-
Talanta Aug 2023Unsaturated lipids play an essential role in living organisms, and their different isomers show significant functional differences. Therefore, in situ characterization...
Unsaturated lipids play an essential role in living organisms, and their different isomers show significant functional differences. Therefore, in situ characterization of unsaturated lipids in tissues needs to be extended to isomer level. However, the exposure of tissue sections to an open environment for a long time may cause cell autolysis or corruption, and current unsaturated lipid imaging methods still face challenges in efficiency. This paper proposes an imaging method based on photoepoxidation coupled with air-flow-assisted desorption electrospray ionization mass spectrometry (AFADESI-MS) to rapidly realize the spatial characterization of unsaturated lipids at the isomer level. The technique has a fast response speed, high epoxide yield (>80%), and high diagnostic ion abundance. After 0.5 min of photoepoxidation, the derivation product yield ratio reached 24.6%. This method rapidly identified six glycerophospholipid isomers containing an 18:1 acyl chain in normal rat liver tissue. Then the imaging method was applied in nude mice lung cancer tissue and human thyroid cancer tissue, with only 3 min photoepoxidation. Results successfully characterized the location and range of unsaturated lipid isomers and revealed their enrichment in tumor tissue. In addition, the experiment shows that the variational trend of the ratio of unsaturated lipid isomers in different types of tumor samples is different. Based on the advantages of efficiency and convenience, this method is prospective for screening unsaturated lipid markers and pathological research of related diseases.
Topics: Mice; Rats; Humans; Animals; Lipids; Mice, Nude; Prospective Studies; Isomerism; Spectrometry, Mass, Electrospray Ionization; Neoplasms
PubMed: 37196400
DOI: 10.1016/j.talanta.2023.124643 -
Acta Biochimica Polonica Nov 2023Previously, the direct interactions of Bβ26-42 fibrin residues with prothrombin were demonstrated. It was also shown that forming prothrombin complexes with E- or...
Previously, the direct interactions of Bβ26-42 fibrin residues with prothrombin were demonstrated. It was also shown that forming prothrombin complexes with E- or DDE-fragments causes non-enzymatic prothrombin activation. The direct measuring of the prothrombin level in the blood plasma of patients with acute myocardial infarction (AMI) allowed us to find a situation where such an activation can occur in vivo. Blood coagulation parameters in the blood plasma of patients with AMI were measured at 2 hours, three days, and seven days after the thrombolysis by streptokinase accompanied with intravenous administration of anticoagulants: unfractionated high molecular weight heparin (HMWH) and low-molecular-weight heparin (LMWH). The prothrombin level in the blood plasma of patients with AMI was normal before thrombolytic therapy and substantially decreased after streptokinase administration. This effect was prominent in the case of concomitant anticoagulant therapy with LMWH and was not observed when HMWH was applied. It can be explained by the fact that LMWH preferentially inhibits factor Xa, while the HMWH is an effective inhibitor of both factor Xa and thrombin. This observation suggested that the prothrombin level decrease was caused by the thrombin-like activity and possible autolysis of prothrombin by thrombin. Also, thrombolytic therapy with streptokinase caused the accumulation of fibrin degradation products (FDPs), some of which were able to bind prothrombin. The dramatic decrease of prothrombin level in the blood plasma of patients with AMI during thrombolysis allowed us to conclude the non-enzymatic prothrombin activation with the following autolysis of prothrombin that contributes to the pathology.
Topics: Humans; Prothrombin; Heparin, Low-Molecular-Weight; Thrombin; Factor Xa; Myocardial Infarction; Heparin; Streptokinase; Thrombolytic Therapy; Anticoagulants
PubMed: 38011253
DOI: 10.18388/abp.2020_6962 -
Frontiers in Microbiology 2023readily forms biofilms on host tissues and medical devices, enabling its persistence in chronic infections and resistance to antibiotic therapy. The accessory gene...
