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Methods in Molecular Biology (Clifton,... 2024The proteomic approach plays a key role to characterize a biological system at any given time. In recent years, advances in proteomics have led to an increasing...
The proteomic approach plays a key role to characterize a biological system at any given time. In recent years, advances in proteomics have led to an increasing application in all biological fields, including plant matrices and associated microbiome studies. However, extracting adequate protein samples remains the most critical step for any plant proteomics study. The protein extraction protocols proposed for the phyllosphere involve an initial leaf washing step; however, this is an approach only applicable if interest is restricted to epiphytes. A metaproteomic approach is required to obtain an overall picture and consequently an extraction that considers proteins derived from the plant, epiphytic and endophytic microorganisms. The most commonly used extractions for plant tissue involve the use of phenol or TCA-acetone. However, for efficient protein recovery is essential to remove interfering components abundant in plant tissues, such as polysaccharides, lipids, and phenolic compounds. A well-proven protocol on the basis of a combination of TCA-acetone and phenol extraction is presented here, obtaining some cleaned protein pellets, suitable for electrophoresis and subsequent proteomics studies. Important points for the success of this protocol are (i) a proper sampling and sample preparation, (ii) maintaining samples at a low temperature during extraction and using protease inhibitors, (iii) an initial step in TCA-acetone to remove part of the interfering substances, and (iv) careful recovery of the phenolic phase. Furthermore, the protocol is timesaving and can be completed in one working day.
Topics: Plant Proteins; Proteomics; Plant Leaves; Acetone; Phenol; Plants; Trichloroacetic Acid
PubMed: 38941013
DOI: 10.1007/978-1-0716-3910-8_5 -
Journal of Bacteriology Jun 2024Responding to changes in oxygen levels is critical for aerobic microbes. In , low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes,...
UNLABELLED
Responding to changes in oxygen levels is critical for aerobic microbes. In , low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆ strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆. However, stabilization of FixT in ∆n strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient .
IMPORTANCE
The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.
PubMed: 38940598
DOI: 10.1128/jb.00237-24 -
Sheng Li Xue Bao : [Acta Physiologica... Jun 2024The secretory leukocyte protease inhibitor (SLPI) is mainly produced by immune cells and various epithelial cells, and is regulated by a variety of cytokines, such as... (Review)
Review
The secretory leukocyte protease inhibitor (SLPI) is mainly produced by immune cells and various epithelial cells, and is regulated by a variety of cytokines, such as transforming growth factor β1, interleukin 1β and tumor necrosis factor α. In addition to commonly known anti-protease activity, it has been found in recent years that SLPI plays essential roles in anti-apoptosis, regulating cell cycle, cell differentiation and proliferation, and inhibiting inflammatory response. SLPI can also assist the immune system to clear pathogens/damaged cells by enhancing the phagocytic function of phagocytes, so as to ameliorate tissue damage and promote repair. Moreover, recent studies have shown that the change of SLPI level in the serum of patients post cardiovascular surgery has a high diagnostic value in predicting the occurrence of acute kidney injury, suggesting that SLPI is involved in ischemia-reperfusion (IR) induced acute kidney injury. In this review, we summarized the expression, regulation, signaling pathways and associated biological events of SLPI in different organ injury models, and also discussed and evaluated the potential role of SLPI in renoprotection against IR induced acute kidney injury and its potential as a new biomarker.
Topics: Acute Kidney Injury; Humans; Reperfusion Injury; Animals; Secretory Leukocyte Peptidase Inhibitor; Signal Transduction
PubMed: 38939941
DOI: No ID Found -
Annals of Ibadan Postgraduate Medicine Apr 2024A major modifiable risk factor for atherosclerotic cardiovascular disease is abnormalities in lipid and lipoprotein metabolism which are frequently seen in HIV as well...
BACKGROUND AND OBJECTIVE
A major modifiable risk factor for atherosclerotic cardiovascular disease is abnormalities in lipid and lipoprotein metabolism which are frequently seen in HIV as well as its treatment. Apo-E is a protein that is important in plasma lipid homeostasis and its genetic alleles have been shown to contribute to lipid abnormalities. We examined for the effect of Apo-E gene polymorphisms on plasma lipid levels in PLHIV on protease inhibitor therapy.
METHODS
This was a cross-sectional study conducted among adult persons living with HIV. Lipid profile, Apo-B and Apo-A were measured in fasting plasma. Amplification and analysis of Apo-E genotypes were determined using the Seeplex Apo-E ACE genotyping kit. Differences in quantitative values were compared with non-parametric analysis methods.
