Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Overexpression of MsDREB1C Modulates Growth and Improves Forage Quality in Tetraploid Alfalfa (Medicago sativa L.).

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa (Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf-stem ratio (LSR) and repression of several lignin-synthesis genes (MsNST, MsPAL1, MsC4H, and Ms4CL). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage.

Interoperability of phenome-wide multimorbidity patterns: a comparative study of two large-scale EHR systems.

Background: Electronic health records (EHR) are increasingly used for studying multimorbidities. However, concerns about accuracy, completeness, and EHRs being primarily designed for billing and administration raise questions about the consistency and reproducibility of EHR-based multimorbidity research. Methods: Utilizing phecodes to represent the disease phenome, we analyzed pairwise comorbidity strengths using a dual logistic regression approach and constructed multimorbidity as an undirected weighted graph. We assessed the consistency of the multimorbidity networks within and between two major EHR systems at local (nodes and edges), meso (neighboring patterns), and global (network statistics) scales. We present case studies to identify disease clusters and uncover clinically interpretable disease relationships. We provide an interactive web tool and a knowledge base combing data from multiple sources for online multimorbidity analysis. Findings: Analyzing data from 500,000 patients across Vanderbilt University Medical Center and Mass General Brigham health systems, we observed a strong correlation in disease frequencies ( Kendall's τ = 0.643) and comorbidity strengths (Pearson ρ = 0.79). Consistent network statistics across EHRs suggest a similar structure of multimorbidity networks at various scales. Comorbidity strengths and similarities of multimorbidity connection patterns align with the disease genetic correlations. Graph-theoretic analyses revealed a consistent core-periphery structure, implying efficient network clustering through threshold graph construction. Using hydronephrosis as a case study, we demonstrated the network's ability to uncover clinically relevant disease relationships and provide novel insights. Interpretation: Our findings demonstrate the robustness of large-scale EHR data for studying complex disease interactions. The alignment of multimorbidity patterns with genetic data suggests the potential utility for uncovering shared etiology of diseases. The consistent core-periphery network structure offers a strategic approach to analyze disease clusters. This work also sets the stage for advanced disease modeling, with implications for precision medicine. Funding: VUMC Biostatistics Development Award, UL1 TR002243, R21DK127075, R01HL140074, P50GM115305, R01CA227481.

The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC.

OBJECTIVE: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. METHODS: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. RESULTS: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. CONCLUSIONS: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.

Illumina and Nanopore sequencing in culture-negative samples from suspected lower respiratory tract infection patients.

Objective: To evaluate the diagnostic value of metagenomic sequencing technology based on Illumina and Nanopore sequencing platforms for patients with suspected lower respiratory tract infection whose pathogen could not be identified by conventional microbiological tests. Methods: Patients admitted to the Respiratory and Critical Care Medicine in Shanghai Ruijin Hospital were retrospectively studied from August 2021 to March 2022. Alveolar lavage or sputum was retained in patients with clinically suspected lower respiratory tract infection who were negative in conventional tests. Bronchoalveolar lavage fluid (BALF) samples were obtained using bronchoscopy. Sputum samples were collected, while BALF samples were not available due to bronchoscopy contraindications. Samples collected from enrolled patients were simultaneously sent for metagenomic sequencing on both platforms. Results: Thirty-eight patients with suspected LRTI were enrolled in this study, consisting of 36 parts of alveolar lavage and 2 parts of sputum. According to the infection diagnosis, 31 patients were confirmed to be infected with pathogens, while 7 patients were diagnosed with non-infectious disease. With regard to the diagnosis of infectious diseases, the sensitivity and specificity of Illumina and Nanopore to diagnose infection in patients were 80.6% vs. 93.5% and 42.9 vs. 28.6%, respectively. In patients diagnosed with bacterial, Mycobacterium, and fungal infections, the positive rates of Illumina and Nanopore sequencer were 71.4% vs. 78.6%, 36.4% vs. 90.9%, and 50% vs. 62.5%, respectively. In terms of pathogen diagnosis, the sensitivity and specificity of pathogens detected by Illumina and Nanopore were 55.6% vs. 77.8% and 42.9% vs. 28.6%, respectively. Among the patients treated with antibiotics in the last 2 weeks, 61.1% (11/18) and 77.8% (14/18) cases of pathogens were accurately detected by Illumina and Nanopore, respectively, among which 8 cases were detected jointly. The consistency between Illumina and diagnosis was 63.9% (23/36), while the consistency between Nanopore and diagnosis was 83.3% (30/36). Between Illumina and Nanopore sequencing methods, the consistency ratio was 55% (22/42) based on pathogen diagnosis. Conclusion: Both platforms play a certain value in infection diagnosis and pathogen diagnosis of CMT-negative suspected LRTI patients, providing a theoretical basis for clinical accurate diagnosis and symptomatic treatment. The Nanopore platform demonstrated potential advantages in the identification of Mycobacterium and could further provide another powerful approach for patients with suspected Mycobacterium infection.

