Dopamine and its precursor levodopa inactivate SARS-CoV-2 main protease by forming a quinoprotein.

Many studies show either the absence, or very low levels of, SARS-CoV-2 viral RNA and/or antigen in the brain of COVID-19 patients. Reports consistently indicate an abortive infection phenomenon in nervous cells despite the fact that they contain the SARS-CoV-2 receptor, ACE2. Dopamine levels in different brain regions are in the range of micromolar to millimolar concentrations. We have shown that sub-micromolar to low micromolar concentrations of dopamine or its precursor (levodopa) time- and dose-dependently inhibit the activity of SARS-CoV-2 main protease (Mpro), which is vital for the viral life cycle, by forming a quinoprotein. Thiol detection coupled with the assessment of Mpro activity suggests that among the 12 cysteinyl thiols, the active site, Cys145-SH, is preferentially conjugated to the quinone derived from the oxidation of dopamine or levodopa. LC-MS/MS analyses show that the Cys145-SH is covalently conjugated by dopamine- or levodopa-o-quinone. These findings help explain why SARS-CoV-2 causes inefficient replication in many nerve cell lines. It is well recognized that inhaled pulmonary drug delivery is the most robust therapy pathway for lung diseases. CVT-301 (orally inhaled levodopa) was approved by the FDA as a drug for Parkinson's patients prior to the outbreak of COVID-19 in 2018. Based on the fact that SARS-CoV-2 causes inefficient replication in the CNS with abundant endogenous Mpro inhibitor in addition to the current finding that levodopa has an Mpro-inhibitory effect somewhat stronger than dopamine, we should urgently investigate the use of CVT-301 as a lung-targeting, COVID-19, Mpro inhibitor.

Association between labor epidural analgesia and gut microbiota: A prospective cohort study.

Background: Labor epidural analgesia (LEA) may influence gut microbiota. We explored the association between LEA and gut microbiota for both mothers and their newborns. Methods: In this prospective cohort study, parturients aged 25-35 years with a gestational age of 37-42 weeks and planned vaginal delivery were recruited. Twenty-one parturients received LEA (the LEA group), and 24 did not (the control group). Maternal and neonatal fecal samples were collected, and the gut microbiota profiles were analyzed using the 16S rRNA gene sequencing. The impact of LEA on gut microbiota was assessed using the general liner models. Results: We showcased the gut microbiota profile from the phyla to species levels based on data on 45 mother-newborn dyads. The results of α- and ß-diversity suggested significant changes in gut microbiota between the LEA and control groups. After adjusting for baseline confounders, the administration of LEA had positive correlations with R. ilealis (ß = 91.87, adjusted P = 0.007) in mothers; LEA also had negative correlations with A. pittii (ß = -449.36, adjusted P = 0.015), P. aeruginosa (ß = -192.55, adjusted P = 0.008), or S. maltophilia (ß = -142.62, adjusted P = 0.001) in mothers, and with Muribaculaceae (ß = -2702.77, adjusted P = 0.003) in neonates. Conclusion: LEA was associated with changes in maternal and neonatal gut microbiota, and future studies are still required to assess their impact on clinical outcomes and explore the mechanisms.

Discovery of Highly Potent Solute Carrier 13 Member 5 (SLC13A5) Inhibitors for the Treatment of Hyperlipidemia.

In the face of escalating metabolic disease prevalence, largely driven by modern lifestyle factors, this study addresses the critical need for novel therapeutic approaches. We have identified the sodium-coupled citrate transporter (NaCT or SLC13A5) as a target for intervention. Utilizing rational drug design, we developed a new class of SLC13A5 inhibitors, anchored by the hydroxysuccinic acid scaffold, refining the structure of PF-06649298. Among these, LBA-3 emerged as a standout compound, exhibiting remarkable potency with an IC50 value of 67 nM, significantly improving upon PF-06649298. In vitro assays demonstrated LBA-3's efficacy in reducing triglyceride levels in OPA-induced HepG2 cells. Moreover, LBA-3 displayed superior pharmacokinetic properties and effectively lowered triglyceride and total cholesterol levels in diverse mouse models (PCN-stimulated and starvation-induced), without detectable toxicity. These findings not only spotlight LBA-3 as a promising candidate for hyperlipidemia treatment but also exemplify the potential of targeted molecular design in advancing metabolic disorder therapeutics.

