Orchestrating information across tissues via a novel multitask GAT framework to improve quantitative gene regulation relation modeling for survival analysis.

Survival analysis is critical to cancer prognosis estimation. High-throughput technologies facilitate the increase in the dimension of genic features, but the number of clinical samples in cohorts is relatively small due to various reasons, including difficulties in participant recruitment and high data-generation costs. Transcriptome is one of the most abundantly available OMIC (referring to the high-throughput data, including genomic, transcriptomic, proteomic and epigenomic) data types. This study introduced a multitask graph attention network (GAT) framework DQSurv for the survival analysis task. We first used a large dataset of healthy tissue samples to pretrain the GAT-based HealthModel for the quantitative measurement of the gene regulatory relations. The multitask survival analysis framework DQSurv used the idea of transfer learning to initiate the GAT model with the pretrained HealthModel and further fine-tuned this model using two tasks i.e. the main task of survival analysis and the auxiliary task of gene expression prediction. This refined GAT was denoted as DiseaseModel. We fused the original transcriptomic features with the difference vector between the latent features encoded by the HealthModel and DiseaseModel for the final task of survival analysis. The proposed DQSurv model stably outperformed the existing models for the survival analysis of 10 benchmark cancer types and an independent dataset. The ablation study also supported the necessity of the main modules. We released the codes and the pretrained HealthModel to facilitate the feature encodings and survival analysis of transcriptome-based future studies, especially on small datasets. The model and the code are available at http://www.healthinformaticslab.org/supp/.

Challenges of COVID-19 Case Forecasting in the US, 2020-2021.

During the COVID-19 pandemic, forecasting COVID-19 trends to support planning and response was a priority for scientists and decision makers alike. In the United States, COVID-19 forecasting was coordinated by a large group of universities, companies, and government entities led by the Centers for Disease Control and Prevention and the US COVID-19 Forecast Hub (https://covid19forecasthub.org). We evaluated approximately 9.7 million forecasts of weekly state-level COVID-19 cases for predictions 1-4 weeks into the future submitted by 24 teams from August 2020 to December 2021. We assessed coverage of central prediction intervals and weighted interval scores (WIS), adjusting for missing forecasts relative to a baseline forecast, and used a Gaussian generalized estimating equation (GEE) model to evaluate differences in skill across epidemic phases that were defined by the effective reproduction number. Overall, we found high variation in skill across individual models, with ensemble-based forecasts outperforming other approaches. Forecast skill relative to the baseline was generally higher for larger jurisdictions (e.g., states compared to counties). Over time, forecasts generally performed worst in periods of rapid changes in reported cases (either in increasing or decreasing epidemic phases) with 95% prediction interval coverage dropping below 50% during the growth phases of the winter 2020, Delta, and Omicron waves. Ideally, case forecasts could serve as a leading indicator of changes in transmission dynamics. However, while most COVID-19 case forecasts outperformed a naïve baseline model, even the most accurate case forecasts were unreliable in key phases. Further research could improve forecasts of leading indicators, like COVID-19 cases, by leveraging additional real-time data, addressing performance across phases, improving the characterization of forecast confidence, and ensuring that forecasts were coherent across spatial scales. In the meantime, it is critical for forecast users to appreciate current limitations and use a broad set of indicators to inform pandemic-related decision making.

The reduction of the m6A methyltransferase METTL3 in granulosa cells is related to the follicular cysts in pigs.

Follicular cysts are a common reproductive disorder in domestic animals that cause considerable economic losses to the farming industry. Effective prevention and treatment methods are lacking because neither the pathogenesis nor formation mechanisms of follicular cysts are well-understood. In this study, we first investigated the granulosa cells (GCs) of cystic follicles isolated from pigs. We observed a significant reduction in the expression of methyltransferase-like 3 (METTL3). Subsequent experiments revealed that METTL3 downregulation in GCs caused a decrease in m6A modification of pri-miR-21. This reduction further inhibited DGCR8 recognition and binding to pri-miR-21, dampening the synthesis of mature miR-21-5p. Additionally, the decrease in miR-21-5p promotes IL-1ß expression in GCs. Elevated IL-1ß activates the NFκB pathway, in turn upregulating apoptotic genes TNFa and BAX/BCL2. The subsequent apoptosis of GCs and inhibition of autophagy causes downregulation of CYP19A1 expression. These processes lower oestrogen secretion and contribute to follicular cyst formation. In conclusion, our findings provide a foundation for understanding and further exploring the mechanisms of follicular-cyst development in farm animals. This work has important implications for treating ovarian disorders in livestock and could potentially be extended to humans.

