Gel Electrophoresis Research Articles

The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

The ubiquitin receptors RPN10 and RPN13 harbor multiple activities including ubiquitin binding; however, solid evidence connecting a particular activity to specific in vivo functions is scarce. Through complementation, the ubiquitin-binding site-truncated Arabidopsis RPN10 (N215) rescued the growth defects of rpn10-2, supporting the idea that the ubiquitin-binding ability of RPN10 is dispensable and N215, which harbors a vWA domain, is fully functional. Instead, a structural role played by RPN10 in the 26S proteasomes is likely vital in vivo. A site-specific variant, RPN10-11A, that likely has a destabilized vWA domain could partially rescue the rpn10-2 growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with rpn10-2 26S proteasomes showed that the loss of RPN10 reduced the abundance of double-capped proteasomes, induced the integration of specific subunit paralogues, and increased the association of ECM29, a well-known factor critical for quality checkpoints by binding and inhibiting aberrant proteasomes. Extensive Y2H and GST-pulldown analyses identified RPN2-binding residues on RPN13 that overlapped with ubiquitin-binding and UCH2-binding sites in the RPN13 C-terminus (246–254). Interestingly, an analysis of homozygous rpn10-2 segregation in a rpn13-1 background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13–RPN2 association in vivo.

Primer reporte de Bemisia tabaci (Gennadius, 1889) (Hemiptera: Aleyrodidae), biotipos B y Q, en soja Glycine max (L.) Merrill (Fabales: Fabaceae) en Paraguay

Whiteflies (Hemiptera: Aleyrodidae) are significant agricultural pests worldwide, primarily because they act as vectors for various plant viruses, including geminiviruses. Additionally, whitefly activity fosters the development of fungal colonies, such as sooty mold (Capnodium spp.), which grows on the sugary secretions (honeydew) produced by these insects. The presence of sooty mold reduces the plant’s photosynthetic capacity, further worsening the negative impact on its growth and development. Together, these factors make whitefly control a challenge for farmers, who must implement integrated pest management strategies to mitigate both the direct damage and the associated diseases that whiteflies can spread. Although approximately 1,200 species of whiteflies have been described, fewer than 40 are considered primary pests, with Bemisia tabaci (Gennadius, 1889) and Trialeurodes vaporariorum (Westwood, 1889) responsible for the greatest agricultural losses worldwide. The emergence of B. tabaci biotypes has prompted significant research, as these biotypes have been linked to economic damage to crops in several nations of the Americas, including Argentina and Brazil. In Paraguay, there is a need to identify local biotypes of B. tabaci led to the collection of whitefly samples from soybean plots in Capitán Miranda and Natalio, Itapúa, Paraguay. DNA was extracted using the Wizard genomic purification kit, followed by PCR amplification of the mitochondrial cytochrome oxidase I (mtCOI) region using specific primers for B. tabaci and T. vaporariorum. Agarose gel electrophoresis confirmed the presence of B and Q biotypes of B. tabaci in both Paraguayan locations, with no detection of T. vaporariorum. Sequencing of the PCR products revealed a 100% match with known sequences for B. tabaci and its biotypes, as confirmed by GenBank BLAST analysis. This research highlights the critical importance of continuously monitoring the population dynamics and geographical distribution of different biotypes. While the expansion of a polyphagous and dynamic pest like the whitefly cannot always be prevented, constant monitoring enables early detection of its spread and allows for the adjustment of management strategies tailored to each region. These measures will not eliminate the pest but will help mitigate damage and manage the problem sustainably and efficiently. The study also emphasizes the need to the effectiveness of insecticides, as resistance has been reported in other countries—an issue not yet explored in Paraguay.

Freshwater jellyfish in northeastern Argentina: a risk to human health.