The association of infant urinary adrenal steroids with the risk of childhood asthma development.

BACKGROUND: Adrenal steroids play important roles in early-life development. However, our understanding of the effects of perinatal adrenal steroids on the development of childhood asthma is incomplete. OBJECTIVE: To evaluate the associations between early-life adrenal steroid levels and childhood asthma. METHODS: Participants included the Infant Susceptibility to Pulmonary Infections and Asthma following Respiratory Syncytial Virus Exposure birth cohort children with untargeted urinary metabolomics data measured during early infancy (n = 264) and nasal immune mediator data measured concurrently at age 2 to 6 months (n = 76). A total of 11 adrenal steroid compounds were identified using untargeted metabolomics and 6 asthma-relevant nasal immune mediators from multiplex assays were a priori selected. Current asthma at ages 5 and 6 years was ascertained using validated questionnaires. Associations were tested using logistic and linear regression with confounders adjustment. RESULTS: Pregnenetriol disulfate (adjusted odds ratio [aOR] = 0.20, 95% CI = 0.06-0.68) and 3a,21-dihydroxy-5b-pregnane-11,20-dione-21-glucuronide (aOR = 0.34, 95% CI = 0.14-0.75) were inversely associated with childhood asthma at 5 and 6 years after multiple testing adjustment. There was a significant interaction effect of pregnanediol-3-glucuronide by biological sex assigned at birth (aOR = 0.11, 95% CI = 0.02-0.51, for those with female sex) on childhood asthma. Pregnenetriol disulfate was inversely associated with granulocyte-macrophage colony-stimulating factor (ß = -0.45, q-value = 0.05). 3a,21-dihydroxy-5b-pregnane-11,20-dione 21-glucuronide was inversely associated with interleukin [IL]-4 (ß = -0.29, q-value = 0.02), IL-5 (ß = -0.35, q-value = 0.006), IL-13 (ß = -0.26, q-value = 0.02), granulocyte-macrophage colony-stimulating factor (ß = -0.35, q-value = 0.006), and fibroblast growth factor-ß (ß = -0.24, q-value = 0.01) after multiple testing adjustment. CONCLUSION: The inverse association between adrenal steroids downstream of progesterone and 17-hydroxypregnenolone and the odds of childhood asthma and nasopharyngeal type 2 immune biomarkers suggest that increased early-life adrenal steroids may suppress type 2 inflammation and protect against the development of childhood asthma.

ß-Nicotinamide mononucleotide improves chilled ram sperm quality in vitro by reducing oxidative stress damage.

OBJECTIVE: The present study aimed to investigate the effect of ß-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4°C in vitro. METHODS: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4°C. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4°C. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4°C. RESULTS: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4°C. CONCLUSION: Ram sperm quality can be maintained during storage at 4°C with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4°C in vitro.

Natural variation in OsMYB8 confers diurnal floret opening time divergence between indica and japonica subspecies.

The inter-subspecific indica-japonica hybrid rice confer potential higher yield than the widely used indica-indica intra-subspecific hybrid rice. Nevertheless, the utilization of this strong heterosis is currently hindered by asynchronous diurnal floret opening time (DFOT) of indica and japonica parental lines. Here, we identify OsMYB8 as a key regulator of rice DFOT. OsMYB8 induces the transcription of JA-Ile synthetase OsJAR1, thereby regulating the expression of genes related to cell osmolality and cell wall remodeling in lodicules to promote floret opening. Natural variations of OsMYB8 promoter contribute to its differential expression, thus differential transcription of OsJAR1 and accumulation of JA-Ile in lodicules of indica and japonica subspecies. Furthermore, introgression of the indica haplotype of OsMYB8 into japonica effectively promotes DFOT in japonica. Our findings reveal an OsMYB8-OsJAR1 module that regulates differential DFOT in indica and japonica, and provide a strategy for breeding early DFOT japonica to facilitate breeding of indica-japonica hybrids.

