VNS improves VSMC metabolism and arteriogenesis in infarcted hearts through m/n-AChR-Akt-SDF-1α in adult male rats.

Vagal nerve stimulation (VNS) provides a novel therapeutic strategy for injured hearts by activating cholinergic anti-inflammatory pathways. However, little information is available on the metabolic pattern and arteriogenesis of VSMCs after MI. VNS has been shown to stimulate the expression of CPT1α, CPT1ß, Glut1, Glut4 and SDF-1α in coronary VSMCs, decreasing the number of CD68-positive macrophages while increasing CD206-positive macrophages in the infarcted hearts, leading to a decrease in TNF-α and IL-1ß accompanied by a reduced ratio of CD68- and CD206-positive cells, which were dramatically abolished by atropine and mecamylamine in vivo. Knockdown of SDF-1α substantially abrogated the effect of VNS on macrophagecell alteration and inflammatory factors in infarcted hearts. Mechanistically, ACh induced SDF-1α expression in VSMCs in a dose-dependent manner. Conversely, atropine, mecamylamine, and a PI3K/Akt inhibitor completely eliminated the effect of ACh on SDF-1α expression. Functionally, VNS promoted arteriogenesis and improved left ventricular performance, which could be abolished by Ad-shSDF-1α. Thus, VNS altered the VSMC metabolism pattern and arteriogenesis to repair the infarcted heart by inducing SDF-1α expression, which was associated with the m/nAChR-Akt signaling pathway.

A Dihydroartemisinin-Loaded Nanoreactor Motivates Anti-Cancer Immunotherapy by Synergy-Induced Ferroptosis to Activate Cgas/STING for Reprogramming of Macrophage.

Infiltration of tumor-associated macrophages (TAM) characterized by an M2 phenotype is an overriding feature in malignant tumors. Reprogramming TAM is the most cutting-edge strategy for cancer therapy. In the present study, an iron-based metal-organic framework (MOF) nanoreactor loaded with dihydroartemisinin (DHA) is developed, which provides high uptake by TAM and retains their viability, thus effectively addressing the inefficiency of the DHA at low concentrations. Impressively, DHA@MIL-101 can selectively accumulate in tumor tissues and remodel TAM to the M1 phenotype. The results of RNA sequencing further suggest that this nanoreactor may regulate ferroptosis, a DNA damage signaling pathway in TAM. Indeed, the outcomes confirm that DHA@MIL-101 triggers ferroptosis in TAM. In addition, the findings reveal that DNA damage induced by DHA nanoreactors activates the intracellular cGAS sensor, resulting in the binding of STING to IRF3 and thereby up-regulating the immunogenicity. In contrast, blocking ferroptosis impairs DHA@MIL-101-induced activation of STING signaling and phenotypic remodeling. Finally, it is shown that DHA nanoreactors deploy anti-tumor immunotherapy through ferroptosis-mediated TAM reprogramming. Taken together, immune efficacy is achieved through TAM's remodeling by delivering DHA and iron ions into TAM using nanoreactors, providing a novel approach for combining phytopharmaceuticals with nanocarriers to regulate the immune microenvironment.

Vagus nerve stimulation-induced stromal cell-derived factor-l alpha participates in angiogenesis and repair of infarcted hearts.

