Single-cell RNA sequencing reveals the immunoregulatory roles of PegIFN-α in patients with chronic hepatitis B.

BACKGROUND AND AIMS: Chronic hepatitis B (CHB) is caused by HBV infection and affects the lives of millions of people worldwide by causing liver inflammation, cirrhosis, and liver cancer. Interferon-alpha (IFN-α) therapy is a conventional immunotherapy that has been widely used in CHB treatment and achieved promising therapeutic outcomes by activating viral sensors and interferon-stimulated genes (ISGs) suppressed by HBV. However, the longitudinal landscape of immune cells of CHB patients and the effect of IFN-α on the immune system are not fully understood. APPROACH AND RESULTS: Here, we applied single-cell RNA sequencing (scRNA-seq) to delineate the transcriptomic landscape of peripheral immune cells in CHB patients before and after PegIFN-α therapy. Notably, we identified three CHB-specific cell subsets, pro-inflammatory (Pro-infla) CD14+ monocytes, Pro-infla CD16+ monocytes and IFNG+ CX3CR1- NK cells, which highly expressed proinflammatory genes and positively correlated with HBsAg. Furthermore, PegIFN-α treatment attenuated percentages of hyperactivated monocytes, increased ratios of long-lived naive/memory T cells and enhanced effector T cell cytotoxicity. Finally, PegIFN-α treatment switched the transcriptional profiles of entire immune cells from TNF-driven to IFN-α-driven pattern and enhanced innate antiviral response, including virus sensing and antigen presentation. CONCLUSIONS: Collectively, our study expands the understanding of the pathological characteristics of CHB and the immunoregulatory roles of PegIFN-α, which provides a new powerful reference for the clinical diagnosis and treatment of CHB.

Inactivated vaccines prevent severe COVID-19 in patients infected with the Delta variant: A comparative study of the Delta and Alpha variants from China.

The Delta variant has gradually replaced the Alpha variant as the major strain of SARS-COV-2 infection worldwide. We extracted the clinical characteristics and outcomes information about 381 hospitalized patients infected with Delta variant and compared them with 856 patients diagnosed with Alpha variant infection in Zhejiang Province. The majority (85.3%) of patients infected with the Delta variant had received inactivated vaccine. The patients' condition was generally mild. Most of them were mild (35.7%) and common (62.7%) types. Only six patients (1.5%) were severe/critical types. During the follow-up period, patients infected with the Delta variant had longer hospital stays than the Alpha variant (24 [21-26] vs. 18 [14-24], p < 0.001). In addition, the unvaccinated patients infected with the Delta variant had a higher proportion of severe/critical cases than vaccinated patients (11.11% vs. 0.92%, p = 0.024) and a higher usage rate of glucocorticoids (38.89 vs. 14.77%, p = 0.017) and antibiotics (55.56% vs. 32.31%, p = 0.042) during hospitalization. The vaccine's efficacy against severe COVID-19 did not diminish over time for patients who received two doses of the inactivated vaccine. The disease types and clinical manifestations were generally mild in patients infected with the Delta variant, possibly associated with widespread vaccination with inactivated vaccines in China.

Pegylated interferon-α-2b combined with tenofovir disoproxil fumarate, granulocyte-macrophage colony-stimulating factor, and hepatitis B vaccine treatment for naïve HBeAg-positive chronic hepatitis B patients: A prospective, multicenter, randomized controlled study.