Although cnidarians are mostly marine organisms, the occurrence of freshwater jellyfish frequently arouses the interest of ecologists, due to their sudden and unusual appearances in natural and artificial water bodies around the world. This study describes a series of cases compatible with cnidarian envenomation that occurred coincidentally in time and space with the presence of Craspedacusta sowerbii Lankester, 1880 jellyfish in the province of Misiones (El Saltito stream) in January 2022. A year later, its presence was confirmed in another watercourse of this province (Cazador stream). Based on these findings, its possible toxicological consequences in humans were evaluated by characterizing the jellyfish extract using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymatic/toxic activities. We revealed the presence of proteolytic enzymes and cytolytic toxin(s), which-by means of activating the inflammatory cascade-could explain the stinging skin lesions observed in the suspected cases described. In addition to recording for the first time the occurrence of this cnidarian in northeastern Argentina, this work provides, for the first time, a toxinological explanation for the clinical observations after contact with the most widespread freshwater jellyfish species in the world, giving support to health professionals in the diagnosis and management of such accidents/envenomation.

Integrated membranes system for water application in microbiology/molecular biology

An integrated membrane filtration system was developed to make water purity suitable for microbiology/molecular biology research. Water samples were collected from outlets in different buildings of the National Research Center and analyzed for their composition before filtration. An integrated membrane system was developed based on mathematical modeling. Flat sheet membranes were produced, including microfiltration, ultrafiltration, and nanofiltration membranes. The flat sheet membranes were converted to a spiral wound module filter to simulate the local market filters and applied in the integrated membrane system that was designed and installed. The produced water was analyzed and compared to molecular-grade water used in different molecular biology/microbiology applications. Both PCR, agarose gel electrophoresis, bacterial liquid cultures, and viral propagation indicated that treated water using the herein-developed system exhibited comparable performance to the molecular grade water provided with imported reagent kits. So, this research can offer a promising solution for producing high-quality water suitable for sensitive laboratory applications.

Molecular Characteristics Demonstrate the Occurrence of Phylogenetic Similar Isolates of <i>Vibrio alginolyticus</i> and <i>Vibrio parahaemolyticus</i> in Aquatic Environments

Vibrio alginolyticus is an opportunistic fish pathogen with a potential to cause septicemia and often associated with other Vibrio infections, particularly Vibrio parahaemolyticus, and can pose a significant threat to aquatic health. This study investigated the biochemical and phylogenetic characteristics of V. alginolyticus isolated from various aquaculture farms, focusing on its prevalence, hemolysin genes, genetic relatedness, and antibiotic susceptibility. Out of 92 brackish water aquaculture farm samples, 16 isolates were biochemically confirmed as V. alginolyticus, with 12 subsequently confirmed by polymerase chain reaction targeting collagenase gene. Molecular analysis of the thermolabile hemolysin gene (tlh) via specific PCR amplified a 450 bp fragment in 8 isolates, confirming the presence of the tlh gene. Pulsed Field Gel Electrophoresis (PFGE) typing differentiated tlh-positive and tlh-negative V. alginolyticus isolates with 92% genetic similarity. The isolates exhibited proteolytic, lipolytic, and lecithinase activities. Notably, the isolates showed intermediate resistance to most of the tested antibiotics, indicating exposure to antimicrobial agents. This study provides evidence of the presence of similar phylogenetical isolates of V. alginolyticus and V. parahaemolyticus in aquatic environments. Furthermore, this study emphasizes the importance of expanding surveillance programs by incorporating strain-specific characterization to better understand and control vibriosis in aquaculture.

Optimization of extraction for efficient recovery of kenaf seed protein isolates: evaluation of physicochemical and techno-functional characteristics.