readily forms biofilms on host tissues and medical devices, enabling its persistence in chronic infections and resistance to antibiotic therapy. The accessory gene regulator (Agr) quorum sensing system plays a key role in regulating biofilm formation. This study reveals the widely used fluoroquinolone antibiotic, ciprofloxacin, strongly stimulates biofilm formation in methicillin-resistant , methicillin-sensitive , and clinical isolates with diverse genetic backgrounds. Crystal violet staining indicated that ciprofloxacin induced a remarkable 12.46- to 15.19-fold increase in biofilm biomass. Confocal laser scanning microscopy revealed that ciprofloxacin induced denser biofilms. Phenotypic assays suggest that ciprofloxacin may enhance polysaccharide intercellular adhesin production, inhibit autolysis, and reduce proteolysis during the biofilm development, thus promoting initial adhesion and enhancing biofilm stability. Mechanistically, ciprofloxacin significantly alters the expression of various biofilm-related genes (, , , , , ) and regulators (, ). Gene knockout experiments revealed that deletion of , rather than , abolishes the ciprofloxacin-induced enhancement of biofilm formation, underscoring the key role of . Thermal shift assays showed ciprofloxacin binds purified AgrC protein, thereby inhibiting the Agr system. Molecular docking results further support the potential interaction between ciprofloxacin and AgrC. In summary, subinhibitory concentrations of ciprofloxacin stimulate biofilm formation via an -dependent pathway. This inductive effect may facilitate local infection establishment and bacterial persistence, ultimately leading to therapeutic failure.
PubMed: 38179460
DOI: 10.3389/fmicb.2023.1328947 -
Journal of Biomolecular Structure &... Aug 2023Rand protease is a serine protease that shared common characteristics with members of the MEROPS S8 subtilisin family. It is thermostable, highly stable in organic...
Effect of cysteine mutation at Ca coordinating residues to the autolysis, folding and hydrophobicity of full length and mature Rand protease: molecular dynamics simulation and essential dynamics.
Rand protease is a serine protease that shared common characteristics with members of the MEROPS S8 subtilisin family. It is thermostable, highly stable in organic solvent and broad in specificity. Many structures of homologous protein solved by X-ray crystallography and NMR have been deposited to Protein Data Bank (PDB) which allowed this study to rely on structure prediction by deep learning to build three-dimensional (3D) structure of full length and mature Rand protease (flRP and mRP). In silico cysteine mutation to 7 predicted high affinity Ca coordinating residues were introduced, and the mutants were subjected to molecular dynamics simulation to study its effect on flRP and mRP. MD simulation showed a marked increase in flexibility of the pro-peptide segment indicating the impact of single cysteine substitution at high affinity Ca coordinating residues to autolysis of flRP. MD simulation for mRP reaffirmed the role of Ca coordinating sites in providing stability to Rand protease. In addition, these residues also affect the autolysis, folding and hydrophobicity of RP. Essential dynamics observed large contribution of the first few eigenvectors of flRP, mRP and their high affinity Ca coordinating residues mutants to the TMSF values which indicates that these values account for a large portion of the overall atomic fluctuations. These results have given a more comprehensive understanding on the role of cysteine substituted Ca coordinating surface loop to the structure of flRP and mRP which are important in contributing to the structural stability of subtilisin.Communicated by Ramaswamy H. Sarma.
PubMed: 37608543
DOI: 10.1080/07391102.2023.2249105 -
Bioorganic Chemistry Mar 2024The scope of bioengineering is expanding from the design of single strain to the microbial communities, allowing for the division-of-labor in synthesizing the...
The scope of bioengineering is expanding from the design of single strain to the microbial communities, allowing for the division-of-labor in synthesizing the multi-protein systems. Predicting the composition of the final product during the biomanufacturing process, however, can be difficult. Consortia-based manufacturing has the potential to boost production efficiency, but this benefit primarily holds in the upstream. The current format of downstream process heavily relies on the centralized facility, and is not economical and flexible to address the demands in small-scale. Here, we present a concise and manageable platform to enable the multi-protein system assembly. We engineer a self-lysis microbial consortium, where each strain lyses autonomously at high densities and produces a single protein component. The product fraction can be precisely tuned by varying the inoculation ratio. Utilizing this platform, we assemble a classical 34-component PURE (protein synthesis using recombinant elements) system. We have further optimized the downstream process of the biomanufacturing by incorporating the porous structure of polymeric materials. The encapsulated autolysis consortium can produce and release the proteins while maintaining the cell factories enclosed in the materials by exporting the multi-protein system for collection. Our research provides a novel approach to the flexible and controllable production of multi-protein systems, opening up new possibilities for pathway assembly and portable biomanufacturing.
