his partner. Altogether, our findings provide necessary evidences to support the idea that the homozygous LOF variant in TDRD6 induces male infertility with severe OAT, suggesting that TDRD6 could be a useful genetic diagnostic target for male infertility.

Purpose Mutations in signal transducer and activator of transcription 1 (STAT1) cause a broad spectrum of disease phenotypes. Heterozygous STAT1 loss-of-function (LOF) mutations cause Mendelian susceptibility to mycobacterial diseases (MSMD) infection, which is attributable to impaired IFN-γ signaling. The identification of novel mutations may extend the phenotypes associated with autosomal dominant (AD) STAT1 deficiency. Methods Five patients with heterozygous STAT1 variations were recruited and their clinical and immunologic phenotypes were analyzed, with particular reference to JAK-STAT1 signaling pathways. Results Four, heterozygous STAT1 deficiency mutations were identified, three of which were novel mutations. Two of the mutations were previously unreported mRNA splicing mutations in AD STAT1 -deficient patients. Patients with heterozygous STAT1 deficiency suffered not only mycobacterial infection, but also intracellular non-mycobacterial bacterial infection and congenital multiple malformations. AD-LOF mutation impaired IFN-γ-mediated STAT1 phosphorylation, gamma-activated sequence (GAS), and IFN-stimulated response element (ISRE) transcription activity and IFN-induced gene expression to different extents, which might account for the diverse clinical manifestations observed in these patients. Conclusion The infectious disease susceptibility and phenotypic spectrum of patients with AD STAT1 -LOF are broader than simply MSMD. The susceptibility to infections and immunological deficiency phenotypes, observed in AD-LOF patients, confirms the importance of STAT1 in host–pathogen interaction and immunity. However, variability in the nature and extent of these phenotypes suggests that functional analysis is required to identify accurately novel, heterozygous STAT1 mutations, associated with pathogenicity. Aberrant splice of STAT1 RNA could result in AD-LOF for STAT1 signaling which need more cases for confirmation.

Male infertility is a major reproductive disorder, which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality. To date, five male patients with biallelic loss-of-function (LOF) variants of PARN-like ribonuclease domain-containing exonuclease 1 (PNLDC1) have been reported to experience infertility with nonobstructive azoospermia. The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia (OAT) in a patient from a Chinese Han family. Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant (NM_173516.2, c.142C>T, p.Gln48Ter) in PNLDC1. Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype, including microcephaly, head tapering, and globozoospermia. Consistently, peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome. Furthermore, the disomy rate of chromosomes in the patient's spermatozoa was significantly increased compared with that of a fertile control sample. We reported an LOF variant of the PNLDC1 gene responsible for OAT.

The JAK/STAT signaling pathway plays a key role in cytokine signaling and is involved in development, immunity, and tumorigenesis for nearly any cell. At first glance, the JAK/STAT signaling pathway appears to be straightforward. However, on closer examination, the factors influencing the JAK/STAT signaling activity, such as cytokine diversity, receptor profile, overlapping JAK and STAT specificity among non-redundant functions of the JAK/STAT complexes, positive regulators (e.g., cooperating transcription factors), and negative regulators (e.g., SOCS, PIAS, PTP), demonstrate the complexity of the pathway’s architecture, which can be quickly disturbed by mutations. The JAK/STAT signaling pathway has been, and still is, subject of basic research and offers an enormous potential for the development of new methods of personalized medicine and thus the translation of basic molecular research into clinical practice beyond the use of JAK inhibitors. Gain-of-function and loss-of-function mutations in the three immunologically particularly relevant signal transducers STAT1, STAT3, and STAT6 as well as JAK1 and JAK3 present themselves through individual phenotypic clinical pictures. The established, traditional paradigm of loss-of-function mutations leading to immunodeficiency and gain-of-function mutation leading to autoimmunity breaks down and a more differentiated picture of disease patterns evolve. This review is intended to provide an overview of these specific syndromes from a clinical perspective and to summarize current findings on pathomechanism, symptoms, immunological features, and therapeutic options of STAT1, STAT3, STAT6, JAK1, and JAK3 loss-of-function and gain-of-function diseases.