Key phytochemicals contributing to the bitterness of quinoa.

Despite its nutritional components and potential health benefits, the bitterness of quinoa seed limits its utilization in the food industry. Saponins are believed to be the main cause of the bitterness, but it is still uncertain which specific compound is responsible. This study aimed to isolate the main components contributing to the bitterness in quinoa seed by solvent extraction and various column chromatography techniques guided by sensory evaluation. Five compounds were identified by mass spectrometry and nuclear magnetic resonance analyses, with the dose-over-threshold factors from 29.03 to 198.89. The results confirmed that triterpenoids are responsible for the bitter taste in quinoa seed, with phytolaccagenic acid derivatives being the primary contributor. Additionally, kaempferol 3-O-(2″, 6″-di-O-α-rhamnopyranosyl)-ß-galactopyranoside (namely mauritianin), was demonstrated for the first time to be associated with the bitterness of quinoa. This study could provide new insight into the bitter compound identification in quinoa.

Stratified Prognostic Comparison Between Stage IIB-IVA Cervical Adenocarcinoma and Squamous Cell Carcinoma: A SEER Database-Based Study.

Objective: In current most observational studies, the prognosis of cervical adenocarcinoma is worse than that of cervical squamous cell carcinoma. However, most of the current studies are holistic and lack more detailed staging and grouping analysis of the prognosis of the two types of cervical tumors. Patients and Methods: Inclusion from the SEER database of stage IIB-IVA cervical squamous cell carcinoma and cervical adenocarcinoma patients who did not undergo surgery from 2000 to 2019, underwent radiotherapy/chemotherapy/radiotherapy and chemotherapy/no treatment, and then propensity score matching (PSM) was performed to eliminate confounding factors between cervical squamous cell carcinoma and cervical adenocarcinoma patients with the same stage and treatment method. After matching the original data and propensity score, logarithmic rank test and chi square test were used to evaluate the survival benefits of different stages and treatment methods for patients using Kaplan Meier curve. The prognosis of two types of cervical tumors under the same treatment method was compared, and factors that may cause poor prognosis were analyzed, excluding confounding factors. Results: A total of 10,057 patients were included in this study, and survival analysis showed a significant correlation between the treatment method used and patient prognosis (P<0.05). However, for patients who received radiotherapy or no special treatment, OS and CSS were only related to tumor stage and not to tumor type. In patients undergoing radiotherapy and chemotherapy, the OS and CSS of stage IIIA and IVA patients are not related to tumor pathological characteristics, while the OS of stage IIB patients is not related to tumor properties after PSM. Conclusion: In patients undergoing radiotherapy and chemotherapy, the OS and CSS of stage IIIA and IVA patients were not related to histological type, while the OS of stage IIB patients was not related to histological type after PSM.

Nanopore-based metagenomics analysis reveals microbial presence in amniotic fluid: A prospective study.

Background: Current research on amniotic fluid (AF) microbiota yields contradictory data, necessitating an accurate, comprehensive, and scientifically rigorous evaluation. Objective: This study aimed to characterise the microbial features of AF and explore the correlation between microbial information and clinical parameters. Methods: 76 AF samples were collected in this prospective cohort study. Fourteen samples were utilised to establish the nanopore metagenomic sequencing methodology, whereas the remaining 62 samples underwent a final statistical analysis along with clinical information. Negative controls included the operating room environment (OE), surgical instruments (SI), and laboratory experimental processes (EP) to elucidate the background contamination at each step. Simultaneously, levels of five cytokines (IL-1ß, IL-6, IL-8, TNF-α, MMP-8) in AF were assessed. Results: Among the 62 AF samples, microbial analysis identified seven without microbes and 55 with low microbial diversity and abundance. No significant clinical differences were observed between AF samples with and without microbes. The correlation between microbes and clinical parameters in AF with normal chromosomal structure revealed noteworthy findings. In particular, the third trimester exhibited richer microbial diversity. Pseudomonas demonstrated higher detection rates and relative abundance in the second trimester and Preterm Birth (PTB) groups. S. yanoikuyae in the PTB group exhibited elevated detection frequencies and relative abundance. Notably, Pseudomonas negatively correlated with activated partial thromboplastin time (APTT) (r = -0.329, P = 0.016), while Staphylococcus showed positive correlations with APTT (r = 0.395, P = 0.003). Furthermore, Staphylococcus negatively correlated with birth weight (r = -0.297, P = 0.034). Conclusion: Most AF samples exhibited low microbial diversity and abundance. Certain microbes in AF may correlate with clinical parameters such as gestational age and PTB. However, these associations require further investigation. It is essential to expand the sample size and undertake more comprehensive research to elucidate the clinical implications of microbial presence in AF.

