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Microorganisms May 2022Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of...
Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains- BL23 and GG-in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.
PubMed: 35744660
DOI: 10.3390/microorganisms10061142 -
International Journal of Molecular... Nov 2021Calpains are a family of Ca‑dependent cysteine proteases that participate in various cellular processes. Calpain 3 (CAPN3) is a classical calpain with unique... (Review)
Review
Calpains are a family of Ca‑dependent cysteine proteases that participate in various cellular processes. Calpain 3 (CAPN3) is a classical calpain with unique N‑terminus and insertion sequence 1 and 2 domains that confer characteristics such as rapid autolysis, Ca‑independent activation and Na activation of the protease. CAPN3 is the only muscle‑specific calpain that has important roles in the promotion of calcium release from skeletal muscle fibers, calcium uptake of sarcoplasmic reticulum, muscle formation and muscle remodeling. Studies have indicated that recessive mutations in CAPN3 cause limb‑girdle muscular dystrophy (MD) type 2A and other types of MD; eosinophilic myositis, melanoma and epilepsy are also closely related to CAPN3. In the present review, the characteristics of CAPN3, its biological functions and roles in the pathogenesis of a number of disorders are discussed.
Topics: Animals; Calpain; Enzyme Activation; Humans; Models, Biological; Muscle, Skeletal; Muscular Diseases; Organ Specificity
PubMed: 34549305
DOI: 10.3892/ijmm.2021.5036 -
Toxins Feb 2023The relative lack of marine venom pharmaceuticals can be anecdotally attributed to difficulties in working with venomous marine animals, including how to maintain venom... (Review)
Review
The relative lack of marine venom pharmaceuticals can be anecdotally attributed to difficulties in working with venomous marine animals, including how to maintain venom bioactivity during extraction and purification. The primary aim of this systematic literature review was to examine the key factors for consideration when extracting and purifying jellyfish venom toxins to maximise their effectiveness in bioassays towards the characterisation of a single toxin.An up-to-date database of 119 peer-reviewed research articles was established for all purified and semi-purified venoms across all jellyfish, including their level of purification, LD50, and the types of experimental toxicity bioassay used (e.g., whole animal and cell lines). We report that, of the toxins successfully purified across all jellyfish, the class Cubozoa (i.e., and ) was most highly represented, followed by Scyphozoa and Hydrozoa. We outline the best practices for maintaining jellyfish venom bioactivity, including strict thermal management, using the "autolysis" extraction method and two-step liquid chromatography purification involving size exclusion chromatography. To date, the box jellyfish has been the most effective jellyfish venom model with the most referenced extraction methods and the most isolated toxins, including CfTX-A/B. In summary, this review can be used as a resource for the efficient extraction, purification, and identification of jellyfish venom toxins.
Topics: Animals; Cnidarian Venoms; Scyphozoa; Cubozoa; Cell Line; Chromatography, Gel
PubMed: 36977061
DOI: 10.3390/toxins15030170 -
Journal of Veterinary Diagnostic... Mar 2023We provide here an overview of the state of applied techniques in the estimation of the early period of the postmortem interval (PMI). The biological methods included... (Review)
Review
We provide here an overview of the state of applied techniques in the estimation of the early period of the postmortem interval (PMI). The biological methods included consist of body cooling, CSF potassium, body cooling combined with CSF potassium, and tissue autolysis. For each method, we present its application in human and veterinary medicine and provide current methodology, strengths, and weaknesses, as well as target areas for improvement. We examine current and future molecular methods as they pertain to DNA and primarily to messenger RNA degradation for the estimation of the PMI, as well as the use of RNA in aging wounds, aging blood stains, and the identification of body fluids. Various types of RNA have different lengths, structures, and functions in cells. These differences in RNAs determine various intrinsic properties, such as their half-lives in cells, and, hence, their decay rate as well as their unique use for specific forensic tests. Future applications and refinements of RNA-based techniques provide opportunities for the use of molecular methods in the estimation of PMI and other general forensic applications.
Topics: Humans; Animals; Postmortem Changes; Forensic Pathology; Autopsy; Potassium; RNA
PubMed: 36744749
DOI: 10.1177/10406387231153930 -
BioRxiv : the Preprint Server For... Jan 2022Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic....
Integrated Autolysis, DNA Hydrolysis and Precipitation Enables an Improved Bioprocess for Q-Griffithsin, a Broad-Spectrum Antiviral and Clinical-Stage anti-COVID-19 Candidate.