Kenaf seeds are a rich source of protein; however, finding the best extraction method is crucial to obtaining high-quality protein from these underutilized seeds. This research devised an optimized extraction process for best recovery of kenaf seeds protein using response surface methodology. The key parameters affecting the yield and protein content were optimized, including extraction pH (2-11), seed:water ratio (5:1-50:1), temperature (30-90 °C), and duration (20-360 min). The physicochemical and techno-functional properties of kenaf seed protein isolates (KSPIs) were examined. A maximum protein yield of 12.05 g/100 g with purity level 91.94 g/100 g was obtained using an optimized extraction with pH 11.0, seed:water ratio 50:1, 360 min duration, and temperature 50 °C. The oil and water retention capacities of KSPI were 1.14 mL g-1 and 1.37 mL g-1 respectively. After 30 min at pH 7, KSPIs demonstrated remarkable emulsion capacity (83.12%) and stability (75.63%), along with high foaming capacity (106%) and stability (18.3%). As per high-performance liquid chromatography analysis, arginine, glutamic acid, leucine, phenylalanine, and lysine were the most abundant amino acids detected in KPSIs. The KSPIs' globular protein structure was successfully verified using analytical approaches, including Fourier transform infrared spectroscopy, protein fraction ratios, and differential scanning calorimetry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that KPSI has a molecular weight distribution ranging from 10 kDa to 50 kDa. The results of this study support the application of the proposed response-surface-methodology-optimized extraction method for efficient recovery of high-quality kenaf seed proteins that meet the necessary physicochemical and techno-functional requirements. © 2024 Society of Chemical Industry.

ISOLATION AND MOLECULAR IDENTIFICATION OF INDIGENOUS BACTERIOCIN-PRODUCING WEISSELLA CIBARIA

Globally, over 6.22 million deaths are associated with antibiotic resistance. Bacteriocins, a set of antimicrobial peptides synthesized on the ribosomes, are widely viewed as a potential answer to this issue. This is due to their pore-forming ability and antimicrobial activity against antibiotic-resistant pathogens. The aim of this study is to isolate bacteriocin-producing Weissella cibaria and evaluate its antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Streptococcus sp., Candida sp. and Rhizopus stolonifer. Weissella cibaria man1 was isolated by inoculating deMan Rogosa Sharpe (MRS) broth with small pieces of ripe Mangifera indica (mango), 24-hour incubation at 370C, 10-fold serial dilution and plating on MRS agar. Molecular identification was achieved by DNA extraction, amplification of the 16S rRNA gene through polymerase chain reaction (PCR), agarose gel electrophoresis, gene sequencing, and BLASTN homology searches in the National Center for Biotechnology Information (NCBI). Antimicrobial activity of the bacteriocin was determined by agar well diffusion assay. Mangifera indica (mango) was found to harbor bacteriocin-producing Weissella cibaria man1. The bacteriocin (weissellicin man1) exhibited a broad spectrum of antimicrobial activity. Weissellicin man1 suppressed the growth of several target pathogens (Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Candida sp. and Rhizopus stolonifer) but had no inhibitory action against Escherichia coli, Streptococcus sp., Staphylococcus aureus. In conclusion, weissellicin man1 from Weissella cibaria man1 has a broad-spectrum of antimicrobial action. These findings will facilitate further evaluation of the antimicrobial potency of weissellicin man1.

Removal mechanisms of pentachlorophenol in a horizontal-flow anaerobic immobilized biomass reactor (HAIB) inoculated with an indigenous estuarine sediment microbiota: adsorption and biodegradation processes.

Pentachlorophenol (PCP) is a highly toxic and carcinogenic compound with significant environmental impact, necessitating effective treatment technologies. This study evaluates PCP removal mechanisms, including adsorption and biodegradation, during the startup of a horizontal-flow anaerobic immobilized biomass reactor (HAIB), and examines the impact of PCP concentration on microbial diversity using denaturing gradient gel electrophoresis (DGGE). The primary mechanism for PCP removal in the HAIB was adsorption, effectively described by the Freundlich isotherm model. Adsorption efficiency ranged from 86 to 104% for PCP concentrations between 0.2 and 5.0mg/L, and 46% to 64% for concentrations between 0.098 and 0.05mg/L. Additionally, PCP degradation intermediates such as 2,3-DCP and 2,6-DCP were detected, indicating that biodegradation also occurred in the HAIB. Organic matter degradation averaged 81 ± 9%, and methane content in the biogas averaged 46 ± 9%, confirming the anaerobic process. No inhibition of microbial activity was observed due to PCP toxicity, even at a PCP load of 5mg PCP/g STV per day. While the archaeal community showed only slight changes, with similarity coefficients ranging from 88 to 95%, the bacterial community was significantly affected by PCP, with similarity coefficients ranging from 18 to 50%. Bacterial groups were responsible for the initial PCP degradation, while the archaeal community was involved in metabolizing the resulting byproducts. The use of indigenous inoculum from the Santos-São Vicente estuary demonstrated its potential for effective PCP removal. Polyurethane foam proved to be an effective support material, enhancing the adsorption process and reducing PCP toxicity to the microbial consortium. This study provides valuable insights into PCP adsorption and biodegradation mechanisms in HAIB, highlighting the effectiveness of indigenous inoculum and polyurethane foam for PCP removal.

