Efficacy and Safety of an mRNA-Based RSV PreF Vaccine in Older Adults.

BACKGROUND: Respiratory syncytial virus (RSV) can cause substantial morbidity and mortality among older adults. An mRNA-based RSV vaccine, mRNA-1345, encoding the stabilized RSV prefusion F glycoprotein, is under clinical investigation. METHODS: In this ongoing, randomized, double-blind, placebo-controlled, phase 2-3 trial, we randomly assigned, in a 1:1 ratio, adults 60 years of age or older to receive one dose of mRNA-1345 (50 µg) or placebo. The two primary efficacy end points were the prevention of RSV-associated lower respiratory tract disease with at least two signs or symptoms and with at least three signs or symptoms. A key secondary efficacy end point was the prevention of RSV-associated acute respiratory disease. Safety was also assessed. RESULTS: Overall, 35,541 participants were assigned to receive the mRNA-1345 vaccine (17,793 participants) or placebo (17,748). The median follow-up was 112 days (range, 1 to 379). The primary analyses were conducted when at least 50% of the anticipated cases of RSV-associated lower respiratory tract disease had occurred. Vaccine efficacy was 83.7% (95.88% confidence interval [CI], 66.0 to 92.2) against RSV-associated lower respiratory tract disease with at least two signs or symptoms and 82.4% (96.36% CI, 34.8 to 95.3) against the disease with at least three signs or symptoms. Vaccine efficacy was 68.4% (95% CI, 50.9 to 79.7) against RSV-associated acute respiratory disease. Protection was observed against both RSV subtypes (A and B) and was generally consistent across subgroups defined according to age and coexisting conditions. Participants in the mRNA-1345 group had a higher incidence than those in the placebo group of solicited local adverse reactions (58.7% vs. 16.2%) and of systemic adverse reactions (47.7% vs. 32.9%); most reactions were mild to moderate in severity and were transient. Serious adverse events occurred in 2.8% of the participants in each trial group. CONCLUSIONS: A single dose of the mRNA-1345 vaccine resulted in no evident safety concerns and led to a lower incidence of RSV-associated lower respiratory tract disease and of RSV-associated acute respiratory disease than placebo among adults 60 years of age or older. (Funded by Moderna; ConquerRSV ClinicalTrials.gov number, NCT05127434.).

A new variant of the colistin resistance gene MCR-1 with co-resistance to ß-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to ß-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to ß-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

USP28 Serves as a Key Suppressor of Mitochondrial Morphofunctional Defects and Cardiac Dysfunction in the Diabetic Heart.

BACKGROUND: The majority of people with diabetes are susceptible to cardiac dysfunction and heart failure, and conventional drug therapy cannot correct diabetic cardiomyopathy progression. Herein, we assessed the potential role and therapeutic value of USP28 (ubiquitin-specific protease 28) on the metabolic vulnerability of diabetic cardiomyopathy. METHODS: The type 2 diabetes mouse model was established using db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced mice. Cardiac-specific knockout of USP28 in the db/db background mice was generated by crossbreeding db/m and Myh6-Cre+/USP28fl/fl mice. Recombinant adeno-associated virus serotype 9 carrying USP28 under cardiac troponin T promoter was injected into db/db mice. High glucose plus palmitic acid-incubated neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes were used to imitate diabetic cardiomyopathy in vitro. The molecular mechanism was explored through RNA sequencing, immunoprecipitation and mass spectrometry analysis, protein pull-down, chromatin immunoprecipitation sequencing, and chromatin immunoprecipitation assay. RESULTS: Microarray profiling of the UPS (ubiquitin-proteasome system) on the basis of db/db mouse hearts and diabetic patients' hearts demonstrated that the diabetic ventricle presented a significant reduction in USP28 expression. Diabetic Myh6-Cre+/USP28fl/fl mice exhibited more severe progressive cardiac dysfunction, lipid accumulation, and mitochondrial disarrangement, compared with their controls. On the other hand, USP28 overexpression improved systolic and diastolic dysfunction and ameliorated cardiac hypertrophy and fibrosis in the diabetic heart. Adeno-associated virus serotype 9-USP28 diabetic mice also exhibited less lipid storage, reduced reactive oxygen species formation, and mitochondrial impairment in heart tissues than adeno-associated virus serotype 9-null diabetic mice. As a result, USP28 overexpression attenuated cardiac remodeling and dysfunction, lipid accumulation, and mitochondrial impairment in high-fat diet/streptozotocin-induced type 2 diabetes mice. These results were also confirmed in neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes. RNA sequencing, immunoprecipitation and mass spectrometry analysis, chromatin immunoprecipitation assays, chromatin immunoprecipitation sequencing, and protein pull-down assay mechanistically revealed that USP28 directly interacted with PPARα (peroxisome proliferator-activated receptor α), deubiquitinating and stabilizing PPARα (Lys152) to promote Mfn2 (mitofusin 2) transcription, thereby impeding mitochondrial morphofunctional defects. However, such cardioprotective benefits of USP28 were largely abrogated in db/db mice with PPARα deletion and conditional loss-of-function of Mfn2. CONCLUSIONS: Our findings provide a USP28-modulated mitochondria homeostasis mechanism that involves the PPARα-Mfn2 axis in diabetic hearts, suggesting that USP28 activation or adeno-associated virus therapy targeting USP28 represents a potential therapeutic strategy for diabetic cardiomyopathy.