AIMS: We aim to explore the role and mechanism of vagus nerve stimulation (VNS) in coronary endothelial cells and angiogenesis in infarcted hearts. METHODS AND RESULTS: Seven days after rat myocardial infarction (MI) was prepared by ligation of the left anterior descending coronary artery, the left cervical vagus nerve was treated with electrical stimulation 1 h after intraperitoneal administration of the α7-nicotinic acetylcholine inhibitor mecamylamine or the mAChR inhibitor atropine or 3 days after local injection of Ad-shSDF-1α into the infarcted heart. Cardiac tissue acetylcholine (ACh) and serum ACh, tumour necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) levels were detected by ELISA to determine whether VNS was successful. An inflammatory injury model in human coronary artery endothelial cells (HCAECs) was established by lipopolysaccharide and identified by evaluating TNF-α, IL-1ß and IL-6 levels and tube formation. Immunohistochemistry staining was performed to evaluate CD31-positive vessel density and stromal cell-derived factor-l alpha (SDF-1α) expression in the MI heart in vivo and the expression and distribution of SDF-1α, C-X-C motif chemokine receptor 4 and CXCR7 in HCAECs in vitro. Western blotting was used to detect the levels of SDF-1α, V-akt murine thymoma viral oncogene homolog (AKT), phosphorylated AKT (pAKT), specificity protein 1 (Sp1) and phosphorylation of Sp1 in HCAECs. Left ventricular performance, including left ventricular systolic pressure, left ventricular end-diastolic pressure and rate of the rise and fall of ventricular pressure, should be evaluated 28 days after VNS treatment. VNS was successfully established for MI therapy with decreases in serum TNF-α, IL-1ß and IL-6 levels and increases in cardiac tissue and serum ACh levels, leading to increased SDF-1α expression in coronary endothelial cells of MI hearts, triggering angiogenesis of MI hearts with increased CD31-positive vessel density, which was abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF-1α by shRNA. ACh promoted SDF-1α expression and its distribution along with the branch of the formed tube in HCAECs, resulting in an increase in the number of tubes formed in HCAECs. ACh increased the levels of pAKT and phosphorylation of Sp1 in HCAECs, resulting in inducing SDF-1α expression, and the specific effects could be abolished by mecamylamine, atropine, the PI3K/AKT blocker wortmannin or the Sp1 blocker mithramycin. Functionally, VNS improved left ventricular performance, which could be abolished by Ad-shSDF-1α. CONCLUSIONS: VNS promoted angiogenesis to repair the infarcted heart by inducing SDF-1α expression and redistribution along new branches during angiogenesis, which was associated with the m/nAChR-AKT-Sp1 signalling pathway.

Modified LIX®84I-Based Polymer Inclusion Membranes for Facilitating the Transport Flux of Cu(II) and Variations of Their Physical-Chemical Characteristics.

A unique facilitation on the transport flux of Cu(II) was investigated by using modified polymer inclusion membranes (PIMs). LIX®84I-based polymer inclusion membranes (LIX®-based PIMs) using poly(vinyl chloride) (PVC) as support, 2-nitrophenyl octyl ether (NPOE) as plasticizer and Lix84I as carrier were modified by reagents with different polar groups. The modified LIX®-based PIMs showed an increasing transport flux of Cu(II) with the help of ethanol or Versatic acid 10 modifiers. The metal fluxes with the modified LIX®-based PIMs were observed varying with the amount of modifiers, and the transmission time was cut by half for the modified LIX®-based PIM cast with Versatic acid 10. The physical-chemical characteristics of the prepared blank PIMs with different Versatic acid 10 were further characterized by using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), contract angle measurements and electro-chemical impedance spectroscopy (EIS). The characterization results indicated that the modified LIX®-based PIMs cast with Versatic acid 10 appeared to be more hydrophilic with increasing membrane dielectric constant and electrical conductivity that allowed better accessibility of Cu(II) across PIMs. Hence, it was deduced that hydrophilic modification might be a potential method to improve the transport flux of the PIM system.

The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

BACKGROUND: Clinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells. METHOD: A gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation. RESULTS: For the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iß levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion. CONCLUSIONS: The spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F.

NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress.

Doxorubicin (DOX) is a commonly used antitumor drug, but its application has been limited because of its strong cardiac damage. This study aims to explore the role of NSUN2 in DOX-induced heart injury. C57BL/6J mice were intraperitoneally injected with 20 mg/Kg DOX to induce heart injury. After 3 days, the cardiac function, cardiac histopathology, myocardial apoptosis, and the expression level of NSUN2 were detected. In vitro, H9C2 cells were transfected with NSUN2 siRNA or overexpressed lentivirus and then treated with 500 ng/ml DOX. After 24 h, the changes in reactive oxygen species (ROS), apoptosis, and NSUN2 expression were detected. After DOX treatment, both in vitro and in vivo experiments showed that the cardiac function decreased, the number of apoptotic cells increased, and the expression level of NSUN2 increased. Interfering the expression of NSUN2 by siRNA promoted DOX-induced heart injury, while overexpression of NSUN2 could inhibit DOX-induced heart injury. Further study showed that NSUN2 promoted antioxidative stress by upregulating the Nrf2 protein level. In addition, NSUN2 overexpression could increase the half-life of Nrf2 mRNA. m5C RNA methylation immunoprecipitation (MeRIP) also showed that the level of Nrf2 m5C mRNA was significantly increased in NSUN2 overexpressed group when compared to the GFP group. NSUN2 enhances the expression of Nrf2 by promoting Nrf2 mRNA m5C modification and enhances its antioxidative stress effect to alleviate DOX-induced myocardial injury.

Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

We aimed to explore whether superfluous sympathetic activity affects myoblast differentiation, fusion, and myofiber types using a continuous single-dose isoprenaline exposure model in vitro and to further confirm the role of distinct NFATs in ISO-mediated effects. Compared with delivery of single and interval single, continuous single-dose ISO most obviously diminished myotube size while postponing myoblast differentiation/fusion in a time- and dose-dependent pattern, accompanied by an apparent decrease in nuclear NFATc1/c2 levels and a slight increase in nuclear NFATc3/c4 levels. Overexpression of NFATc1 or NFATc2, particularly NFATc1, markedly abolished the inhibitory effects of ISO on myoblast differentiation/fusion, myotube size and Myh7 expression, which was attributed to a remarkable increase in the nuclear NFATc1/c2 levels and a reduction in the nuclear NFATc4 levels and the associated increase in the numbers of MyoG and MEF2C positive nuclei within more than 3 nuclei myotubes, especially in MEF2C. Moreover, knockdown of NFATc3 by shRNA did not alter the inhibitory effect of ISO on myoblast differentiation/fusion or myotube size but partially recovered the expression of Myh7, which was related to the slightly increased nuclear levels of NFATc1/c2, MyoG and MEF2C. Knockdown of NFATc4 by shRNA prominently increased the number of MyHC +, MyoG or MEF2C + myoblast cells with 1 ~ 2 nuclei, causing fewer numbers and smaller myotube sizes. However, NFATc4 knockdown further deteriorated the effects of ISO on myoblast fusion and myotube size, with more than 5 nuclei and Myh1/2/4 expression, which was associated with a decrease in nuclear NFATc2/c3 levels. Therefore, ISO inhibited myoblast differentiation/fusion and myotube size through the NFAT-MyoG-MEF2C signaling pathway.

ANGPTL8 is a negative regulator in pathological cardiac hypertrophy.

Pathological cardiac hypertrophy is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the role of angiopoietin-like protein 8 (ANGPTL8) in pathological cardiac hypertrophy. We found that serum ANGPTL8 levels were significantly increased in hypertensive patients with cardiac hypertrophy and in mice with cardiac hypertrophy induced by Ang II or TAC. Furthermore, the secretion of ANGPTL8 from the liver was increased during hypertrophic processes, which were triggered by Ang II. In the Ang II- and transverse aortic constriction (TAC)-induced mouse cardiac hypertrophy model, ANGPTL8 deficiency remarkably accelerated cardiac hypertrophy and fibrosis with deteriorating cardiac dysfunction. Accordingly, both recombinant human full-length ANGPTL8 (rANGPTL8) protein and ANGPTL8 overexpression significantly mitigated Ang II-induced cell enlargement in primary neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Mechanistically, the antihypertrophic effects of ANGPTL8 depended on inhibiting Akt and GSK-3ß activation, and the Akt activator SC-79 abolished the antihypertrophic effects of rANGPTL8 in vitro. Moreover, we demonstrated that ANGPTL8 directly bound to the paired Ig-like receptor PIRB (LILRB3) by RNA-seq and immunoprecipitation-mass screening. Remarkably, the antihypertrophic effects of ANGPTL8 were largely blocked by anti-LILRB3 and siRNA-LILRB3. Our study indicated that ANGPTL8 served as a novel negative regulator of pathological cardiac hypertrophy by binding to LILRB3 (PIRB) and inhibiting Akt/GSK3ß activation, suggesting that ANGPTL8 may provide synergistic effects in combination with AT1 blockers and become a therapeutic target for cardiac hypertrophy and heart failure.

