Proteomics Investigation of Diverse Serological Patterns in COVID-19.

Serum antibodies IgM and IgG are elevated during Coronavirus Disease 2019 (COVID-19) to defend against viral attacks. Atypical results such as negative and abnormally high antibody expression were frequently observed whereas the underlying molecular mechanisms are elusive. In our cohort of 144 COVID-19 patients, 3.5% were both IgM and IgG negative, whereas 29.2% remained only IgM negative. The remaining patients exhibited positive IgM and IgG expression, with 9.3% of them exhibiting over 20-fold higher titers of IgM than the others at their plateau. IgG titers in all of them were significantly boosted after vaccination in the second year. To investigate the underlying molecular mechanisms, we classed the patients into four groups with diverse serological patterns and analyzed their 2-year clinical indicators. Additionally, we collected 111 serum samples for TMTpro-based longitudinal proteomic profiling and characterized 1494 proteins in total. We found that the continuously negative IgM and IgG expression during COVID-19 were associated with mild inflammatory reactions and high T cell responses. Low levels of serum IgD, inferior complement 1 activation of complement cascades, and insufficient cellular immune responses might collectively lead to compensatory serological responses, causing overexpression of IgM. Serum CD163 was positively correlated with antibody titers during seroconversion. This study suggests that patients with negative serology still developed cellular immunity for viral defense and that high titers of IgM might not be favorable to COVID-19 recovery.

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.

The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

An Unusual Ferryl Intermediate and Its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO.

N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Mesenchymal stem cell-derived exosomes promote tissue repair injury in rats with liver trauma by regulating gut microbiota and metabolism.

OBJECTIVE: The effects of mesenchymal stem cells (MSCs) and MSC-derived exosomes (MSC-exos) on serum metabolites and intestinal microbiota in rats after liver trauma were discussed. METHODS: Adult Wistar Albino rats were assigned into control, model (liver trauma), MSCs, and MSC-exos groups (n = 6). The study examined changes in the inflammatory environment in liver tissues were analyzed by histological examination and analysis of macrophage phenotypes. Alterations in serum metabolites were determined by untargeted metabonomics, and gut microbiota composition was characterized by 16S rDNA sequencing. Correlations between specific gut microbiota, metabolites, and inflammatory response were calculated using Spearman correlation analysis. RESULTS: Rats with liver trauma after MSCs and MSC-exos treatment exhibited attenuated inflammatory infiltration and necrosis in liver tissues. MSCs and MSC-exos treatment reduced the proportion of M1 macrophages, accompanied by a decrease in inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) levels. Furthermore, MSCs and MSC-exos treatment expanded the proportion of M2 macrophages, accompanied by an increase in arginase-1 (Arg-1) and interleukin-10 (IL-10) levels. The beneficial effects of MSC-exo treatment on rats with liver trauma were superior to those of MSC treatment. The composition and abundance of the gut microbiota and metabolites were altered in pathological rats, whereas MSC and MSC-exo intervention partially restored specific gut microbiota and metabolite alterations. At the phylum level, alterations in Bacteroidota, Proteobacteria, and Verrucomicrobiota were observed after MSC and MSC-exo intervention. At the genus level, Intestinimonas, Alistipes, Aerococcus, Faecalibaculum, and Lachnospiraceae_ND3007_group were the main differential microbiota. 6-Methylnicotinamide, N-Methylnicotinamide, Glutathione, oxidized, ISOBUTYRATE, ASCORBATE, EICOSAPENTAENOATE, GLYCEROL 3-PHOSPHATE, and Ascorbate radical were selected as important differential metabolites. There was a clear correlation between Ascorbate, Intestinimonas/Faecalibaculum and inflammatory cytokines. CONCLUSION: MSC-exos promoted the repair of tissue damage in rats with liver trauma by regulating serum metabolites and intestinal microbiota, providing new insights into how MSC-exos reduced inflammation in rats with liver trauma.

Cytotoxic withanolides from the leaves of Datura stramonium L.