RESULTS
Eighty-four persons were recruited into the study, 75% of whom were virally suppressed. The 3 homozygous genotypes had significantly different levels of low-density lipoprotein cholesterol (LDL-C), Apolipoprotein B (Apo-B) and Apolipoprotein A1 (Apo-A1). Persons with apo ε2/ε2 had higher LDL-C compared to those with apo ε3/ε3 (3.26 (3.61) mmol/L vs. 2.76 (1.28) mmol/L, p = 0.010). Those with apo ε4/ε4 had lower Apo-A1 compared to those with apo ε3/ε3 (0.84 (0.48) g/dL vs. 1.27 (0.70) g/dL, p =0.009). Compared with the same group, the heterozygous genotype, apo ε2/ε3 had lower triglyceride levels :1.33 (0.65) mmol/ L vs. 1.86 (1.11) mmol/L, p = 0.045.
CONCLUSION
Polymorphisms in the Apo-E gene may have significant influences on plasma lipid and apolipoprotein levels in PLHIV on PI therapy. This may have implications for the assessment of risk for cardiovascular disease.
PubMed: 38939889
DOI: No ID Found -
Journal of Cardiothoracic Surgery Jun 2024We aimed to assess the efficacy of the neutrophil elastase inhibitor, sivelestat, in the treatment of sepsis-induced acute respiratory distress syndrome (ARDS) and... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
We aimed to assess the efficacy of the neutrophil elastase inhibitor, sivelestat, in the treatment of sepsis-induced acute respiratory distress syndrome (ARDS) and septic cardiomyopathy (SCM).
METHODS
Between January 2019 and December 2021, we conducted a randomized trial on patients who had been diagnosed with sepsis-induced acute respiratory distress syndrome (ARDS) and septic cardiomyopathy (SCM) at Wuhan Union Hospital. The patients were divided into two groups by random envelop method, the Sivelestat group and the Control group. We measured the serum concentrations of Interleukin (IL)-6, IL-8, Tumor necrosis factor-α (TNF-α), and High-mobility group box 1 (HMGB1) at five time points, which were the baseline, 12 h, 24 h, 48 h, and 72 h after admission to the ICU. We evaluated the cardiac function by sonography and the heart rate variability (HRV) with 24-hour Holter recording between the time of admission to the intensive care unit (ICU) and 72 h after Sivelestat treatment.
RESULTS
From January 2019 to December 2021, a total of 70 patients were included in this study. The levels of IL-6, IL-8, and TNF-α were significantly lower in the Sivelestat group at different time points (12 h, 24 h, 48 h, and 72 h). HMGB1 levels were significantly lower at 72 h after Sivelestat treatment (19.46 ± 2.63pg/mL vs. 21.20 ± 2.03pg/mL, P = 0.003). The stroke volume (SV), tricuspid annular plane systolic excursion (TAPSE), early to late diastolic transmitral flow velocity (E/A), early (e') and late (a') diastoles were significantly low in the Control group compared with the Sivelestat group. Tei index was high in the Control group compared with the Sivelestat group (0.60 ± 0.08 vs. 0.56 ± 0.07, P = 0.029). The result of HRV showed significant differences in standard deviation of normal-to-normal intervals (SDNN), low frequency (LF), and LF/HF (high frequency) between the two groups.
CONCLUSIONS
Sivelestat can significantly reduce the levels of serum inflammatory factors, improve cardiac function, and reduce heart rate variability in patients with Sepsis-induced ARDS and SCM.
Topics: Humans; Male; Female; Glycine; Cardiomyopathies; Sepsis; Middle Aged; Respiratory Distress Syndrome; Sulfonamides; Treatment Outcome; Aged; Serine Proteinase Inhibitors
PubMed: 38937755
DOI: 10.1186/s13019-024-02835-3 -
Immunology and Allergy Clinics of North... Aug 2024The role of contact system activation has been clearly established in the pathogenesis of hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH). C1 inhibitor... (Review)
Review
The role of contact system activation has been clearly established in the pathogenesis of hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH). C1 inhibitor (C1INH)-protease complexes, levels of functional C1INH, plasma kallikrein activation, and cleavage of high-molecular-weight kininogen have each been associated with disease activity. More recently, HAE with normal levels of C1INH (HAE-nl-C1INH) has been recognized. Six genetic mutations have been identified which are linked to HAE-nl-C1INH phenotypes. The majority of individuals with HAE-nl-C1INH fall into the unknown category. There is substantial evidence that bradykinin generation underlies the recurrent attacks of swelling in some of these cohorts.
Topics: Humans; Bradykinin; Biomarkers; Complement C1 Inhibitor Protein; Angioedema; Angioedemas, Hereditary; Mutation
PubMed: 38937015
DOI: 10.1016/j.iac.2024.03.011 -
In Vivo (Athens, Greece) 2024Reports regarding the association of remdesivir use for the treatment of Coronavirus disease 2019 (COVID-19) with the development of acute kidney injury (AKI) are...
BACKGROUND/AIM
Reports regarding the association of remdesivir use for the treatment of Coronavirus disease 2019 (COVID-19) with the development of acute kidney injury (AKI) are inconsistent, and the associations between the use of other antivirals and AKI remain unclear. Therefore, this study investigated whether the use of antiviral drugs for the treatment of COVID-19 is a risk factor for the development of AKI.