ATG7-mediated autophagy facilitates embryonic stem cell exit from naive pluripotency and marks commitment to differentiation.

Macroautophagy/autophagy is a conserved cellular mechanism to degrade unneeded cytoplasmic proteins and organelles to recycle their components, and it is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Whereas autophagy is essential for early development of embryos, no information exists regarding its functions during the transition from naive-to-primed pluripotency. Here, by using an in vitro transition model of ESCs to epiblast-like cells (EpiLCs), we find that dynamic changes in ATG7-dependent autophagy are critical for the naive-to-primed transition, and are also necessary for germline specification. RNA-seq and ATAC-seq profiling reveal that NANOG acts as a barrier to prevent pluripotency transition, and autophagy-dependent NANOG degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers. Mechanistically, we found that autophagy receptor protein SQSTM1/p62 translocated into the nucleus during the pluripotency transition period and is preferentially associated with K63 ubiquitinated NANOG for selective protein degradation. In vivo, loss of autophagy by ATG7 depletion disrupts peri-implantation development and causes increased chromatin association of NANOG, which affects neuronal differentiation by competitively binding to OTX2-specific neuroectodermal development-associated regions. Taken together, our findings reveal that autophagy-dependent degradation of NANOG plays a critical role in regulating exit from the naive state and marks distinct cell fate allocation during lineage specification.Abbreviations: 3-MA: 3-methyladenine; EpiLC: epiblast-like cell; ESC: embryonic stem cell; PGC: primordial germ cell.

Platelets are highly efficient and efficacious carriers for tumor-targeted nano-drug delivery.

The present work aims to prove the concept of tumor-targeted drug delivery mediated by platelets. Doxorubicin (DOX) attached to nanodiamonds (ND-DOX) was investigated as the model payload drug of platelets. In vitro experiments first showed that ND-DOX could be loaded in mouse platelets in a dose-dependent manner with a markedly higher efficiency and capacity than free DOX. ND-DOX-loaded platelets (Plt@ND-DOX) maintained viability and ND-DOX could be stably held in the platelets for at least 4 hr. Next, mouse Lewis lung cancer cells were found to activate Plt@ND-DOX and thereby stimulate cargo unloading of Plt@ND-DOX. The unloaded ND-DOX was taken up by co-cultured cancer cells which consequently exhibited loss of viability, proliferation suppression and apoptosis. In vivo, Plt@ND-DOX displayed significantly prolonged blood circulation time over ND-DOX and DOX in mice, and Lewis tumor grafts demonstrated infiltration, activation and cargo unloading of Plt@ND-DOX in the tumor tissue. Consequently, Plt@ND-DOX effectively reversed the growth of Lewis tumor grafts which exhibited significant inhibition of cell proliferation and apoptosis. Importantly, Plt@ND-DOX displayed a markedly higher therapeutic potency than free DOX but without the severe systemic toxicity associated with DOX. Our findings are concrete proof of platelets as efficient and efficacious carriers for tumor-targeted nano-drug delivery with the following features: 1) large loading capacity and high loading efficiency, 2) good tolerance of cargo drug, 3) stable cargo retention and no cargo unloading in the absence of stimulation, 4) prolonged blood circulation time, and 5) excellent tumor distribution and tumor-activated drug unloading leading to high therapeutic potency and few adverse effects. Platelets hold great potential as efficient and efficacious carriers for tumor-targeted nano-drug delivery.

Patient-specific iPSC-derived cardiomyocytes reveal abnormal regulation of FGF16 in a familial atrial septal defect.

AIMS: Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD. METHODS AND RESULTS: Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time. CONCLUSIONS: In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Histone Lysine Methylation and Long Non-Coding RNA: The New Target Players in Skeletal Muscle Cell Regeneration.