Recent Advances of Bio-Based Hydrogel Derived Interfacial Evaporator for Sustainable Water and Collaborative Energy Storage Applications.

Solar interfacial evaporation strategy (SIES) has shown great potential to deal with water scarcity and energy crisis. Biobased hydrogel derived interfacial evaporator can realize efficient evaporation due to the unique structure- properties relationship. As such, increasing studies have focused on water treatment or even potential accompanying advanced energy storage applications with respect of efficiency and mechanism of bio-based hydrogel derived interfacial evaporation from microscale to molecular scale. In this review, the interrelationship between efficient interfacial evaporator and bio-based hydrogel is first presented. Then, special attention is paid on the inherent molecular characteristics of the biopolymer related to the up-to-date studies of promising biopolymers derived interfacial evaporator with the objective to showcase the unique superiority of biopolymer. In addition, the applications of the bio-based hydrogels are highlighted concerning the aspects including water desalination, water decontamination atmospheric water harvesting, energy storage and conversion. Finally, the challenges and future perspectives are given to unveil the bottleneck of the biobased hydrogel derived SIES in sustainable water and other energy storage applications.

METTL14 mediates nerve growth factor-stimulated testosterone synthesis in porcine theca cells.

Ovarian theca cells produce testosterone, which acts as a vital precursor substance for synthesizing estrogens during follicular development. Nerve growth factor (NGF) has been shown to participate in reproductive physiology, specifically to follicular development and ovulation. There is currently no available data on the impact of NGF on testosterone synthesis in porcine theca cells. Furthermore, m6A modification is the most common internal modification in eukaryotic mRNAs that are closely associated with female gametogenesis, follicle development, ovulation, and other related processes. It is also uncertain whether the three main enzymes associated with m6A, such as Writers, Erasers and Readers, play a role in this process. The present study, with an in vitro culture model, investigated the effect of NGF on testosterone synthesis in porcine theca cells and the role of Writers-METTL14 in this process. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. This study will help to further elucidate the mechanisms by which NGF regulates follicular development and provide new therapeutic targets for ovary-related diseases in female animals.

GW9 determines grain size and floral organ identity in rice.

Grain weight is an important determinant of grain yield. However, the underlying regulatory mechanisms for grain size remain to be fully elucidated. Here, we identify a rice mutant grain weight 9 (gw9), which exhibits larger and heavier grains due to excessive cell proliferation and expansion in spikelet hull. GW9 encodes a nucleus-localized protein containing both C2H2 zinc finger (C2H2-ZnF) and VRN2-EMF2-FIS2-SUZ12 (VEFS) domains, serving as a negative regulator of grain size and weight. Interestingly, the non-frameshift mutations in C2H2-ZnF domain result in increased plant height and larger grain size, whereas frameshift mutations in both C2H2-ZnF and VEFS domains lead to dwarf and malformed spikelet. These observations indicated the dual functions of GW9 in regulating grain size and floral organ identity through the C2H2-ZnF and VEFS domains, respectively. Further investigation revealed the interaction between GW9 and the E3 ubiquitin ligase protein GW2, with GW9 being the target of ubiquitination by GW2. Genetic analyses suggest that GW9 and GW2 function in a coordinated pathway controlling grain size and weight. Our findings provide a novel insight into the functional role of GW9 in the regulation of grain size and weight, offering potential molecular strategies for improving rice yield.

Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A.

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide. KEY POINTS: • Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation. • In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections. • Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.

Sleep behaviors predicted sleep disturbances among Chinese health science students: a cross-sectional study.

