Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

CSB-independent, XPC-dependent transcription-coupled repair in Drosophila.

Drosophila melanogaster has been extensively used as a model system to study ionizing radiation and chemical-induced mutagenesis, double-strand break repair, and recombination. However, there are only limited studies on nucleotide excision repair in this important model organism. An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, we recently found that the Drosophila S2 cell line performs TCR comparable to human cells. Here, we have extended this work to Drosophila at all its developmental stages. We find TCR takes place throughout the life cycle of the organism. Moreover, we find that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.

NSUN2-Mediated m5C Methylation Impairs Endometrial Receptivity.

Impaired endometrial decidualization is the primary cause of recurrent implantation failure (RIF). RNA methylation modification, especially NSUN family mediated m5C, is crucial for various physiological events, such as maternal-to-zygotic transition, gametogenesis, embryonic development, organismal lifespan, and cell cycle. However, the regulatory mechanisms between NSUN family mediated m5C modification and RIF remain unknown. We acquired NSUN2 expression data of 15 human endometrium samples at proliferative and secretory stages from reproductive cell atlas. The overall pattern of m5C sites and genes was elucidated through m5C-BS-seq, whereas the overall m5C levels in different groups were revealed by dot blot assay. BrdU and western blotting assays were carried out to evaluate the role of NSUN2 in proliferation and autophagy. The effects of NSUN2-mediated m5C modification on embryo attachment were evaluated by an in vitro model of a confluent monolayer of Ishikawa cells cocultured with BeWo spheroids, and its downstream targets were evaluated by real-time reverse-transcription PCR and western blotting in Ishikawa cells. The molecular mechanism for NSUN2 regulating its downstream targets' expression was determined by Cut&Tag and coimmunoprecipitation assays. NSUN2 was increased in SOX9+ cells and widespread in epithelial cell type at the proliferative stage by previous single-cell RNA sequencing data. NSUN2 overexpression (NSUN2OE) in the Ishikawa cell line elevated m5C levels and promoted cell proliferation and autophagy. NSUN2OE reduced attachment efficiency of BeWo cell spheres. Overexpressed NSUN2 was found to increase STAT1 and MMP14 mRNA expressions by inducing exon skipping. NSUN2 interacted with CLDN4 through m5C modification, and NSUN2OE or NSUN2 knockdown resulted in a similar variation tendency of CLDN4. Overexpression of NSUN2 increased CLDN4 H3K9ac modification by downregulating SIRT4 expression at the protein level, leading to the upregulation of CLDN4 mRNA expression. Our results uncovered a novel intricate regulatory mechanism between NSUN2-mediated m5C and RIF and suggested a potential new therapeutic strategy for RIF.

Association between triglyceride-glucose index and intracranial/extracranial atherosclerotic stenosis: findings from a retrospective study.

OBJECTIVE: The association of the triglyceride-glucose (TyG) index with intracranial atherosclerotic stenosis (ICAS) and extracranial atherosclerotic stenosis (ECAS) is unclear. This study aimed to investigate the relationship of TyG index with the distribution and severity of ICAS and ECAS. METHOD: Patients who underwent digital subtraction angiography (DSA) for evaluating ICAS/ECAS in Zhongnan Hospital of Wuhan University from January 2017 to October 2021 were retrospectively enrolled in our study. Clinical characteristics, DSA data, blood routine, lipid profile and fasting glucose were recorded. The association of TyG index and ICAS/ECAS status were investigated in four aspects: location and distribution of stenosis, stenosis severity and whether stenosis is symptomatic. Logistic regression models were used to evaluate the association. Restricted cubic splines were constructed to model the non-linear relationship between the TyG index and different arterial stenosis status. RESULTS: Among 1129 included patients, the median age was 62 (IQR 55-68) years, and 71.3% were male. The median TyG index was 8.81 (8.40, 9.21). Elevated TyG index was significantly associated with ICAS, combined ICAS/ECAS, anterior circulation stenosis, posterior circulation stenosis, combined anterior/posterior circulation stenosis, severe stenosis, both asymptomatic and symptomatic stenosis. This association was maintained after adjusting for age, sex, smoking, drinking, medical history of hypertension and stroke, platelet, total cholesterol, high-density lipoprotein, and low-density lipoprotein. Multivariable-adjusted spline regression models showed that a progressively increasing risk of arterial stenosis was related to an elevated TyG index. CONCLUSION: Elevated TyG index was associated with ICAS/ECAS. TyG index might be a useful indicator of ICAS and severe stenosis.