Satellite stem cell availability and high regenerative capacity have made them an ideal therapeutic approach for muscular dystrophies and neuromuscular diseases. Adult satellite stem cells remain in a quiescent state and become activated upon muscular injury. A series of molecular mechanisms succeed under the control of epigenetic regulation and various myogenic regulatory transcription factors myogenic regulatory factors, leading to their differentiation into skeletal muscles. The regulation of MRFs via various epigenetic factors, including DNA methylation, histone modification, and non-coding RNA, determine the fate of myogenesis. Furthermore, the development of histone deacetylation inhibitors (HDACi) has shown promising benefits in their use in clinical trials of muscular diseases. However, the complete application of using satellite stem cells in the clinic is still not achieved. While therapeutic advancements in the use of HDACi in clinical trials have emerged, histone methylation modulations and the long non-coding RNA (lncRNA) are still under study. A comprehensive understanding of these other significant epigenetic modulations is still incomplete. This review aims to discuss some of the current studies on these two significant epigenetic modulations, histone methylation and lncRNA, as potential epigenetic targets in skeletal muscle regeneration. Understanding the mechanisms that initiate myoblast differentiation from its proliferative state to generate new muscle fibres will provide valuable information to advance the field of regenerative medicine and stem cell transplant.

Common mtDNA variations at C5178a and A249d/T6392C/G10310A decrease the risk of severe COVID-19 in a Han Chinese population from Central China.

BACKGROUND: Mitochondria have been shown to play vital roles during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) development. Currently, it is unclear whether mitochondrial DNA (mtDNA) variants, which define mtDNA haplogroups and determine oxidative phosphorylation performance and reactive oxygen species production, are associated with COVID-19 risk. METHODS: A population-based case-control study was conducted to compare the distribution of mtDNA variations defining mtDNA haplogroups between healthy controls (n = 615) and COVID-19 patients (n = 536). COVID-19 patients were diagnosed based on molecular diagnostics of the viral genome by qPCR and chest X-ray or computed tomography scanning. The exclusion criteria for the healthy controls were any history of disease in the month preceding the study assessment. MtDNA variants defining mtDNA haplogroups were identified by PCR-RFLPs and HVS-I sequencing and determined based on mtDNA phylogenetic analysis using Mitomap Phylogeny. Student's t-test was used for continuous variables, and Pearson's chi-squared test or Fisher's exact test was used for categorical variables. To assess the independent effect of each mtDNA variant defining mtDNA haplogroups, multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) with adjustments for possible confounding factors of age, sex, smoking and diseases (including cardiopulmonary diseases, diabetes, obesity and hypertension) as determined through clinical and radiographic examinations. RESULTS: Multivariate logistic regression analyses revealed that the most common investigated mtDNA variations (> 10% in the control population) at C5178a (in NADH dehydrogenase subunit 2 gene, ND2) and A249d (in the displacement loop region, D-loop)/T6392C (in cytochrome c oxidase I gene, CO1)/G10310A (in ND3) were associated with a reduced risk of severe COVID-19 (OR = 0.590, 95% CI 0.428-0.814, P = 0.001; and OR = 0.654, 95% CI 0.457-0.936, P = 0.020, respectively), while A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) were related to an increased risk of severe COVID-19 (OR = 2.336, 95% CI 1.179-4.608, P = 0.015; OR = 2.033, 95% CI 1.242-3.322, P = 0.005; OR = 3.040, 95% CI 1.522-6.061, P = 0.002; and OR = 2.890, 95% CI 1.199-6.993, P = 0.018, respectively). CONCLUSIONS: This is the first study to explore the association of mtDNA variants with individual's risk of developing severe COVID-19. Based on the case-control study, we concluded that the common mtDNA variants at C5178a and A249d/T6392C/G10310A might contribute to an individual's resistance to developing severe COVID-19, whereas A4833G, A4715G, T3394C and G5417A/C16257a/C16261T might increase an individual's risk of developing severe COVID-19.

The interaction between PDCD4 and YB1 is critical for cervical cancer stemness and cisplatin resistance.

