STING activation promotes inflammatory response and delays skin wound healing in diabetic mice.

Sustained inflammatory responses delay wound repair in diabetic skin. The stimulator of interferon genes (STING) plays a vital role in the innate immune responses. However, its function in diabetic skin wound repair, and the underlying mechanism remains unclear. Here, we reported that STING activation is a pathogenic marker that correlates with delayed wound repair in diabetic skin. Firstly, we found that STING expression is enhanced in the epidermis of STZ induced diabetes mouse model and db/db mouse model. Consistently, we also found that STING expression was upregulated in keratinocytes with the high-glucose (HG) treatment. Moreover, silencing of STING accelerated wound healing in vitro. In vivo, inhibition of STING by c176 inhibited inflammatory response in the epidermis and accelerated wound healing in diabetic skin. In addition, we found that autophagy dysfunction is correlated with the expression of STING in epidermis of diabetic mice. Induction of autophagy by rapamycin significantly reduced STING expression in keratinocytes. Collectively, these results indicated that defects of autophagy might lead to the activation of STING and finally delay the diabetic wound healing.

Transcriptomics of different tissues of blueberry and diversity analysis of rhizosphere fungi under cadmium stress.

Blueberry (Vaccinium ssp.) is a perennial shrub belonging to the family Ericaceae, which is highly tolerant of acid soils and heavy metal pollution. In the present study, blueberry was subjected to cadmium (Cd) stress in simulated pot culture. The transcriptomics and rhizosphere fungal diversity of blueberry were analyzed, and the iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and cadmium (Cd) content of blueberry tissues, soil and DGT was determined. A correlation analysis was also performed. A total of 84 374 annotated genes were identified in the root, stem, leaf and fruit tissue of blueberry, of which 3370 were DEGs, and in stem tissue, of which 2521 were DEGs. The annotation data showed that these DEGs were mainly concentrated in a series of metabolic pathways related to signal transduction, defense and the plant-pathogen response. Blueberry transferred excess Cd from the root to the stem for storage, and the highest levels of Cd were found in stem tissue, consistent with the results of transcriptome analysis, while the lowest Cd concentration occurred in the fruit, Cd also inhibited the absorption of other metal elements by blueberry. A series of genes related to Cd regulation were screened by analyzing the correlation between heavy metal content and transcriptome results. The roots of blueberry rely on mycorrhiza to absorb nutrients from the soil. The presence of Cd has a significant effect on the microbial community composition of the blueberry rhizosphere. The fungal family Coniochaetaceae, which is extremely extremelytolerant, has gradually become the dominant population. The results of this study increase our understanding of the plant regulation mechanism for heavy metals, and suggest potential methods of soil remediation using blueberry.

Applied evolution: Dual dynamic regulations-based approaches in engineering intracellular malonyl-CoA availability.

Malonyl-CoA is an important building block for microbial synthesis of numerous pharmaceutically interesting or fatty acid-derived compounds including polyketides, flavonoids, phenylpropanoids and fatty acids. However, the tightly regulated intracellular malonyl-CoA availability often impedes overall product formation. Here, in order to unleash this tightly cellular behavior, we present evolution: dual dynamic regulations-based approaches to write artificial robust and dynamic function into intricate cellular background. Firstly, a conserved core domain based evolutionary principles were incorporated into genome mining to explore the biosynthetic diversities of discrete acetyl-CoA carboxylase (ACC) families, as malonyl-CoA is solely derived from carboxylation of acetyl-CoA by ACC in most organisms. A comprehensive phylogenomic and further experimental analysis, which included genomes of 50 strains throughout representative species, was performed to recapitulate the evolutionary history and reveal that previously unnoticed ACC families from Salmonella enterica exhibited the highest activities among all the candidates. A set of orthogonal and bi-functional quorum-sensing (QS)-based regulation tools were further designed and connected with T7 RNA polymerase as genetic amplifier to achieve dual dynamic control in a high dynamic range, which allowed us to efficiently activate and repress different sets of genes dynamically and independently. These genetic circuits were then combined with ACC of S. enterica and CRISPRi system to reprogram central metabolism that rewired the tightly regulated malonyl-CoA pathway to a robust and autonomous behavior, leading to a 29-fold increase of malony-CoA availability. We applied this dual regulation tool to successfully synthesizing malonyl-CoA-derived compound (2S)-naringenin, and achieved the highest production (1073.8 mg/L) reported to date associate with dramatic decreases of by-product formation. Notably, the whole fermentation presents as an autonomous behavior, totally eliminating human supervision and inducer supplementation. Hence, the constructed evolution: dual dynamic regulations-based approaches pave the way to develop an economically viable and scalable procedure for microbial production of malonyl-CoA derived compounds.