PURPOSE: Healthy sleep is essential for individuals' physiological and psychological health. Health science students experience a high prevalence of sleep disturbances which may be due to maladaptive behaviors. This study aimed to examine the associations of sleep behaviors including sleep hygiene and bedtime procrastination with the associations of sleep disturbances (e.g., poor sleep quality, insomnia, and short sleep). METHODS: This cross-sectional study included health science students from a medical university in Shanghai, China. Sleep disturbances included poor sleep quality, insomnia, and short sleep. They were measured by the Pittsburgh Sleep Quality Index (PSQI), Insomnia Severity Index (ISI), and one question "How many hours of sleep did you usually get during the past week?", respectively. Sleep behaviors included sleep hygiene and bedtime procrastination measured by the Sleep Hygiene Index (SHI) and Bedtime Procrastination Scale (BPS), respectively. Logistic regression was performed while controlling for potential confounders. RESULTS: A total of 464 health science students participated. Poorer overall sleep hygiene and more bedtime procrastination were independently associated with higher odds of poor sleep quality (OR=1.065, 95% CI 1.028-1.103; OR=1.040, 95% CI 1.006-1.075, respectively) and insomnia (OR=1.059, 95% CI 1.018-1.101; OR=1.093, 95% CI 1.049-1.139, respectively). More bedtime procrastination was associated with higher odds of short sleep (OR=1.148, 95% CI 1.093-1.206). Commonly reported specific sleep behaviors, such as "Going to bed later than intended", "Doing other things than sleep at bedtime", and "Easily stopping what I am doing at bedtime", were also related to higher odds of sleep disturbances. CONCLUSIONS: Sleep hygiene and bedtime procrastination were strong predictors of sleep disturbances. Tailored interventions targeting specific sleep behaviors are warranted to clarify their effect on sleep disturbances.

Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones.

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

3-(Methylthio)Propionic Acid from Bacillus thuringiensis Berliner Exhibits High Nematicidal Activity against the Root Knot Nematode Meloidogyne incognita (Kofoid and White) Chitwood.

Root knot nematodes cause serious damage to global agricultural production annually. Given that traditional chemical fumigant nematicides are harmful to non-target organisms and the environment, the development of biocontrol strategies has attracted significant attention in recent years. In this study, it was found that the Bacillus thuringiensis Berliner strain NBIN-863 exhibits strong fumigant nematicidal activity and has a high attraction effect on Meloidogyne incognita (Kofoid and White) Chitwood. Four volatile organic compounds (VOCs) produced by NBIN-863 were identified using solid-phase microextraction and gas chromatography-mass spectrometry. The nematicidal activity of four VOCs, namely, N-methylformamide, propenamide, 3-(methylthio)propionic acid, and phenylmalonic acid, was detected. Among these compounds, 3-(methylthio)propionic acid exhibited the highest direct contact nematicidal activity against M. incognita, with an LC50 value of 6.27 µg/mL at 24 h. In the fumigant bioassay, the mortality rate of M. incognita treated with 1 mg/mL of 3-(methylthio)propionic acid for 24 h increased to 69.93%. Furthermore, 3-(methylthio)propionic acid also exhibited an inhibitory effect on the egg-hatching of M. incognita. Using chemotaxis assays, it was determined that 3-(methylthio)propionic acid was highly attractive to M. incognita. In pot experiments, the application of 3-(methylthio)propionic acid resulted in a reduction in gall numbers, decreasing the number of galls per gram of tomato root from 97.58 to 6.97. Additionally, the root length and plant height of the treated plants showed significant increases in comparison with the control group. The current study suggests that 3-(methylthio)propionic acid is a novel nematicidal virulence factor of B. thuringiensis. Our research provides evidence for the potential use of NBIN-863 or its VOCs in biocontrol against root knot nematodes.

Genomic and Comparative Transcriptomic Analyses Reveal Key Genes Associated with the Biosynthesis Regulation of Okaramine B in Penicillium daleae NBP-49626.

