-
Journal of Gastrointestinal and Liver... Jun 2024
Response to Letter to Editor on "Reduction of Fecal Calprotectin Levels Induced by a Short Course of Escherichia Coli Nissle is Associated with a Lower Likelihood of Disease Flares in Patients with Ulcerative Colitis in Clinical Remission".
Topics: Humans; Leukocyte L1 Antigen Complex; Colitis, Ulcerative; Feces; Escherichia coli; Remission Induction; Symptom Flare Up; Probiotics; Biomarkers
PubMed: 38944863
DOI: 10.15403/jgld-5660 -
NPJ Biofilms and Microbiomes Jun 2024Gut metaproteomics can provide direct evidence of microbial functions actively expressed in the colonic environments, contributing to clarify the role of the gut... (Meta-Analysis)
Meta-Analysis
Gut metaproteomics can provide direct evidence of microbial functions actively expressed in the colonic environments, contributing to clarify the role of the gut microbiota in human physiology. In this study, we re-analyzed 10 fecal metaproteomics datasets of healthy individuals from different continents and countries, with the aim of identifying stable and variable gut microbial functions and defining the contribution of specific bacterial taxa to the main metabolic pathways. The "core" metaproteome included 182 microbial functions and 83 pathways that were identified in all individuals analyzed. Several enzymes involved in glucose and pyruvate metabolism, along with glutamate dehydrogenase, acetate kinase, elongation factors G and Tu and DnaK, were the proteins with the lowest abundance variability in the cohorts under study. On the contrary, proteins involved in chemotaxis, response to stress and cell adhesion were among the most variable functions. Random-effect meta-analysis of correlation trends between taxa, functions and pathways revealed key ecological and molecular associations within the gut microbiota. The contribution of specific bacterial taxa to the main biological processes was also investigated, finding that Faecalibacterium is the most stable genus and the top contributor to anti-inflammatory butyrate production in the healthy gut microbiota. Active production of other mucosal immunomodulators facilitating host tolerance was observed, including Roseburia flagellin and lipopolysaccharide biosynthetic enzymes expressed by members of Bacteroidota. Our study provides a detailed picture of the healthy human gut microbiota, contributing to unveil its functional mechanisms and its relationship with nutrition, immunity, and environmental stressors.
Topics: Humans; Gastrointestinal Microbiome; Proteomics; Bacteria; Feces; Bacterial Proteins; Healthy Volunteers; Proteome; Metabolic Networks and Pathways
PubMed: 38944645
DOI: 10.1038/s41522-024-00526-4 -
Protein Expression and Purification Jun 2024Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently...
Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways.
PubMed: 38944221
DOI: 10.1016/j.pep.2024.106537 -
The Journal of Biological Chemistry Jun 2024L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in...
L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in intracellular biosynthesis and recycling of GDP-L-fucose for fucosylation. However, in certain mammalian species, L-fucose is efficiently broken down to pyruvate and lactate in a poorly understood metabolic pathway. In the 1970s, L-fucose dehydrogenase, an enzyme responsible for the initial step of this pathway, was partially purified from pig and rabbit livers and characterized biochemically. However, its molecular identity remained elusive until recently. This study reports the purification, identification, and biochemical characterization of the mammalian L-fucose dehydrogenase. The enzyme was purified from rabbit liver approximately 340-fold. Mass spectrometry analysis of the purified protein preparation identified mammalian hydroxysteroid 17-β dehydrogenase 14 (HSD17B14) as the sole candidate enzyme. Rabbit and human HSD17B14 were expressed in HEK293T and Escherichia coli, respectively, purified and demonstrated to catalyze the oxidation of L-fucose to L-fucono-1,5-lactone, as confirmed by mass spectrometry and NMR analysis. Substrate specificity studies revealed that L-fucose is the preferred substrate for both enzymes. The human enzyme exhibited a catalytic efficiency for L-fucose that was 359-fold higher than its efficiency for estradiol. Additionally, recombinant rat HSD17B14 exhibited negligible activity towards L-fucose, consistent with the absence of L-fucose metabolism in this species. The identification of the gene encoding mammalian L-fucose dehydrogenase provides novel insights into the substrate specificity of enzymes belonging to the 17-β-hydroxysteroid dehydrogenase family. This discovery also paves the way for unraveling the physiological functions of the L-fucose degradation pathway, which remains enigmatic.
PubMed: 38944119
DOI: 10.1016/j.jbc.2024.107501 -
Developmental Cell Jun 2024We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest...