Six novel withanolides, along with nine known related compounds were isolated from the leaves of Datura stramonium L. The structures and absolute configurations of the new withanolides were elucidated by employing various spectral techniques and comparing them with those previously reported in the literature. In addition, four withanlides demonstrated interesting cytotoxic activity on LN229 cells with IC50 <20 µM.

Causal relationship between Helicobacter pylori infection and IgA nephropathy: a bidirectional two-sample mendelian randomization study.

IgA nephropathy (IgAN) is one of the most common primary glomerulonephritis, and serum Helicobacter pylori (H. pylori) antibody levels are increased in patients with IgA N, but the role of H. pylori infection in the pathogenesis of IgAN is unclear. In this study, we investigated whether there is a causal relationship and reverse causality between IgAN and H. pylori infection by using a bidirectional two-sample Mendelian randomization (MR) analysis. This study was estimated using inverse variance weighted (IVW), MR-Egger and weighted median methods, with the IVW method having the strongest statistical efficacy. Seven common serum H. pylori antibodies were selected as exposure factors for positive MR analysis. The results showed that there was no evidence of a causal relationship between H. pylori infection and IgAN. Reverse MR analysis showed that there was also no evidence that the occurrence of IgAN leads to an increased risk of H. pylori infection.

Characteristics of inpatients with newly diagnosed multiple myeloma in hematology, nephrology, and orthopedic departments.

To describe the characteristics of multiple myeloma (MM) by retrospectively analyzing data from patients with MM in hematology, nephrology, and orthopedic departments, we selected inpatients diagnosed with MM for the first time who were admitted to the hematology department of Taizhou Hospital of Zhejiang Province between January 1, 2017, and June 30, 2021 ([Formula: see text]) and those admitted to the nephrology ([Formula: see text]) and orthopedic ([Formula: see text]) departments of Taizhou Hospital of Zhejiang Province between January 1, 2010, and June 30, 2021. For patients with MM initially diagnosed in the nephrology or orthopedic departments, age- and sex-matched patients without MM were randomly selected in a 1:1 ratio. Conditional logistic regression models were used to assess the risk of MM in hospitalized patients in the nephrology and orthopedic departments. This study included 200 patients, with a median age of 65 years. There were differences among patients in the three departments with respect to fracture, degree of anemia, proteinuria levels, red blood cell (RBC) count, hemoglobin levels, total protein (TP) levels, albumin levels, and laboratory indicators of renal function. TP levels > 68.45 g/L were independent risk factors for patients in the nephrology department (odds ratio (OR) = 1.01, 95% confidence interval (CI) = 1.001-1.024, P = 0.04), and RBC count < 3.79 × 1012/L was an independent risk factor for orthopedic inpatients (OR = 0.49, 95% CI 0.268-0.913, P = 0.02). Therefore, nephrologists should pay attention to the TP level of hospitalized patients to facilitate the early identification of MM in patients with chronic kidney disease. Orthopedic surgeons should pay attention to RBC counts in patients with fractures, and patients with low RBC counts should avoid unnecessary surgery and move to specialist care as early as possible.

New chromones from the roots of Saposhnikovia divaricata (Turcz.) Schischk with anti-inflammatory activity.

Fifteen new chromones, sadivamones A-E (1-5), cimifugin monoacetate (6), sadivamones F-N (7-15), together with fifteen known chromones (16-30), were isolated from the ethyl acetate portions of 70% ethanol extract of Saposhnikovia divaricata (Turcz.) Schischk roots. The structures of the isolates were determined using 1D/2D NMR data and electron circular dichroism (ECD) calculations. Meanwhile, LPS induced RAW264.7 inflammatory cell model was used to determine the potential anti-inflammatory activity of all the isolated compounds in vitro. The results showed that compounds 2, 8, 12-13, 18, 20-22, 24, and 27 significantly inhibited the production of lipopolysaccharide (LPS)-induced NO in macrophages. To determine the signaling pathways involved in the suppression of NO production by compounds 8, 12 and 13, we investigated ERK and c-Jun N-terminal protein kinase (JNK) expression by western blot analysis. Further mechanistic studies demonstrated that compounds 12 and 13 inhibited the phosphorylation of ERK and the activation of ERK and JNK signaling in RAW264.7 cells via MAPK signaling pathways. Taken together, compounds 12 and 13 may be valuable candidates for the treatment of inflammatory diseases.