Restricted production of fungal secondary metabolites hinders the ability to conduct comprehensive research and development of novel biopesticides. Okaramine B from Penicillium demonstrates remarkable insecticidal efficacy; however, its biosynthetic yield is low, and its regulatory mechanism remains unknown. The present study found that the yield difference was influenced by fermentation modes in okaramine-producing strains and performed genomic and comparative transcriptome analysis of P. daleae strain NBP-49626, which exhibits significant features. The NBP-49626 genome is 37.4 Mb, and it encodes 10,131 protein-encoding genes. Up to 5097 differentially expressed genes (DEGs) were identified during the submerged and semi-solid fermentation processes. The oka gene cluster, lacking regulatory and transport genes, displayed distinct transcriptional patterns in response to the fermentation modes and yield of Okaramine B. Although transcription trends of most known global regulatory genes are inconsistent with those of oka, this study identified five potential regulatory genes, including two novel Zn(II)2Cys6 transcription factors, Reg2 and Reg19. A significant correlation was also observed between tryptophan metabolism and Okaramine B yields. In addition, several transporter genes were identified as DEGs. These results were confirmed using real-time quantitative PCR. This study provides comprehensive information regarding the regulatory mechanism of Okaramine B biosynthesis in Penicillium and is critical to the further yield improvement for the development of insecticides.

A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase.

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOXStr) from Streptomyces viridosporus R111 and catalase (KatGEsc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOXStr r) and catalase (KatGEsc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOXStr and KatGEsc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.

A centromere map based on super pan-genome highlights the structure and function of rice centromeres.

Rice (Oryza sativa) is a significant crop worldwide with a genome shaped by various evolutionary factors. Rice centromeres are crucial for chromosome segregation, and contain some unreported genes. Due to the diverse and complex centromere region, a comprehensive understanding of rice centromere structure and function at the population level is needed. We constructed a high-quality centromere map based on the rice super pan-genome consisting of a 251-accession panel comprising both cultivated and wild species of Asian and African rice. We showed that rice centromeres have diverse satellite repeat CentO, which vary across chromosomes and subpopulations, reflecting their distinct evolutionary patterns. We also revealed that long terminal repeats (LTRs), especially young Gypsy-type LTRs, are abundant in the peripheral CentO-enriched regions and drive rice centromere expansion and evolution. Furthermore, high-quality genome assembly and complete telomere-to-telomere (T2T) reference genome enable us to obtain more centromeric genome information despite mapping and cloning of centromere genes being challenging. We investigated the association between structural variations and gene expression in the rice centromere. A centromere gene, OsMAB, which positively regulates rice tiller number, was further confirmed by expression quantitative trait loci, haplotype analysis and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 methods. By revealing the new insights into the evolutionary patterns and biological roles of rice centromeres, our finding will facilitate future research on centromere biology and crop improvement.

Proteomic profiles and the function of RBP4 in endometrium during embryo implantation phases in pigs.

BACKGROUND: Endometrial receptivity plays a vital role in the success of embryo implantation. However, the temporal proteomic profile of porcine endometrium during embryo implantation is still unclear. RESULTS: In this study, the abundance of proteins in endometrium on days 9, 10, 11, 12, 13, 14, 15 and 18 of pregnancy (D9, 10, 11, 12, 13, 14, 15 and 18) was profiled via iTRAQ technology. The results showed that 25, 55, 103, 91, 100, 120, 149 proteins were up-regulated, and 24, 70, 169, 159, 164, 161, 198 proteins were down-regulated in porcine endometrium on D10, 11, 12, 13, 14, 15 and 18 compared with that on D9, respectively. Among these differentially abundance proteins (DAPs), Multiple Reaction Monitoring (MRM) results indicated that S100A9, S100A12, HRG and IFI6 were differentially abundance in endometrial during embryo implantation period. Bioinformatics analysis showed that the proteins differentially expressed in the 7 comparisons were involved in important processes and pathways related to immunization, endometrial remodeling, which have a vital effect on embryonic implantation. CONCLUSION: Our results reveal that retinol binding protein 4 (RBP4) could regulate the cell proliferation, migration and apoptosis of endometrial epithelial cells and endometrial stromal cells to affect embryo implantation. This research also provides resources for studies of proteins in endometrium during early pregnancy.

A mitochondrial FUNDC1/HSC70 interaction organizes the proteostatic stress response at the risk of cell morbidity.