The cardioprotective potential of soluble urokinase Plasminogen Activator Receptor (suPAR) in myocardial ischemia

Abstract Background and aims: Increased circulating soluble urokinase plasminogen activator receptor (suPAR) levels are associated with adverse cardiovascular outcomes, however, any cardiovascular-related functions remain unknown. This study aimed to (i) document haemodynamic profiles in isolated rat hearts under the influence of suPAR, and to (ii) explore any mechanisms triggered by suPAR following myocardial ischaemia-reperfusion. Methods We undertook a 4-pronged approach (Figure 1). A Langendorff isolated rat heart model was used to perfuse all hearts with Krebs-Henseleit solution flowing retrogradely through the aorta at 37°C. A balloon inserted into the left ventricle allowed measurements of ventricular pressure, and a pressure transducer placed above the aorta recorded perfusion pressure for coronary flow rates. The experimental protocol consisted an initial 30-minute stabilisation period, followed by 40 minutes of ischemia via buffer flow cessation. Hearts were subsequently recovered during 90 minutes of reperfusion, receiving either suPAR treatment or perfusion buffer as controls. An inhibitor arm comprising co-infusion of suPAR and a monoclonal antibody capable of binding to suPAR was also assessed. Troponin T was measured in outflow perfusates collected at specific intervals. Proteins from hearts in treatment groups were separated using gel electrophoresis, with Western blotting to visualise the phosphorylation status of kinases (Akt, STAT). High-flow respirometry and fluorometry were investigated in cardiac mitochondria to determine respiration rates and ATP production in ischaemia reperfusion under the influence of suPAR. Statistical analysis was conducted with ANOVA using SPSS. Results Hearts receiving suPAR infusion at reperfusion (n = 16) showed a significant increase in developed pressure (p = .017) and lower perfusion pressure (p = .02) compared to controls (n = 16), revealing that suPAR-treated hearts recovered with better contractility and vasodilatory properties post-ischaemia. suPAR’s haemodynamic profiles in the antibody group were similar to controls, attenuating suPAR-induced effects (n = 8). Outflow effluents in suPAR-treated hearts obtained >3 fold lower serial Troponin T values than controls (p < .001). Heart tissues receiving suPAR treatment revealed a 1.7-fold increase in Akt (p = .001) and a 3.8-fold increase in STAT3 phosphorylation compared to controls (p < .001) and the inhibitor group (p = .04). Finally, suPAR increased mitochondrial ATP production by 28% (p = .013) compared to controls after ischaemia. Conclusion This is a world-first demonstration of suPAR’s cardioprotective influences in isolated rat hearts recovering from ischaemia. suPAR’s ability to promote cardiac contractility and vasodilation was associated with lesser injury as shown by Troponin T reduction. Mechanistically, suPAR infusion upregulated two key cardioprotective pathways, which may also function to protect cardiac mitochondria.Figure 1 - Schematic of Methods

Exploring microbial diversity in Kermanshah province’s Kermanshahi oil through DGGE and sequencing analysis