Visualization and Analysis of Infectious Disease Research on Nursing Care Based on CiteSpace in China.

Objective: To analyze the relevant research publications on infectious disease nursing in China to understand the current research status of infectious disease in nursing. Methods: Retrieve relevant literature on infectious disease in nursing from the establishment of the Chinese Biomedical Literature Database, China National Knowledge Infrastructure (CNKI), VIP Database, and Wanfang Database until May 10, 2021. Conduct bibliometric analysis using CiteSpace software. Key words were analyzed using cluster analysis. Results: A total of 4693 relevant literature on infectious disease research in nursing care were included in this study. The overall number of publications on infectious disease research in nursing showed an increasing trend, with a peak in 2010. There were 324 papers funded by scientific research funds, mainly from provincial-level fund projects. The core journal with the most published articles was Nursing Research. The research on infectious disease in nursing mainly focused on various aspects of infectious disease in nursing and infection control. CiteSpace cluster analysis of keywords showed that a total of six clusters were formed: infectious diseases, infectious disease care, health education, mental health, infectious disease nurses, and etiology. After 2015, high-mutation keywords included "quality nursing" and "infection control". Conclusion: Chinese research on infectious disease research in nursing closely follows clinical reality and has developed rapidly. Currently, research focuses on infectious disease research in nursing and infection control. Future research trends will further broaden the depth and breadth of the research, enhance research on infection control and quality nursing, and improve the breadth and depth of the research.

Microbial-Transferred Metabolites and Improvement of Biological Activities of Green Tea Catechins by Human Gut Microbiota.

Green tea catechins (GTCs) are dietary polyphenols with broad bioactivities that undergo extensive microbial metabolism in the human gut. However, microbial-transferred metabolites and their health benefits are not fully understood. Herein, the microbial metabolism of GTCs by human fecal microbiota and dynamic alteration of the microbiota were integrally investigated via in vitro anaerobic fermentation. The results showed that the human gut microbiota exhibited a strong metabolic effect on GTCs via UHPLC-MS/MS analysis. A total of 35 microbial-transferred metabolites were identified, far more than were identified in previous studies. Among them, five metabolites, namely EGCG quinone, EGC quinone, ECG quinone, EC quinone, and mono-oxygenated EGCG, were identified for the first time in fermented GTCs with the human gut microbiota. Consequently, corresponding metabolic pathways were proposed. Notably, the antioxidant, α-amylase, and α-glucosidase inhibitory activities of the GTCs sample increased after fermentation compared to those of the initial unfermented sample. The results of the 16S rRNA gene sequence analysis showed that the GTCs significantly altered gut microbial diversity and enriched the abundancy of Eubacterium, Flavonifractor, etc., which may be further involved in the metabolisms of GTCs. Thus, these findings contribute to a better understanding of the interactions between GTCs and gut microbiota, as well as the health benefits of green tea consumption.

The E3 ligase TRIM22 restricts SARS-CoV-2 replication by promoting proteasomal degradation of NSP8.

Replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is mediated by a complex of non-structural proteins (NSPs), of which NSP7 and NSP8 serve as subunits and play a key role in promoting the activity of RNA-dependent RNA polymerase (RdRp) of NSP12. However, the stability of subunits of the RdRp complex has rarely been reported. Here, we found that NSP8 was degraded by the proteasome in host cells, and identified tripartite motif containing 22 (TRIM22) as its E3 ligase. The interferon (IFN) signaling pathway was activated upon viral invasion into host cells, and TRIM22 expression increased. TRIM22 interacted with NSP8 and ubiquitinated it at Lys97 via K48-type ubiquitination. TRIM22 overexpression significantly reduced viral RNA and protein levels. Knockdown of TRIM22 enhanced viral replication. This study provides a new explanation for treating patients suffering from SARS-CoV-2 with IFNs and new possibilities for drug development targeting the interaction between NSP8 and TRIM22.IMPORTANCENon-structural proteins (NSPs) play a crucial role in the replication of severe acute respiratory syndrome coronavirus 2, facilitating virus amplification and propagation. In this study, we conducted a comprehensive investigation into the stability of all subunits comprising the RNA-dependent RNA polymerase complex. Notably, our results reveal for the first time that NSP8 is a relatively unstable protein, which is found to be readily recognized and degraded by the proteasome. This degradation process is mediated by the host E3 ligase tripartite motif containing 22 (TRIM22), which is also a member of the interferon stimulated gene (ISG) family. Our study elucidates a novel mechanism of antiviral effect of TRIM22, which utilizes its own E3 ubiquitin ligase activity to hinder viral replication by inducing ubiquitination and subsequent degradation of NSP8. These findings provide new ideas for the development of novel therapeutic strategies. In addition, the conserved property of NSP8 raises the possibility of developing broad antiviral drugs targeting the TRIM22-NSP8 interaction.

Drosophila eIF3f1 mediates host immune defense by targeting dTak1.

Eukaryotic translation initiation factors have long been recognized for their critical roles in governing the translation of coding RNAs into peptides/proteins. However, whether they harbor functional activities at the post-translational level remains poorly understood. Here, we demonstrate that eIF3f1 (eukaryotic translation initiation factor 3 subunit f1), which encodes an archetypal deubiquitinase, is essential for the antimicrobial innate immune defense of Drosophila melanogaster. Our in vitro and in vivo evidence indicate that the immunological function of eIF3f1 is dependent on the N-terminal JAMM (JAB1/MPN/Mov34 metalloenzymes) domain. Mechanistically, eIF3f1 physically associates with dTak1 (Drosophila TGF-beta activating kinase 1), a key regulator of the IMD (immune deficiency) signaling pathway, and mediates the turnover of dTak1 by specifically restricting its K48-linked ubiquitination. Collectively, these results provide compelling insight into a noncanonical molecular function of a translation initiation factor that controls the post-translational modification of a target protein.

The application of nanopore targeted sequencing for pathogen diagnosis in bronchoalveolar lavage fluid of patients with pneumonia: a prospective multicenter study.

OBJECTIVE: To evaluate the value of nanopore targeted sequencing in diagnosing pneumonia pathogens. METHODS: This large-scale multicentre prospective study performed in 8 hospitals across China from April to October 2022. Hospitalised patients with a diagnosis of pneumonia at admission were included. Complete clinical data were collected, and bronchoalveolar lavage fluid were obtained from each patient. These samples underwent simultaneous testing using conventional microbial testing, metagenomic next-generation sequencing, and nanopore targeted sequencing. RESULTS: A total of 218 patients were included. Among the 168 cases of pulmonary infection, 246 strains of pathogens were confirmed. Nanopore targeted sequencing outperformed conventional microbial testing, identifying more pathogens with a sensitivity increase of 47.9% (77.2% vs. 29.3%). Metagenomic next-generation sequencing had a sensitivity of 82.9%. Total of 70.1% patients had consistent results in both metagenomic next-generation sequencing and nanopore targeted sequencing. Nanopore targeted sequencing exhibited significantly higher sensitivity in detecting Pneumocystis jiroveci, cytomegalovirus, Mycobacterium tuberculosis, Nontuberculous mycobacteria, Streptococcus pneumoniae, and Mycoplasma pneumoniae compared to conventional microbial testing. However, metagenomic next-generation sequencing demonstrated higher sensitivity than nanopore targeted sequencing for Aspergillus (88.5% vs. 53.8%). Regarding the detection of co-infections, nanopore targeted sequencing displayed significantly higher sensitivity than conventional microbial testing (76.7% vs. 28.7%) and was on par with metagenomic next-generation sequencing (76.7% vs. 82.9%). CONCLUSION: Nanopore targeted sequencing performs equally well as metagenomic next-generation sequencing in bronchoalveolar lavage fluid for pathogen diagnosis in pneumonia, both methods showing higher sensitivity than conventional microbial testing. Nanopore targeted sequencing can be considered a reliable method for diagnosing pathogens in pneumonia.