Epigenetic modification in liver fibrosis: Promising therapeutic direction with significant challenges ahead.

Liver fibrosis, characterized by scar tissue formation, can ultimately result in liver failure. It's a major cause of morbidity and mortality globally, often associated with chronic liver diseases like hepatitis or alcoholic and non-alcoholic fatty liver diseases. However, current treatment options are limited, highlighting the urgent need for the development of new therapies. As a reversible regulatory mechanism, epigenetic modification is implicated in many biological processes, including liver fibrosis. Exploring the epigenetic mechanisms involved in liver fibrosis could provide valuable insights into developing new treatments for chronic liver diseases, although the current evidence is still controversial. This review provides a comprehensive summary of the regulatory mechanisms and critical targets of epigenetic modifications, including DNA methylation, histone modification, and RNA modification, in liver fibrotic diseases. The potential cooperation of different epigenetic modifications in promoting fibrogenesis was also highlighted. Finally, available agonists or inhibitors regulating these epigenetic mechanisms and their potential application in preventing liver fibrosis were discussed. In summary, elucidating specific druggable epigenetic targets and developing more selective and specific candidate medicines may represent a promising approach with bright prospects for the treatment of chronic liver diseases.

MORE FLORET1 controls anther development by negatively regulating key tapetal genes in both diploid and tetraploid rice.

Polyploid hybrid rice (Oryza sativa) has great potential for increasing yields. However, hybrid rice depends on male fertility and its regulation, which is less well studied in polyploid rice than in diploid rice. We previously identified a MYB transcription factor, MORE FLORET1 (MOF1), whose mutation causes male sterility in neo-tetraploid rice. MOF1 expression in anthers peaks at anther Stage 7 (S7) and progressively decreases to low levels at S10. However, it remains unclear how the dynamics of MOF1 expression contribute to male fertility. Here, we carefully examined anther development in both diploid and tetraploid mof1 rice mutants, as well as lines ectopically expressing MOF1 in a temporal manner. MOF1 mutations caused delayed degeneration of the tapetum and middle layer of anthers and aberrant pollen wall organization. Ectopic MOF1 expression at later stages of anther development led to retarded cytoplasmic reorganization of tapetal cells. In both cases, pollen grains were aborted and seed production was abolished, indicating that precise control of MOF1 expression is essential for male reproduction. We demonstrated that five key tapetal genes, CYP703A3 (CYTOCHROME P450 HYDROXYLASE 703A3), OsABCG26 (Oryza sativa ATP BINDING CASSETTE G26), PTC1 (PERSISTENT TAPETAL CELL1), PKS2 (POLYKETIDE SYNTHASE 2), and OsABCG15 (Oryza sativa ATP BINDING CASSETTE G15), exhibit expression patterns opposite to those of MOF1 and are negatively regulated by MOF1. Moreover, DNA affinity purification sequencing (DAP-seq), luciferase activity assays, and electrophoretic mobility shift assays indicated that MOF1 binds directly to the PKS2 promoter for transcriptional repression. Our results provide a mechanistic basis for the regulation of male reproduction by MOF1 in both diploid and tetraploid rice. This study will facilitate the development of polyploid male sterile lines, which are useful for breeding of polyploid hybrid rice.