BackgroundGhee, known as “roghane heiwâni,” or “Kermanshahi oil” is a traditional fermented butter-like product highly esteemed for its nutritional value. Ghee is prepared using traditional methods and has substantial potential as a reservoir of probiotic microorganisms. Previous research delved into isolating and identifying lactic acid bacteria (LAB) in Kermanshahi through culture and PCR sequencing. This study seeks to elucidate the microbial profiles and diversity within Kermanshahi using culture, Denaturing Gradient Gel Electrophoresis (DGGE), and sequencing methodologies.MethodsTwenty samples of Kermanshahi oil were meticulously gathered from diverse locales across Kermanshah province. These samples were cultivated under specialized conditions in MRS and M17 environments spanning 24 to 72 h. Following DNA extraction, amplification of the 16SrRNA gene sequences was performed, culminating in sequencing for conclusive identification of the isolates. Furthermore, the DGGE technique was directly employed to separate and identify various species present in the oil samples utilizing bioinformatics software.ResultsSequencing outcomes revealed a diverse array of microorganisms among the isolates, with Lactobacillus constituting 43%, Streptococcus comprising 27.6%, Enterococcus at 4.61%, and yeasts at 7.6%. Other species exhibited lower frequencies, encompassing Rhizobium, Bacillus coagulans, and Staphylococcus hominis.ConclusionsThe isolation of a diverse spectrum of probiotic microorganisms underscores their potential utility in the realm of industrial dairy product production. These findings allude to the possibility of integrating these valuable microorganisms, which have historically been associated with traditional products, into the contemporary dairy industry. As consumer interest in probiotic-enriched products surges, the insights gained from this study pave the way for harnessing the benefits of Kermanshahi-derived probiotics.

Molecular surveillance of A. baumannii in intensive care units: exploration of transmission chains

Abstract Background Multidrug-resistant Acinetobacter baumannii (MDR-Ab) is one of the main causes of healthcare associated infections (HAIs). During the SARS-CoV-2 pandemic there was an increase in MDR-Ab infections, especially in intensive care units (ICUs). This study aimed to assess the potential spread or emergence of specific clusters of MDR-Ab across four different ICUs at the Umberto I teaching hospital of Rome. Methods From January 2020 to January 2022 microbiological surveillance was conducted in four ICUs: two dedicated to COVID-19 patients (ICU-1C, ICU-2C) and two to non-COVID-19 patients (ICU-1R, ICU-2R). The genetic relatedness between A. baumannii isolates was assessed using pulsed-field gel electrophoresis (PFGE). Illumina whole genome sequencing was conducted on 26 representative isolates. Results In total, 178 A. baumannii isolates were obtained from 129 COVID-19 patients and 49 non-COVID-19 patients. The isolates were classified into 17 PFGE pulsotypes, being two major (A, B) and five intermediate (C, D, E, H, Q). Clone A was present in all ICUs, while Clone B was present only in ICU-1C and ICU-2R. Overall, 117 isolates belonged to clone A and exhibited a MDR phenotype; all of them were placed within the international clonal lineage II. All isolates showed carbapenems resistance primarily attributed to the presence of the blaOXA-23 gene, while aminoglycosides resistance observed in almost all isolates was attributed to the presence of the armA gene. Small outbreaks involving intermediate pulsotypes were detected between ICU-1R and ICU-2R, ICU-1C and ICU-2R, ICU-1C and ICU-2C, ICU-1C and ICU-1R. Conclusions The observed outbreaks could be attributed to a decline in attention to normal care practices for the prevention of HAIs during the COVID-19 pandemic, favouring the spread of MDR-Ab. Therefore, it is recommended the strengthen of control measures and the implementation of long-term strategies targeting MDR microorganisms in the ICUs. Key messages • Acinetobacter baumannii was frequently found infecting patients across four different ICUs at the Umberto I teaching hospital of Rome. • Acinetobacter baumanni outbreaks and multidrug resistance were observed. Therefore, the implementation of long-term strategies targeting MDR microorganisms in the ICUs is highly recommended.

Clinical epidemiology and impact of Haemophilus influenzae airway infections in adults with cystic fibrosis