Both protein quality and mitochondrial quality are vital for the cellular activity, and impaired proteostasis and mitochondrial dysfunction are common etiologies of aging and age-related disorders. Here, we report that the mitochondrial outer membrane protein FUNDC1 interacts with the chaperone HSC70 to promote the mitochondrial translocation of unfolded cytosolic proteins for degradation by LONP1 or for formation of non-aggresomal mitochondrion-associated protein aggregates (MAPAs) upon proteasome inhibition in cultured human cells. Integrative approaches including csCLEM, Apex, and biochemical analysis reveal that MAPAs contain ubiquitinated cytosolic proteins, autophagy receptor p62, and mitochondrial proteins. MAPAs are segregated from mitochondria in a FIS1-dependent manner and can subsequently be degraded via autophagy. Although the FUNDC1/HSC70 pathway promotes the degradation of unfolded cytosolic proteins, excessive accumulation of unfolded proteins on the mitochondria prior to MAPA formation impairs mitochondrial integrity and activates AMPK, leading to cellular senescence. We suggest that human mitochondria organize cellular proteostatic response at the risk of their own malfunction and cell lethality.

MALAT1 knockdown alleviates the pyroptosis of microglias in diabetic cerebral ischemia via regulating STAT1 mediated NLRP3 transcription.

BACKGROUND: Dysregulated long non-coding RNAs participate in the development of diabetic cerebral ischemia. This study aimed to investigate the underlying mechanism of lncRNA MALAT1 in diabetic cerebral ischemia. METHOD: Middle cerebral artery occlusion (MCAO) was performed to establish diabetic cerebral I/R in vivo. TTC and neurological deficits assessment were performed to assess cerebral ischemic injury. LDH was conducted to detect cytotoxicity. RT-qPCR and western blotting assays were applied to determine mRNA and protein expression. Flow cytometry was performed to detect the pyroptosis of BV2 cells. Immunofluorescence and FISH were conducted for subcellular localization of MALAT1 and STAT1. ELISA was performed to determine cytokine release. Dual luciferase reporter, RIP, and ChIP assays were used to validate the interaction between STAT1 and MALAT1/NLRP3. Diabetes aggravated cerebral injury in vivo and in vitro. Diabetic cerebral ischemia induced inflammatory response and inflammation-induced cell pyroptosis. RESULT: MALAT1 was overexpressed in diabetic cerebral ischemia models in vivo and in vitro. However, knockdown of MALAT1 suppressed inflammatory response and the pyroptosis of BV2 cells. Moreover, MALAT1 interacted with STAT1 to transcriptionally activate NLRP3. Knockdown of STAT1 significantly reversed the effects of MALAT1. Furthermore, STAT1 promotes the MALAT1 transcription. MALAT1 interacts with STAT1 to promote the pyroptosis of microglias induced by diabetic cerebral ischemia through activating NLRP3 transcription. CONCLUSION: Thus, knockdown of MALAT1 may be a potential promising therapy target for diabetic cerebral ischemia.

NLG1, encoding a mitochondrial membrane protein, controls leaf and grain development in rice.

BACKGROUND: Mitochondrion is the key respiratory organ and participate in multiple anabolism and catabolism pathways in eukaryote. However, the underlying mechanism of how mitochondrial membrane proteins regulate leaf and grain development remains to be further elucidated. RESULTS: Here, a mitochondria-defective mutant narrow leaf and slender grain 1 (nlg1) was identified from an EMS-treated mutant population, which exhibits narrow leaves and slender grains. Moreover, nlg1 also presents abnormal mitochondria structure and was sensitive to the inhibitors of mitochondrial electron transport chain. Map-based cloning and transgenic functional confirmation revealed that NLG1 encodes a mitochondrial import inner membrane translocase containing a subunit Tim21. GUS staining assay and RT-qPCR suggested that NLG1 was mainly expressed in leaves and panicles. The expression level of respiratory function and auxin response related genes were significantly down-regulated in nlg1, which may be responsible for the declination of ATP production and auxin content. CONCLUSIONS: These results suggested that NLG1 plays an important role in the regulation of leaf and grain size development by maintaining mitochondrial homeostasis. Our finding provides a novel insight into the effects of mitochondria development on leaf and grain morphogenesis in rice.

DNMT3A mutation promotes leukemia development through NAM-NAD metabolic reprogramming.