We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions.
PubMed: 38944040
DOI: 10.1016/j.devcel.2024.06.002 -
Analytical Chemistry Jun 2024Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand-protein affinity...
Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand-protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep-Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1-2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.
PubMed: 38943570
DOI: 10.1021/acs.analchem.4c02067 -
Cell Communication and Signaling : CCS Jun 2024The neonatal mammalian heart exhibits considerable regenerative potential following injury through cardiomyocyte proliferation, whereas mature cardiomyocytes withdraw...
BACKGROUND
The neonatal mammalian heart exhibits considerable regenerative potential following injury through cardiomyocyte proliferation, whereas mature cardiomyocytes withdraw from the cell cycle and lose regenerative capacities. Therefore, investigating the mechanisms underlying neonatal cardiomyocyte proliferation and regeneration is crucial for unlocking the regenerative potential of adult mammalian heart to repair damage and restore contractile function following myocardial injury.
METHODS
The Tudor staphylococcal nuclease (Tudor-SN) transgenic (TG) or cardiomyocyte-specific knockout mice (Myh6-Tudor-SN ) were generated to investigate the role of Tudor-SN in cardiomyocyte proliferation and heart regeneration following apical resection (AR) surgery. Primary cardiomyocytes isolated from neonatal mice were used to assess the influence of Tudor-SN on cardiomyocyte proliferation in vitro. Affinity purification and mass spectrometry were employed to elucidate the underlying mechanism. H9c2 cells and mouse myocardia with either overexpression or knockout of Tudor-SN were utilized to assess its impact on the phosphorylation of Yes-associated protein (YAP), both in vitro and in vivo.
RESULTS
We previously identified Tudor-SN as a cell cycle regulator that is highly expressed in neonatal mice myocardia but downregulated in adults. Our present study demonstrates that sustained expression of Tudor-SN promotes and prolongs the proliferation of neonatal cardiomyocytes, improves cardiac function, and enhances the ability to repair the left ventricular apex resection in neonatal mice. Consistently, cardiomyocyte-specific knockout of Tudor-SN impairs cardiac function and retards recovery after injury. Tudor-SN associates with YAP, which plays important roles in heart development and regeneration, inhibiting phosphorylation at Ser 127 and Ser 397 residues by preventing the association between Large Tumor Suppressor 1 (LATS1) and YAP, correspondingly maintaining stability and promoting nuclear translocation of YAP to enhance the proliferation-related genes transcription.
CONCLUSION
Tudor-SN regulates the phosphorylation of YAP, consequently enhancing and prolonging neonatal cardiomyocyte proliferation under physiological conditions and promoting neonatal heart regeneration after injury.
Topics: Animals; Myocytes, Cardiac; Regeneration; Phosphorylation; Cell Proliferation; YAP-Signaling Proteins; Animals, Newborn; Adaptor Proteins, Signal Transducing; Mice; Cell Cycle Proteins; Heart; Mice, Knockout; Rats
PubMed: 38943195
DOI: 10.1186/s12964-024-01715-6 -
BMC Microbiology Jun 2024Carbapenemase-producing Klebsiella pneumoniae (CRKP) presents a significant challenge to antimicrobial therapy, especially when compounded by resistance to colistin. The...
Molecular characterization and epidemiological investigation of colistin resistance in carbapenem-resistant Klebsiella pneumoniae in a tertiary care hospital in Tehran, Iran.
BACKGROUND
Carbapenemase-producing Klebsiella pneumoniae (CRKP) presents a significant challenge to antimicrobial therapy, especially when compounded by resistance to colistin. The objective of this study was to explore molecular epidemiological insights into strains of clinical K. pneumoniae that produce carbapenemases and exhibit resistance to colistin. Eighty clinical isolates of CRKP were obtained from Milad Hospital in Tehran, Iran. Antimicrobial susceptibility and colistin broth disk elution were determined. PCR assays were conducted to examine the prevalence of resistance-associated genes, including bla, bla, bla, bla, bla and mcr-1 to -10. Molecular typing (PFGE) was used to assess their spread.
RESULTS
Colistin resistance was observed in 27 isolates (33.7%) using the Broth Disk Elution method. Among positive isolates for carbapenemase genes, the most frequent gene was bla, identified in 36 strains (45%). The mcr-1 gene was detected in 3.7% of the obtained isolates, with none of the other of the other mcr genes detected in the studied isolates.