Cascaded-TOARNet: A cascaded framework based on mixed attention and multiscale information for thoracic OARs segmentation.

BACKGROUND: Accurate and automated segmentation of thoracic organs-at-risk (OARs) is critical for radiotherapy treatment planning of thoracic cancers. However, this has remained a challenging task for four major reasons: (1) thoracic OARs have diverse morphologies; (2) thoracic OARs have low contrast with the background; (3) boundaries of thoracic OARs are blurry; (4) class imbalance issue caused by small organs. PURPOSE: To overcome the above challenges and achieve accurate and automated segmentation of thoracic OARs on thoracic CT. METHODS: A novel cascaded framework based on mixed attention and multiscale information for thoracic OARs segmentation, called Cascaded-TOARNet. This cascaded framework comprises two stages: localization and segmentation. During the localization stage, TOARNet locates each organ to crop the regions of interest (ROIs). During the segmentation stage, TOARNet accurately segments the ROIs, and the segmentation results are merged into a complete result. RESULTS: We evaluated our proposed method and other common segmentation methods on two public datasets: the AAPM Thoracic Auto-Segmentation Challenge dataset and the Segmentation of Thoracic Organs at Risk (SegTHOR) dataset. Our method demonstrated superior performance, achieving a mean Dice score of 92.6% on the SegTHOR dataset and 90.8% on the AAPM dataset. CONCLUSIONS: This segmentation method holds great promise as an essential tool for enhancing the efficiency of thoracic radiotherapy planning.

Tailoring flavin-based photosensitizers for efficient photooxidative coupling of benzylic amines.

Photooxidative coupling of benzylic amines using naturally abundant O2 as an oxidant under visible light irradiation is an alternative green approach to synthesis imines and is of both fundamental and practical significance. We investigated the photophysical properties of flavin (FL) that is a naturally available sensitizer and its derivatives, i.e. 9-bromoflavin (MB-FL), 7,8-dibromoflavin (DB-FL) and 10-phenylflavin (Ph-FL), as well as the performance of these FL-based sensitizers (FLPSs) in the photooxidative coupling of benzylic amines to imines combining experimental and theoretical efforts. We showed that chemical functionalization with Br and phenyl effectively improves the photophysical properties of these FLPSs, in terms of absorption in the visible light range, singlet oxygen quantum yields, triplet lifetime, etc. Apart from nearly quantitative selectivity for the production of imines, the performance of DB-FL is superior to those of other FLPSs, and it is among the best photocatalysts for imine synthesis. Specifically, 0.5 mol% DB-FL is capable of converting 91% of 0.2 mmol benzylamine and more than 80% of 0.2 mmol fluorobenzylic amine derivatives into their corresponding imines in 5 h batch runs. Mechanistic investigation finely explained the observed photophysical properties of FLPSs and highlighted the dominant role of electron transfer in FLPS sensitized coupling of benzylic amines to imines. This work not only helps to understand the pathways for photocatalysis with FLPSs but also paves the way for the design of novel and efficient PSs to promote organic synthesis.

Copine-6 is a Ca2+ sensor for activity-induced AMPA receptor exocytosis.

The recruitment of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors underlies the strengthening of neuronal connectivity during learning and memory. This process is triggered by N-methyl-D-aspartate (NMDA) receptor-dependent postsynaptic Ca2+ influx. Synaptotagmin (Syt)-1 and -7 have been proposed as Ca2+ sensors for AMPA receptor exocytosis but are functionally redundant. Here, we identify a cytosolic C2 domain-containing Ca2+-binding protein, Copine-6, that forms a complex with AMPA receptors. Loss of Copine-6 expression impairs activity-induced exocytosis of AMPA receptors in primary neurons, which is rescued by wild-type Copine-6 but not Ca2+-binding mutants. In contrast, Copine-6 loss of function does not affect steady-state expression or tetrodotoxin-induced synaptic upscaling of surface AMPA receptors. Loss of Syt-1/Syt-7 significantly reduces Copine-6 protein expression. Interestingly, overexpression of wild-type Copine-6, but not the Ca2+-binding mutants, restores activity-dependent exocytosis of AMPA receptors in Syt-1/Syt-7 double-knockdown neurons. We conclude that Copine-6 is a postsynaptic Ca2+ sensor that mediates AMPA receptor exocytosis during synaptic potentiation.

DNA damage repair-related gene signature for identifying the immune status and predicting the prognosis of hepatocellular carcinoma.

The heterogeneity of hepatocellular carcinoma (HCC) poses a challenge for accurate prognosis prediction. DNA damage repair genes (DDRGs) have an impact on a wide range of malignancies. However, the relevance of these genes in HCC prognosis has received little attention. In this study, we aimed to develop a prognostic signature to identify novel therapy options for HCC. We acquired mRNA expression profiles and clinical data for HCC patients from The Cancer Genome Atlas (TCGA) database. A polygenic prognostic model for HCC was constructed using selection operator Cox analysis and least absolute shrinkage. The model was validated using International Cancer Genome Consortium (ICGC) data. Overall survival (OS) between the high-risk and low-risk groups was compared using Kaplan‒Meier analysis. Independent predictors of OS were identified through both univariate and multivariate Cox analyses. To determine immune cell infiltration scores and activity in immune-related pathways, a single-sample gene set enrichment analysis was performed. The protein and mRNA expression levels of the prognostic genes between HCC and normal liver tissues were also examined by immunohistochemistry (IHC), immunofluorescence (IF) and quantitative real-time PCR (qRT-PCR). A novel ten-gene signature (CHD1L, HDAC1, KPNA2, MUTYH, PPP2R5B, NEIL3, POLR2L, RAD54B, RUVBL1 and SPP1) was established for HCC prognosis prediction. Patients in the high-risk group had worse OS than those in the low-risk group. Receiver operating characteristic curve analysis confirmed the predictive ability of this prognostic gene signature. Multivariate Cox analysis showed that the risk score was an independent predictor of OS. Functional analysis revealed a strong association with cell cycle and antigen binding pathways, and the risk score was highly correlated with tumor grade, tumor stage, and types of immune infiltrate. High expression levels of the prognostic genes were significantly correlated with increased sensitivity of cancer cells to antitumor drugs. IHC, IF and qRT-PCR all indicated that the prognostic genes were highly expressed in HCC relative to normal liver tissue, consistent with the results of bioinformatics analysis. Ten DDRGs were utilized to create a new signature for identifying the immunological state of HCC and predicting prognosis. In addition, blocking these genes could represent a promising treatment.

Discovery of Biaryl Amide Derivatives as Potent, Selective, and Orally Bioavailable RORγt Agonists for Cancer Immunotherapy.

The master transcription factor receptor retinoic acid receptor-related orphan receptor γt (RORγt) regulates the differentiation of T-helper 17 (Th17) cells and the production of interleukin-17 (IL-17). Activation of RORγt+ T cells in the tumor microenvironment promotes immune infiltration to more effectively inhibit tumor growth. Therefore, RORγt agonists provide a reachable approach to cancer immunotherapy. Herein, a series of biaryl amide derivatives as novel RORγt agonists were designed, synthesized, and evaluated. Starting from the reported RORγt inverse agonist GSK805 (1), "functionality switching" and structure-based drug optimization led to the discovery of a promising RORγt agonist lead compound 14, which displayed potent and selective RORγt agonist activity and significantly improved metabolic stability. With excellent in vivo pharmacokinetic profiles, compound 14 demonstrated robust efficacy in preclinical tumor models of mouse B16F10 melanoma and LLC lung adenocarcinoma. Taken together, current studies indicate that 14 deserves further investigation as a potential lead RORγt agonist for cancer immunotherapy.

Identification of ferroptosis-related genes in type 2 diabetes mellitus based on machine learning.