The tumour suppressor factor p53 plays an essential role in regulating numerous cellular processes, including the cell cycle, DNA repair, apoptosis, autophagy, cell metabolism and immune response. TP53 is the most commonly mutated gene in human cancers. These mutations are primarily non-synonymous changes that produce mutant p53 proteins characterized by loss of function, a dominant negative effect on p53 tetramerisation and gain of function (GOF). GOF mutations not only disrupt the tumour-suppressive activities of p53 but also endow the mutant proteins with new oncogenic properties. Recent studies analysing different pathogenic features of mutant p53 in cancer-derived cell lines have demonstrated that restoring wild-type p53, rather than removing GOF mutations, reduces cancer cell growth. These findings suggest that therapeutic strategies for reactivating wild-type p53 function in cancer cells may bring a greater benefit than approaches halting mutant p53. This approach could involve the use of small molecules, gene therapy and other methods to re-establish wild-type p53 activity. This review describes the complexity of the biological activities of different p53 mutants and summarizes the current therapeutic approaches to restore p53 function.

Under dehydration in plants, antagonistic activities of histone 3 lysine 4 (H3K4) methyl-transferase and histone demethylase maintain a dynamic and homeostatic state of gene expression by orientating transcriptional reprogramming toward growth or stress tolerance. However, the histone demethylase that specifically controls histone methylation homeostasis under dehydration stress remains unknown. Here, we document that a histone demethylase, JMJ17, belonging to the KDM5/JARID1 family, plays crucial roles in response to dehydration stress and abscisic acid (ABA) in Arabidopsis thaliana. jmj17 loss-of-function mutants displayed dehydration stress tolerance and ABA hypersensitivity in terms of stomatal closure. JMJ17 specifically demethylated H3K4me1/2/3 via conserved iron-binding amino acids in vitro and in vivo. Moreover, H3K4 demethylase activity of JMJ17 was required for dehydration stress response. Systematic combination of genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) analyses revealed that a loss-of-function mutation in JMJ17 caused an ectopic increase in genome-wide H3K4me3 levels and activated a plethora of dehydration stress-responsive genes. Importantly, JMJ17 bound directly to the chromatin of OPEN STOMATA 1 (OST1) and demethylated H3K4me3 for the regulation of OST1 mRNA abundance, thereby modulating the dehydration stress response. Our results demonstrate a new function of a histone demethylase under dehydration stress in plants.

Key messageThe WRKY transcription factor WRKY12 negatively regulates Cd tolerance in Arabidopsis via the glutathione-dependent phytochelatin synthesis pathway by directly targeting GSH1 and indirectly repressing phytochelatin synthesis-related gene expression.Cadmium (Cd) is a widespread pollutant toxic to plants. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway plays key roles in Cd detoxification. However, its regulatory mechanism remains largely unknown. Here, we showed a previously unknown function of the WRKY transcription factor WRKY12 in the regulation of Cd tolerance by repressing the expression of PC synthesis-related genes. The expression of WRKY12 was inhibited by Cd stress. Enhanced Cd tolerance was observed in the WRKY12 loss-of-function mutants, whereas increased Cd sensitivity was found in the WRKY12-overexpressing plants. Overexpression and loss-of-function of WRKY12 were associated respectively with increased and decreased Cd accumulation by repressing or releasing the expression of the genes involved in the PC synthesis pathway. Transient expression assay showed that WRKY12 repressed the expression of GSH1, GSH2, PCS1, and PCS2. Further analysis indicated that WRKY12 could directly bind to the W-box of the promoter in GSH1 but not in GSH2, PCS1, and PCS2 in vivo. Together, our results suggest that WRKY12 directly targets GSH1 and indirectly represses PC synthesis-related gene expression to negatively regulate Cd accumulation and tolerance in Arabidopsis.