UNLABELLED
Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral currently in clinical trials for COVID-19. Q-Griffithsin is produced at high titer in and purified to anticipated clinical grade without conventional chromatography or the need for any fixed downstream equipment. The process is thus both low-cost and highly flexible, facilitating low sales prices and agile modifications of production capacity, two key features for pandemic response. The simplicity of this process is enabled by a novel unit operation that integrates cellular autolysis, autohydrolysis of nucleic acids, and contaminant precipitation, giving essentially complete removal of host cell DNA as well as reducing host cell proteins and endotoxin by 3.6 and 2.4 log units, respectively. This unit operation can be performed rapidly and in the fermentation vessel, such that Q-GRFT is obtained with 100% yield and >99.9% purity immediately after fermentation and requires only a flow-through membrane chromatography step for further contaminant removal. Using this operation or variations of it may enable improved bioprocesses for a range of other high-value proteins in .
HIGHLIGHTS
Integrating autolysis, DNA hydrolysis and precipitation enables process simplificationAutolysis reduces endotoxin release and burden to purificationQ-Griffithsin recovered from fermentation vessel at >99.9% purity and 100% yieldQ-Griffithsin purified to anticipated clinical grade without conventional chromatographyThe resulting bioprocess is 100% disposables-compatible, scalable, and low-cost.
PubMed: 35018377
DOI: 10.1101/2021.12.30.474602 -
Journal of Dairy Science Dec 2020The importance of starter cultures to cheese manufacture and ripening is well known. Starters are inoculated into cheese milk at a level of ∼10 cfu/mL either from a... (Review)
Review
The importance of starter cultures to cheese manufacture and ripening is well known. Starters are inoculated into cheese milk at a level of ∼10 cfu/mL either from a bulk culture or using commercial direct-to-vat cultures. Before ripening, starters grow in the milk to reach populations of 10 to 10 cfu/g of curd depending on processing variables such as cook temperature, inclusion of washing steps, degree of partitioning with curds and whey, and importantly salt addition rate. Inherent strain-related properties also determine final populations in the curd following manufacture and include temperature sensitivity, salt sensitivity, presence of prophage, autolytic and permeabilization properties (which are influenced by processing steps), presence and type of cell envelope proteinase, and metabolic activity. Ripening of important industrial cheese varieties such as Cheddar, Dutch, Swiss, and Italian-type cheese varieties is characterized by extended storage under temperature-controlled conditions enabling characteristic flavor and texture development to occur. Over ripening, microbiological, biochemical and enzymatic changes occur with a decline in starter viability, release of intracellular enzymes, hydrolysis of proteins, carbohydrates and lipids, and formation of a range of volatile and nonvolatile flavor components. Recent reports suggest that starter strains may be present during the later stages of ripening and therefore their potential role needs to be reconsidered. This review will focus on our current understanding of starter viability and vitality during cheese ripening and will also review the area of starter permeabilization, autolysis, and enzyme release.
Topics: Animals; Cheese; Food Handling; Food Microbiology; Lactobacillales; Milk; Taste
PubMed: 33010919
DOI: 10.3168/jds.2020-18960 -
Annals of Translational Medicine Sep 2022Traumatic spinal cord injury (SCI) is a major clinical concern and a life-changing neurological condition with substantial socioeconomic implications. The initial...
BACKGROUND
Traumatic spinal cord injury (SCI) is a major clinical concern and a life-changing neurological condition with substantial socioeconomic implications. The initial mechanical force applied to the spinal cord at the time of injury is known as the primary injury. After the primary injury, ischemia and hypoxia induce cell death and autolysis, which are associated with the release of a group of inflammatory factors and biologically active substances, such as superoxide dismutase (SOD), malonaldehyde (MDA), lactate dehydrogenase (LDH), and tumor necrosis factor-α (TNF-α). These processes are called the secondary injury, and may lead to an excess of extracellular glutamate (Glu), which in turn promotes the neuronal injuries. Muscone has been shown to have anti-inflammatory effects in the treatment of brain diseases and other diseases. However, to date, no study has examined the effects of muscone in the treatment of SCI.
METHODS
Astrocytes were separated and purified by the method of short-term exposure combining with differential sticking wall. Astrocyte was identified by glial fibers acidic protein (GFAP) selecting cell immunochemical staining. A mechanical-chemical damage (MCD) model was established via the primary spinal astrocytes of rats, and treatment was administered with different concentrations of muscone. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) was detected at 6, 12, 24, 48 and 72 h. SOD, MDA, LDH, TNF-alpha and intracellular calcium was detected at 3, 6 and 12 h. Glu in supernatant was detected respectively at 3, 6 and 12 h by enzyme-linked immunosorbent assay (ELISA) method. Intracellular calcium was detected respectively at 3, 6 and 12 h by flow cytometry method. MRNA expression of excitatory amino acid transporters (EAATs) and GFAP were detected by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) method and protein expression of those by western blot at 6 h.
