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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 -
Foods (Basel, Switzerland) May 2024Yeast, crucial in beer production, holds great potential owing to its ability to transform into a valuable by-product resource, known as brewer's spent yeast (BSY), with...
Yeast, crucial in beer production, holds great potential owing to its ability to transform into a valuable by-product resource, known as brewer's spent yeast (BSY), with potentially beneficial physiological effects. This study aimed to compare the composition and soluble polysaccharide content of Brewer's spent yeast with those of cultured yeast strains, namely (SC) and . (SB), to facilitate the utilization of BSY as an alternative source of functional polysaccharides. BSY exhibited significantly higher carbohydrate content and lower crude protein content than SC and SB cells. The residues recovered through autolysis were 53.11%, 43.83%, and 44.99% for BSY, SC, and SB, respectively. Notably, the polysaccharide content of the BSY residue (641.90 μg/mg) was higher than that of SC (553.52 μg/mg) and SB (591.56 μg/mg). The yields of alkali-extracted water-soluble polysaccharides were 33.62%, 40.76%, and 42.97% for BSY, SC, and SB, respectively, with BSY comprising a comparable proportion of water-soluble saccharides made with SC and SB, including 49.31% mannan and 20.18% β-glucan. Furthermore, BSY demonstrated antioxidant activities, including superoxide dismutase (SOD), ABTS, and DPPH scavenging potential, suggesting its ability to mitigate oxidative stress. BSY also exhibited a significantly higher total phenolic compound content, indicating its potential to act as an effective functional food material.
PubMed: 38790867
DOI: 10.3390/foods13101567 -
Antibiotics (Basel, Switzerland) Apr 2024TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species,...
TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species, and absence of eukaryotic homologs. Previous studies have elucidated the role of the essential staphylococcal protein, TsaB/YeaZ, in binding DNA to mediate the transcription of the - operon, responsible for encoding key enzymes involved in the biosynthesis of branched-chain amino acids-namely isoleucine, leucine, and valine (ILV). However, the regulation of ILV biosynthesis does not account for the essentiality of TsaB/YeaZ for bacterial growth. In this study, we investigated the impact of TsaB/YeaZ depletion on bacterial morphology and gene expression profiles using electron microscopy and deep transcriptomic analysis, respectively. Our results revealed significant alterations in bacterial size and surface smoothness upon TsaB/YeaZ depletion. Furthermore, we pinpointed specific genes and enriched biological pathways significantly affected by TsaB/YeaZ during the early and middle exponential phases and early stationary phases of growth. Crucially, our research uncovered a regulatory role for TsaB/YeaZ in bacterial autolysis. These discoveries offer fresh insights into the multifaceted biological functions of TsaB/YeaZ within .
PubMed: 38786122
DOI: 10.3390/antibiotics13050393 -
Nature Communications May 2024Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by invasive behavior and a compromised immune response, presenting treatment challenges....
Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by invasive behavior and a compromised immune response, presenting treatment challenges. Surgical debulking of GBM fails to address its highly infiltrative nature, leaving neoplastic satellites in an environment characterized by impaired immune surveillance, ultimately paving the way for tumor recurrence. Tracking and eradicating residual GBM cells by boosting antitumor immunity is critical for preventing postoperative relapse, but effective immunotherapeutic strategies remain elusive. Here, we report a cavity-injectable bacterium-hydrogel superstructure that targets GBM satellites around the cavity, triggers GBM pyroptosis, and initiates innate and adaptive immune responses, which prevent postoperative GBM relapse in male mice. The immunostimulatory Salmonella delivery vehicles (SDVs) engineered from attenuated Salmonella typhimurium (VNP20009) seek and attack GBM cells. Salmonella lysis-inducing nanocapsules (SLINs), designed to trigger autolysis, are tethered to the SDVs, eliciting antitumor immune response through the intracellular release of bacterial components. Furthermore, SDVs and SLINs administration via intracavitary injection of the ATP-responsive hydrogel can recruit phagocytes and promote antigen presentation, initiating an adaptive immune response. Therefore, our work offers a local bacteriotherapy for stimulating anti-GBM immunity, with potential applicability for patients facing malignancies at a high risk of recurrence.
Topics: Glioblastoma; Animals; Mice; Salmonella typhimurium; Male; Neoplasm Recurrence, Local; Brain Neoplasms; Humans; Cell Line, Tumor; Mice, Inbred C57BL; Pyroptosis; Adaptive Immunity; Immunity, Innate; Hydrogels; Immunotherapy
PubMed: 38762500
DOI: 10.1038/s41467-024-48606-5 -
Journal of Agricultural and Food... May 2024The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and...
The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.
Topics: Calpain; Oxidation-Reduction; Animals; Aldehydes; Tandem Mass Spectrometry; Malondialdehyde; Muscle, Skeletal; Meat; Swine
PubMed: 38743679
DOI: 10.1021/acs.jafc.4c00335 -
Journal of Animal Science Jan 2024Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are...
Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.
Topics: Calpain; Calcium-Binding Proteins; Animals; Oxidation-Reduction; Hydrogen Peroxide; Swine; Calcium; Ethylmaleimide; Humans
PubMed: 38738874
DOI: 10.1093/jas/skae135 -
Archives of Plastic Surgery May 2024Reconstructive surgeons face challenges when considering limb salvage methods for the treatment of diabetic foot ulcers (DFUs). In this article, we present our...