CONCLUSION
To stop the spread of resistant K. pneumoniae and prevent the evolution of mcr genes, it is imperative to enhance surveillance, adhere rigorously to infection prevention protocols, and implement antibiotic stewardship practices.
Topics: Colistin; Iran; Klebsiella pneumoniae; Humans; Klebsiella Infections; Tertiary Care Centers; Anti-Bacterial Agents; Microbial Sensitivity Tests; Bacterial Proteins; beta-Lactamases; Carbapenems; Drug Resistance, Bacterial; Carbapenem-Resistant Enterobacteriaceae; Molecular Epidemiology
PubMed: 38943054
DOI: 10.1186/s12866-024-03376-4 -
Cellular and Molecular Life Sciences :... Jun 2024Cetuximab resistance has been a major challenge for head and neck squamous cell carcinoma (HNSCC) patients receiving targeted therapy. However, the mechanism that causes...
Cetuximab resistance has been a major challenge for head and neck squamous cell carcinoma (HNSCC) patients receiving targeted therapy. However, the mechanism that causes cetuximab resistance, especially microRNA (miRNA) regulation, remains unclear. Growing evidence suggests that miRNAs may act as "nuclear activating miRNAs" for targeting promoter regions or enhancers related to target genes. This study elucidates a novel mechanism underlying cetuximab resistance in HNSCC involving the nuclear activation of KDM7A transcription via miR-451a. Herein, small RNA sequencing, quantitative real-time polymerase chain reaction (qRT‒PCR) and fluorescence in situ hybridization (FISH) results provided compelling evidence of miR-451a nuclear enrichment in response to cetuximab treatment. Chromatin isolation via RNA purification, microarray analysis, and bioinformatic analysis revealed that miR-451a interacts with an enhancer region in KDM7A, activating its expression and further facilitating cetuximab resistance. It has also been demonstrated that the activation of KDM7A by nuclear miR-451a is induced by cetuximab treatment and is AGO2 dependent. Logistic regression analyses of 87 HNSCC samples indicated the significance of miR-451a and KDM7A in the development of cetuximab resistance. These discoveries support the potential of miR-451a and KDM7A as valuable biomarkers for cetuximab resistance and emphasize the function of nuclear-activating miRNAs.
Topics: Humans; MicroRNAs; Cetuximab; Drug Resistance, Neoplasm; Squamous Cell Carcinoma of Head and Neck; Head and Neck Neoplasms; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Jumonji Domain-Containing Histone Demethylases; Argonaute Proteins; Animals; Mice; Cell Nucleus; Female; Mice, Nude
PubMed: 38943031
DOI: 10.1007/s00018-024-05324-x -
Scientific Reports Jun 2024Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, causes a spectrum of symptoms ranging from mild upper to severe lower...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, causes a spectrum of symptoms ranging from mild upper to severe lower respiratory tract infections. However, the dynamics of nucleocapsid (N) protein antigenemia and RNAemia are not fully understood. We conducted a cohort study involving 117 patients with clinically confirmed COVID-19, focusing on the kinetics of antigenemia and RNAemia and their association with various clinical characteristics. The patients had a median age of 66.0 years (52.0-79.0 years), with a gender distribution of 46.2% male and 53.8% female. Antigenemia reached 100% in fatal cases during the first week after admission. The sensitivity/specificity of antigenemia for diagnosis were 64.7%/73.0% at admission, 69.1%/100% in Week 1, and 66.3%/100% in Week 2. Additionally, the rates of antigenemia in asymptomatic patients were 27.3% upon admission and 22.0% in Week 1, respectively; however, no antigenemia was in samples collected in Week 2. Viral RNAemia was not detected in asymptomatic patients, but RNAemia viral loads were elevated in fatal cases. Kaplan-Meier survival curves demonstrated a higher mortality rate when antigenemia concentrations were elevated in the follow-up samples (P = 0.005). Our study provides a comprehensive analysis of the kinetics of viral N-protein antigenemia and RNAemia according to disease severity and clinical classification. Our findings suggest that highest concentrations of antigenemia in fatal cases occur in the first week after admission, indicating that early elevated antigenemia may serve as a marker of mortality risk.
Topics: Humans; Male; COVID-19; Female; Middle Aged; Aged; SARS-CoV-2; RNA, Viral; Severity of Illness Index; Antigens, Viral; Coronavirus Nucleocapsid Proteins; Cohort Studies; Phosphoproteins
PubMed: 38942808
DOI: 10.1038/s41598-024-65489-0