Topics: Bioengineering; Microbial Consortia; Proteins
PubMed: 38266324
DOI: 10.1016/j.bioorg.2024.107117 -
Archives of Pathology & Laboratory... May 2024The National Institutes of Health Genotype-Tissue Expression (GTEx) project was developed to elucidate how genetic variation influences gene expression in multiple...
CONTEXT.—
The National Institutes of Health Genotype-Tissue Expression (GTEx) project was developed to elucidate how genetic variation influences gene expression in multiple normal tissues procured from postmortem donors.
OBJECTIVE.—
To provide critical insight into a biospecimen's suitability for subsequent analysis, each biospecimen underwent quality assessment measures that included evaluation for underlying disease and potential effects introduced by preanalytic factors.
DESIGN.—
Electronic images of each tissue collected from nearly 1000 postmortem donors were evaluated by board-certified pathologists for the extent of autolysis, tissue purity, and the type and abundance of any extraneous tissue. Tissue-specific differences in the severity of autolysis and RNA integrity were evaluated, as were potential relationships between these markers and the duration of postmortem interval and rapidity of death.
RESULTS.—
Tissue-specific challenges in the procurement and preservation of the nearly 30 000 tissue specimens collected during the GTEx project are summarized. Differences in the degree of autolysis and RNA integrity number were observed among the 40 tissue types evaluated, and tissue-specific susceptibilities to the duration of postmortem interval and rapidity of death were observed.
CONCLUSIONS.—
Ninety-five percent of tissues were of sufficient quality to support RNA sequencing analysis. Biospecimens, annotated whole slide images, de-identified clinical data, and genomic data generated for GTEx represent a high-quality and comprehensive resource for the scientific community that has contributed to its use in approximately 1695 articles. Biospecimens and data collected under the GTEx project are available via the GTEx portal and authorized access to the Database of Genotypes and Phenotypes; procedures and whole slide images are available from the National Cancer Institute.
PubMed: 38797720
DOI: 10.5858/arpa.2023-0467-OA -
Comparative Biochemistry and... Jun 2024Sea cucumber is a valuable seafood product and autolysis is the main concern for the aquaculture industry. This study employed proteomics and transcriptomics to...
Sea cucumber is a valuable seafood product and autolysis is the main concern for the aquaculture industry. This study employed proteomics and transcriptomics to investigate the autolysis mechanism of sea cucumbers. The fresh sea cucumber was exposed to UV light to induce autolysis. The body wall samples were cut off to analyze by proteomics and transcriptomics. The angiotensin-converting enzyme (ACE) inhibitor of teprotide and the activator of imatinib were gastric gavage to live sea cucumbers, respectively, to identify the regulation target. Autolysis occurrence was evaluated by appearance, soluble peptide, and hydroxyproline content. Four gene-protein pairs were ACE, AJAP10923, Heme-binding protein 2-like, and Ficolin-2-like. Only the ACE protein and gene changed synchronously and a significant down-regulation of ACE occurred in the autolysis sea cucumbers. Teprotide led to a 1.58-fold increase in the TCA-soluble protein content and a 1.57-fold increase in hydroxyproline content. No significant differences were observed between imatinib-treated sea cucumbers and fresh ones regarding TCA-soluble protein content or hydroxyproline levels (P > 0.05). ACE inhibitor accelerated the autolysis of sea cucumber, but ACE activator inhibited the autolysis. Therefore, ACE can serve as a regulatory target for autolysis in sea cucumbers.
PubMed: 38906042
DOI: 10.1016/j.cbd.2024.101274 -
BMC Microbiology Mar 2024Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an...
BACKGROUND
Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated.
RESULTS
AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC.
CONCLUSIONS
In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.