Reconstructive surgeons face challenges when considering limb salvage methods for the treatment of diabetic foot ulcers (DFUs). In this article, we present our experience with autologous fat grafting as a viable alternative in cases where flap reconstruction is difficult. We encountered a 78-year-old female patient with a nonhealing DFU who had multiple comorbidities, including renal failure and severe peripheral arterial disease. During the initial multidisciplinary meeting, due to extensive necrosis and osteomyelitis, amputation was recommended. However, the patient expressed a strong preference for a salvage procedure and refused amputation. After careful consideration, we opted to reconstruct the patient's foot using three-dimensional bioprinted autologous minimally manipulated homologous adipose tissue. The AMHAT was engrafted well without complications such as autolysis, graft failure, or infection. After the operation, the large defect with partial bone exposure was covered with healthy granulation tissue. The size of the wound decreased to less than half its original size after 6 weeks of surgery, and it decreased to less than 25% after 12 weeks of surgery. The AMHAT may be an appealing treatment option for diabetic foot patients who are unsuitable for flap reconstruction due to comorbidities.
PubMed: 38737843
DOI: 10.1055/a-2263-7957 -
Microbiology Spectrum Jun 2024infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and pathogenesis studies are eagerly required for CDI therapy. The major...
infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in pathogenesis; however, a lack of isogenic mutants has greatly hampered analysis of SlpA functions. In this study, the whole gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of in resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCE infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic mutants. Surprisingly, the first deletion strain was generated in this study via CRISPR-Cas9, further negating the previous thought about being essential. Results in this study will provide direct proof for roles of SlpA in pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.
Topics: Clostridioides difficile; Bacterial Proteins; Humans; Gene Deletion; Clostridium Infections; Biofilms; Anti-Bacterial Agents; Virulence; CRISPR-Cas Systems; Bacterial Adhesion; Membrane Glycoproteins
PubMed: 38709045
DOI: 10.1128/spectrum.04005-23 -
Frontiers in Microbiology 2024Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The...
INTRODUCTION
Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The objectives of this research were to investigate changes in microbial diversity and community structure during the growth of Pholiota nameko and to analyze the adaptability of the dominant strains to their respective habitats through metabolic.
METHODS
Specifically, we conducted second-generation sequencing of the 16S rRNA gene (Illumina) on samples obtained from these stages. In addition, we isolated and characterized endophytes present in Pholiota nameko, focusing on examining the impact of dominant endophyte genera on autolysis. We also conducted a metabolic pathway analysis.
RESULTS AND DISCUSSION
The results unveiled 578,414 valid sequences of Pholiota nameko endophytic fungi. At the phylum level, the dominant taxa were Basidiomycota, Ascomycota, Zoopagomycota, and Mucoromycota. At the genus level, the dominant taxa observed were Pholiota, Inocybe, Fusarium, and Hortiboletus. For endophytic bacteria, we obtained 458,475 valid sequences. The dominant phyla were Proteobacteria, TM6, Firmicutes, and Bacteroidetes, while the dominant genera were Edaphobacter, Xanthomonas, Burkholderia, and Pseudomonas. Moreover, we identified the isolated strains in Pholiota nameko using 16S rDNA, and most of them were found to belong to the genus Pseudomonas, with Pseudomonas putida being the most prevalent strain. The findings revealed that the Pseudomonas putida strain has the ability to slow down the breakdown of soluble proteins and partially suppress the metabolic processes that generate superoxide anion radicals in Pholiota nameko, thereby reducing autolysis. Additionally, our results demonstrated that molybdenum enzyme-mediated anaerobic oxidative phosphorylation reactions were the primary energy metabolism pathway in the Pseudomonas putida strain. This suggests that the molybdenum cofactor synthesis pathway might be the main mechanism through which Pholiota nameko adapts to its complex and diverse habitats.
PubMed: 38690362
DOI: 10.3389/fmicb.2024.1319886 -
International Journal of Biological... May 2024Recent advancements in enzyme research have unveiled a new proteoform of bovine trypsin, expanding our understanding of this well-characterized enzyme. While generally...
Recent advancements in enzyme research have unveiled a new proteoform of bovine trypsin, expanding our understanding of this well-characterized enzyme. While generally similar to other trypsins, this novel proteoform comprises three polypeptide chains, marking a significant difference in activity, kinetic properties, and conformational stability. Compared with the already known bovine trypsin proteoforms, the results showed a lower: activity, k and k.K and protein 'foldedness' ratio for the new proteoform. Molecular autolysis, a common feature in trypsin and chymotrypsin, has been explored through comparative physical chemistry properties with other proteoforms. This new proteoform of trypsin not only enriches the existing enzyme repertoire but also promises to shed light on the intricate physiological pathway for enzyme inactivation. Our results suggest that the new trypsin proteoform is one of the likely final pathways for enzyme inactivation in a physiological environment. This discovery opens up new avenues for further research into the functional implications of this new trypsin proteoform.
Topics: Trypsin; Animals; Cattle; Kinetics; Enzyme Stability; Protein Conformation
PubMed: 38670206
DOI: 10.1016/j.ijbiomac.2024.131860