A comprehensive comparison of molecular and phenotypic profiles between hepatitis B virus (HBV)-infected and non-HBV-infected hepatocellular carcinoma by multi-omics analysis.

Hepatitis B virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC). An interesting question is how different are the molecular and phenotypic profiles between HBV-infected (HBV+) and non-HBV-infected (HBV-) HCCs? Based on the publicly available multi-omics data for HCC, including bulk and single-cell data, and the data we collected and sequenced, we performed a comprehensive comparison of molecular and phenotypic features between HBV+ and HBV- HCCs. Our analysis showed that compared to HBV- HCCs, HBV+ HCCs had significantly better clinical outcomes, higher degree of genomic instability, higher enrichment of DNA repair and immune-related pathways, lower enrichment of stromal and oncogenic signaling pathways, and better response to immunotherapy. Furthermore, in vitro experiments confirmed that HBV+ HCCs had higher immunity, PD-L1 expression and activation of DNA damage response pathways. This study may provide insights into the profiles of HBV+ and HBV- HCCs, and guide rational therapeutic interventions for HCC patients.

Altered epitopes enhance macrophage-mediated anti-tumour immunity to low-immunogenic tumour mutations.

Personalized neoantigen therapy has shown long-term and stable efficacy in specific patient populations. However, not all patients have sufficient levels of neoantigens for treatment. Although somatic mutations are commonly found in tumours, a significant portion of these mutations do not trigger an immune response. Patients with low mutation burdens continue to exhibit unresponsiveness to this treatment. We propose a design paradigm for neoantigen vaccines by utilizing the highly immunogenic unnatural amino acid p-nitrophenylalanine (pNO2Phe) for sequence alteration of somatic mutations that failed to generate neoepitopes. This enhances the immunogenicity of the mutations and transforms it into a suitable candidate for immunotherapy. The nitrated altered epitope vaccines designed according to this paradigm is capable of activating circulating CD8+ T cells and inducing immune cross-reactivity against autologous mutated epitopes in different MHC backgrounds (H-2Kb, H-2Kd, and human HLA-A02:01), leading to the elimination of tumour cells carrying the mutation. After immunization with the altered epitopes, tumour growth was significantly inhibited. It is noteworthy that nitrated epitopes induce tumour-infiltrating macrophages to differentiate into the M1 phenotype, surprisingly enhancing the MHC II molecule presenting pathway of macrophages. Nitrated epitope-treated macrophages have the potential to cross-activate CD4+ and CD8+ T cells, which may explain why pNO2Phe can enhance the immunogenicity of epitopes. Meanwhile, the immunosuppressive microenvironment of the tumour is altered due to the activation of macrophages. The nitrated neoantigen vaccine strategy enables the design of vaccines targeting non-immunogenic tumour mutations, expanding the pool of potential peptides for personalized and shared novel antigen therapy. This approach provides treatment opportunities for patients previously ineligible for new antigen vaccine therapy.

Cascaded Nanozyme Based pH-Responsive Oxygenation for Targeted Eradication of Resistant Helicobacter Pylori.

Nanozymes, as substitutes for natural enzymes, are constructed as cascade catalysis systems for biomedical applications due to their inherent catalytic properties, high stability, tunable physicochemical properties, and environmental responsiveness. Herein, a multifunctional nanozyme is reported to initiate cascade enzymatic reactions specific in acidic environments for resistant Helicobacter pylori (H. pylori) targeting eradication. The cobalt-coated Prussian blue analog based FPB-Co-Ch NPs displays oxidase-, superoxide dismutase-, peroxidase-, and catalase- mimicking activities that trigger • O 2 - ${\mathrm{O}}_2^ - {\bm{\ }}$ and H2O2 to supply O2, thereby killing H. pylori in the stomach. To this end, chitosan is modified on the surface to exert bacterial targeted adhesion and improve the biocompatibility of the composite. In the intestinal environment, the cascade enzymatic activities are significantly inhibited, ensuring the biosafety of the treatment. In vitro, sensitive and resistant strains of H. pylori are cultured and the antibacterial activity is evaluated. In vivo, murine infection models are developed and its success is confirmed by gastric mucosal reculturing, Gram staining, H&E staining, and Giemsa staining. Additionally, the antibacterial capacity, anti-inflammation, repair effects, and biosafety of FPB-Co-Ch NPs are comprehensively investigated. This strategy renders a drug-free approach that specifically targets and kills H. pylori, restoring the damaged gastric mucosa while relieving inflammation.

A pioneer nematode effector suppresses plant reactive oxygen species burst by interacting with the class III peroxidase.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease, which can devastate the pine forest ecosystem. Usually, plant cells generate reactive oxygen species (ROS) as a defensive substance or signalling molecules to resist the infection of nematodes. However, little is known about how B. xylophilus effectors mediate the plant ROS metabolism. Here, we identified a pioneer B. xylophilus Prx3-interacting effector 1 (BxPIE1) expressed in the dorsal gland cells and the intestine. Silencing of the BxPIE1 gene resulted in reduced nematode reproduction and a delay in disease progression during parasitic stages, with the upregulation of pathogenesis-related (PR) genes PtPR-3 (class Ⅳ chitinase) and PtPR-9 (peroxidase). The protein-protein interaction assays further demonstrated that BxPIE1 interacts with a Pinus thunbergii class III peroxidase (PtPrx3), which produces H2O2 under biotic stress. The expression of BxPIE1 and PtPrx3 was upregulated during the infection stage. Furthermore, BxPIE1 effectively inhibited H2O2 generating from class III peroxidase and ascorbate can recover the virulence of siBxPIE1-treated B. xylophilus by scavenging H2O2. Taken together, BxPIE1 is an important virulence factor, revealing a novel mechanism utilized by nematodes to suppress plant immunity.

New monoterpene phenol dimers from the fruits of Psoralea corylifolia.

Three new monoterpene phenol dimers, bisbakuchiols V-X (1-3), and two bakuchiol ethers (4 and 5), along with four known compounds (6-9) were isolated from the fruits of Psoralea corylifolia. Their structures were elucidated based on extensive spectral analysis. The absolute configurations of 1, 2, 4, and 5 were specified by quantum chemical calculations of ECD spectra.

The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

Phototherapy and Mechanism Exploration of Biofilm and Multidrug-Resistant Helicobacter pylori by Bacteria-Targeted NIR Photosensitizer.

Helicobacter pylori (H. pylori) infection has been the leading cause of gastric cancer development. In recent years, the resistance of H. pylori against antibiotic treatment has been a great challenge for most countries worldwide. Since biofilm formation is one of the reasons for the antibiotic resistance of H. pylori, and phototherapy has emerged as a promisingly alternative antibacterial treatment, herein the bacteria-targeted near-infrared (NIR) photosensitizer (T780T-Gu) by combining positively-charged guanidinium (Gu) with an efficient phototherapeutic agent T780T is developed. The proposed molecule T780T-Gu exhibits synergistic photothermal therapy/photodynamic therapy effect against both H. pylori biofilms and multidrug-resistant (MDR) clinical strains. More importantly, the phototherapy mechanism of T780T-Gu acquired by the RNA-seq analysis indicates that structural deficiency as well as a decrease in metabolism and defense activity are the possible reasons for the efficient H. pylori phototherapy.

The Use of Metformin and Postoperative Insulin Pump Were Predictive Factors for Outcomes of Diabetic Colorectal Cancer Patients after Surgery.

PURPOSE: This present study aims to explore the influence of metformin and postoperative insulin pump use on colorectal cancer (CRC) patients with type II diabetes mellitus (T2DM) who received surgery in terms of short-term and long-term outcomes. METHODS: 613 CRC patients who had comorbid T2DM and received surgery at a single clinical center from Jan, 2011 to Dec, 2021 were included in this study. Univariate and multivariate logistic regression analyses were used to find predictive factors for overall complications and major complications. Cox regression analyses was used to find prognostic factors for overall survival (OS) and disease-free survival (DFS). All statistical analysis was performed using SPSS (version 22.0) software. The Kaplan-Meier curve was used to show the OS and DFS between the insulin pump group and the no insulin pump group. RESULTS: Multivariate logistic regression analysis reported that lower body mass index (BMI) (p < 0.01, OR = 0.922, 95% CI = 0.870-0.977) and metformin use (p = 0.03, OR = 0.643, 95% CI = 0.431-0.959) were independent protective factors for overall complications, and insulin pump after surgery (p < 0.01, OR = 3.991, 95% CI = 2.434-6.544) was an independent risk factor for overall complications. As for major complications, metformin use (p = 0.042, OR = 0.274, 95% CI = 0.079-0.956) and insulin pump after surgery (p = 0.03, OR = 2.892, 95% CI = 1.107-7.552) remained independent protective factors and independent risk factors, respectively. Moreover, in Cox regression analyses, age (OS: p < 0.01, HR = 1.032, 95% CI = 1.008-1.057; DFS: p < 0.01, HR = 1.030, 95% CI = 1.008-1.052), tumor stage (OS: p < 0.01, HR = 1.709, 95% CI = 1.244-2.346; DFS: p < 0.01, HR = 1.696, 95% CI = 1.276-2.254), and Insulin pump after surgery (OS: p < 0.01, HR = 2.923, 95% CI = 1.887-4.527; DFS: p < 0.01, HR = 2.671, 95% CI = 1.779-4.009) were independent prognostic factors for both OS and DFS. After comparing the OS and DFS between the insulin pump group and the no insulin pump group, patients who received postoperative insulin pump had worse OS and DFS in all tumor node metastasis (TNM) stages (p < 0.01). CONCLUSION: Diabetic CRC patients who used metformin had a lower risk of postoperative complications. However, there was no difference from patients not using metformin in terms of survival. Furthermore, patients receiving postoperative insulin pump had more postoperative complications and worse survival in all TNM stages.

Do colorectal cancer patients with a postoperative stoma have sexual problems? A pooling up analysis of 2566 patients.

PURPOSE: This study aimed to evaluate the effect of the stoma on sexual function in colorectal cancer patients. METHODS: Search strategy was conducted in four databases including PubMed, Embase, the Cochrane Library, and Medline on Nov 20, 2022. After that, the quality of eligible studies was assessed by the Newcastle Ottawa Scale (NOS) and the Agency for Healthcare Research Quality (AHRQ). In order to evaluate the sexual function between the stoma group and the non-stoma group, mean values and standard deviation were pooled for scores in questionnaires, and odds ratio and 95% confidence intervals were pooled for self-designed problems. Results were shown in forest plots; Egger's test and the trim and fill analysis were used for assessing the publication bias. All the data analyses above were performed by STATA 16.0 software. RESULTS: Six cohort studies and three cross-sectional studies were included for this pooling up analysis. As for the European Organization for Research and Treatment of Cancer (EORTC) quality of life questionnaire (QLQ-C38), male patients with stomas had more sexual problems (mean differences = 0.32, 95% confidence intervals = 0.01 to 0.63, I2 = 67.80%, P = 0.05); however, other indicators including sexual functioning, sexual enjoyment, and female sexual problems had little difference between the two groups. As for other questionnaires and problems, stoma did not have an impact on sexual function. CONCLUSION: The stoma after surgery was not significantly associated with the sexual function of female patients with colorectal cancer and was associated with worse scores on only sexual problems for males.

Two Novel Bursaphelenchus xylophilus Kunitz Effector Proteins Using Different Infection and Survival Strategies to Suppress Immunity in Pine.

Pine wilt disease, caused by Bursaphelenchus xylophilus, results in tremendous economic loss in conifer production every year. To disturb the host immune responses, plant pathogens secrete a mass of effector proteins that facilitate the infection process. Although several effectors of B. xylophilus have been identified, detailed mechanisms of their functions remain largely unexplored. Here, we reveal two novel B. xylophilus Kunitz effectors, named BxKU1 and BxKU2, using different infection strategies to suppress immunity in Pinus thunbergii. We found that both BxKU1 and BxKU2 could suppress PsXEG1-triggered cell death and were present in the nucleus and cytoplasm in Nicotiana benthamiana. However, they had different three-dimensional structures and various expression patterns in B. xylophilus infection. In situ hybridization experiments showed that BxKU2 was expressed in the esophageal glands and ovaries, whereas BxKU1 was only expressed in the esophageal glands of females. We further confirmed that the morbidity was significantly decreased in P. thunbergii infected with B. xylophilus when BxKU1 and BxKU2 were silenced. The silenced BxKU2I, but not BxKU1, affected the reproduction and feeding rate of B. xylophilus. Moreover, BxKU1 and BxKU2 targeted to different proteins in P. thunbergii, but they all interacted with thaumatin-like protein 4 (TLP4) according to yeast two-hybrid screening. Collectively, our study showed that B. xylophilus could incorporate two Kunitz effectors in a multilayer strategy to counter immune response in P. thunbergii, which could help us better understand the interaction between plant and B. xylophilus.

A comparative study on laparoscopic and open surgical approaches for perforated peptic ulcer repair: efficacy and outcomes analysis.

PURPOSE: This study aimed to compare the clinical outcomes of the clinical outcomes of laparoscopic and open sutures for peptic ulcer perforation (PPU). MATERIALS AND METHODS: PubMed, EMBASE, and Cochrane Library databases were searched for eligible studies from inception to March 31, 2023. Odds ratios (OR) and 95% confidence intervals (Cl) were also calculated. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the included studies. This study was performed using the Stata (V.16.0) software. RESULTS: A total of 29 studies involving 17,228 patients were included in this study. In terms of postoperative outcomes, the laparoscopic group had a shorter postoperative hospital stay (MD = -0.29, 95%CI = -0.44 to -0.13, P = 0.00), less blood loss (MD = -0.45, 95%CI = -0.82 to -0.08, P = 0.02), fewer wound infection (OR = 0.20, 95%CI = 0.17 to 0.24, P = 0.00), fewer pneumonia (OR = 0.59, 95%CI = 0.41 to 0.87, P = 0.01), fewer respiratory complications (OR = 0.26, 95%CI = 0.13 to 0.55, P = 0.00) and lower postoperative morbidity (OR = 0.51, 95%CI = 0.33 to 0.78, P = 0.00). The laparoscopic group had a lower mortality rate (OR = 0.36, 95%CI = 0.27 to 0.49, P = 0.00) than the open group. We also found that the laparoscopic group had a higher overall complication rate than the open group (OR = 0.45, 95%CI = 0.34 to 0.60, P = 0.00). CONCLUSION: Laparoscopic repair was associated with a lower risk of mortality than open repair in patients with PPU. Laparoscopic repair may be a better option in patients with PPU.

The predictive value of advanced lung cancer inflammation index for short-term outcomes and prognosis of colorectal cancer patients who underwent radical surgery.

PURPOSE: The purpose of this study was to investigate the predictive value of advanced lung cancer inflammation index (ALI) for short-term outcomes and prognosis of colorectal cancer (CRC) patients who underwent radical surgery. METHODS: CRC patients who underwent radical resection were included from Jan 2011 to Jan 2020 in our single clinical centre. Short-term outcomes, overall survival (OS), and disease-free survival (DFS) were compared in different groups. Cox analysis was conducted to identify independent risk factors for OS and DFS. RESULTS: A total of 4010 patients who underwent radical CRC surgery were enrolled in the current study. As a result, the low ALI group had longer operation time (p = 0.02), more intra-operative blood loss (p < 0.01), longer postoperative hospital stay (p < 0.01), and more overall complications (p < 0.01). Moreover, ALI (p < 0.01, OR = 0.679, 95% CI = 0.578-0.798) was an independent risk factor for overall complications. As for survival, the low ALI group had worse OS in all TNM stages (p < 0.01), stage II (p < 0.01) and stage III (p < 0.01). Similarly, the low ALI group had worse DFS in all TNM stages (p < 0.01), stage II (p < 0.01), and stage III (p < 0.01). In Cox analysis, ALI was an independent risk factors for OS (p < 0.01, HR = 0.707, 95% CI = 0.589-0.849) and DFS (p < 0.01, HR = 0.732, 95% CI = 0.622-0.861). CONCLUSION: Lower ALI was associated with more postoperative complications, worse OS, and DFS for CRC patients who underwent radical surgery. Furthermore, ALI was an independent risk factor for overall complications, OS, and DFS. Surgeons should pay close attention to patients with low ALI before surgery and make clinical strategies cautiously.

Can natural environments enhance acute effects of rehabilitation exercise for older adults? A pilot randomized controlled trial.

The added value of natural environments in rehabilitation exercise is unclear. This study aimed to investigate whether there are more acute health benefits for older adults after a single rehabilitation exercise session performed in an outdoor natural environment compared with an indoor environment. Twenty-two nursing home residents were randomly assigned to the outdoor (n = 11, 79.5 ± 2.1 years) or indoor group (n = 11, 78.8 ± 5.2 years). Performance test outcomes were measured pre- and post-training session. The indoor group had a significantly higher blood pressure, greater increase in heart rate, higher perceived exercise intensity and physiological fatigue than the outdoor group. The combination of rehabilitation exercise with an outdoor natural environment may reduce exercise fatigue and improve cardiovascular health in older adults, with greater acute health benefits compared with an indoor environment. Rehabilitation exercise in the natural environments may be a highly valued environmental intervention for physiotherapy in older adults.

Enhanced virulence of genetically engineered Autographa californica nucleopolyhedrovirus owing to accelerated viral DNA replication aided by inserted ascovirus genes.

Genetic engineering technology is an ideal method to improve insecticidal efficiency by combining the advantages of different pathogenic microorganisms. Thus, six ascovirus genes were introduced into the genomic DNA of Autographa californica nucleopolyhedrovirus (AcMNPV) to possibly transfer the intrinsically valuable insecticidal properties from ascovirus to baculovirus. The viral budded virus (BV) production and viral DNA replication ability of AcMNPV-111 and AcMNPV-165 were significantly stronger than that of AcMNPV-Egfp (used as the wild-type virus in this study), whereas AcMNPV-33 had reduced ones. AcMNPV-111 and AcMNPV-165 also exhibited excellent insecticidal efficiency in the in vivo bioassays: AcMNPV-111 showed a 24.1% decrease in the LT50 value and AcMNPV-165 exhibited a 56.3% decrease in the LD50 value compared with AcMNPV-Egfp against the 3rd instar of Spodoptera exigua larvae, respectively. Furthermore, the size of the occlusion bodies (OBs) of AcMNPV-33, AcMNPV-111, and AcMNPV-165 were significantly increased compared to that of AcMNPV-Egfp. AcMNPV-111 and AcMNPV-165 had stable virulence against the 2nd to 4th instars tested larvae and higher OB yield than AcMNPV-Egfp in the 3rd and 4th instar larvae. Correlation and regression analyses indicated that it is better to use 5 OBs/larva virus to infect the 2nd instar larvae to produce AcMNPV-111 and 50 OBs/larva virus to infect the 3rd instar larvae to produce AcMNPV-165. The results of this study obtained recombinant viruses with enhanced virulence and exhibited a diversity of ascovirus gene function based on the baculovirus platform, which provided a novel strategy for the improvement of baculovirus as a biological insecticide.

Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O-Linked ß-N-Acetylglucosamine Strategy.

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked ß-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.

The Bursaphelenchus xylophilus effector BxML1 targets the cyclophilin protein (CyP) to promote parasitism and virulence in pine.

BACKGROUND: Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD) that has caused enormous ecological and economic losses in China. The mechanism in the interaction between nematodes and pine remains unclear. Plant parasitic nematodes (PPNs) secrete effectors into host plant tissues. However, it is poorly studied that role of effector in the infection of pine wood nematode (PWN). RESULTS: We cloned, characterized and functionally validated the B. xylophilus effector BxML1, containing an MD-2-related lipid-recognition (ML) domain. This protein inhibits immune responses triggered by the molecular pattern BxCDP1 of B. xylophilus. An insitu hybridization assay demonstrated that BxML1 was expressed mainly in the dorsal glands and intestine of B. xylophilus. Subcellular localization analysis showed the presence of BxML1 in the cytoplasm and nucleus. Furthermore, number of B. xylophilus and morbidity of pine were significantly reduced in Pinus thunbergii infected with B. xylophilus when BxML was silenced. Using yeast two-hybrid (Y2H) and coimmunoprecipitation (CoIP) assays, we found that the BxML1 interacts with cyclophilin protein PtCyP1 in P. thunbergii. CONCLUSIONS: This study illustrated that BxML1 plays a critical role in the B. xylophilus-plant interaction and virulence of B. xylophilus.

Development and validation of a liquid chromeatography-tandem mass spectrometry method for simultaneous quantification of medium- and long-chain saturated fatty acids in hamster plasma samples.

RATIONALE: Saturated fatty acids (SFAs) are associated with many diseases in humans. Developing a reliable analytical method to analyze SFAs in plasma is essential to understand their biological activities. An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method has been developed for the quantification of medium- and long-chain SFAs (M/LCSFAs) in hamster plasma. METHODS: We compared three methods (DOLE, Folch and MTBE) for extracting M/LCSFAs from plasma. The M/LCSFA derivatives were separated using a C18 column. The method was validated and applied to analyze M/LCSFA concentrations in normal-fat diet (NFD) and high-fat diet (HFD) hamster plasma. RESULTS: Among the three extraction methods, the DOLE method had the highest extraction recovery and was simple to operate with a short incubation time. All of the calibration curves exhibited good linear relationships (r ≥ 0.9958). The results for selectivity, accuracy, precision, matrix effects and recovery were all within the acceptance criteria. In HFD hamster plasma, the concentration of M/LCSFAs with even-carbon chain length was significantly increased. CONCLUSIONS: A simple, robust and reproducible method for the simultaneous quantification of M/LCSFAs by UHPLC/MS/MS was developed and validated. The method gave successfully quantification of M/LCSFAs in plasma samples from NFD and HFD hamsters.

Development and validation of an LC-MS/MS method for simultaneous determination of remdesivir and its hydrolyzed metabolite and nucleoside, and its application in a pharmacokinetic study of normal and diabetic nephropathy mice.

Remdesivir (RDV), a phosphoramidate prodrug, has broad-spectrum antiviral activity. It is the first antiviral drug approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Remdesivir is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, the phosphatase inhibitor PhosSTOP and carboxylesterase inhibitor 5,5'-dithiobis-2-nitrobenzoic acid were used to improve stability of RDV in mouse blood. We developed a rapid and sensitive LC-MS/MS method to simultaneously quantify RDV, RM-442 and RN in mouse blood. Chromatographic separation was achieved by gradient elution on an Acquity HSS T3 column. The run time was 3.2 min. The linearity ranges of the analytes were 0.5-1,000 ng/ml for RDV and 5-10,000 ng/ml for both RM-442 and RN. The method had an acceptable precision (RSD < 8.4% for RDV, RSD < 10.7% for RM-442 and RSD < 7.2% for RN) and accuracy (91.0-106.3% for RDV, 92.5-98.6% for RM-442 and 87.5-98.4% for RN). This method was successfully applied to analyze RDV, RM-442 and RN in the blood of normal and diabetic nephropathy DBA/2 J mice after intravenous injection of RDV at 20 mg/kg. The area under the concentration-time curve of RN between the normal and diabetic nephropathy mice showed a significant difference (P < 0.01).

Time-series gene expression patterns and their characteristics of Beauveria bassiana in the process of infecting pest insects.

Beauveria bassiana has been widely used as an important biological control fungus for agricultural and forest pests, and clarifying the interaction mechanism between B. bassiana and its host will help to better exert the efficacy of the mycoinsecticide. Here, we proposed a novel pattern analysis (PA) method for analyzing time-series data and applied it to a transcriptomic data set of B. bassiana infecting Galleria mellonella. We screened out 14 patterns including 868 genes, which had some characteristics that were not inferior to differentially expressed genes (DEGs). Compared with the previous analysis of this data set, we had three novel discoveries during B. bassiana infection, including overall downregulation of gene expression, the more critical first 24 h, and enrichment of regulatory functions of downregulated genes. Our new PA method promises to be an important complement to DEGs analysis for time-series transcriptomic data, and our findings enrich our knowledge of molecular mechanisms of fungal-host interactions.