BackgroundHaemophilus influenzae is prevalent within the airways of persons with cystic fibrosis (pwCF). H. influenzae is often associated with pulmonary exacerbations (PEx) in pediatric cohorts, but in adults, studies have yielded conflicting reports around the impact(s) on clinical outcomes such as lung function decline. Accordingly, we sought to discern the prevalence, natural history, and clinical impact of H. influenzae in adult pwCF.MethodsThis single-centre retrospective cohort study reviewed all adult pwCF with H. influenzae sputum cultures between 2002 and 2016. From this cohort, persistently infected subjects (defined as: ≥2 samples with the same pulsotype and > 50% sputum culture-positive for H. influenzae in each year) were matched (1:2) to controls without H. influenzae. Demographic and clinical status (baseline health or during periods of PEx) were obtained at each visit that H. influenzae was cultured. Yearly biobank isolates were typed using pulsed-field gel electrophoresis (PFGE) to assess relatedness.ResultsOver the study period, 30% (n = 70/240) of pwCF were culture positive for H. influenzae, of which 38 (54%) were culture-positive on multiple occasions and 12 (17%) had persistent infection. One hundred and thirty-seven isolates underwent PFGE, with 94 unique pulsotypes identified. Two (1.5%) were serotype f with the rest non-typeable (98.5%). H. influenzae isolation was associated with an increased risk of PEx (RR = 1.61 [1.14–2.27], p = 0.006), however, this association was lost when we excluded those who irregularly produced sputum (i.e. only during a PEx). Annual lung function decline did not differ across cohorts.ConclusionsIsolation of H. influenzae was common amongst adult pwCF but often transient. H. influenzae infection was not associated with acute PEx or chronic lung function decline.

Contribution of haptoglobin phenotypic variation to the presence of hyperhomocysteinemia in type 2 diabetics with and without angiopathy.

The genetic polymorphism of haptoglobin (Hp) has been associated with several cardiovascular risk factors, but a possible relationship between Hp phenotypic variation and increased levels of homocysteine (Hcy) and cysteine (Cy) is still unknown. The objective of this study is to evaluate the relationship between the Hp polymorphism and hyperhomocysteinemia (HHcy) and hypercysteinemia (HCy) in type 2 diabetics (T2D) with and without angiopathy (AGP). A case-control study was carried out on 293 adults: Group I (GI) - 75 subjects with T2D and AGP; Group II (GII) - 75 subjects with T2D without AGP; Group III (GIII) - 143 controls. Plasma levels of Hcy, Cy and vitamin B6 were measured by high performance liquid chromatography (HPLC) and vitamins B9 and B12 determined by electrochemiluminescence (ECL). The Hp polymorphism was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peroxidase staining. The results were analyzed in SPSS®, version 26.0 with a significance of 95%. Mean Hcy concentrations were significantly lower in carriers of the Hp2-2 phenotype (6.14 µM; p = 0.046) compared to the other genotypes. The presence of Hp2-1 is associated with an approximately 3.3 times greater probability of occurrence of HHcy (p = 0.015) and 3.7 times greater probability occurrence of HCy (p = 0.021) in T2D with AGP. The presence of the Hp2-1 phenotype is associated with the predisposition of HHcy and HCy in individuals with T2D and AGP, possibly through a positive heterosis mechanism. Carriers of the Hp2-2 phenotype appear to have a greater activation of the transsulfuration pathway in the Hcy cycle and consequent protection for its accumulation.

Evaluation of physical and chemical isolation methods to extract and purify Campylobacter jejuni extracellular polymeric substances

The pathogenic bacterium Campylobacter jejuni is a major food safety concern as it can form biofilms that increase its survival and infective potential. Biofilms consist of microbial cells and extracellular matrix (ECM), which is made of water and extracellular polymeric substances (EPS), which are critical for structural integrity and pathogenicity. The aim of this study was to optimize a protocol for the isolation of C. jejuni ECM. We employed eight physical and chemical isolation methods to extract and purify ECM, followed by different qualitative and quantitative analyses using gel electrophoresis and spectroscopy. This comprehensive approach enabled the evaluation of ECM composition in terms of polysaccharides, proteins, and extracellular DNA. The isolation methods resulted in different yields and purities of the extracted ECM components. Centrifugation in combination with chemical treatments proved to be most effective, isolating higher concentrations of polysaccharides and proteins. Additionally, extraction with ether solution facilitated the recovery of high-molecular-weight extracellular DNA. Overall, we provide a refined methodology for ECM extraction from C. jejuni. As polysaccharides and proteins participate in biofilm stability and microbial communication, and extracellular DNA participates in genetic exchange and virulence, our study contributes towards a better understanding of the persistence of this pathogen in the food industry.