Topics: N-Acetylmuramoyl-L-alanine Amidase; Staphylococcus
PubMed: 38459514
DOI: 10.1186/s12866-024-03231-6 -
Frontiers in Plant Science 2023Chitin/polysaccharide deacetylases belong to the carbohydrate esterases family 4 (CE4 enzymes). They play a crucial role in modifying the physiochemical characteristics... (Review)
Review
Chitin/polysaccharide deacetylases belong to the carbohydrate esterases family 4 (CE4 enzymes). They play a crucial role in modifying the physiochemical characteristics of structural polysaccharides and are also involved in a wide range of biological processes such as fungal autolysis, spore formation, cell wall formation and integrity, and germling adhesion. These enzymes are mostly common in fungi, marine bacteria, and a limited number of insects. They facilitate the deacetylation of chitin which is a structural biopolymer that is abundantly found in fungal cell walls and spores and also in the cuticle and peritrophic matrices of insects. The deacetylases exhibit specificity towards a substrate containing a sequence of four GlcNAc units, with one of these units being subjected to deacetylation. Chitin deacetylation results in the formation of chitosan, which is a poor substrate for host plant chitinases, therefore it can suppress the host immune response triggered by fungal pathogens and enhance pathogen virulence and colonization. This review discusses plant pathogenic fungal chitin/polysaccharide deacetylases including their structure, substrate specificity, biological roles and some recently discovered chitin deacetylase inhibitors that can help to mitigate plant fungal diseases. This review provides fundamental knowledge that will undoubtedly lead to the rational design of novel inhibitors that target pathogenic fungal chitin deacetylases, which will also aid in the management of plant diseases, thereby safeguarding global food security.
PubMed: 38264029
DOI: 10.3389/fpls.2023.1335646 -
MSphere Jan 2024The emergence of antibiotic-resistant and biofilm-producing isolates presents major challenges for treating staphylococcal infections. Biofilm inhibition is an...
The emergence of antibiotic-resistant and biofilm-producing isolates presents major challenges for treating staphylococcal infections. Biofilm inhibition is an important anti-virulence strategy. In this study, a novel maleimide-diselenide hybrid compound (YH7) was synthesized and demonstrated remarkable antimicrobial activity against methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) in both planktonic cultures and biofilms. The minimum inhibitory concentration (MIC) of YH7 for isolates was 16 µg/mL. Quantification of biofilms demonstrated that the sub-MIC (4 µg/mL) of YH7 significantly inhibits biofilm formation in both MSSA and MRSA. Confocal laser scanning microscopy analysis further confirmed the biofilm inhibitory potential of YH7. YH7 also significantly suppressed bacterial adherence to A549 cells. Moreover, YH7 treatment significantly inhibited colonization in nasal tissue of mice. Preliminary mechanistic studies revealed that YH7 exerted potent biofilm-suppressing effects by inhibiting polysaccharide intercellular adhesin (PIA) synthesis, rather than suppressing bacterial autolysis. Real-time quantitative PCR data indicated that YH7 downregulated biofilm formation-related genes (, , and ) and the global regulatory gene , which promotes PIA synthesis. The -dependent antibiofilm potential of YH7 was validated by constructing NCTC8325 knockout and complementation strains. Importantly, YH7 demonstrated a low potential to induce drug resistance in and exhibited non-toxic to rabbit erythrocytes, A549, and BEAS-2B cells at antibacterial concentrations. toxicity assays conducted on further confirmed that YH7 is biocompatible. Overall, YH7 demonstrated potent antibiofilm activity supports its potential as an antimicrobial agent against biofilm-related infections. IMPORTANCE Biofilm-associated infections, characterized by antibiotic resistance and persistence, present a formidable challenge in healthcare. Traditional antibacterial agents prove inadequate against biofilms. In this study, the novel compound YH7 demonstrates potent antibiofilm properties by impeding the adhesion and the polysaccharide intercellular adhesin production of . Notably, its exceptional efficacy against both methicillin-resistant and methicillin-susceptible strains highlights its broad applicability. This study highlights the potential of YH7 as a novel therapeutic agent to address the pressing issue of biofilm-driven infections.
Topics: Animals; Mice; Rabbits; Staphylococcus aureus; Methicillin-Resistant Staphylococcus aureus; Methicillin; Anti-Bacterial Agents; Staphylococcal Infections; Biofilms
PubMed: 38170984
DOI: 10.1128/msphere.00564-23