Cancer multidrug resistance (MDR) is existence in stem cell-like cancer cells characterized by stemness including high-proliferation and self-renewal. Programmed cell death 4 (PDCD4), as a proapoptotic gene, whether it engaged in cancer stemness and cisplatin resistance is still unknown. Here we showed that PDCD4 expressions in Hela/DDP (cisplatin resistance) cells were lower than in parental Hela cells. Moreover, the levels of drug resistance genes and typical stemness markers were markedly elevated in Hela/DDP cells. In vivo, xenograft tumor assay confirmed that knockdown of PDCD4 accelerated the grafted tumor growth. In vitro, colony formation and MTT assay demonstrated that PDCD4 overexpression inhibited cells proliferation in conditions with or without cisplatin. By contrast, PDCD4 deficiency provoked cell proliferation and cisplatin resistance. On mechanism, PDCD4 decreased the protein levels of pAKT and pYB1, accompanied by reduced MDR1 expression. Correspondingly, luciferase reporter assay showed PDCD4 regulated MDR1 promoter activity entirely relied on YB1. Furthermore, Ch-IP, GST-pulldown, and Co-IP assays provided novel evidence that PDCD4 could directly bind with YB1 by the nucleolar localization signal (NOLS) segment, causing the reduced YB1 binding into the MDR1 promoter region through blocking YB1 nucleus translocation, triggering the decreased MDR1 transcription. Taken together, PDCD4-pAKT-pYB1 forms the integrated molecular network to regulate MDR1 transcription during the process of stemness-associated cisplatin resistance.

Efficient Delivery of Chlorin e6 by Polyglycerol-Coated Iron Oxide Nanoparticles with Conjugated Doxorubicin for Enhanced Photodynamic Therapy of Melanoma.

Chlorin e6 (Ce6) is a promising photosensitizer for tumor photodynamic therapy (PDT). However, the efficacy of Ce6 PDT is limited by Ce6's poor water solubility, rapid blood clearance, and inadequate accumulation in the tumor tissue. This problem is tackled in this work, wherein functionalized superparamagnetic iron oxide nanoparticles (IO-NPs) were used as carriers to deliver Ce6 to melanoma. The IO-NPs were coated with polyglycerol (PG) to afford good aqueous solubility. The chemotherapeutic agent doxorubicin (DOX) was attached to the PG coating via the hydrazone bond to afford affinity to the cell membrane and thereby promote the cell uptake. The hydrophobic nature of DOX also induced the aggregation of IO-NPs to form nanoclusters. Ce6 was then loaded onto the IO nanoclusters through physical adsorption and coordination with surface iron atoms, yielding the final composites IO-PG-DOX-Ce6. In vitro experiments showed that IO-PG-DOX-Ce6 markedly increased Ce6 uptake in mouse melanoma cells, leading to much-enhanced photocytotoxicity characterized by intensified reactive oxygen species production, loss of viability, DNA damage, and stimulation of tumor cell immunogenicity. In vivo experiments corroborated the in vitro findings and demonstrated prolonged blood clearance of IO-PG-DOX-Ce6. Importantly, IO-PG-DOX-Ce6 markedly increased the Ce6 distribution and retention in mouse subcutaneous melanoma grafts and significantly improved the efficacy of Ce6-mediated PDT. No apparent vital organ damage was observed at the same time. In conclusion, the IO-PG-DOX NPs provide a simple and safe delivery platform for efficient tumor enrichment of Ce6, thereby enhancing antimelanoma PDT.

Sensorineural Hearing Loss and Mitochondrial Apoptosis of Cochlear Spiral Ganglion Neurons in Fibroblast Growth Factor 13 Knockout Mice.

Deafness is known to occur in more than 400 syndromes and accounts for almost 30% of hereditary hearing loss. The molecular mechanisms underlying such syndromic deafness remain unclear. Furthermore, deafness has been a common feature in patients with three main syndromes, the BÖrjeson-Forssman-Lehmann syndrome, Wildervanck syndrome, and Congenital Generalized Hirsutism, all of which are characterized by loss-of-function mutations in the Fgf13 gene. Whether the pathogenesis of deafness in these syndromes is associated with the Fgf13 mutation is not known. To elucidate its role in auditory function, we generated a mouse line with conditional knockout of the Fgf13 gene in the inner ear (Fgf13 cKO). FGF13 is expressed predominantly in the organ of Corti, spiral ganglion neurons (SGNs), stria vascularis, and the supporting cells. Conditional knockout of the gene in the inner ear led to sensorineural deafness with low amplitude and increased latency of wave I in the auditory brainstem response test but had a normal distortion product otoacoustic emission threshold. Fgf13 deficiency resulted in decreased SGN density from the apical to the basal region without significant morphological changes and those in the number of hair cells. TUNEL and caspase-3 immunocytochemistry assays showed that apoptotic cell death mediated the loss of SGNs. Further detection of apoptotic factors through qRT-PCR suggested the activation of the mitochondrial apoptotic pathway in SGNs. Together, this study reveals a novel role for Fgf13 in auditory function, and indicates that the gene could be a potential candidate for understanding deafness. These findings may provide new perspectives on the molecular mechanisms and novel therapeutic targets for treatment deafness.