Cytonuclear Interactions and Subgenome Dominance Shape the Evolution of Organelle-Targeted Genes in the Brassica Triangle of U.

The interaction and coevolution between nuclear and cytoplasmic genomes are one of the fundamental hallmarks of eukaryotic genome evolution and, 2 billion yr later, are still major contributors to the formation of new species. Although many studies have investigated the role of cytonuclear interactions following allopolyploidization, the relative magnitude of the effect of subgenome dominance versus cytonuclear interaction on genome evolution remains unclear. The Brassica triangle of U features 3 diploid species that together have formed 3 separate allotetraploid species on similar evolutionary timescales, providing an ideal system for understanding the contribution of the cytoplasmic donor to hybrid polyploid. Here, we investigated the evolutionary pattern of organelle-targeted genes in Brassica carinata (BBCC) and 2 varieties of Brassica juncea (AABB) at the whole-genome level, with particular focus on cytonuclear enzyme complexes. We found partial evidence that plastid-targeted genes experience selection to match plastid genomes, but no obvious corresponding signal in mitochondria-targeted genes from these 2 separately formed allopolyploids. Interestingly, selection acting on plastid genomes always reduced the retention rate of plastid-targeted genes encoded by the B subgenome, regardless of whether the Brassica nigra (BB) subgenome was contributed by the paternal or maternal progenitor. More broadly, this study illustrates the distinct selective pressures experienced by plastid- and mitochondria-targeted genes, despite a shared pattern of inheritance and natural history. Our study also highlights an important role for subgenome dominance in allopolyploid genome evolution, even in genes whose function depends on separately inherited molecules.

Humoral Immunogenicity of mRNA-1345 RSV Vaccine in Older Adults.

BACKGROUND: The mRNA-1345 vaccine demonstrated efficacy against RSV disease with acceptable safety in adults ≥60 years in the ConquerRSV trial. Here, humoral immunogenicity results from the trial are presented. METHODS: This phase 2/3 trial randomly assigned adults (≥60 years) to mRNA-1345 50-µg encoding prefusion F (preF) glycoprotein (n = 17,793) vaccine or placebo (n = 17,748). RSV-A and RSV-B neutralizing antibody (nAb) and preF binding antibody (bAb) levels at baseline and day 29 post-vaccination were assessed in a per-protocol immunogenicity subset ([PPIS]; mRNA-1345, n = 1515; placebo, n = 333). RESULTS: Day 29 nAb geometric mean titers (GMTs) increased 8.4-fold against RSV-A and 5.1-fold against RSV-B from baseline. Seroresponses (4-fold rise from baseline) in the mRNA-1345 groups were 74.2% and 56.5% for RSV-A and RSV-B, respectively. Baseline GMTs were lower among participants who met the seroresponse criteria than those who did not. mRNA-1345 induced preF bAbs at day 29, with a pattern similar to nAbs. Day 29 antibody responses across demographic and risk subgroups were generally consistent with the overall PPIS. CONCLUSION: mRNA-1345 enhanced RSV-A and RSV-B nAbs and preF bAbs in adults (≥60 years) across various subgroups, including those at risk for severe disease, consistent with its demonstrated efficacy in the prevention of RSV disease.