Visible Light-Induced 1,2-Diphenyldisulfane-Mediated Defluoroborylation of Polyfluoroarenes.

A green and practical protocol of defluoroborylation of polyfluoroarenes with stable and readily accessible NHC-borane was developed, using 1,2-diphenyldisulfane as a hydrogen atom transfer (HAT) and single electron transfer (SET) reagent precursor under visible-light irradiation, leading to the concise formation of value-added fluorinated organoboron scaffolds. Mechanism studies revealed the method underwent a boryl radical addition reaction with polyfluoroarene, followed by successive single electron transfer pathways and defluorination of the C-F bond to offer the targeted product. This unprecedented platform relies on 1,2-diphenyldisulfane and base without using expensive photocatalysts, highlighting the methodology has promising application value to prepare borylated polyfluoroarene compounds.

Lewis acid catalyzed Markovnikov hydrobromination and hydrochlorination of alkynes using TMSX (X = Br, Cl).

Markovnikov hydrobromination and hydrochlorination of alkynes were achieved using TMSX (X = Br, Cl) instead of corrosive HX (X = Br, Cl) as the bromination and chlorination reagents. Mn(OAc)2·4H2O was used as the hydrobromination catalyst for electron-neutral/rich alkynes. For the hydrobromination of electron-deficient alkynes and hydrochlorination of alkynes, Zn(OAc)2·2H2O was employed as the catalyst. Mechanistic studies suggested that the in situ formed TMS-substituted alkyne might be a reactive intermediate and the proton of the terminal alkyne should be a hydrogen source for the hydrohalogenation reaction.

1 H-NMR revealed pyruvate as a differentially abundant metabolite in the venom glands of Apis cerana and Apis mellifera.

As a common defense mechanism in Hymenoptera, bee venom has complex components. Systematic and comprehensive analysis of bee venom components can aid in early evaluation, accurate diagnosis, and protection of organ function in humans in cases of bee stings. To determine the differences in bee venom composition and metabolic pathways between Apis cerana and Apis mellifera, proton nuclear magnetic resonance (1 H-NMR) technology was used to detect the metabolites in venom samples. A total of 74 metabolites were identified and structurally analyzed in the venom of A. cerana and A. mellifera. Differences in the composition and abundance of major components of bee venom from A. cerana and A. mellifera were mapped to four main metabolic pathways: valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; alanine, aspartate and glutamate metabolism; and the tricarboxylic acid cycle. These findings indicated that the synthesis and metabolic activities of proteins or polypeptides in bee venom glands were different between A. cerana and A. mellifera. Pyruvate was highly activated in 3 selected metabolic pathways in A. mellifera, being much more dominant in A. mellifera venom than in A. cerana venom. These findings indicated that pyruvate in bee venom glands is involved in various life activities, such as biosynthesis and energy metabolism, by acting as a precursor substance or intermediate product.

Molecular mechanism of the repressive phase of the mammalian circadian clock.

The mammalian circadian clock consists of a transcription-translation feedback loop (TTFL) composed of CLOCK-BMAL1 transcriptional activators and CRY-PER transcriptional repressors. Previous work showed that CRY inhibits CLOCK-BMAL1-activated transcription by a "blocking"-type mechanism and that CRY-PER inhibits CLOCK-BMAL1 by a "displacement"-type mechanism. While the mechanism of CRY-mediated repression was explained by both in vitro and in vivo experiments, the CRY-PER-mediated repression in vivo seemed in conflict with the in vitro data demonstrating PER removes CRY from the CLOCK-BMAL1-E-box complex. Here, we show that CRY-PER participates in the displacement-type repression by recruiting CK1δ to the nucleus and mediating an increased local concentration of CK1δ at CLOCK-BMAL1-bound promoters/enhancers and thus promoting the phosphorylation of CLOCK and dissociation of CLOCK-BMAL1 along with CRY from the E-box. Our findings bring clarity to the role of PER in the dynamic nature of the repressive phase of the TTFL.