Chemical profiling and mechanistic studies of Zhi-Shang-Feng granules against influenza virus by high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high-resolution mass spectrometry in combination with network pharmacology analysis.

Zhi-Shang-Feng Granules are used in the clinical treatment of influenza to relieve headaches, chills and fever, bronchitis, nasal congestion, neuralgia and other symptoms. To decipher the components responsible for therapeutic effects of Zhi-Shang-Feng g ranules against influenza virus, an analytical method based on high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high resolution mass spectrometry was developed and the chemical profile of Zhi-Shang-Feng granules was characterized. Then, the identified components were used to conduct network pharmacological analysis and determine the potential mechanism of Zhi-Shang-Feng Granules. As a result, 177 compounds were putatively identified through comprehensive analysis by liquid chromatography coupled with high-resolution mass spectrometry, of which 23 compounds were unambiguously confirmed with reference standards. Components in Zhi-Shang-Feng Granules were found to specifically act on different enzymes, G-protein-coupled receptors, ion channels and transporters in the immune, endocrine, nervous, and circulatory systems. The potential mechanism was related to several biological processes, including cell growth and death, pattern recognition receptor signalling, signalling by interleukins, and lipid metabolism. The combination of chemical profile characterization and network construction provided useful insight into the overall chemical composition of Zhi-Shang-Feng granules and revealed their potential anti-infection, anti-inflammatory and immunoregulatory mechanisms against influenza virus infected disease.

Residues of sulfoxaflor and its metabolites in floral and extrafloral nectar from Hibiscus rosa-sinensis L. (Malvaceae) with or without co-application of tebuconazole.

Systemic pesticide exposure through nectar is a growing global concern linked to loss of insect diversity, especially pollinators. The insecticide sulfoxaflor and the fungicide tebuconazole are currently widely used systemic pesticides which are toxic to certain pollinators. However, their metabolisms in floral or extrafloral nectar under different application methods have not yet been well studied. Hibiscus rosa-sinensis was exposed to sulfoxaflor and tebuconazole via soil drenching and foliar spraying. Sulfoxaflor, tebuconazole, and their main metabolites in floral and extrafloral nectar, soil, and leaves were identified and quantified using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ MS). The chemical compositions of unexposed and contaminated H. rosa-sinensis floral nectar or extrafloral nectar were compared using regular biochemical methods. The activities of two pesticide detoxifying enzymes, glutathione-s-transferase and nitrile hydratase, in H. rosa-sinensis nectar were examined using LC-MS and spectrophotometry. The floral nectar proteome of H. rosa-sinensis was analysed using high-resolution orbitrap-based MS/MS analysis to screen for sulfoxaflor and tebuconazole detoxifying enzymes. H. rosa-sinensis can absorb sulfoxaflor and tebuconazole through its roots or leaf surfaces and secrete them into floral nectar and extrafloral nectar. Both sulfoxaflor and tebuconazole and their major metabolites were present at higher concentrations in extrafloral nectar than in floral nectar. X11719474 was the dominant metabolite of sulfoxaflor in the nectars we studied. Compared with soil application, more sulfoxaflor and tebuconazole remained in their original forms in floral nectar and extrafloral nectar after foliar application. Sulfoxaflor and tebuconazole exposure did not modify the chemical composition of floral or extrafloral nectar. No active components, including proteins in the nectar, were detected to be able to detoxify sulfoxaflor.

UHPLC-Orbitrap-Fusion-TMS-Based Metabolomics Study of Phenylpropionamides in the Seed of Cannabis sativa L. against Alzheimer's Disease.

Phenylpropionamides in the seed of Cannabis sativa L. (PHS) have a protective effect on neuroinflammation and antioxidant activity. In this study, the UHPLC-Orbitrap-fusion-TMS-based metabolomics approach was used to analyze the serum samples and identify potential biomarkers in Streptozotocin (STZ) induced Alzheimer's disease (AD) rats. The results revealed that primary bile acid biosynthesis and taurine and hypotaurine metabolism were significantly correlated with STZ-induced AD rats. In addition, the key enzymes in these two pathways were verified at the protein level. The levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD), cysteamine (2-aminoethanethiol) dioxygenase (ADO), 7α-hydroxylase (CYP7A1), and sterol 12α-hydroxylase (CYP8B1) were the key enzymes affecting the two pathways in AD rats compared with the control group (CON). Furthermore, after a high-dose group of phenylpropionamides in the seed of Cannabis sativa L. (PHS-H) was administrated, the levels of CDO1, CSAD, CYP7A1, and CYP8B1 were all callback. These findings demonstrate for the first time that the anti-AD effect of PHS is associated with the regulation of primary bile acid biosynthesis and taurine and hypotaurine metabolism in STZ-induced AD rats.

Phenylpropanoids from Solanum capsicoides and their anti-inflammatory activity.

Two new phenylpropanoids, 4-O-(1''-O-cis-caffeoyl)-ß-glucopyran osyl-1-allyl-3-methoxy-benzene (1), 4'-O-(1''-O-cis-caffeoyl)-ß-glucopyranosyl-hydroxymegastigm-4-en-3-one (2), together with nine known compounds were obtained from the leaves of Solanum capsicoides. Their structures were elucidated based on spectroscopic methods, and comparing spectral data with those in literature. Meanwhile, their anti-inflammatory activities were evaluated on (LPS)-induced RAW 246.7 cells, and 1, 9, and 10 showed better inhibitory effects with IC50 values of 17.19 ± 1.12, 18.15 ± 0.47, and 19.8 ± 0.95 µM, respectively.

Partial response to pralsetinib in an advanced pulmonary sarcomatoid carcinoma patient harboring a KIF5B-RET rearrangement: a case report.

BACKGROUND: Pulmonary sarcomatoid carcinoma (PSC) is a rare and unconventional non-small-cell lung cancer (NSCLC) that appears to be aggressive, with a poor prognosis and response to conventional treatment. Approximately 30% of PSCs have potentially targetable genomic alterations, but few studies have involved RET gene fusions, and corresponding targeted therapies are lacking. CASE PRESENTATION: In this report, we describe a patient with PSC harboring a KIF5B-RET gene fusion who was initially diagnosed with stage IVb lung cancer. Due to the poor performance status, the patient was unable to tolerate any radiotherapy or chemotherapy. Based on the next-generation sequencing (NGS) result of RET gene fusion, the patient was treated with pralsetinib. Two months after the treatment, the patient achieved a partial response. CONCLUSIONS: Our case indicates that RET is one of the main driver oncogenes of PSC and provides useful information for precise RET inhibitor administration in the future. Thus, the use of comprehensive genomic profiling may provide important treatment options for PSC.

Cytotoxic Sesquiterpenoids from Atractylodes chinensis (DC.) Koidz.

Four new sesquiterpenoids named atrchiterpenes A-D (1-4), a new natural product (5), and twelve known compounds (6-17) were isolated from Atractylodes chinensis (DC.) Koidz. Compound 1 was a rare N-containing eudesmane-type sesquiterpenoid. Structure elucidation was performed by spectroscopic techniques, including 1D, 2D NMR spectra, and HR-ESI-MS. Compounds 6-11, 14, and 17 were reported from Atractylodes for the first time. All the isolated compounds were evaluated for cytotoxicity activity. Compound 16 showed moderate cytotoxicity against HepG2 cells with an IC50 value of 5.81±0.47.

Chemical Constituents of the Roots of Schisandra chinensis.

One new phenylpropanoid schineolignin D (1), one new sesquiterpene (-)-(7S,10S)-3,11,12,13-tetrahydroxycalamenene (2), one new quinic acid 4-(E)-O-coumaroylquinic acid ethyl ester (3), and seven known compounds 4-10 were separated from the roots of Schisandra chinensis. The chemical structures of all compounds were characterized by NMR spectroscopic experiments. All compounds were assessed for their neuroprotective effects on PC12 cell lines induced by H2 O2 . Compounds 1, 3-4, and 7 showed statistically significant neuroprotective activities with the negative control group at 12.5 µM.

Effects of contralateral nephrectomy timing and ischemic conditions on kidney fibrosis after unilateral kidney ischemia-reperfusion injury.

Acute kidney injury (AKI) is an important cause of chronic kidney disease (CKD), but the underlying mechanisms are unclear. Animal models are tools for studying the AKI-CKD progression. Kidney ischemia-reperfusion injury (IRI) models, especially the unilateral IRI (uIRI) model with delayed contralateral kidney resection, are commonly used to induce fibrotic progression to CKD after AKI. However, in previous studies, we found that details of the operation had a significant impact on the long-term outcomes of the kidney in this uIRI model. In this study, we investigated the effects of resection timing of the contralateral intact kidney, core body temperatures during ischemia, and time length of kidney ischemia on kidney function, histological injury and kidney fibrosis after AKI, using a mouse uIRI model with delayed contralateral nephrectomy. The results showed that all these parameters significantly affected the AKI-CKD transition. The post-AKI fibrosis worsened and the survival rate declined with a longer interval between contralateral nephrectomy and uIRI, higher ischemic body temperature, or longer ischemic duration when the other two variables were fixed. In conclusion, in the uIRI model with delayed contralateral nephrectomy, kidney fibrosis after AKI is influenced by many factors. Strictly controlling the experimental conditions is very important for the stability and consistency of the model.

Aromatic glycosides from the aerial part of Bupleurum chinense.

A new aromatic glycoside (1) and twelve (2-13) known aromatic glycosides were isolated from the n-butyl alcohol of the 70% EtOH extract of the aerial part of Bupleurum chinense. Among them, compounds 4-7, 9, 11-13 were isolated from Umbelliferae plants for the first time. Their structures were elucidated by NMR and MS spectroscopy, and the absolute configuration of compound 1 was elucidated by single-crystal X-ray diffraction. Compounds 6 and 10 showed moderate cytotoxic activities on MGC-803 cell lines with IC50 values of 8.63 ± 0.08 and 13.65 ± 1.73 µM, respectively.

A new ent-kaurane diterpenoid from the pericarps of Datura metel.

A new ent-kaurane diterpenoid, named kaurane daturoside A (1), was isolated from the 70%-EtOH extract of dried pericarps of Datura metel L., along with six known terpenoids, 16α,17-dihydroxy-ent-kauran-19-diglycoside (2), cyclosieversioside F (3), astragaloside II (4), ginsenoside Rg1 (5), astrojanoside A (6), celerioside E (7). The isolated structures were elucidated by means of spectroscopic analyses, and the compounds 2, 3, 7 were separated from Solanaceae for the first time. Meanwhile, among isolates, compounds 2 and 5 exhibited anti-inflammatory activities against LPS-activated RAW264.7 cells (IC50<11.00 µM).

Two new quinic acid derivatives from the leaves of Schisandra chinensis.

Two new (1-2) and three known quinic acid derivatives (3-5) were isolated from the leaves of Schisandra chinensis (Turcz) Baill. The structures of the compounds were determined by spectroscopic methods, especially the NMR techniques, and also by comparison with reported data in the literature. The cytotoxicity activities of these compounds were evaluated on human tumor cell lines LN229 and three of them showed a certain activity.

Transcriptional Regulation of Endogenous Retroviruses and Their Misregulation in Human Diseases.

Endogenous retroviruses (ERVs), deriving from exogenous retroviral infections of germ line cells occurred millions of years ago, represent ~8% of human genome. Most ERVs are highly inactivated because of the accumulation of mutations, insertions, deletions, and/or truncations. However, it is becoming increasingly apparent that ERVs influence host biology through genetic and epigenetic mechanisms under particular physiological and pathological conditions, which provide both beneficial and deleterious effects for the host. For instance, certain ERVs expression is essential for human embryonic development. Whereas abnormal activation of ERVs was found to be involved in numbers of human diseases, such as cancer and neurodegenerative diseases. Therefore, understanding the mechanisms of regulation of ERVs would provide insights into the role of ERVs in health and diseases. Here, we provide an overview of mechanisms of transcriptional regulation of ERVs and their dysregulation in human diseases.