Controllable Synthesis of Transferable Ultrathin Bi2Ge(Si)O5 Dielectric Alloys with Composition-Tunable High-κ Properties.

Two-dimensional (2D) alloys hold great promise to serve as important components of 2D transistors, since their properties allow continuous regulation by varying their compositions. However, previous studies are mainly limited to the metallic/semiconducting ones as contact/channel materials, but very few are related to the insulating dielectrics. Here, we use a facile one-step chemical vapor deposition (CVD) method to synthesize ultrathin Bi2SixGe1-xO5 dielectric alloys, whose composition is tunable over the full range of x just by changing the relative ratios of the GeO2/SiO2 precursors. Moreover, their dielectric properties are highly composition-tunable, showing a record-high dielectric constant of >40 among CVD-grown 2D insulators. The vertically grown nature of Bi2GeO5 and Bi2SixGe1-xO5 enables polymer-free transfer and subsequent clean van der Waals integration as the high-κ encapsulation layer to enhance the mobility of 2D semiconductors. Besides, the MoS2 transistors using Bi2SixGe1-xO5 alloy as gate dielectrics exhibit a large Ion/Ioff (>108), ideal subthreshold swing of ∼61 mV/decade, and a small gate hysteresis (∼5 mV). Our work not only gives very few examples on controlled CVD growth of insulating dielectric alloys but also expands the family of 2D single-crystalline high-κ dielectrics.

LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor α degradation and LITAF activation.

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.

Novel mitophagy inducer alleviates lupus nephritis by reducing myeloid cell activation and autoantigen presentation.

Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE), but its mechanism of onset remains unclear. Since impaired mitophagy has been implicated in multiple organs in SLE, we hypothesized that mitophagy dysfunction is critical in the development of LN and that pharmacologically targeting mitophagy would ameliorate this disease. Therefore, lupus-prone MRL/MpJ-Faslpr (MRL/lpr) and NZBWF1/J mice were treated with a novel mitophagy inducer, UMI-77, during their onset of LN. This treatment effectively mitigated kidney inflammation and damage as assessed by histology and flow cytometry. Furthermore, dendritic cell (DC)-T-cell coculture assay indicated that UMI-77 treatment attenuated DC function that would drive T-cell proliferation but did not directly influence the potent T-cell proliferation in lupus mice. UMI-77 also restored mitochondrial function and attenuated proinflammatory phenotypes in lupus DCs. Adoptive transfer of DCs from MRL/lpr mice augmented serum anti-dsDNA IgG, urine protein and T-cell infiltration of the kidney in MRL/MpJ mice, which could be prevented by either treating lupus donors in vivo or lupus DCs directly with UMI-77. UMI-77 also restored mitochondrial function in myeloid cells from patients with LN in vitro as evidenced by increased ATP levels. Thus, enhancing mitophagy in SLE restrains autoimmunity and limits kidney inflammation for LN development. Hence, our findings suggest targeting mitophagy as a tangible pathway to treat LN.

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

Clinical and genetic architecture of a large cohort with auditory neuropathy.

Auditory neuropathy (AN) is a unique type of language developmental disorder, with no precise rate of genetic contribution that has been deciphered in a large cohort. In a retrospective cohort of 311 patients with AN, pathogenic and likely pathogenic variants of 23 genes were identified in 98 patients (31.5% in 311 patients), and 14 genes were mutated in two or more patients. Among subgroups of patients with AN, the prevalence of pathogenic and likely pathogenic variants was 54.4% and 56.2% in trios and families, while 22.9% in the cases with proband-only; 45.7% and 25.6% in the infant and non-infant group; and 33.7% and 0% in the bilateral and unilateral AN cases. Most of the OTOF gene (96.6%, 28/29) could only be identified in the infant group, while the AIFM1 gene could only be identified in the non-infant group; other genes such as ATP1A3 and OPA1 were identified in both infant and non-infant groups. In conclusion, genes distribution of AN, with the most common genes being OTOF and AIFM1, is totally different from other sensorineural hearing loss. The subgroups with different onset ages showed different genetic spectrums, so did bilateral and unilateral groups and sporadic and familial or trio groups.

HTRA1-driven detachment of type I collagen from endoplasmic reticulum contributes to myocardial fibrosis in dilated cardiomyopathy.

BACKGROUND: The aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear. METHODS AND RESULTS: RNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson's analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM. CONCLUSIONS: The findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion.

Toxoplasma gondii Induces Pyroptosis in Human Placental Trophoblast and Amniotic Cells by Inducing ROS Production and Activation of Cathepsin B and NLRP1/NLRP3/NLRC4/AIM2 Inflammasome.

Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1ß and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.

Association between different insulin resistance surrogates and all-cause mortality in patients with coronary heart disease and hypertension: NHANES longitudinal cohort study.

BACKGROUND: Studies on the relationship between insulin resistance (IR) surrogates and long-term all-cause mortality in patients with coronary heart disease (CHD) and hypertension are lacking. This study aimed to explore the relationship between different IR surrogates and all-cause mortality and identify valuable predictors of survival status in this population. METHODS: The data came from the National Health and Nutrition Examination Survey (NHANES 2001-2018) and National Death Index (NDI). Multivariate Cox regression and restricted cubic splines (RCS) were performed to evaluate the relationship between homeostatic model assessment of IR (HOMA-IR), triglyceride glucose index (TyG index), triglyceride glucose-body mass index (TyG-BMI index) and all-cause mortality. The recursive algorithm was conducted to calculate inflection points when segmenting effects were found. Then, segmented Kaplan-Meier analysis, LogRank tests, and multivariable Cox regression were carried out. Receiver operating characteristic (ROC) and calibration curves were drawn to evaluate the differentiation and accuracy of IR surrogates in predicting the all-cause mortality. Stratified analysis and interaction tests were conducted according to age, gender, diabetes, cancer, hypoglycemic and lipid-lowering drug use. RESULTS: 1126 participants were included in the study. During the median follow-up of 76 months, 455 participants died. RCS showed that HOMA-IR had a segmented effect on all-cause mortality. 3.59 was a statistically significant inflection point. When the HOMA-IR was less than 3.59, it was negatively associated with all-cause mortality [HR = 0.87,95%CI (0.78, 0.97)]. Conversely, when the HOMA-IR was greater than 3.59, it was positively associated with all-cause mortality [HR = 1.03,95%CI (1.00, 1.05)]. ROC and calibration curves indicated that HOMA-IR was a reliable predictor of survival status (area under curve = 0,812). No interactions between HOMA-IR and stratified variables were found. CONCLUSION: The relationship between HOMA-IR and all-cause mortality was U-shaped in patients with CHD and hypertension. HOMA-IR was a reliable predictor of all-cause mortality in this population.

Efficacy of Rituximab for Minimal Change Disease and Focal Segmental Glomerulosclerosis with Frequently Relapsing or Steroid-Dependent Nephrotic Syndrome in Adults: A Chinese Multicenter Retrospective Study.

INTRODUCTION: Rituximab has been proven effective and safe in pediatric patients with frequently relapsing or steroid-dependent nephrotic syndrome (FR/SDNS). We aimed to analyze the efficacy and safety of rituximab in adult FR/SDNS patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). METHODS: A retrospective cohort study at three nephrology centers in China included adult FR/SDNS patients with biopsy-proven MCD or FSGS. Primary outcomes were relapse frequency and first relapse-free survival time. Adverse events were well recorded, and logistic regression analyses were used to investigate the risk factors of relapse. RESULTS: Eighty-one patients (age, 25.0 years; interquartile range, 20.0-40.5; 67% males; 82.7% MCD) received an average rituximab dose of 1,393.8 ± 618.7 mg/2 years during the 2-year follow-up period. The relapse frequency, calculated as the ratio of relapse times to follow-up years, significantly decreased after rituximab treatment (0.04 [0.00, 0.08] vs. 1.71 [1.00, 2.45], p < 0.001). The first relapse-free survival time was 16.7 ± 8.0 months. Fifty-seven patients (70.4%) achieved cessation of corticosteroids and immunosuppressants within 3 months after the first rituximab infusion. Adverse events were mostly mild, and no severe treatment-related adverse events were observed. Low serum albumin level before rituximab and high CD56+CD16+ natural killer cell count after rituximab were independent risk factors of relapse within 2 years after rituximab treatment. CONCLUSION: Rituximab was proven an effective and safe treatment option for adult FR/SDNS patients with MCD or FSGS in maintaining disease remission and minimizing corticosteroid exposure.

PFKFB3-driven vascular smooth muscle cell glycolysis promotes vascular calcification via the altered FoxO3 and lactate production.

A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR-26a-5p and miR-26b-5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR-26a-5p or miR-26b-5p was seen in these samples. Overexpression of miR-26a/b-5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD3 -overloaded mice in vivo or attenuated high phosphate (Pi)-induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA-seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion-mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3-mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3-driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.

Molecular detection and genetic characteristics of porcine circovirus 3 and porcine circovirus 4 in central China.

Porcine circoviruses (PCVs) are a significant cause of concern for swine health, with four genotypes currently recognized. Two of these, PCV3 and PCV4, have been detected in pigs across all age groups, in both healthy and diseased animals. These viruses have been associated with various clinical manifestations, including porcine dermatitis and nephropathy syndrome (PDNS) and respiratory and enteric signs. In this study, we detected PCV3 and PCV4 in central China between January 2022 and February 2023. We tested fecal swabs and tissue samples from growing-finishing and suckling pigs with or without respiratory and systemic manifestations and found the prevalence of PCV3 to be 15.15% (15/99) and that of PCV3/PCV4 coinfection to be 4.04% (4/99). This relatively low prevalence might be attributed to the fact that most of the clinical samples were collected from pigs exhibiting respiratory signs, with only a few samples having been obtained from pigs with diarrhea. In some cases, PCV2 was also detected, and the coinfection rates of PCV2/3, PCV2/4, and PCV2/3/4 were 6.06% (6/99), 5.05% (5/99), and 3.03% (3/99), respectively. The complete genomic sequences of four PCV3 and two PCV4 isolates were determined. All four of the PCV3 isolates were of subtype PCV3b, and the two PCV4 isolates were of subtype PCV4b. Two mutations (A24V and R27K) were found in antibody recognition domains of PCV3, suggesting that they might be associated with immune escape. This study provides valuable insights into the molecular epidemiology and evolution of PCV3 and PCV4 that will be useful in future investigations of genotyping, immunogenicity, and immune evasion strategies.

Developing deep learning-based strategies to predict the risk of hepatocellular carcinoma among patients with nonalcoholic fatty liver disease from electronic health records.

OBJECTIVE: The accuracy of deep learning models for many disease prediction problems is affected by time-varying covariates, rare incidence, covariate imbalance and delayed diagnosis when using structured electronic health records data. The situation is further exasperated when predicting the risk of one disease on condition of another disease, such as the hepatocellular carcinoma risk among patients with nonalcoholic fatty liver disease due to slow, chronic progression, the scarce of data with both disease conditions and the sex bias of the diseases. The goal of this study is to investigate the extent to which the aforementioned issues influence deep learning performance, and then devised strategies to tackle these challenges. These strategies were applied to improve hepatocellular carcinoma risk prediction among patients with nonalcoholic fatty liver disease. METHODS: We evaluated two representative deep learning models in the task of predicting the occurrence of hepatocellular carcinoma in a cohort of patients with nonalcoholic fatty liver disease (n = 220,838) from a national EHR database. The disease prediction task was carefully formulated as a classification problem while taking censorship and the length of follow-up into consideration. RESULTS: We developed a novel backward masking scheme to deal with the issue of delayed diagnosis which is very common in EHR data analysis and evaluate how the length of longitudinal information after the index date affects disease prediction. We observed that modeling time-varying covariates improved the performance of the algorithms and transfer learning mitigated reduced performance caused by the lack of data. In addition, covariate imbalance, such as sex bias in data impaired performance. Deep learning models trained on one sex and evaluated in the other sex showed reduced performance, indicating the importance of assessing covariate imbalance while preparing data for model training. CONCLUSIONS: The strategies developed in this work can significantly improve the performance of hepatocellular carcinoma risk prediction among patients with nonalcoholic fatty liver disease. Furthermore, our novel strategies can be generalized to apply to other disease risk predictions using structured electronic health records, especially for disease risks on condition of another disease.

PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy.

Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 µM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

Canagliflozin alleviates pulmonary hypertension by activating PPARγ and inhibiting its S225 phosphorylation.