Engineering the pathway in Escherichia coli for the synthesis of medium-chain-length polyhydroxyalkanoates consisting of both even- and odd-chain monomers.

BACKGROUND: Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) containing various chain length monomers from C6 to C14 have more applications besides sustainable and environmental-friendly biomaterials owing to their superior physical and mechanical properties. We engineered a reversed fatty acid ß-oxidation pathway in Escherichia coli that can synthesize mcl-PHA directly from glucose and achieved high yield. However, there were only even-chain monomers in the biosynthetic polymers. The need for mcl-PHA harboring both even- and odd-chain monomers with better and wider utility impels us to develop the biosynthetic routes for the production of the novel and unnatural mcl-PHA through rewiring the basic metabolism. RESULTS: In the present study, a propionate assimilation and metabolic route was integrated into the reversed fatty acid ß-oxidation in order to produce mcl-PHA consisting of both even- and odd-numbered monomers. The content of odd-numbered monomers in mcl-PHA was improved with the increased propionate addition. After further deletion of pyruvate oxidase (PoxB) and pyruvate formate-lyase (PflB), the metabolically engineered chassis E. coli LZ08 harboring pQQ05 and pZQ06 (overexpression of prpP and prpE genes from Ralstonia eutropha H16) innovatively accumulated 6.23 wt% mcl-PHA containing odd-chain monomers ranging from 7 to 13 carbon atoms about 20.03 mol%. CONCLUSIONS: This is the first successful report on production of mcl-PHA harboring both even- and odd-chain monomers (C6-C14) synthesized from glucose and propionate in recombinant E. coli. This present study achieved the highest yield of de novo production of mcl-PHA containing odd-numbered monomers in E. coli at shake-flask fermentation level. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of odd-chain monomers based on market demand. The synthetic pathway can provide a promising platform for production of other value-added chemicals and biomaterials that use acetyl-CoA and propionyl-CoA as versatile precursors and can be extended to other microorganisms as intelligent cell factories.

Engineering of Escherichia coli for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from glucose.

The copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has the potential to serve as a biodegradable tissue engineering material. However, the production of this kind of copolymer still suffers from high cost and uncertainty. We describe here the design of metabolic pathways to synthesize P(HB-co-HHx) directly from glucose using recombinant Escherichia coli. By combining the BktB-dependent condensation pathway with the inverted ß-oxidation cycle pathway, we were able to synthesize a P(HB-co-HHx) copolymer with a 10 mol% HHx fraction in recombinant E. coli. After optimizing the host strain and employing thioesterase mutant strains, the engineered E. coli produced 12.9 wt% P(HB-co-HHx) with a 13.2 mol% 3HHx fraction.

Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid ß-oxidation cycle in Escherichia coli.

Polyhydroxyalkanoates that contain the medium-chain-length monomers (mcl-PHAs) have a wide range of applications owing to their superior physical and mechanical properties. A challenge to synthesize such mcl-PHAs from unrelated and renewable sources is exploiting the efficient metabolic pathways that lead to the formation of precursor (R)-3-hydroxyacyl-CoA. Here, by engineering the reversed fatty acid ß-oxidation cycle, we were able to synthesize mcl-PHAs in Escherichia coli directly from glucose. After deletion of the major thioesterases, the engineered E. coli produced 6.62wt% of cell dry weight mcl-PHA heteropolymers. Furthermore, when a low-substrate-specificity PHA synthase from Pseudomonas stutzeri 1317 was employed, recombinant E. coli synthesized 12.10wt% of cell dry weight scl-mcl PHA copolymers, of which 21.18mol% was 3-hydroxybutyrate and 78.82mol% was medium-chain-length monomers. The reversed fatty acid ß-oxidation cycle offered an efficient metabolic pathway for mcl-PHA biosynthesis in E. coli and can be further optimized.