Chromosome-level genome of black cutworm provides novel insights into polyphagy and seasonal migration in insects.

BACKGROUND: The black cutworm, Agrotis ipsilon, is a serious global underground pest. Its distinct phenotypic traits, especially its polyphagy and ability to migrate long distances, contribute to its widening distribution and increasing difficulty of control. However, knowledge about these traits is still limited. RESULTS: We generated a high-quality chromosome-level assembly of A. ipsilon using PacBio and Hi-C technology with a contig N50 length of ~ 6.7 Mb. Comparative genomic and transcriptomic analyses showed that detoxification-associated gene families were highly expanded and induced after insects fed on specific host plants. Knockout of genes that encoded two induced ABC transporters using CRISPR/Cas9 significantly reduced larval growth rate, consistent with their contribution to host adaptation. A comparative transcriptomic analysis between tethered-flight moths and migrating moths showed expression changes in the circadian rhythm gene AiCry2 involved in sensing photoperiod variations and may receipt magnetic fields accompanied by MagR and in genes that regulate the juvenile hormone pathway and energy metabolism, all involved in migration processes. CONCLUSIONS: This study provides valuable genomic resources for elucidating the mechanisms involved in moth migration and developing innovative control strategies.

Being Stung Once or Twice by Bees (Apis mellifera L.) Slightly Disturbed the Serum Metabolome of SD Rats to a Similar Extent.

In most cases, the number of honeybee stings received by the body is generally small, but honeybee stings can still cause serious allergic reactions. This study fully simulated bee stings under natural conditions and used 1H Nuclear Magnetic Resonance (1H NMR) to analyze the changes in the serum metabolome of Sprague-Dawley (SD) rats stung once or twice by honeybees to verify the impact of this mild sting on the body and its underlying mechanism. The differentially abundant metabolites between the blank control rats and the rats stung by honeybees included four amino acids (aspartate, glutamate, glutamine, and valine) and four organic acids (ascorbic acid, lactate, malate, and pyruvate). There was no separation between the sting groups, indicating that the impact of stinging once or twice on the serum metabolome was similar. Using the Principal Component Discriminant Analysis ( PCA-DA) and Variable Importance in Projection (VIP) methods, glucose, lactate, and pyruvate were identified to help distinguish between sting groups and non-sting groups. Metabolic pathway analysis revealed that four metabolic pathways, namely, the tricarboxylic acid cycle, pyruvate metabolism, glutamate metabolism, and alanine, aspartate, and glutamate metabolism, were significantly affected by bee stings. The above results can provide a theoretical basis for future epidemiological studies of bee stings and medical treatment of patients stung by honeybees.

A Drosophila Model Reveals the Potential Role for mtt in Retinal Disease.

Congenital stationary night blindness (CSNB) is a genetically heterogeneous inherited retinal disorder, caused by over 300 mutations in 17 different genes. While there are numerous fly models available for simulating ocular diseases, most are focused on mimicking retinitis pigmentosa (RP), with animal models specifically addressing CSNB limited to mammals. Here, we present a CSNB fly model associated with the mtt gene, utilizing RNA interference (RNAi) to silence the mtt gene in fly eyes (homologous to the mammalian GRM6 gene) and construct a CSNB model. Through this approach, we observed significant defects in the eye structure and function upon reducing mtt expression in fly eyes. This manifested as disruptions in the compound eye lens structure and reduced sensitivity to light responses. These results suggest a critical role for mtt in the function of fly adult eyes. Interestingly, we found that the mtt gene is not expressed in the photoreceptor neurons of adult flies but is localized to the inner lamina neurons. In summary, these results underscore the crucial involvement of mtt in fly retinal function, providing a framework for understanding the pathogenic mechanisms of CSNB and facilitating research into potential therapeutic interventions.

Characterization of a membrane Fcγ receptor in largemouth bass (Micropterus saloumoides) and its response to bacterial challenge.