Comprehensive analysis of cuproptosis-related genes in immune infiltration and development of a novel diagnostic model for acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) represents a diverse range of conditions characterized by high incidence and mortality rates, and it is mainly associated with immune-mediated mechanisms and mitochondrial metabolism dysfunction. Cuproptosis, a recently identified form of programmed cell death dependent on copper, is closely linked to mitochondrial respiration and contributes to various diseases. Our study aimed to investigate the involvement of cuproptosis-related genes (CRGs) in AKI. METHODS: Identification of CRGs was conducted using differential expression analysis, and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using human sequencing profiles. Utilizing CIBERSORT algorithm, receiver operating characteristic (ROC) curve analysis, nomogram development, and decision curve analysis (DCA), the association among immune scores, CRGs, and the diagnostic value of these genes was explored. RESULTS: Notably, six CRGs (FDX1, DLD, DLAT, DBT, PDHA1, and ATP7A) were identified as significant differentiators between AKI and non-AKI groups. The ROC curve, based on these six genes, demonstrated an AUC value of 0.917, which was further validated using an additional dataset with an AUC value of 0.902. Nomogram and DCA further confirmed the accuracy of the model in predicting the risk of AKI. CONCLUSION: This study elucidated the role of cuproptosis in AKI and revealed the association between CRGs and infiltrated immune cells through comprehensive bioinformatic techniques. The six-gene cuproptosis-related signature exhibited remarkable predictive efficiency for AKI.

Effects of early recovery of renal function on adverse renal outcomes and mortality in patients with acute kidney injury: a systematic review and meta-analysis.

AIM: This study intended to scrutinize the effect of RFR time on adverse renal outcomes and mortality and try to define the cutoff of early RFR. METHODS: We conducted a literature search from database inception to February 2023. Outcome measures incorporated the progression of CKD, delivery of RRT, incidence of composite renal outcomes, and mortality. And pooled results were depicted as odds ratio (OR) and 95% confidence interval (CI). RESULTS: A total of 11 studies were finally selected (507,989 patients, mean follow-up, 3.89 years). The results exhibited that the crude mortality was lower in patients with early RFR (OR = 0.39, 95% CI: 0.16-0.95, P = 0.037). In addition, patients with early RFR had a lower incidence of progression to CKD (OR = 0.38, 95% CI: 0.17-0.85, P < 0.018), RRT (OR = 0.37, 95% CI: 0.20-0.71, P = 0.03), and composite renal outcomes (OR = 0.18, 95% CI: 0.15-0.20, P < 0.001). CKD progression-related events were significantly higher in patients whose renal function recovered after 7 days (OR = 0.69, 95% CI: 0.47-1.09, P = 0.112) than in those whose renal function recovered within 7 days (OR = 0.23, 95% CI: 0.06-0.92, P = 0.038), and the risk of RRT was lower in patients who recovered within 7 days (OR = 0.32, 95% CI: 0.15-0.66, P = 0.002) than in those who recovered after 7 days (OR = 0.72, 95% CI: 0.17-3.09, P = 0.654) or longer. CONCLUSION: Patients with early RFR had a lower risk of CKD progression, RRT, and composite renal outcomes, as well as lower crude mortality than those without early recovery, despite no marked difference in 30-day, 90-day, and 1-year mortality. We speculated that 7 days may be used as a cutoff for early RFR.

Functional Characterization of the MsFKF1 Gene Reveals Its Dual Role in Regulating the Flowering Time and Plant Height in Medicago sativa L.

Alfalfa (M. sativa), a perennial legume forage, is known for its high yield and good quality. As a long-day plant, it is sensitive to changes in the day length, which affects the flowering time and plant growth, and limits alfalfa yield. Photoperiod-mediated delayed flowering in alfalfa helps to extend the vegetative growth period and increase the yield. We isolated a blue-light phytohormone gene from the alfalfa genome that is an ortholog of soybean FKF1 and named it MsFKF1. Gene expression analyses showed that MsFKF1 responds to blue light and the circadian clock in alfalfa. We found that MsFKF1 regulates the flowering time through the plant circadian clock pathway by inhibiting the transcription of E1 and COL, thus suppressing FLOWERING LOCUS T a1 (FTa1) transcription. In addition, transgenic lines exhibited higher plant height and accumulated more biomass in comparison to wild-type plants. However, the increased fiber (NDF and ADF) and lignin content also led to a reduction in the digestibility of the forage. The key genes related to GA biosynthesis, GA20OX1, increased in the transgenic lines, while GA2OX1 decreased for the inactive GA transformation. These findings offer novel insights on the function of MsFKF1 in the regulation of the flowering time and plant height in cultivated M. sativa. These insights into MsFKF1's roles in alfalfa offer potential strategies for molecular breeding aimed at optimizing flowering time and biomass yield.

Unbiased characterization of atrial fibrillation phenotypic architecture provides insight to genetic liability and clinically relevant outcomes.