Hepatitis B surface antigen (HBsAg) loss or seroconversion is an ideal treatment endpoint for patients with chronic hepatitis B but is rarely achievable in  hepatitis B e-antigen (HBeAg)-positive patients using existing treatment strategies. In this study, the effect of pegylated interferon (peg-IFN) alfa-2b plus tenofovir disoproxil fumarate (TDF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and hepatitis B vaccine was evaluated. This randomized controlled trial was conducted at nine liver centers in Chinese university hospitals from May 2018 to July 2020. Patients (n = 303) enrolled were randomly administered peg-IFN-α-2b combined with TDF, GM-CSF, and hepatitis B vaccine (experimental group); peg-IFN-α-2b plus TDF (control group 2); or interferon-α-2b alone (control group 1). The primary efficacy endpoint was HBsAg seroconversion at 48 weeks and the secondary endpoint included safety. No differences in baseline HBsAg levels were observed among the groups. The primary endpoint was achieved in three (3.0%), one (1.03%), and one (1.19%) patient in the experimental group, control group 2, and control group 1, respectively. The incidence of HBsAg seroconversion at week 48 was not significantly different among the three groups (p = 0.629). However, the decrease in serum levels of HBsAg at week 48 was significantly higher in the experimental and control group 2 compared with that in control group 1 (p = 0.008 and 0.006, respectively). No significant difference between the experimental and control group 2 was observed (p = 0.619). Adverse events were not significantly different among the groups except for the lower incidence of neutropenia in the experimental group. Peg-IFN-α-2b combined with TDF, GM-CSF, and hepatitis B vaccine is not superior to peg-IFN-α-2b combined with TDF in HBeAg-positive naïve patients. Clinical Trials Registration: ChiCTR1800016173.

PointSite: A Point Cloud Segmentation Tool for Identification of Protein Ligand Binding Atoms.

Accurate identification of ligand binding sites (LBS) on a protein structure is critical for understanding protein function and designing structure-based drugs. As the previous pocket-centric methods are usually based on the investigation of pseudo-surface-points outside the protein structure, they cannot fully take advantage of the local connectivity of atoms within the protein, as well as the global 3D geometrical information from all the protein atoms. In this paper, we propose a novel point clouds segmentation method, PointSite, for accurate identification of protein ligand binding atoms, which performs protein LBS identification at the atom-level in a protein-centric manner. Specifically, we first transfer the original 3D protein structure to point clouds and then conduct segmentation through Submanifold Sparse Convolution based U-Net. With the fine-grained atom-level binding atoms representation and enhanced feature learning, PointSite can outperform previous methods in atom Intersection over Union (atom-IoU) by a large margin. Furthermore, our segmented binding atoms, that is, atoms with high probability predicted by our model can work as a filter on predictions achieved by previous pocket-centric approaches, which significantly decreases the false-positive of LBS candidates. Besides, we further directly extend PointSite trained on bound proteins for LBS identification on unbound proteins, which demonstrates the superior generalization capacity of PointSite. Through cascaded filter and reranking aided by the segmented atoms, state-of-the-art performance can be achieved over various canonical benchmarks, CAMEO hard targets, and unbound proteins in terms of the commonly used DCA criteria.

High body mass index is a significant risk factor for the progression and prognosis of imported COVID-19: a multicenter, retrospective cohort study.

BACKGROUND: Coronavirus disease 2019(COVID-19) has spread worldwide. The present study aimed to characterize the clinical features and outcomes of imported COVID-19 patients with high body mass index (BMI) and the independent association of BMI with disease severity. METHODS: In this retrospective cohort study, 455 imported COVID-19 patients were admitted and discharged in Zhejiang province by February 28, 2020. Epidemiological, demographic, clinical, laboratory, radiological, treatment, and outcome data were collected, analyzed and compared between patients with BMI ≥ 24and < 24. RESULTS: A total of 268 patients had BMI < 24, and 187 patients had BMI ≥ 24. Those with high BMI were mostly men, had a smoking history, fever, cough, and sputum than those with BMI < 24. A large number of patients with BMI ≥ 24 were diagnosed as severe/critical types. Some biochemical indicators were significantly elevated in patients with BMI ≥ 24. Also, acute liver injury was the most common complication in these patients. The median days from illness onset to severe acute respiratory syndrome coronavirus 2 detection, duration of hospitalization, and days from illness onset to discharge were significantly longer in patients with BMI ≥ 24 than those with BMI < 24. High BMI, exposure to Wuhan, any coexisting medical condition, high temperature, C-reactive protein (CRP), and increased lactate dehydrogenase (LDH) were independent risk factors for severe/critical COVID-19. After adjusting for age, sex and above factors, BMI was still independently associated with progression to severe/critical illness (P = 0.0040). Hemoglobin, alanine aminotransferase (ALT), CRP, and serum creatinine (Scr) were independent risk factors associated with high BMI. CONCLUSIONS: Contrasted with the imported COVID-19 patients with BMI < 24, high proportion of COVID-19 patients with BMI ≥ 24 in our study, especially those with elevated CRP and LDH, developed to severe type, with longer hospitalization duration and anti-virus course. Thus, high BMI is a risk factor for the progression and prognosis of imported COVID-19.