Fc receptors (FcRs), specific to the Fc portion of immunoglobulin (Ig), are required to regulate immune responses against pathogenic infections. However, FcγR is a member of FcRs family, whose structure and function remains to be elucidated in teleost fish. In this study, the FcγRII, from largemouth bass (Micropterus saloumoides), named membrane MsFcγRII (mMsFcγRII), was cloned and identified. The opening reading frame (ORF) of mMsFcγRII was 750 bp, encoding 249 amino acids with a predicted molecular mass of 27 kDa. The mMsFcγRII contained a signal peptide, two Ig domains, a transmembrane domain, and an intracellular region, which was highly homology with FcγR from other teleost fish. The mRNA expression analysis showed that mMsFcγRII was widely distributed in all tested tissues and with the highest expression level in spleen. After bacterial challenge, the expression of mMsFcγRII was significantly upregulated in vivo (spleen and head kidney), as well as in vitro (leukocytes from head kidney). The subcellular localization assay revealed that mMsFcγRII was mostly observed on the membrane of HEK293T cells which were transfected with mMsFcγRII overexpression plasmid. Flow cytometric analysis showed that natural mMsFcγRII protein was highly expressed in head kidney lymphocytes. Moreover, indirect immunofluorescence assay and pull-down assay indicated that mMsFcγRII could bind to IgM purified from largemouth bass serum. These results suggested that mMsFcγRII was likely to play an influential role in the immune response against pathogens and provided valuable insights for studying the function of FcRs in teleost.

A novel homozygous mutation in ACTL7A leads to male infertility.

Male infertility, a global public health problem, exhibits complex pathogenic causes and genetic factors deserve further discovery and study. We identified a novel homozygous missense mutation c.224A > C (p.D75A) in ACTL7A gene in two infertile brothers with teratozoospermia by whole-exome sequencing (WES). In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) showed fertilization failure of the two affected couples. The three-dimensional (3D) models showed that a small section of α-helix transformed into random coil in the mutant ACTL7A protein and mutant amino acid lacked a hydrogen bond with Ser170 amino acid. Immunofluorescence revealed that ACTL7A protein was degraded in sperms of patients. Transmission electron microscopy (TEM) analysis of sperms from the infertile patients showed that the irregular perinuclear theca (PT) and acrosomal ultrastructural defects. Furthermore, ACTL7A mutation caused abnormal localization and reduced the expression of PLCZ1 in sperms of the patients, which may be the key reasons for the fertilization failure after ICSI. Our findings expand the spectrum of ACTL7A mutations and provide novel theoretical basis for genetic counseling.

Isolated cryptococcal osteomyelitis of the sacrum in an immunocompetent patient: a case report and literature review.

BACKGROUND: Cryptococcus neoformans, an opportunistic fungal pathogen, seldom causes infection in immunocompetent people. Cryptococcal osteomyelitis is an uncommon condition in which Cryptococcus invades the bone. It usually occurs as part of a disseminated infection and rarely in isolation. The spine has been reported as the most common site of cryptococcal osteomyelitis; however, isolated case of sacrum involvement in immunocompetent patients has never been reported. CASE PRESENTATION: We report the case of a 37-year-old man without underlying disease who presented with progressive low back and sacrococcygeal pain. The patient was initially diagnosed with sacral tumour by a local doctor, and subsequently, after admission, was diagnosed with sacral tuberculosis. He was empirically treated with antitubercular drugs. The patient failed to respond to antitubercular drugs and complained of worsening low back pain. Additionally, he developed persistent radiating pain and numbness in his legs. For further diagnosis, we performed a computed tomography-guided puncture biopsy of the sacrum, which revealed granulomatous inflammation with massive macrophage infiltration and special staining revealed a fungal infection. We performed sacral debridement and drainage and obtained purulent specimens for pathological examination and microbial culture. Microbial identification and drug susceptibility tests revealed a Cryptococcus neoformans infection sensitive to fluconazole. Postoperatively, the persistent radiating pain and numbness in the legs resolved. After 12 consecutive weeks of antifungal therapy, all his symptoms resolved. The patient remained without any signs of recurrence at the 8-month follow-up. CONCLUSION: We reported a rare case of isolated sacrum cryptococcal osteomyelitis in an immunocompetent patient. Furthermore, we identified and reviewed 18 published cases of spine cryptococcal osteomyelitis. Immunocompetent individuals are also at risk for cryptococcal osteomyelitis. Clinical manifestation and imaging are insufficient to diagnose cryptococcal osteomyelitis of the spine, and invasive examinations, such as puncture biopsy and fungal examinations, are needed. Antifungal therapy yields satisfactory results for the treatment of cryptococcal osteomyelitis of the spine, however, if the infective lesion is large, especially when it compresses the spinal cord and nerves, a regimen combining aggressive surgery with antifungal therapy is indispensable.