PATIENTS AND METHODS
This study analyzed 176,197 reports submitted to the Japanese Adverse Event Reporting Database between 2020 and 2022. Reporting odds ratios (RORs) and 95% confidence intervals (95%CIs) for AKI that were associated with the use of antiviral drugs in patients with COVID-19 were calculated after adjusting for potential confounders.
RESULTS
Overall, 5,879 of the reports analyzed were associated with AKI. Signs of AKI were detected with the use of remdesivir [crude ROR (cROR)=2.45; 95%CI=1.91-3.14] and nirmatrelvir/ritonavir (cROR=6.07; 95%CI=4.06-9.06). These results were maintained even after adjusting for potential confounders [remdesivir: adjusted ROR (aROR)=2.18; 95%CI=1.69-2.80, nirmatrelvir/ritonavir: aROR=5.24; 95%CI=3.48-7.90]. However, when analyzing data stratified by reporting year, the association between remdesivir and AKI appeared to diminish over time and was not sustained.
CONCLUSION
Nirmatrelvir/ritonavir use may be associated with developing AKI. This knowledge may be useful in helping patients with COVID-19 avoid AKI complications.
Topics: Acute Kidney Injury; Humans; Antiviral Agents; COVID-19 Drug Treatment; Adenosine Monophosphate; Alanine; SARS-CoV-2; COVID-19; Female; Male; Ritonavir; Middle Aged; Aged; Risk Factors; Adult; Drug Combinations; Adenosine
PubMed: 38936945
DOI: 10.21873/invivo.13637 -
In Vivo (Athens, Greece) 2024Vildagliptin is one of the dipeptidyl peptidase-4 (DPP-4) inhibitors that have been shown to improve hyperglycemia in clinical trials among patients with type 2...
BACKGROUND/AIM
Vildagliptin is one of the dipeptidyl peptidase-4 (DPP-4) inhibitors that have been shown to improve hyperglycemia in clinical trials among patients with type 2 diabetes. However, few studies have examined the efficacy of vildagliptin in patients with diabetic kidney disease (DKD).
PATIENTS AND METHODS
Eight patients with DKD received oral vildagliptin 50-100 mg/day. The duration of diabetes was 6.7±5.9 years and observation period was 23.6±9.8 months. Changes in fasting blood glucose, and hemoglobin A1c (HbA1c), estimated glomerular filtration rate (eGFR), and urine protein-to-creatinine ratio (UPCR) were studied before and after the administration of vildagliptin.
RESULTS
Vildagliptin treatment significantly decreased fasting blood glucose and HbA1c, compared to baseline (132±56 mg/dl, p=0.036, 6.0±0.3, p=0.041, respectively). UPCR tended to be decreased, albeit without statistical significance. However, eGFR was decreased after the administration of vildagliptin. No significant adverse effects were observed in all patients during the study.
CONCLUSION
Although the sample size was limited and the observation period was brief, vildagliptin was found to be an effective and reasonably well-tolerated treatment for patients with DKD.
Topics: Humans; Vildagliptin; Diabetes Mellitus, Type 2; Male; Female; Diabetic Nephropathies; Middle Aged; Aged; Glomerular Filtration Rate; Blood Glucose; Dipeptidyl-Peptidase IV Inhibitors; Glycated Hemoglobin; Adamantane; Treatment Outcome; Pyrrolidines; Nitriles; Creatinine
PubMed: 38936943
DOI: 10.21873/invivo.13635 -
Microbial Pathogenesis Jun 2024Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an...
Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens.
PubMed: 38936638
DOI: 10.1016/j.micpath.2024.106762 -
Plant, Cell & Environment Jun 2024In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis....
In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis. Their targeting and abundance is essential for optimal movement responses. However, our knowledge how Chlamydomonas achieves this is still at its infancy. Here we show that ChR1 internalization occurs via light-stimulated endocytosis. Prior or during endocytosis ChR1 is modified and forms high molecular mass complexes. These are the solely detectable ChR1 forms in extracellular vesicles and their abundance therein dynamically changes upon illumination. The ChR1-containing extracellular vesicles are secreted via the plasma membrane and/or the ciliary base. In line with this, ciliogenesis mutants exhibit increased ChR1 degradation rates. Further, we establish involvement of the cysteine protease CEP1, a member of the papain-type C1A subfamily. ΔCEP1-knockout strains lack light-induced ChR1 degradation, whereas ChR2 degradation was unaffected. Low light stimulates CEP1 expression, which is regulated via phototropin, a SPA1 E3 ubiquitin ligase and cyclic AMP. Further, mutant and inhibitor analyses revealed involvement of the small GTPase ARL11 and SUMOylation in ChR1 targeting to the eyespot and cilia. Our study thus defines the degradation pathway of this central photoreceptor of Chlamydomonas and identifies novel elements involved in its homoeostasis and targeting.
PubMed: 38935876
DOI: 10.1111/pce.15017