BACKGROUND: Type 2 diabetes mellitus (T2DM), which has a high incidence and several harmful consequences, poses a severe danger to human health. Research on the function of ferroptosis in T2DM is increasing. This study uses bioinformatics techniques identify new diagnostic T2DM biomarkers associated with ferroptosis. METHODS: To identify ferroptosis-related genes (FRGs) that are differentially expressed between T2DM patients and healthy individuals, we first obtained T2DM sequencing data and FRGs from the Gene Expression Omnibus (GEO) database and FerrDb database. Then, drug-gene interaction networks and competitive endogenous RNA (ceRNA) networks linked to the marker genes were built after marker genes were filtered by two machine learning algorithms (LASSO and SVM-RFE algorithms). Finally, to confirm the expression of marker genes, the GSE76895 dataset was utilized. The protein and RNA expression of some marker genes in T2DM and nondiabetic tissues was also examined by Western blotting, immunohistochemistry (IHC), immunofluorescence (IF) and quantitative real-time PCR (qRT-PCR). RESULTS: We obtained 58 differentially expressed genes (DEGs) associated with ferroptosis. GO and KEGG enrichment analyses showed that these DEGs were significantly enriched in hypoxia and ferroptosis. Subsequently, eight marker genes (SCD, CD44, HIF1A, BCAT2, MTF1, HILPDA, NR1D2, and MYCN) were screened by LASSO and SVM-RFE machine learning algorithms, and a model was constructed based on these eight genes. This model also has high diagnostic power. In addition, based on these eight genes, we obtained 48 drugs and constructed a complex ceRNA network map. Finally, Western blotting, IHC, IF, and qRT-PCR results of clinical samples further confirmed the results of public databases. CONCLUSIONS: The diagnosis and aetiology of T2DM can be greatly aided by eight FRGs, providing novel therapeutic avenues.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Identification and Validation of Active Ingredient in Cerebrotein Hydrolysate-I Based on Pharmacokinetic and Pharmacodynamic Studies.

Cerebrotein hydrolysate-1 (CH-1), a mixture of small peptides, polypeptides, and various amino acids derived from porcine brain, has been widely used in the treatment of cerebral injury. However, the bioactive composition and pharmacokinetics of CH-1 are still unexplored because of their complicated composition and relatively tiny amounts in vivo. Herein, NanoLC Orbitrap Fusion Lumos Tribrid Mass Spectrometer was firstly used to qualitatively analyze the components of CH-1. A total of 1347 peptides were identified, of which 43 peptides were characterized by high mass spectrometry (MS) intensity and identification accuracy. We then innovatively synthesized four main peptides for activity verification, and the results suggested that Pep72 (NYEPPTVVPGGDL) had the strongest neuroprotective effect on both in vivo and in vitro models. Next, a quantitative method for Pep72 was established based on liquid chromatography tandem mass spectrometry (LC-MS/MS) with the aid of Skyline software and then used in its pharmacokinetic studies. The results revealed that Pep72 had a high elimination rate and low exposure in rats. In addition, a hCMEC/D3-based in vitro model was built and firstly used to investigate the transport of Pep72. We found that Pep72 had extremely low blood-brain barrier permeability and was not a substrate of efflux transporters. The biotransformation of Pep72 in rat fresh plasma and tissues was investigated to explore the contradiction between pharmacokinetics and efficacy. A total of 11 main metabolites were structurally identified, with PGGDL and EPPTVPGGDL being the main metabolites of Pep72. Notably, metalloproteinase and cysteine protease were confirmed to be the main enzymes mediating Pep72 metabolism in rat tissues. SIGNIFICANCE STATEMENT: The NanoLC Orbitrap Fusion Lumos Tribrid Mass Spectrometer was firstly applied to discover the components of CH-1, and one main peptide Pep72 (NYEPPTVVPGGDL) was innovatively synthesized and firstly found to have the strongest neuroprotective effect among 1347 peptides identified from CH-1. Our study is the first time to identify and verify the active ingredient of CH-1 from the perspective of pharmacokinetics and pharmacodynamics, and provides a systematic technical platforms and strategies for the active substance research of other protein hydrolysates.