Circadian regulation of c-MYC in mice.

The circadian clock is a global regulatory mechanism that controls the expression of 50 to 80% of transcripts in mammals. Some of the genes controlled by the circadian clock are oncogenes or tumor suppressors. Among these Myc has been the focus of several studies which have investigated the effect of clock genes and proteins on Myc transcription and MYC protein stability. Other studies have focused on effects of Myc mutation or overproduction on the circadian clock in comparison to their effects on cell cycle progression and tumorigenesis. Here we have used mice with mutations in the essential clock genes Bmal1, Cry1, and Cry2 to gain further insight into the effect of the circadian clock on this important oncogene/oncoprotein and tumorigenesis. We find that mutation of both Cry1 and Cry2, which abolishes the negative arm of the clock transcription-translation feedback loop (TTFL), causes down-regulation of c-MYC, and mutation of Bmal1, which abolishes the positive arm of TTFL, causes up-regulation of the c-MYC protein level in mouse spleen. These findings must be taken into account in models of the clock disruption-cancer connection.

Responses of maize (Zea mays L.) seedlings growth and physiological traits triggered by polyvinyl chloride microplastics is dominated by soil available nitrogen.

As a burgeoning pollutant, microplastics (MPs) has elicited global concern. However, ecological effects and mechanisms of MPs on plant-soil system are still poorly understood. In the present study, the impacts of polyvinyl chloride microplastics (PVC-MPs) on maize (Zea mays L.) seedlings growth and physiological traits and soil properties were discussed through a 30-day pot experiment. Results showed that PVC-MPs had greater toxicity effect on seedlings shoot biomass than root biomass. To defense the impact of PVC-MPs, the superoxide dismutase and catalase activities in seedlings leaf were stimulated. Moreover, the adhesion of MPs on soil particles increased, and soil microorganism, enzymes, and nutrients were altered significantly with increasing content of PVC-MPs. Notably, soil nitrate nitrogen decreased significantly with increasing content of PVC-MPs, whereas soil ammonium nitrogen was promoted under lower contents (0.1% and 1%) of PVC-MPs. Redundancy analysis indicated that soil nitrate nitrogen and ammonium nitrogen can explain 87.4% and 7.7% of variation in maize seedlings growth and physiological traits, respectively. These results display that maize seedlings shoot is more susceptible to the impact of PVC-MPs and soil available nitrogen is the primary limiting factor on maize seedlings growth and physiological traits triggered by PVC-MPs. Impacts of PVC-MPs on maize seedlings growth and physiological traits by nitrogen depletion lead to the possible yield and economic loess and potential risks due to the over use of nitrogen fertilizers.

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides).

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5 g) were injected intraperitoneally with Nocardia seriolae (105 CFU/100 µl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC > |2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM+ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM+ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.

Circadian clock, carcinogenesis, chronochemotherapy connections.

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.

Actl7a deficiency in mice leads to male infertility and fertilization failure.

Mutations in the Actl7a gene have been reported to lead to male infertility; however, the detailed mechanism of this phenomenon remains unknown. In this study, we constructed Actl7a gene knockout (KO) mice and found that Actl7a deficiency led to malformed formation of sperm acrosomes, male infertility, fertilization failure during in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and reduced sperm-zona pellucida (ZP) binding ability. Moreover, we found that the localization of the zona pellucida binding protein (ZPBP) was altered in the sperm of Actl7a homozygous KO male mice, which may affect the sperm-zona pellucida binding ability. ACTL7A and ZPBP could form complex, which may be involved in acrosomal formation. Further studies found that localization and expression of the PLCZ1 protein were abnormal in misshapen sperm, leading to reduced calcium oscillations in oocytes. Herein, we provide more detailed mechanisms underlining Actl7a deficiency and male infertility.

Two novel STAT1 mutations cause Mendelian susceptibility to mycobacterial disease.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations.