Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

Fortification of Maize Tortilla with an Optimized Chickpea Hydrolysate and Its Effect on DPPIV Inhibition Capacity and Physicochemical Characteristics

Chickpea hydrolysates have shown bioactivity towards type 2 diabetes by inhibiting dipeptidyl peptidase (DPPIV) activity. The objective was to compare the effect of adding different levels of an optimized bromelain hydrolysate from chickpea isolated protein on DPPIV inhibition capacity and physicochemical properties of maize tortilla. White and blue maize tortillas, with no added chickpea hydrolysates were compared with fortified tortillas at the levels of 5%, 10%, and 15% w/w. Changes in color (L* a* b*, hue angle, and ΔE), texture (hardness, cohesiveness, and puncture force), and moisture were tested. Soluble protein determination and SDS-PAGE electrophoresis were used to characterize the protein profiles, and LC-MS-MS was used to sequence the peptides. DPPIV inhibition was evaluated before and after simulated gastrointestinal digestion. Peptides in the hydrolysates had high hydrophobicity (7.97–27.05 kcal * mol −1) and pI (5.18–11.13). Molecular docking of peptides showed interaction with DPPIV with an energy of affinity of –5.8 kcal/mol for FDLPAL in comparison with vildagliptin (−6.2 kcal/mol). The lowest fortification level increased soluble protein in 105% (8 g/100 g tortilla). DPPIV inhibition of white maize tortilla increased from 11% (fresh control) to 91% (15% fortification), and for blue tortilla from 26% to 95%. After simulated digestion, there was not a difference between blue or maize tortillas for DPPIV inhibition. Fortification of maize tortilla with chickpea hydrolysate inhibits DPPIV and can potentially be used in the prevention and management of type 2 diabetes. However, due to observed physicochemical changes of the fortified tortilla, sensory properties and consumer acceptance need to be evaluated.

CORRELATION OF THE PROTEIN DETERMINATION IN 24-HOUR URINE WITH THE PROTEIN / CREATININE INDEX IN PEDIATRIC PATIENTS WHO GO TO THE EXTERNAL CONSULTATION OF THE PEDIATRIC NEPHROLOGY SERVICE AT THE NAVAL MEDICAL CENTER

Introduction : Proteinuria increases infant morbidity and mortality, constituting a risk for the development of chronic kidney disease. Medical follow-up is necessary to monitor kidney function, as well as to prevent long-term deterioration of kidney function. Proteinuria can be benign or a serious systemic disorder, the one that is persistent accelerates the decrease in the glomerular filtration rate and key in the progression of kidney diseases. There are various ways of measuring urinary proteins, between the protein / creatinine index and in 24-hour urine, which present a variable correlation according to what is described in the literature. Objective: To determine the correlation of 24-hour protein and protein / creatinine index in pediatric patients who attend the outpatient clinic of the pediatric nephrology service at the Naval Medical Center. Material and methods : Descriptive cross-sectional study. Performed in the Pediatric service and Pediatric Nephrology outpatient service at the Naval Medical Center. Study population: Children with a history of proteinuria treated by the Pediatric Nephrology service at the Naval Medical Center. General description of the study : The patients were selected from the electronic file that they attended the Pediatric Nephrology service. In the clinical evaluation, weight and height were taken, and a physical examination was performed, as well as a 24-hour protein determination in 24-hour urine, 24-hour creatinine clearance in urine in addition to creatinine and proteins in isolated urine. Statistical analysis : Descriptive statistics were performed, according to the measurement scale of the variables and the correlation between the variables was determined using the Spearman or Pearson test according to the type of distribution of the quantitative variables. Results : 107 were included. Taking into account the 24-hour urine collection protein measurement, only 4 patients (7.4%) had proteinuria in nephrotic ranges and for the CPI measurement only in 3 (5.6%). With both measurement methods, the same proportion of significant proteinuria was observed, corresponding to 13 patients (24.1%), obtaining in the first case a spearman correlation coefficient of r = 0.752, p = 0.000, and for the second an r = 0.911, p: 0.000. Conclusions: The protein / creatinine index had a positive correlation with 24-hour proteinuria in those pediatric patients who presented abnormal results in urine studies and kidney stones (32%), chronic kidney disease and kidney transplantation (18%) and obesity / overweight. Likewise it allows to establish a directly proportional association. There is a positive correlation between the protein / creatinine index and the quantification of proteinuria in 24 hours after adjusting for creatinine per kilogram of weight. And the protein / creatinine ratio is a good candidate for the diagnosis of chronic kidney disease in pediatric patients and its use is justified.

Kaempferol attenuates the effects of XIST/miR-130a/STAT3 on inflammation and extracellular matrix degradation in osteoarthritis

Aim: To investigate whether kaempferol exhibited protective effects on osteoarthritis chondrocytes by modulating the XIST/miR-130a/STAT3 axis. Methods: qRT-PCR and western blot assays were used for gene and protein determination. Dual luciferase reporter and RNA immunoprecipitation assays were employed to study the interaction between miRNA and lncRNA or genes. Results: Kaempferol decreased proinflammatory cytokine production and extracellular matrix degradation in C28/I2 cells. Additionally, kaempferol ameliorated XIST expression and enhanced miR-130a expression. XIST interacted with miR-130a, and STAT3 was identified as a target of miR-130a. Knockdown of XIST expression suppressed proinflammatory cytokine production and extracellular matrix degradation in C28/I2 cells. Overexpression of STAT3 rescued the effects of XIST knockdown. Conclusion: Kaempferol inhibited inflammation and extracellular matrix degradation by modulating the XIST/miR-130a/STAT3 axis in chondrocytes.

Kaempferol ameliorates the regulatory effects of PVT1/miR-214 on epithelial–mesenchymal transition through the PAK4/β-catenin axis in SRA01/04 cells

Aim: To investigate whether kaempferol exhibits a protective effect on high glucose-induced epithelial–mesenchymal transition (EMT) by mediating the PVT1/ miR-214 and PAK4/β-catenin pathways in SRA01/04 cells. Methods & methods: qRT-PCR and western blot assays were used for gene and protein determination, and migration and invasion assays were conducted. A coimmunoprecipitation assay was used for determining protein interactions. Results: High glucose effectively upregulated PVT1 expression, downregulated miR-214 expression and promoted cell migration and invasion. Kaempferol attenuated high glucose-induced EMT by increasing PVT1 expression and decreasing miR-214 expression. PAK4 was identified as a direct target of miR-214. PAK4 overexpression could rescue the effects of PVT1 deficiency on SRA01/04 cells. Conclusion: Kaempferol ameliorated the regulatory effects of PVT1/ miR-214 on high glucose-induced EMT through PAK4/β-catenin in SRA01/04 cells.

Pitfalls of Accurate Protein Determination inside PLGA Nanoparticles Using the Micro BCA Assay

Cancer is one of the leading causes of death in the world and protein therapeutics play an important role in combating this disease. Novel nanocarriers are needed for optimal delivery, enhanced therapeutic effect, and protection of proteins. Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles are commonly used, since they are non-toxic, biodegradable, and allow for the sustained release of the active pharmaceutical ingredient (API). Accurate quantification of the therapeutic inside these nanocarriers is essential for further development and precise in vivo experiments, especially for determining the correct therapeutic dose. Bicinchoninic acid (BCA) assay is one of the most popular methods of protein quantification, known for its low sensitivity to common surfactants. However, large discrepancies between published results are often observed, with determined protein encapsulation efficiencies (EE) varying from 20 to 80%. We investigate the interference of excipients or the combination of excipients, on accurate EE determination of PLGA nanoparticles using the micro BCA assay. The EE was determined using multiple methods: by measuring the un-encapsulated protein (indirect approach) and directly by extracting the protein using sodium hydroxide and dimethyl sulfoxide. We show differences between the methods, highlight the most common pitfalls, and show the importance of using correct standards in assessing EE.

Proteoglycan from Bacillus sp. BS11 Inhibits the Inflammatory Response by Suppressing the MAPK and NF-κB Pathways in Lipopolysaccharide-Induced RAW264.7 Macrophages

Inflammation is involved in the pathogenesis of many debilitating diseases. Proteoglycan isolated from marine Bacillus sp. BS11 (EPS11) was shown to have anticancer activity, but its anti-inflammatory potential remains elusive. In the present study, the anti-inflammatory effects and mechanism of EPS11 were evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage model. Biochemical characterization showed that the total sugar content and protein content of EPS11 were 49.5% and 30.2% respectively. EPS11 was composed of mannose, glucosamine, galactosamine, glucose, galactose, rhamnose, and glucuronic acid. Its molecular weight was determined to be 3.06 × 105 Da. The protein determination of EPS11 was also performed. EPS11 displayed a strong anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages in vitro, which significantly suppressed inflammatory cytokines and mediators (such as NO, TNF-α, IL-6 and IL-1β, and COX-2). Western blot analysis indicated that EPS11 could downregulate the expression of many key proteins in mitogen-activated protein kinases (MAPKs) and transcription factor nuclear factor-κB (NF-κB) signaling pathways. In particular, EPS11 almost completely inhibited the expression of NF-κB P65, which indicated that EPS11 acted primarily on the NF-κB pathways. These findings offer new insights into the molecular mechanism underlying the anti-inflammatory effect of EPS11.