Exploring the action mechanism of Gegensan in the treatment of alcoholic liver disease based on network pharmacology and bioinformatics.

Gegensan (GGS) has been reported for the treatment of alcoholic liver disease (ALD), but its therapeutic mechanism is still unclear. This paper aims to determine the therapeutic mechanism and targets of action of GGS on alcoholic liver disease utilizing network pharmacology and bioinformatics. The active ingredients in GGS were screened in the literature and databases, and common targets of ALD were then obtained from public databases to construct the network diagram of traditional Chinese medicine-active ingredient targets. Based on the common targets, Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to find target enrichment pathways, and the core targets were screened out by combining differential analysis and protein-protein interaction network analysis. Molecular docking was performed to verify the binding effect between the core targets and the corresponding active ingredients. ALD and GGS have 84 common targets, corresponding to 91 active ingredients. After subsequent differential analysis and protein-protein interaction network analysis, 10 core targets were identified. Gene Ontology and KEGG enrichment analyses showed that the main BPs corresponding to the common targets included the response to lipopolysaccharide, inflammatory response, etc. The KEGG pathways involved in the regulation of the common targets included the lipid-atherosclerosis pathway and the alcoholic liver disease pathway, etc. Further molecular docking showed that the core targets CYP1A1, CYP1A2, CXCL8, ADH1C, MMP1, SERPINE1, COL1A1, APOB, MMP1, and their corresponding 4 active ingredients, Naringenin, Kaempferol, Quercetin, and Stigmasterol, have a greater docking potential. The above results suggest that GGS can regulate lipid metabolism and inflammatory response in the ALD process, and alleviate the lipid accumulation and oxidative stress caused by ethanol. This study analyzed the core targets and mechanisms of action of GGS on ALD, which provides certain theoretical support for the further development of GGS in the treatment of ALD, and provides a reference for the subsequent research on the treatment of ALD.

NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Reconstruction the feedback regulation of amino acid metabolism to develop a non-auxotrophic L-threonine producing Corynebacterium glutamicum.

L-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation. The GRAS (generally regarded as safe) industrial workhorse Corynebacterium glutamicum is an attractive chassis for L-threonine production. However, the present L-threonine production in C. glutamicum cannot meet the requirement of industrialization due to the relatively low production level of L-threonine and the accumulation of large amounts of by-products (such as L-lysine, L-isoleucine, and glycine). Herein, to enhance the L-threonine biosynthesis in C. glutamicum, releasing the aspartate kinase (LysC) and homoserine dehydrogenase (Hom) from feedback inhibition by L-lysine and L-threonine, respectively, and overexpressing four flux-control genes were performed. Next, to reduce the formation of by-products L-lysine and L-isoleucine without the cause of an auxotrophic phenotype, the feedback regulation of dihydrodipicolinate synthase (DapA) and threonine dehydratase (IlvA) was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by L-lysine and L-isoleucine, respectively. The resulting strain maintained the capability of synthesizing enough amounts of L-lysine and L-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids. To further enhance L-threonine production and reduce the by-product glycine, L-threonine exporter and homoserine kinase were overexpressed. Finally, the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L (17.2% higher) L-threonine with a productivity of 1.20 g/L/h (108.0% higher) in fed-batch fermentation, along with significantly reduced by-product accumulation, representing the record for L-threonine production in C. glutamicum. In this study, we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic L-threonine producing C. glutamicum. The main end by-products including L-lysine, L-isoleucine, and glycine were almost eliminated in fed-batch fermentation of the engineered C. glutamicum strain. This strategy can also be used for engineering producing strains for other amino acids and derivatives.

Polyhydroxyalkanoic acids from structurally-unrelated carbon sources in Escherichia coli.