RESULTS
Muscone reduced the levels of LDH, TNF-α, and MDA after injury, and upregulated the level of SOD. Muscone also reduced the density of extracellular Glu and suppressed the intracellular calcium level. Additionally, it decreased the expression levels of EAATs and GFAPs.
CONCLUSIONS
Muscone has a protective effect on astrocytes in a MCD and inhibits astrocytes' proliferation.
PubMed: 36172099
DOI: 10.21037/atm-22-3848 -
Biomolecular Concepts Sep 2017Amphibian metamorphosis has historically attracted a good deal of scientific attention owing to its dramatic nature and easy observability. However, the genetic... (Review)
Review
Amphibian metamorphosis has historically attracted a good deal of scientific attention owing to its dramatic nature and easy observability. However, the genetic mechanisms of amphibian metamorphosis have not been thoroughly examined using modern techniques such as gene cloning, DNA sequencing, polymerase chain reaction or genomic editing. Here, we review the current state of knowledge regarding molecular mechanisms underlying tadpole tail resorption.
Topics: Animals; Anura; Autolysis; Metamorphosis, Biological; Models, Biological; Musculoskeletal Physiological Phenomena; Phagocytosis; Reptilian Proteins; Species Specificity; Tail; Xenopus
PubMed: 28873065
DOI: 10.1515/bmc-2017-0022 -
BioMed Research International 2021Autolysis is an internal phenomenon following the death of an organism that leads to the degradation of tissues. In order to explore the initial stages of autolysis and...
Autolysis is an internal phenomenon following the death of an organism that leads to the degradation of tissues. In order to explore the initial stages of autolysis and attempt to establish reference standards for tissue changes after death, we studied the rapidly autolyzing tissue of the crayfish hepatopancreas. Samples from the hepatopancreas of crayfish were examined 0, 5, 10, 30, 60, and 120 minutes after death. Histological and ultrapathological examinations and evaluations and apoptotic cell counts were conducted to determine the initiation time and degree of autolysis. The results showed that autolysis in the hepatopancreas of crayfish began within 5 minutes. Initially, autolysis manifested in the swelling of hepatic tubular cells and the widening of mesenchyme. Cells undergoing autolysis showed severe organelle necrolysis. Based on these observations, tissue samples should be collected and preserved within five minutes to avoid interfering with histopathological diagnoses.
Topics: Animals; Apoptosis; Astacoidea; Autolysis; Body Size; Body Weight; Hepatopancreas; Microscopy, Electron, Transmission; Seafood; Time Factors
PubMed: 33521126
DOI: 10.1155/2021/2345878 -
Scientific Reports Oct 2022Currently, there is no specific pharmaceutical agent for treating acute pancreatitis (AP). Somatostatin and its analogues have been used to prevent the autolysis of the... (Meta-Analysis)
Meta-Analysis
Currently, there is no specific pharmaceutical agent for treating acute pancreatitis (AP). Somatostatin and its analogues have been used to prevent the autolysis of the pancreas in AP, however, their effectiveness has not been confirmed. This investigation aimed to examine the efficacy of ulinastatin, a protease inhibitor, combined with somatostatin analogues in the treatment of AP. We conducted a systematic database search in 4 databases to identify randomized controlled trials in which the efficacy of ulinastatin in combination with somatostatin analogue was compared to somatostatin analogue alone in patients with AP. Since the patient populations of analysed papers were slightly different, we used random effect models to pool odds ratios (OR) and mean differences (MD) and the corresponding 95% confidence intervals (CI). A total of 9 articles comprising 1037 patients were included in the meta-analysis. The combination therapy significantly reduced the complication rates for acute respiratory distress syndrome, acute kidney injury, and multiple organ dysfunction. Symptoms were relieved threefold with the combination therapy compared to somatostatin alone, and combination therapy significantly shortened the length of hospital stay. The decrease in mortality was not statistically significant..
Topics: Humans; Acute Disease; Pancreatitis; Protease Inhibitors; Randomized Controlled Trials as Topic; Somatostatin
PubMed: 36289288
DOI: 10.1038/s41598-022-22341-7