BACKGROUND: DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia (AML) with Arg882His (R882H) as the hotspot mutation. It has been reported that DNMT3A mutation plays a key role in leukemogenesis through hypomethylation of some target genes associated with cell growth and differentiation. In this study, we investigated the function of DNMT3A R882H in the malignant progression of AML by regulating metabolic reprogramming. METHODS: Ultra-High Performance Liquid Chromatography-High Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) was used to detect metabolites in the serum of mice harboring Dnmt3a R878H mutation and the wild-type Dnmt3a. Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) were used to analyze the levels of DNA methylation and mRNA expression of genes in mouse Gr1+ bone marrow cells respectively. The TCGA and GO databases were used to analyze the differential genes between human samples carrying the DNMT3A R882 mutation and the wild-type DNMT3A. Co-immunoprecipitation and immunoblotting were used to illustrate the binding levels of Cyclins-CDKs and CDK inhibitors including CDKN1A and CDKN1B. Flow cytometry was used to analyze the cell differentiation, division, apoptosis and cell cycle. The effect of NAMPT inhibition on leukemia was evaluated by using in vivo fluorescence imaging in NOG mouse model bearing OCI-AML3 cells. RESULTS: DNMT3A mutation caused high expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage synthetic pathway, through DNA hypomethylation, and finally led to abnormal nicotinamide (NAM) metabolism and NAD synthesis. The NAM-NAD metabolic abnormalities caused accelerated cell cycle progression. Inhibition of NAMPT can reduce the binding degree between Cyclins-CDKs, and increase the binding interaction of the CDK inhibitors with Cyclins-CDKs complexes. Moreover, cells with high expression of NAMPT were more sensitive to the NAMPT inhibitor FK866 with a lower IC50. The inhibition of NAMPT can remarkably extend the survival time of tumor-bearing mice and reduce the infiltration of tumor cells. CONCLUSIONS: Taken together, our data showed that DNMT3A mutation caused NAMPT overexpression to induce the reprogramming of NAM-NAD metabolism and contribute to abnormal proliferation, which provided a potential direction for targeted therapy at the metabolic level in AML with DNMT3A mutation.

A dominant variant in apoptosis-related gene XKR8 is relevant to hereditary auditory neuropathy.

BACKGROUND: Auditory neuropathy is an unusual type of hearing loss. At least 40% of patients with this disease have underlying genetic causes. However, in many hereditary auditory neuropathy cases, etiology remains undetermined. METHODS: We collected data and blood samples from a four-generation Chinese family. After excluding relevant variants in known deafness-related genes, exome sequencing was conducted. Candidate genes were verified by pedigree segregation, transcript/protein expression in the mouse cochlea, and plasmid expression studies in HEK 293T cells. Moreover, a mutant mouse model was generated and underwent hearing evaluations; protein localization in the inner ear was also assessed. RESULTS: The clinical features of the family were diagnosed as auditory neuropathy. A novel variant c.710G > A (p.W237X) in apoptosis-related gene XKR8 was identified. Genotyping of 16 family members confirmed the segregation of this variant with the deafness phenotype. Both XKR8 mRNA and XKR8 protein were expressed in the mouse inner ear, predominantly in regions of spiral ganglion neurons; Moreover, this nonsense variant impaired the surface localization of XKR8 in cells. Transgenic mutant mice exhibited late-onset auditory neuropathy, and their altered XKR8 protein localization in the inner ear confirmed the damaging effects of this variant. CONCLUSIONS: We identified a variant in the XKR8 gene that is relevant to auditory neuropathy. The essential role of XKR8 in inner ear development and neural homeostasis should be explored.

RNA silencing of GM-CSF in CAR-T cells reduces the secretion of multiple inflammatory cytokines.

Chimeric antigen receptor T (CAR-T) cell therapy has become a research hotspot in the field of hematological malignancies. However, CAR-T cell therapy can lead to immunotherapy-associated side effects including cytokine release syndrome and neurotoxicity. Gene depletion of GM-CSF in CAR-T cells was found preventive against adverse effects, but additional transfections were required to produce CAR-T cells. In this study, we interrupted GM-CSF expression in CAR-T cells by inserting the GM-CSF shRNA-expression cassette in the CAR vector. Reduction of GM-CSF in CAR-T cells could decrease the level of several proinflammatory cytokines without hampering the killing capacity. The manufacture of GM-CSF knockdown CAR-T cells does not require complicated transfections, which makes it more practical and feasible for clinical application.