Ameliorated biomechanical properties of carotid arteries by puerarin in spontaneously hypertensive rats.

BACKGROUND: An emerging body of evidence indicates that puerarin (PUE) plays an important role in the treatment of angina pectoris, myocardial ischemia-reperfusion injury, hypertension and other cardiovascular diseases, but how PUE affects the vascular remodeling of hypertensive rats has not been reported yet. This study aimed to investigate the effect and mechanism of PUE on carotid arteries of spontaneously hypertensive rats (SHR) to provide the basis for the clinical application of PUE. METHODS: Thirty male SHR and six male Wistar Kyoto rats (WKY) aged 3 months were used in this study, SHR rats were randomly divided into 5 groups, PUE(40 or 80 mg/kg/d, ip) and telmisartan (TELMI) (30 mg/kg/d, ig) were administrated for 3 months. We use DMT myography pressure-diameter system to investigate biomechanical properties of carotid arteries, 10 µM pan-classical transient receptor potential channels (TRPCs) inhibitor SKF96365, 200 nM specific TRPC6 inhibitor SAR7334 and 100 µM Orai1 inhibitor ANCOA4 were used in the mechanical test. RESULTS: PUE can significantly decrease systolic and diastolic blood pressure, long-term administration of PUE resulted in a mild reduction of thickness and inner diameter of carotid artery. PUE ameliorate NE-response and vascular remodeling mainly through inhibiting TRPCs channel activities of VSMC. CONCLUSION: PUE can ameliorate biomechanical remodeling of carotid arteries through inhibiting TRPCs channel activities of VSMC in spontaneously hypertensive rats.

Establishment and characterization of a human embryonic stem cell line carrying a heterozygous GATA4T280M mutation.

Heterozygous T280M mutation in the GATA4 gene, encoding GATA binding protein 4, was recently identified in patients with congenital heart disease (CHD). Here, a human embryonic stem cell line carrying a heterozygous GATA4T280M mutation was generated by using the CRISPR/Cas9 and piggyBac technologies. This GATA4T280M cell line remains normal morphology, pluripotency and karyotype, and could differentiate into all three germ layers both in vivo and in vitro. Thus, the GATA4T280M cell line will be a useful platform to model GATA4-associated CHD through the induction of cardiomyocyte differentiation in vitro.

Gene variations in autism spectrum disorder are associated with alteration of gut microbiota, metabolites and cytokines.

The genetic variations and dysbiosis of gut microbiota are associated with ASD. However, the role of the microbiota in the etiology of ASD in terms of host genetic susceptibility remains unclear. This study aims to systematically explore the interplay between host genetic variation and gut microbiota in ASD children. Whole-exon sequencing was applied to 26 ASD children and 26 matched controls to identify the single nucleotide variations (SNVs) in ASD. Our previous study revealed alteration in gut microbiota and disorder of metabolism activity in ASD for this cohort. Systematic bioinformatic analyses were further performed to identify associations between SNVs and gut microbiota, as well as their metabolites. The ASD SNVs were significantly enriched in genes associated with innate immune response, protein glycosylation process, and retrograde axonal transport. These SNVs were also correlated with the microbiome composition and a broad aspect of microbial functions, especially metabolism. Additionally, the abundance of metabolites involved in the metabolic network of neurotransmitters was inferred to be causally related to specific SNVs and microbes. Furthermore, our data suggested that the interaction of host genetics and gut microbes may play a crucial role in the immune and metabolism homeostasis of ASD. This study may provide valuable clues to investigate the interaction of host genetic variations and gut microbiota in the pathogenesis of ASD.