Pulmonary hypertension (PH) is a progressive fatal disease with no cure. Canagliflozin (CANA), a novel medication for diabetes, has been found to have remarkable cardiovascular benefits. However, few studies have addressed the effect and pharmacological mechanism of CANA in the treatment of PH. Therefore, our study aimed to investigate the effect and pharmacological mechanism of CANA in treating PH. First, CANA suppressed increased pulmonary artery pressure, right ventricular hypertrophy, and vascular remodeling in both mouse and rat PH models. Network pharmacology, transcriptomics, and biological results suggested that CANA could ameliorate PH by suppressing excessive oxidative stress and pulmonary artery smooth muscle cell proliferation partially through the activation of PPARγ. Further studies demonstrated that CANA inhibited phosphorylation of PPARγ at Ser225 (a novel serine phosphorylation site in PPARγ), thereby promoting the nuclear translocation of PPARγ and increasing its ability to resist oxidative stress and proliferation. Taken together, our study not only highlighted the potential pharmacological effect of CANA on PH but also revealed that CANA-induced inhibition of PPARγ Ser225 phosphorylation increases its capacity to counteract oxidative stress and inhibits proliferation. These findings may stimulate further research and encourage future clinical trials exploring the therapeutic potential of CANA in PH treatment.

Nicotinate-curcumin improves NASH by inhibiting the AKR1B10/ACCα-mediated triglyceride synthesis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a prevalent chronic liver condition. However, the potential therapeutic benefits and underlying mechanism of nicotinate-curcumin (NC) in the treatment of NASH remain uncertain. METHODS: A rat model of NASH induced by a high-fat and high-fructose diet was treated with nicotinate-curcumin (NC, 20, 40 mg·kg- 1), curcumin (Cur, 40 mg·kg- 1) and metformin (Met, 50 mg·kg- 1) for a duration of 4 weeks. The interaction between NASH, Cur and Aldo-Keto reductase family 1 member B10 (AKR1B10) was filter and analyzed using network pharmacology. The interaction of Cur, NC and AKR1B10 was analyzed using molecular docking techniques, and the binding energy of Cur and NC with AKR1B10 was compared. HepG2 cells were induced by Ox-LDL (25 µg·ml- 1, 24 h) in high glucose medium. NC (20µM, 40µM), Cur (40µM) Met (150µM) and epalrestat (Epa, 75µM) were administered individually. The activities of ALT, AST, ALP and the levels of LDL, HDL, TG, TC and FFA in serum were quantified using a chemiluminescence assay. Based on the changes in the above indicators, score according to NAS standards. The activities of Acetyl-CoA and Malonyl-CoA were measured using an ELISA assay. And the expression and cellular localization of AKR1B10 and Acetyl-CoA carboxylase (ACCα) in HepG2 cells were detected by Western blotting and immunofluorescence. RESULTS: The results of the animal experiments demonstrated that NASH rat model induced by a high-fat and high-fructose diet exhibited pronounced dysfunction in liver function and lipid metabolism. Additionally, there was a significant increase in serum levels of FFA and TG, as well as elevated expression of AKR1B10 and ACCα, and heightened activity of Acetyl-CoA and Malonyl-CoA in liver tissue. The administration of NC showed to enhance liver function in rats with NASH, leading to reductions in ALT, AST and ALP levels, and decrease in blood lipid and significant inhibition of FFA and TG synthesis in the liver. Network pharmacological analysis identified AKR1B10 and ACCα as potential targets for NASH treatment. Molecular docking studies revealed that both Cur and NC are capable of binding to AKR1B10, with NC exhibiting a stronger binding energy to AKR1B10. Western blot analysis demonstrated an upregulation in the expression of AKR1B10 and ACCα in the liver tissue of NASH rats, accompanied by elevated Acetyl-CoA and Malonyl-CoA activity, and increased levels of FFA and TG. The results of the HepG2 cell experiments induced by Ox-LDL suggest that NC significantly inhibited the expression and co-localization of AKR1B10 and ACCα, while also reduced levels of TC and LDL-C and increased level of HDL-C. These effects are accompanied by a decrease in the activities of ACCα and Malonyl-CoA, and levels of FFA and TG. Furthermore, the impact of NC appears to be more pronounced compared to Cur. CONCLUSION: NC could effectively treat NASH and improve liver function and lipid metabolism disorder. The mechanism of NC is related to the inhibition of AKR1B10/ACCα pathway and FFA/TG synthesis of liver.