Polyhydroxyalkanoates (PHAs) that contain varied monomers with different chain lengths/structures were normally synthesized when a structurally-related precursor was present. The biosynthesis of PHAs from unrelated carbon sources in microorganisms including Escherichia coli met many challenges in the past. Recently, with the development of metabolic engineering and synthetic biology, the production of PHAs from unrelated carbon sources obtained a breakthrough. Polyesters containing 2-hydroxypropionate, 3-hydroxypropionate, 4-hydroxybutyrate, 3-hydroxyvalarate, and medium-chain-length 3-hydroxyalkanoate monomers can all be synthesized in E. coli by integrating exogenous or endogenous pathways and/or genes. This review will summarize the progresses in this area. In addition, the strategies that lead to the production of PHAs with varied monomers and high polymer content in the cell are discussed.

Active ingredient and mechanistic analysis of traditional Chinese medicine formulas for the prevention and treatment of COVID-19: Insights from bioinformatics and in vitro experiments.

Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by a novel coronavirus. Traditional Chinese medicine (TCM) has been proven to have a potential curative effect on COVID-19. This study preliminarily analyzed the existing TCM prescription's key components and action mechanisms for preventing and treating COVID-19 using bioinformatic and experimental methods. Association and clustering analysis reveals that the "HQ + FF + BZ" drug combination had a strong correlation and confidence in 93 TCM prescriptions and may affect the progression of COVID-19 through inflammatory pathways such as the TNF signaling pathway. Further molecular docking revealed that quercetin has a higher affinity for IL6 and IL10 in the TNF signaling pathway associated with COVID-19. In vitro experiments demonstrated that quercetin could effectively reduce the levels of the inflammatory factor IL-6 and increase the anti-inflammatory factor IL-10, alleviating inflammation impact on cells. Our results provide a new understanding of the molecular mechanism of TCM prevention and treatment of COVID-19, which is helpful to the development of new diagnosis and treatment schemes for COVID-19.

Osteocalcin ameliorates cognitive dysfunctions in a mouse model of Alzheimer's Disease by reducing amyloid ß burden and upregulating glycolysis in neuroglia.

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by the accumulation of amyloid ß peptides (Aß) and impaired glucose metabolism in the brain. Osteocalcin (OCN), an osteoblast-derived protein, has been shown to modulate brain functions but whether it has any effect on AD is undetermined. In this study, daily intraperitoneal injection of OCN for 4 weeks ameliorated the anxiety-like behaviors and cognitive dysfunctions in the APP/PS1 transgenic AD mice model, as shown in the increased entries into the central area in open field test, the increased time and entries into open arms in elevated plus maze test, the increased time spent in the light chamber in light-dark transition test, as well as the reduced escape latency and the increased preference for target quadrant in Morris water maze test. Aß burden in the hippocampus and cortex of AD mice was ameliorated by OCN. Besides, OCN improved the neural network function of the brain, mainly in the enhanced power of high gamma band in the medial prefrontal cortex of AD mice. The proliferation of astrocytes in the hippocampus in AD mice was also inhibited by OCN as demonstrated by immunofluorescence. Furthermore, OCN enhanced glycolysis in astrocytes and microglia, as evidenced by elevated glucose consumption, lactate production, and increased extracellular acidification rate. Such an effect was abolished when the receptor of OCN - Gpr158 was knockdown in astrocytes. Our study revealed OCN as a novel therapeutic factor for AD potentially through reducing Aß burden and upregulation of glycolysis in neuroglia.

KLF14 regulates the growth of hepatocellular carcinoma cells via its modulation of iron homeostasis through the repression of iron-responsive element-binding protein 2.

BACKGROUND: Hepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored. METHODS: We generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis. RESULTS: KLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency. CONCLUSION: KLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization. / å¬ç¥žç»ç— ç›¸å ³AIFM1åŸºå› çªå˜å¼•èµ·å‡‹äº¡è¯±å¯¼å› å­äºŒèšä½“å½¢æˆéšœç¢å¹¶å¯¼è‡´ç»†èƒžå‡‹äº¡å¢žåŠ .

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.|T260A, p.|R422W, and p.|R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%|‒|49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%|‒|17.9%, which was significantly higher than that (6.9%|‒|7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

Impaired AIF-CHCHD4 interaction and mitochondrial calcium overload contribute to auditory neuropathy spectrum disorder in patient-iPSC-derived neurons with AIFM1 variant.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment caused by dysfunction of inner hair cells, ribbon synapses, spiral ganglion neurons and/or the auditory nerve itself. Approximately 1/7000 newborns have abnormal auditory nerve function, accounting for 10%-14% of cases of permanent hearing loss in children. Although we previously identified the AIFM1 c.1265 G > A variant to be associated with ANSD, the mechanism by which ANSD is associated with AIFM1 is poorly understood. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. The patient-specific iPSCs were edited via CRISPR/Cas9 technology to generate gene-corrected isogenic iPSCs. These iPSCs were further differentiated into neurons via neural stem cells (NSCs). The pathogenic mechanism was explored in these neurons. In patient cells (PBMCs, iPSCs, and neurons), the AIFM1 c.1265 G > A variant caused a novel splicing variant (c.1267-1305del), resulting in AIF p.R422Q and p.423-435del proteins, which impaired AIF dimerization. Such impaired AIF dimerization then weakened the interaction between AIF and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). On the one hand, the mitochondrial import of ETC complex subunits was inhibited, subsequently leading to an increased ADP/ATP ratio and elevated ROS levels. On the other hand, MICU1-MICU2 heterodimerization was impaired, leading to mCa2+ overload. Calpain was activated by mCa2+ and subsequently cleaved AIF for its translocation into the nucleus, ultimately resulting in caspase-independent apoptosis. Interestingly, correction of the AIFM1 variant significantly restored the structure and function of AIF, further improving the physiological state of patient-specific iPSC-derived neurons. This study demonstrates that the AIFM1 variant is one of the molecular bases of ANSD. Mitochondrial dysfunction, especially mCa2+ overload, plays a prominent role in ANSD associated with AIFM1. Our findings help elucidate the mechanism of ANSD and may lead to the provision of novel therapies.

1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young people. Due to heterogeneity in clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.

MAP3K1 Variant Causes Hyperactivation of Wnt4/ß-Catenin/FOXL2 Signaling Contributing to 46,XY Disorders/Differences of Sex Development.

Background: 46,XY disorders/differences of sex development (46,XY DSD) are congenital conditions that result from abnormal gonadal development (gonadal dysgenesis) or abnormalities in androgen synthesis or action. During early embryonic development, several genes are involved in regulating the initiation and maintenance of testicular or ovarian-specific pathways. Recent reports have shown that MAP3K1 genes mediate the development of the 46,XY DSD, which present as complete or partial gonadal dysgenesis. Previous functional studies have demonstrated that some MAP3K1 variants result in the gain of protein function. However, data on possible mechanisms of MAP3K1 genes in modulating protein functions remain scant. Methods: This study identified a Han Chinese family with the 46,XY DSD. To assess the history and clinical manifestations for the 46,XY DSD patients, the physical, operational, ultra-sonographical, pathological, and other examinations were performed for family members. Variant analysis was conducted using both trio whole-exome sequencing (trio WES) and Sanger sequencing. On the other hand, we generated transiently transfected testicular teratoma cells (NT2/D1) and ovary-derived granular cells (KGN), with mutant or wild-type MAP3K1 gene. We then performed functional assays such as determination of steady-state levels of gender related factors, protein interaction and luciferase assay system. Results: Two affected siblings were diagnosed with 46,XY DSD. Our analysis showed a missense c.556A > G/p.R186G variant in the MAP3K1 gene. Functional assays demonstrated that the MAP3K1R186G variant was associated with significantly decreased affinity to ubiquitin (Ub; 43-49%) and increased affinity to RhoA, which was 3.19 ± 0.18 fold, compared to MAP3K1. The MAP3K1R186G led to hyperphosphorylation of p38 and GSK3ß, and promoted hyperactivation of the Wnt4/ß-catenin signaling. In addition, there was increased recruitment of ß-catenin into the nucleus, which enhanced the expression of pro-ovarian transcription factor FOXL2 gene, thus contributing to the 46,XY DSD. Conclusion: Our study identified a missense MAP3K1 variant associated with 46,XY DSD. We demonstrated that MAP3K1R186G variant enhances binding to the RhoA and improves its own stability, resulting in the activation of the Wnt4/ß-catenin/FOXL2 pathway. Taken together, these findings provide novel insights into the molecular mechanisms of 46,XY DSD and promotes better clinical evaluation.