Decreased B Cells on Admission Associated With Prolonged Viral RNA Shedding From the Respiratory Tract in Coronavirus Disease 2019: A Case-Control Study.

The viral RNA shedding time (VST) for severe acute respiratory syndrome coronavirus 2 has not been well characterized. Clinical data were collected and compared between patients with short and long VSTs (in the lower and upper quartiles, respectively). The probability of recurrent positive reverse-transcription polymerase chain reaction results decreased sharply to 4.8% after 3 consecutive negative results. A series of ≥3 consecutive negative results was suitable as a criterion for the end of viral RNA shedding. The VST for shedding from the respiratory tract was significantly shorter in patients with normal B-cell counts on admission than in those with decreased B-cell counts (median [interquartile range], 11 [9-13] vs 16 [12-20] days, respectively; P = .001).

Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms.

OBJECTIVE: The SARS-CoV-2-infected disease (COVID-19) outbreak is a major threat to human beings. Previous studies mainly focused on Wuhan and typical symptoms. We analysed 74 confirmed COVID-19 cases with GI symptoms in the Zhejiang province to determine epidemiological, clinical and virological characteristics. DESIGN: COVID-19 hospital patients were admitted in the Zhejiang province from 17 January 2020 to 8 February 2020. Epidemiological, demographic, clinical, laboratory, management and outcome data of patients with GI symptoms were analysed using multivariate analysis for risk of severe/critical type. Bioinformatics were used to analyse features of SARS-CoV-2 from Zhejiang province. RESULTS: Among enrolled 651 patients, 74 (11.4%) presented with at least one GI symptom (nausea, vomiting or diarrhoea), average age of 46.14 years, 4-day incubation period and 10.8% had pre-existing liver disease. Of patients with COVID-19 with GI symptoms, 17 (22.97%) and 23 (31.08%) had severe/critical types and family clustering, respectively, significantly higher than those without GI symptoms, 47 (8.14%) and 118 (20.45%). Of patients with COVID-19 with GI symptoms, 29 (39.19%), 23 (31.08%), 8 (10.81%) and 16 (21.62%) had significantly higher rates of fever >38.5°C, fatigue, shortness of breath and headache, respectively. Low-dose glucocorticoids and antibiotics were administered to 14.86% and 41.89% of patients, respectively. Sputum production and increased lactate dehydrogenase/glucose levels were risk factors for severe/critical type. Bioinformatics showed sequence mutation of SARS-CoV-2 with m6A methylation and changed binding capacity with ACE2. CONCLUSION: We report COVID-19 cases with GI symptoms with novel features outside Wuhan. Attention to patients with COVID-19 with non-classic symptoms should increase to protect health providers.

Analysis of Epidemiological and Clinical Features in Older Patients With Coronavirus Disease 2019 (COVID-19) Outside Wuhan.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) has become a large threat to public health in China, with high contagious capacity and varied mortality. This study aimed to investigate the epidemiological and clinical characteristics of older patients with COVID-19 outside Wuhan. METHODS: A retrospective study was performed, with collecting data from medical records of confirmed COVID-19 patients in Zhejiang province from 17 January to 12 February 2020. Epidemiological, clinical, and treatment data were analyzed between older (≥ 60 years) and younger (< 60 years) patients. RESULTS: A total of 788 patients with confirmed COVID-19 were selected; 136 were older patients with corresponding mean age of 68.28 ±â€…7.31 years. There was a significantly higher frequency of women in older patient group compared with younger patients (57.35% vs 46.47%, P = .021). The presence of coexisting medical conditions was significantly higher in older patients compared with younger patients (55.15% vs 21.93%, P < .001), including the rate of hypertension, diabetes, heart disease, and chronic obstructive pulmonary disease. Significantly higher rates of severe clinical type (older vs younger groups: 16.18% vs 5.98%, P < .001), critical clinical type (8.82% vs 0.77%, P < .001), shortness of breath (12.50% vs 3.07%, P < .001), and temperature of > 39.0°C (13.97% vs 7.21%, P = .010) were observed in older patients compared with younger patients. Finally, higher rates of intensive care unit admission (9.56% vs 1.38%, P < .001) and methylprednisolone application (28.68% vs 9.36%, P < .001) were also identified in older patients compared with younger ones. CONCLUSIONS: The specific epidemiological and clinical features of older COVID-19 patients included significantly higher female sex, body temperature, comorbidities, and rate of severe and critical type disease.

Liver Enzyme Elevation in Coronavirus Disease 2019: A Multicenter, Retrospective, Cross-Sectional Study.

INTRODUCTION: Elevated liver enzyme levels are observed in patients with coronavirus disease 2019 (COVID-19); however, these features have not been characterized. METHODS: Hospitalized patients with COVID-19 in Zhejiang Province, China, from January 17 to February 12, 2020, were enrolled. Liver enzyme level elevation was defined as alanine aminotransferase level >35 U/L for men and 25 U/L for women at admission. Patients with normal alanine aminotransferase levels were included in the control group. Reverse transcription polymerase chain reaction was used to confirm severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and patients symptomatic with SARS-CoV-2 infection were defined as patients with COVID-19. Epidemiological, demographic, clinical, laboratory, treatment, and outcome data were collected and compared. RESULTS: Of 788 patients with COVID-19, 222 (28.2%) patients had elevated liver enzyme levels (median [interquartile range {IQR}] age, 47.0 [35.0-55.0] years; 40.5% women). Being male, overweight, and smoking increased the risk of liver enzyme level elevation. The liver enzyme level elevation group had lesser pharyngalgia and more diarrhea than the control group. The median time from illness onset to admission was 3 days for liver enzyme level elevation groups (IQR, 2-6), whereas the median hospitalization time for 86 (38.7%) discharged patients was 13 days (IQR, 11-16). No differences in disease severity and clinical outcomes were noted between the groups. DISCUSSION: We found that 28.2% of patients with COVID-19 presented with elevated liver enzyme levels on admission, which could partially be related to SARS-CoV-2 infection. Male patients had a higher risk of liver enzyme level elevation. With early medical intervention, liver enzyme level elevation did not worsen the outcomes of patients with COVID-19.

Impact of comorbidities on patients with COVID-19: A large retrospective study in Zhejiang, China.

Coronavirus disease 2019 (COVID-19) has become a serious public health problem worldwide. Here, we stratified COVID-19 patients based on their comorbidities to assess their risk of serious adverse outcomes. We collected 856 hospitalized cases diagnosed with COVID-19 from 17 January to 7 February 2020, in Zhejiang Province, and analyzed their comorbidities and composite endpoint (including admission to intensive care unit owing to disease progression, shock, invasive ventilation, and death) to determine the relationship between comorbidities and adverse outcomes. The median age of patients was 46 (36-56) years; 439 (51.3%) were men, 242 (28.3%) had comorbidities, and 152 (17.8%) had two or more comorbidities. The most common comorbidity was hypertension (142 [16.6%]), followed by diabetes (64 [7.5%]). Of the 856 patients, there are 154 (18.0%) severe cases. Thirty-two (3.7%) reached composite endpoints, of which 22 (9.1%) were from the comorbidity group and 10 (1.6%) from the non-comorbidity group (P < .001). After adjusting for age and gender status, the risk of reaching the composite endpoint was higher in the group with comorbidity than in that without comorbidity (hazard ratio [HR] 3.04, 95% confidence interval [CI]: 1.40-6.60). HR values for patients with one, two, and three or more comorbidities were 1.61 (95% CI: 0.44-5.91), 3.44 (95% CI: 1.31-9.08), and 6.90 (95% CI: 2.69-17.69), respectively. COVID-19 patients with comorbidities had worse clinical outcomes as compared with those without any comorbidity. The higher the number of comorbidities, the greater was the risk of serious adverse outcomes.

Fibroblast growth factor 21 facilitates peripheral nerve regeneration through suppressing oxidative damage and autophagic cell death.

Seeking for effective drugs which are beneficial to facilitating axonal regrowth and elongation after peripheral nerve injury (PNI) has gained extensive attention. Fibroblast growth factor 21 (FGF21) is a metabolic factor that regulates blood glucose and lipid homeostasis. However, there is little concern for the potential protective effect of FGF21 on nerve regeneration after PNI and revealing related molecular mechanisms. Here, we firstly found that exogenous FGF21 administration remarkably promoted functional and morphologic recovery in a rat model of sciatic crush injury, manifesting as persistently improved motor and sensory function, enhanced axonal remyelination and regrowth and accelerated Schwann cells (SCs) proliferation. Furthermore, local FGF21 application attenuated the excessive activation of oxidative stress, which was accompanied with the activation of nuclear factor erythroid-2-related factor 2 (Nrf-2) transcription and extracellular regulated protein kinases (ERK) phosphorylation. We detected FGF21 also suppressed autophagic cell death in SCs. Additionally, treatment with the ERK inhibitor U0126 or autophagy inhibitor 3-MA partially abolishes anti-oxidant effect and reduces SCs death. Taken together, these results indicated that the role of FGF21 in remyelination and nerve regeneration after PNI was probably related to inhibit the excessive activation of ERK/Nrf-2 signalling-regulated oxidative stress and autophagy-induced cell death. Overall, our work suggests that FGF21 administration may provide a new therapy for PNI.

TCRß repertoire of memory T cell reveals potential role for Escherichia coli in the pathogenesis of primary biliary cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) is an organ-specific, T cell-mediated autoimmune disease which is characterized by the breakdown of self-tolerance to the highly conserved pyruvate dehydrogenase complex, especially the pyruvate dehydrogenase E2 complex (PDC-E2). However, the molecular mechanism of breakdown of self-tolerance is still unclear. METHODS: A combination of multiplex-PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of memory T cell receptor (TCR) ß-chain repertoire of PBC patient and healthy volunteers. In vitro induction and expansion of human PDC-E2163-176 (human PDC-E2)-specific T cells and E coli PDC-E231-44/134-147/235-248 (E coli PDC-E2)-specific T cells, and identified the human (and E coli) PDC-E2-specific TCRß repertoire by IR-seq. RESULTS: Primary biliary cholangitis patients have shorter complementarity-determining region 3s (CDR3s), and higher degree of sequence overlap in the TCRß repertoire of memory T cell. Moreover, altered insertion patterns and skewed TRBV segment usage were observed in PBC patients. With regard to the pathogenesis, the concentration of E coli was higher in PBC patients' faecal. The frequency of E coli (and human)-specific TCRs was higher in the memory TCRß repertoire of PBC patients compared with healthy controls. Importantly, the TCRß repertoire characteristics were almost identical between E coli PDC-E2-related TCRs and human PDC-E2-related TCRs, including the patterns of TRBV usage, CDR3 length and amino acid composition. CONCLUSION: Our findings comprehensively revealed the TCRß repertoire characterization of PBC patients, and provided a TCR molecular basis to understand the mechanism of cross-recognition between human PDC-E2 and E coli PDC-E2, and the imbalance of immune tolerance in PBC.

Switching Lamivudine with Adefovir Dipivoxil Combination Therapy to Entecavir Monotherapy Provides Better Viral Suppression and Kidney Safety.

Introduction: Most chronic hepatitis B (CHB) patients in China are primitively treated with a combination of lamivudine (LAM) and adefovir dipivoxil (ADV). Although antiviral resistance can be avoided with this combination therapy, using it can have harmful side effects related to ADV, specifically kidney and bone injury. This study was designed to compare viral suppression and kidney safety when switching LAM and ADV combination therapy de novo to entecavir (ETV) monotherapy in patients with CHB and compensated hepatic cirrhosis. Materials and methods: In total, 360 CHB and compensated liver cirrhosis patients who received treatment of LAM and ADV combination therapy for more than 1 year were included in this study. One hundred and eighty patients continued combination therapy to serve as a control group and the other 180 patients were switched to ETV monotherapy to serve as the experimental group. The total course of therapy was 3 years. Laboratory studies were done every 3 months to measure liver and kidney function. Studies included glomerular filtration rate (eGFR), HBV-DNA, urine ß2-microglobulin (ß2-M) and retinol binding protein (RBP). Results: In the experimental group, an HBV-DNA level below 20 IU/ml was found in 77.65%, 85.88%, and 94.77% in years 1, 2, and 3, respectively. In the control group, HBV-DNA levels were below 20 IU/ml in 69.66%, 75.42%, and 85.80% in years 1, 2, and 3, respectively. Low HBV-DNA levels in the experimental group were significantly less common than in the control group on the second and third year; P values were 0.009 and 0.006 for years 2 and 3, respectively. The cumulative genetic mutation rate was 3.49% in the experimental group and 8.88% in the control group (P=0.044). Decreases in eGFR more than 30% from baseline were found in 0%, 0.56%, and 1.74% of patients in the experimental group and 4.49%, 9.14% and 14.79% in patients in the control group in the first, second, and third year, respectively. Serum creatinine more than 50 µmol/L above baseline was found in 0%, 0% and 1.74% of patients in the experimental group and 1.12%, 4.00% and 5.32% of patients in the control group in years 1, 2, and 3, respectively. The urine ß2-M and RBP levels were abnormal more often in the experimental group than in the control group. Conclusion: Switching to ETV monotherapy can decrease HBV-DNA levels, reduce the genetic mutation rate, and prevent renal damage caused by LAM and ADV combination therapy in patients with CHB and compensated liver cirrhosis. Patients receiving LAM and ADV combination therapy de novo should be switched to ETV monotherapy immediately.

Dual Delivery of bFGF- and NGF-Binding Coacervate Confers Neuroprotection by Promoting Neuronal Proliferation.

BACKGROUND/AIMS: Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are essential for proper development, survival, growth, and maintenance of neurons in the central and peripheral nervous systems. However, because bFGF and NGF have short half-life and rapid diffusion rate, they have limited clinical efficacy. Thus, there is an urgent need to develop an effective delivery system to protect bFGF and NGF from proteolysis while maintaining their normal bioactivities. METHODS: To more efficiently deliver bFGF and NGF, we used a coacervate (synthesized with heparin and a biodegradable polycation at mass ratio of 500: 100). The maximal package loads of GFs in coacervate were determined by Western Blotting; release efficiency of bFGF and NGF was measured by ELISA. Additionally, we evaluated the effect of bFGF and NGF on the viability, survival, and proliferation of neurons by MTT assay, BrdU cell proliferation, and calcein staining. RESULTS: Our coacervate incorporated bFGF and NGF and continuously released them for at least three weeks. This enhanced the growth and proliferation of PC12 cells and SH-SY5Y cells. Moreover, co-delivery of bFGF and NGF using coacervate was more neuroprotective than free application of both factors or coacervate delivery of each GF separately. CONCLUSIONS: Dual delivery of bFGF and NGF binding coacervate was neuroprotective via stimulating the growth and proliferation of neurons.

Development of high-performance chemical isotope labeling LC-MS for profiling the human fecal metabolome.

Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively identified based on mass match using MyCompoundID against a Human Metabolome Database and an Evidence-based Metabolome Library, respectively. This represents the most comprehensive profile of the amine/phenol submetabolome ever detected in human fecal samples. The quantitative metabolome profiles of individual samples were shown to be useful to separate different groups of samples, illustrating the possibility of using this method for fecal metabolomics studies.

Perioperative dynamics and significance of amino acid profiles in patients with cancer.

BACKGROUND: Metabolome analysis including amino acid profile is under investigation as an approach in cancer screening. The present study aims to analyze plasma free amino acid (PFAA) profiles in cancer patients and investigate their potential as biomarkers of malignancy. METHODS: Plasma samples from 56 gastric cancer patients, 28 breast cancer patients, 33 thyroid cancer patients, and 137 age-matched healthy controls were collected in the study. PFAA levels were measured and their perioperative alterations were analyzed. Biological effects of ten cancer-related amino acids were further validated in gastric and breast cancer cells. RESULTS: We found that PFAA profiles of cancer patients differed significantly from those of healthy controls. Decreased concentrations of PFAAs were associated with lymph node metastases in gastric cancer. Levels of PFAAs such as aspartate and alanine increased after tumor resection. PFAA levels correlated with clinical tumor markers in gastric cancer patients and pathological immunohistochemistry markers in breast cancer patients. Specifically, alanine, arginine, aspartate and cysteine had proliferative effects on breast cancer cells. Proliferation of gastric cancer cells was promoted by cysteine, but inhibited by alanine and glutamic acid. Furthermore, alanine treatment decreased total and stable fraction of gastric cancer cells, and alanine and glutamic acid induced apoptosis of gastric cancer cells. CONCLUSIONS: PFAA patterns in cancer patients are altered perioperatively. Tumor-related amino acids identified by dynamic study of PFAA patterns may have the potential to be developed as novel biomarkers for diagnosis and prognosis of cancer patients.

LAM add-on ADV combination therapy or ETV monotherapy for CHB patients with suboptimal response to ADV.

INTRODUCTION: Among the available nucleos(t)ide analogues adefovir dipivoxil (ADV) is relatively cheap and widely used in rural area in China. However, there are insufficient data on recommendation for patients with suboptimal response to ADV after 48 weeks of treatment in order to reduce the resistance rate in the long term. The aim of this study was to compare the efficacy and safety of LAM add-on combination therapy versus ETV monotherapy for patients with suboptimal response to ADV. MATERIAL AND METHODS: 136 patients with suboptimal response to ADV were randomly assigned to the add-on LAM with ADV combination therapy (68 patients) group and the ETV monotherapy (68 patients) group. Patients in the add-on group were prescribed 100 mg LAM and 10 mg ADV per day, while the monotherapy group received 0.5 mg ETV per day for 48 weeks. Tests for liver and kidney function, HBV serum markers, HBV DNA load, were performed every 3 months. RESULTS: The mean patient age in LAM add-on group and ETV monotherapy was 38.59 ± 7.65 and 37.56 ± 8.67 years respectively. The HBV DNA undetectable rate in the LAM add-on group and the ETV group were not significant difference at week 4, 12 and 24 (P > 0.05). However, the HBV undetectable rate in the ETV group was higher than that in the LAM add-on group at week 36 and 48 (P = 0.043 for week 36 and P = 0.038 for week 48). There was no significant difference both for HBeAg loss and HBeAg seroconversion between two groups (P > 0.05) at 48 weeks. Meanwhile, our study also demonstrated that the mean eGFR levels in LAM add-on group was decreased from 99.6 ± 8.71 at baseline to 86.4 ± 9.83 at the end of 48 weeks, which was significantly higher than that in the ETV monotherapy group (P < 0.05). 8.8% of patients in LAM add-on group experienced eGFR reduction by 20-30% from baseline at 48 weeks. No patients developed hyposphosphatemia in our study. CONCLUSION: Our study clearly showed that switch to ETV monotherapy was the more effective and more safe than that of LAM add-on combination therapy for patients with suboptimal response to ADV.

High frequency of thrombocytopenia in patients with acute-on-chronic liver failure treated with linezolid.

BACKGROUND: Linezolid is an effective antibiotic reagent for Gram-positive bacterial infection; its most common side effect is thrombocytopenia. However, the incidence of thrombocytopenia in patients with acute-on-chronic liver failure (ACLF) who underwent linezolid therapy was unclear. The present study was to evaluate the incidence of thrombocytopenia in ACLF and non-ACLF patients treated with linezolid and the risk factors of thrombocytopenia in these patients. METHODS: Thirty-five patients with ACLF who had been subjected to intravenous administration of 600 mg linezolid every 12 hours for more than 7 days were categorized as a ACLF treatment (ACLF-T) group, 72 patients without ACLF treated with the same dosage of linezolid were recruited as a non-ACLF treatment (NACLF-T) group, and 70 patients with ACLF without linezolid treatment served as an ACLF control (ACLF-C) group. The incidences of thrombocytopenia in different groups were compared at day 14. Risk factors were investigated using logistic regression analysis. RESULTS: The incidence of thrombocytopenia at day 14 was significantly higher in the ACLF-T group than in the ACLF-C group (20/35 vs 24/70, P=0.025) and in the NACLF-T group (20/35 vs 9/72, P<0.001). Multivariate analysis showed that the ratio of platelet count (day 7/day 0)<1 (OR=10.021; P=0.012) and the baseline platelet count (OR=0.985; P=0.036) were independent risk factors of thrombocytopenia at day 14 of linezolid therapy. CONCLUSIONS: The benefits of linezolid treatment should outweigh the risk of thrombocytopenia in patients with ACLF. Moreover, it is necessary to closely monitor the platelet count during linezolid therapy especially in the patients with decreased platelet count at day 7 of linezolid therapy.

Plasma metabolome analysis for predicting antiviral treatment efficacy in chronic hepatitis B: diagnostic biomarkers and therapeutic insights.

The early and accurate identification of predictive biomarkers for antiviral treatment efficacy remains a significant clinical challenge, particularly in the management of chronic hepatitis B (CHB). This study aimed to assess whether the plasma metabolome could reliably predict the success of antiviral therapy in CHB patients. We conducted a retrospective analysis on 56 treatment-naive CHB patients at the First Affiliated Hospital of Zhejiang University from December 2013 to March 2016. Patients who underwent a 48-week treatment regimen of entecavir (ETV) and interferon-alpha (IFN-α) were randomly assigned to either a discovery cohort (n=29) or a validation cohort (n=27). Based on the outcome of the treatment, patients were classified as HBeAg seroconversion group (High responders, Hrp) or the non-remission group (Low responder, Lrp). Our methodology involved an untargeted analysis of the amine/phenol and carboxylic acid submetabolomes in the CHB patients under treatment, utilizing chemical isotope labeling (CIL) techniques with liquid chromatography-mass spectrometry (LC-MS). Several metabolites were identified as having significant diagnostic potential for distinguishing Hrp from Lrp, with areas under the receiver operating characteristic curve (AUC) exceeding those typical clinical indicators. Notably, four metabolites, namely 2-methyl-3-ketovaleric acid, 2-ketohexanoic acid, 6-oxo-1,4,5,6-tetrahydronicotinic acid, and α-ketoisovaleric acid, demonstrated exceptionally high sensitivity and specificity in both cohorts, nearing 100%. In contrast, the clinical indicators, including HBcAb, log(HBsAg), and HBeAb, demonstrated lower and inconsistent sensitivity and specificity between the discovery and validation cohorts. Using HBcAb as a marker, the sensitivity was 87.5% with 76.9% specificity in the discovery cohort; however, the sensitivity dropped to 46.7% with 91.7% specificity in the validation cohort. Using log(HBsAg), the sensitivity was 84.6% with 69.2% specificity in the discovery cohort, compared to 85.7% sensitivity and 83.3% specificity in the validation cohort. For HBeAb, the separation of Hrp and Lrp had a sensitivity of 87.5% with 69.2% specificity in the discovery cohort, while the validation cohort showed 86.7% sensitivity and 91.7% specificity.

FGF21 inhibits ferroptosis caused by mitochondrial damage to promote the repair of peripheral nerve injury.

Introduction: Ferroptosis is a new type of cell death characterized by lipid peroxidation and iron dependency, representing an emerging disease regulation mechanism. The limited understanding of ferroptosis in peripheral nerve injury (PNI) complicates the management of such injuries. Mitochondrial dysfunction, which contributes to ferroptosis, further exacerbates the challenges of peripheral nerve repair. Methods: In this study, we established an in vitro model of Schwann cells model treated with TBHP and an in vivo sciatic nerve crush injury model in rats. These models were used to investigate the effects of fibroblast growth factor 21 (FGF21) on PNI, both in vitro and in vivo, and to explore the potential mechanisms linking injury-induced ferroptosis and mitochondrial dysfunction. Results: Our findings reveal that PNI triggers abnormal accumulation of lipid reactive oxygen species (ROS) and inactivates mitochondrial respiratory chain complex III, leading to mitochondrial dysfunction. This dysfunction catalyzes the oxidation of excessive polyunsaturated fatty acids, resulting in antioxidant imbalance and loss of ferroptosis suppressor protein 1 (FSP1), which drives lipid peroxidation. Additionally, irregular iron metabolism, defective mitophagy, and other factors contribute to the induction of ferroptosis. Importantly, we found that FGF21 attenuates the abnormal accumulation of lipid ROS, restores mitochondrial function, and suppresses ferroptosis, thus promoting PNI repair. Notably, glutathione peroxidase 4 (GPX4), a downstream target of nuclear factor E2-related factor 2 (Nrf2), and the ERK/Nrf2 pathway are involved in the regulation of ferroptosis by FGF21. Conclusion: FGF21 promotes peripheral nerve repair by inhibiting ferroptosis caused by mitochondrial dysfunction. Therefore, targeting mitochondria and ferroptosis represents a promising therapeutic strategy for effective PNI repair.