Ten Years Later: Still Unchanged Susceptibility to Antibiotics of Listeria monocytogenes from Poultry Processing Environments.

Listeria monocytogenes is still recognized as being commonly susceptible to antibiotics; however, there have been reports of reduced susceptibility in recent years. The significance of this resistance is not clear, in part due to the disparity in the antimicrobial susceptibility testing methods used. EUCAST (European Committee on Antimicrobial Susceptibility Testing) has recently proposed a standardized method for antibiotic susceptibility testing of L. monocytogenes. The aim of this work was to evaluate the susceptibility to 11 antibiotics in clinical use of 50 pulsed-field gel electrophoresis types of L. monocytogenes representing 347 isolates from a poultry industry setting using the EUCAST method and to compare the results with those obtained 10 years before. All poultry strains were sensitive to all the antibiotics tested but one strain was resistant to benzylpenicillin according to the EUCAST criteria. The current findings supported the previous study and confirmed that in certain food-associated L. monocytogenes populations, antibiotic sensitivity has remained stable.

A novel γ-chain mutation p.Asp318His in a Chinese family with dysfibrinogenemia

Background Congenital dysfibrinogenemia is characterized by reduced fibrinogen activity, but normal immunoreactive fibrinogen levels. Here, we present a novel case with an elevated risk of thrombosis. Methods Coagulation assays, gene analysis, in silico tools, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin polymerization, thrombin generation assay, and electron microscopy scanning were utilized to elucidate the pathogenic mechanism. Results The proband manifested with a normal immunologic fibrinogen (2.13 g/l) but reduced functional fibrinogen (0.39 g/l). Subsequent genetic analysis unveiled a novel heterozygous mutation, c.1030G>C (p.Asp318His), in the γ-chain D domain of fibrinogen, which was highly conserved in homologous species and led to enhanced thrombin generation capability. The ability of the proband's fibrinogen to polymerize was significantly impaired, with decreased final turbidity. Scanning electron microscopy indicated that the fibers of the proband were thinner than normal, with smaller pores. Thromboelastography (TEG) results demonstrated prolonged K time, decreased angle value, and a normal confidence interval value in the proband. Conclusion We present a novel case displaying the γAsp318His mutation, which resulted in dysfibrinogenemia.

Comparative Study on Protein Composition and Foam Characteristics of Barley and Wheat Beer

Protein is an important component of beer, and its type, content and molecular weight directly affect the quality of beer, especially the foam quality of beer. Different brands of wheat beer and barley beer available in the market were used for this analysis. The differences in protein composition and foam performance between multi-sample barley and wheat beer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure size exclusion chromatography. Protein significantly influences beer quality, particularly its foam properties. Wheat beer (WB) has 9.52–84.10% more total protein content than barley beer (BB). The primary proteins in both beers are 6.9–20.1 kDa, with WB having 1.04 g/L more of this protein, 60.11% higher than that of BB. It is one of the main different proteins between WB and BB. WB also contains 66.67% more 20.1–32.4 kDa protein compared to BB. This is one of the main differences between WB and BB proteins. Both 6.9–20.1 kDa and 20.1–32.4 kDa proteins enhance beer viscosity and foam properties. Additionally, WB’s > 32.4 kDa protein content is 246.67% higher than BB’s, significantly improving beer hydrophobicity and foam performance. These protein differences are key factors in the superior foam quality of WB.

Methylglyoxal-Induced Modifications in Human Triosephosphate Isomerase: Structural and Functional Repercussions of Specific Mutations

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme’s structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI’s susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

Comparison of Two Different Integration Methods for Quantifying Monoclonal Proteins on Agarose Gel and Capillary Zone Electrophoresis Instruments

Comparison of Two Different Integration Methods for Quantifying Monoclonal Proteins on Agarose Gel and Capillary Zone Electrophoresis Instruments

Evaluation of protective efficacy of recombinant Toxoplasma gondii DDX39 protein vaccine against acute and chronic T. gondii infection in mice

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+T cells rising by 21.9% and CD8+T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response.Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15 to 20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.