Background: Atrial Fibrillation (AF) is a common and clinically heterogeneous arrythmia. Machine learning (ML) algorithms can define data-driven disease subtypes in an unbiased fashion, but whether the AF subgroups defined in this way align with underlying mechanisms, such as high polygenic liability to AF or inflammation, and associate with clinical outcomes is unclear. Methods: We identified individuals with AF in a large biobank linked to electronic health records (EHR) and genome-wide genotyping. The phenotypic architecture in the AF cohort was defined using principal component analysis of 35 expertly curated and uncorrelated clinical features. We applied an unsupervised co-clustering machine learning algorithm to the 35 features to identify distinct phenotypic AF clusters. The clinical inflammatory status of the clusters was defined using measured biomarkers (CRP, ESR, WBC, Neutrophil %, Platelet count, RDW) within 6 months of first AF mention in the EHR. Polygenic risk scores (PRS) for AF and cytokine levels were used to assess genetic liability of clusters to AF and inflammation, respectively. Clinical outcomes were collected from EHR up to the last medical contact. Results: The analysis included 23,271 subjects with AF, of which 6,023 had available genome-wide genotyping. The machine learning algorithm identified 3 phenotypic clusters that were distinguished by increasing prevalence of comorbidities, particularly renal dysfunction, and coronary artery disease. Polygenic liability to AF across clusters was highest in the low comorbidity cluster. Clinically measured inflammatory biomarkers were highest in the high comorbid cluster, while there was no difference between groups in genetically predicted levels of inflammatory biomarkers. Subgroup assignment was associated with multiple clinical outcomes including mortality, stroke, bleeding, and use of cardiac implantable electronic devices after AF diagnosis. Conclusion: Patient subgroups identified by unsupervised clustering were distinguished by comorbidity burden and associated with risk of clinically important outcomes. Polygenic liability to AF across clusters was greatest in the low comorbidity subgroup. Clinical inflammation, as reflected by measured biomarkers, was lowest in the subgroup with lowest comorbidities. However, there were no differences in genetically predicted levels of inflammatory biomarkers, suggesting associations between AF and inflammation is driven by acquired comorbidities rather than genetic predisposition.

A screening study on the detection strain of Coxsackievirus A6: the key to evaluating neutralizing antibodies in vaccines.

The increasing incidence of diseases caused by Coxsackievirus A6 (CV-A6) and the presence of various mutants in the population present significant public health challenges. Given the concurrent development of multiple vaccines in China, it is challenging to objectively and accurately evaluate the level of neutralizing antibody response to different vaccines. The choice of the detection strain is a crucial factor that influences the detection of neutralizing antibodies. In this study, the National Institutes for Food and Drug Control collected a prototype strain (Gdula), one subgenotype D1, as well as 13 CV-A6 candidate vaccine strains and candidate detection strains (subgenotype D3) from various institutions and manufacturers involved in research and development. We evaluated cross-neutralization activity using plasma from naturally infected adults (n = 30) and serum from rats immunized with the aforementioned CV-A6 strains. Although there were differences between the geometric mean titer (GMT) ranges of human plasma and murine sera, the overall trends were similar. A significant effect of each strain on the neutralizing antibody test (MAX/MIN 48.0 ∼16410.3) was observed. Among all strains, neutralization of the S112 strain by 15 different sera resulted in higher neutralizing antibody titers (GMTS112 = 132.0) and more consistent responses across different genotypic immune sera (MAX/MIN = 48.0). Therefore, S112 may serve as a detection strain for NtAb testing in various vaccines, minimizing bias and making it suitable for evaluating the immunogenicity of the CV-A6 vaccine.

Chuanxiong Rhizoma extracts prevent liver fibrosis via targeting CTCF-c-MYC-H19 pathway.