A novel NF2 splicing mutant causes neurofibromatosis type 2 via liquid-liquid phase separation with large tumor suppressor and Hippo pathway.

Neurofibromatosis type 2 is an autosomal dominant multiple neoplasia syndrome and is usually caused by mutations in the neurofibromin 2 (NF2) gene, which encodes a tumor suppressor and initiates the Hippo pathway. However, the mechanism by which NF2 functions in the Hippo pathway isn't fully understood. Here we identified a NF2 c.770-784del mutation from a neurofibromatosis type 2 family. MD simulations showed that this mutation significantly changed the structure of the F3 module of the NF2-FERM domain. Functional assays indicated that the NF2 c.770-784del variant formed LLPS in the cytoplasm with LATS to restrain LATS plasma membrane localization and inactivated the Hippo pathway. Besides, this deletion partly caused a skipping of exon 8 and reduced the protein level of NF2, collectively promoting proliferation and tumorigenesis of meningeal cells. We identified an unrecognized mechanism of LLPS and splicing skipping for the NF2-induced Hippo pathway, which provided new insight into the pathogenesis of neurofibromatosis type 2.

Joint transcriptomic and metabolomic analysis reveals the mechanism of low-temperature tolerance in Hosta ventricosa.

Hosta ventricosa is a robust ornamental perennial plant that can tolerate low temperatures, and which is widely used in urban landscaping design in Northeast China. However, the mechanism of cold-stress tolerance in this species is unclear. A combination of transcriptomic and metabolomic analysis was used to explore the mechanism of low-temperature tolerance in H. ventricosa. A total of 12 059 differentially expressed genes and 131 differentially expressed metabolites were obtained, which were mainly concentrated in the signal transduction and phenylpropanoid metabolic pathways. In the process of low-temperature signal transduction, possibly by transmitting Ca2+ inside and outside the cell through the ion channels on the three cell membranes of COLD, CNGCs and CRLK, H. ventricosa senses temperature changes and stimulates SCRM to combine with DREB through the MAPK signal pathway and Ca2+ signal sensors such as CBL, thus strengthening its low-temperature resistance. The pathways of phenylpropanoid and flavonoid metabolism represent the main mechanism of low-temperature tolerance in this species. The plant protects itself from low-temperature damage by increasing its content of genistein, scopolentin and scopolin. It is speculated that H. ventricosa can also adjust the content ratio of sinapyl alcohol and coniferyl alcohol and thereby alter the morphological structure of its cell walls and so increase its resistance to low temperatures.When subjected to low-temperature stress, H. ventricosa perceives temperature changes via COLD, CNGCs and CRLK, and protection from low-temperature damage is achieved by an increase in the levels of genistein, scopolentin and scopolin through the pathways of phenylpropanoid biosynthesis and flavonoid biosynthesis.

Warm white-light emitting silica films prepared using lead-free double perovskite QDs.

Lead-free double perovskite has attracted widespread attention due to its good stability and non-toxicity. In this work, Cs2AgxNa1-xInCl6 quantum dots were synthesized via a thermal injection method using non-toxic precursors. Based on the wide spectrum of self-bound excitons, the quantum dots achieved white light emission. Bi-doped Cs2AgxNa1-xInCl6 quantum dots with excellent luminescence performance have the same cubic structure, and they have a larger Stokes shift. The cubic perovskite space group is Fm3[combining macron]m, and [NaCl6], [AgCl6] and [InCl6] octahedrons alternately appear in the cubic structure. The photoluminescence quantum yield of Cs2AgxNa1-xInCl6 is improved by doping with a small amount of Bi; the PL QY increased to 57.3% with an obvious emission peak at 600 nm. The stability and luminescence intensity of perovskite QDs were further enhanced by SiO2 coating and a Cs2AgxNa1-xInCl6:Bi-SiO2 thin film was prepared using perhydropolysilazane as the precursor. The materials have huge application potential in the field of white light emission and display.