Objective: Hepatic fibrosis has been widely considered as a conjoint consequence of almost all chronic liver diseases. Chuanxiong Rhizoma (Chuanxiong in Chinese, CX) is a traditional Chinese herbal product to prevent cerebrovascular, gynecologic and hepatic diseases. Our previous study found that CX extracts significantly reduced collagen contraction force of hepatic stellate cells (HSCs). Here, this study aimed to compare the protection of different CX extracts on bile duct ligation (BDL)-induced liver fibrosis and investigate plausible underlying mechanisms. Methods: The active compounds of CX extracts were identified by high performance liquid chromatography (HPLC). Network pharmacology was used to determine potential targets of CX against hepatic fibrosis. Bile duct hyperplasia and liver fibrosis were evaluated by serologic testing and histopathological evaluation. The expression of targets of interest was determined by quantitative real-time PCR (qPCR) and Western blot. Results: Different CX extracts were identified by tetramethylpyrazine, ferulic acid and senkyunolide A. Based on the network pharmacological analysis, 42 overlap targets were obtained via merging the candidates targets of CX and liver fibrosis. Different aqueous, alkaloid and phthalide extracts of CX (CXAE, CXAL and CXPHL) significantly inhibited diffuse severe bile duct hyperplasia and thus suppressed hepatic fibrosis by decreasing CCCTC binding factor (CTCF)-c-MYC-long non-coding RNA H19 (H19) pathway in the BDL-induced mouse model. Meanwhile, CX extracts, especially CXAL and CXPHL also suppressed CTCF-c-MYC-H19 pathway and inhibited ductular reaction in cholangiocytes stimulated with taurocholate acid (TCA), lithocholic acid (LCA) and transforming growth factor beta (TGF-ß), as illustrated by decreased bile duct proliferation markers. Conclusion: Our data supported that different CX extracts, especially CXAL and CXPHL significantly alleviated hepatic fibrosis and bile duct hyperplasia via inhibiting CTCF-c-MYC-H19 pathway, providing novel insights into the anti-fibrotic mechanism of CX.

Efficient One-Pot Synthesis of Uridine Diphosphate Galactose Employing a Trienzyme System.

The limited availability of high-cost nucleotide sugars is a significant constraint on the application of their downstream products (glycosides and prebiotics) in the food or pharmaceutical industry. To better solve the problem, this study presented a one-pot approach for the biosynthesis of UDP-Gal using a thermophilic multienzyme system consisting of GalK, UGPase, and PPase. Under optimal conditions, a 2 h reaction resulted in a UTP conversion rate of 87.4%. In a fed-batch reaction with Gal/ATP = 20 mM:10 mM, UDP-Gal accumulated to 33.76 mM with a space-time yield (STY) of 6.36 g/L·h-1 after the second feeding. In repetitive batch synthesis, the average yield of UDP-Gal over 8 cycles reached 10.80 g/L with a very low biocatalyst loading of 0.002 genzymes/gproduct. Interestingly, Galk (Tth0595) could synthesize Gal-1P using ADP as a donor of phosphate groups, which had never been reported before. This approach possessed the benefits of high synthesis efficiency, low cost, and superior reaction system stability, and it provided new insights into the rapid one-pot synthesis of UDP-Gal and high-value glycosidic compounds.

Driver mutation zygosity is a critical factor in predicting clonal hematopoiesis transformation risk.

Clonal hematopoiesis (CH) can be caused by either single gene mutations (eg point mutations in JAK2 causing CHIP) or mosaic chromosomal alterations (e.g., loss of heterozygosity at chromosome 9p). CH is associated with a significantly increased risk of hematologic malignancies. However, the absolute rate of transformation on an annualized basis is low. Improved prognostication of transformation risk is urgently needed for routine clinical practice. We hypothesized that the co-occurrence of CHIP and mCAs at the same locus (e.g., transforming a heterozygous JAK2 CHIP mutation into a homozygous mutation through concomitant loss of heterozygosity at chromosome 9) might have important prognostic implications for malignancy transformation risk. We tested this hypothesis using our discovery cohort, the UK Biobank (n = 451,180), and subsequently validated it in the BioVU cohort (n = 91,335). We find that individuals with a concurrent somatic mutation and mCA were at significantly increased risk of hematologic malignancy (for example, In BioVU cohort incidence of hematologic malignancies is higher in individuals with co-occurring JAK2 V617F and 9p CN-LOH; HR = 54.76, 95% CI = 33.92-88.41, P < 0.001 vs. JAK2 V617F alone; HR = 44.05, 95% CI = 35.06-55.35, P < 0.001). Currently, the 'zygosity' of the CHIP mutation is not routinely reported in clinical assays or considered in prognosticating CHIP transformation risk. Based on these observations, we propose that clinical reports should include 'zygosity' status of CHIP mutations and that future prognostication systems should take mutation 'zygosity' into account.

Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.