Gel Electrophoresis Research Articles

Decoding halal and jhatka slaughter: novel insights into welfare and protein biomarkers in slow-growing broiler chicken.

The first evidence of blood biochemical and proteomic changes in slow-growing broiler chicken subjected to ritual slaughter like halal (HS) and jhatka (JS) without stunning and commercial slaughter with electrical stunning (ES) was decoded. Significant stress indicators like cortisol and triiodothyronine were markedly elevated in JS birds, whereas increased (P < 0.05) levels of lactate dehydrogenase and creatine kinase were observed in JS and ES birds. Two-dimensional gel electrophoresis coupled to MALDI-TOF MS elucidated the overabundance of glyceraldehyde-3-phosphate dehydrogenase and l-lactate dehydrogenase that are positively correlated with stress in JS broilers. Bioinformatic analysis explored the multifaceted landscape of molecular functions. The study has uncovered that ritual slaughter performed without stunning against commercial slaughter with ES practices elicit varying levels of stress as evident from blood biochemistry and novel protein markers. © 2024 Society of Chemical Industry.

Changes in protein expression in Acacia mearnsii De Wild (black wattle) as a result of exposure to frost-inducing temperatures

Acacia mearnsii De Wild (black wattle) is one of the most important forestry crops in South Africa. The bark of this species contains high-quality tannins, while its wood is used for wood chips, charcoal and mining timber. One of the main problems faced by black wattle farmers is frost damage, thus there is an urgent need to develop frost-tolerant black wattle varieties. A quick method for the screening of frost tolerance variation would be beneficial for black wattle breeders and would result in the need for fewer families to undergo field trials. Frost tolerance in plants is directly related to their ability to undergo cold acclimation in response to changing weather conditions. During the cold acclimation process, many physiological changes occur, including modifications in protein expression. In this study, two-dimensional gel electrophoresis was used to investigate changes in protein expression among 40 black wattle families with varying levels of frost tolerance before and after a cold-stress treatment. The proteomic data generated from this was analysed using multivariate analysis. Six proteins were observed to be upregulated in frosttolerant black wattle families. The proteins were identified and have been previously shown to contribute to the protection of cellular membranes, maintenance of photosynthetic processes and the prevention of protein misfolding and aggregation.

Diagnostic Utility of Multiplex Polymerase Chain Reaction in Patients of Clinically Suspected Pure Neuritic Leprosy by Identifying Mycobacterium leprae in Skin Biopsy Samples and Nasal Swabs.

Pure neuritic leprosy (PNL) often remains underdiagnosed due to the lack of simple, reliable diagnostic tools to detect Mycobacterium leprae. This study aimed to investigate the utility of multiplex polymerase chain reaction (MPCR) in easily accessible and less invasive biopsy sites, including skin biopsy samples and nasal swabs (NSs), to detect M. leprae. A total of 30 (N = 30) clinically suspected and untreated patients with PNL were recruited. Nasal swabs and skin biopsy samples from the innervation territory of an "enlarged nerve" were collected. DNA was extracted and subjected to MPCR (targeting leprae-specific repetitive element [RLEP], 16S rRNA, and SodA genes) and RLEP-PCR (individual gene PCR). The PCR products were analyzed by 3% agarose gel electrophoresis. In 30 patients with clinically suspected PNL, 60% (N = 18) of skin biopsy samples and 53% (N = 16) of NSs were found positive for M. leprae DNA by MPCR, whereas only 23.3% (N = 7) of skin biopsy samples and 10% (N = 3) of NSs were found positive by RLEP-PCR. MPCR demonstrated a greater positivity rate than did RLEP-PCR for detection of M. leprae. Serologic positivity for anti-natural disaccharide-octyl conjugated with bovine serum albumin (ND-O-BSA) antibodies was 80% (16/20), including 35% (7/20) of PNL patients for which the skin MPCR was negative. Both serologic positivity and skin MPCR positivity were observed in 65% of patients (N = 20). Multiplex polymerase chain reaction is a useful tool for detection for M. leprae in skin biopsy samples and NSs in clinically suspected cases of PNL, with the added advantages of being less invasive and technically easier than nerve biopsy.

In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Comparative Linkage of Novel Anti-Tumor Pd(II) Complex with Bio-Macromulecules: Fluorescence, UV-Vis, DFT, Molecular Docking and Molecular Dynamics Simulation Studies.

A novel mononuclear palladium complex, [Pd(dach)(SSA)], where dach and SSA are diaminocyclohexane and sulfosalicylic acid ligands, respectively, has been synthesized and identified utilizing analytical and spectral methods. DFT calculations, namely geometry optimization, MEP, HOMO-LUMO and NBO analysis, have been conducted at B3LYP level by aug-ccpVTZ-PP and 6-311G(d, p) basis sets. NBO and HOMO-LUMO analysis exhibited that the palladium compound is stable. MEP showed the potential sites of molecule for the interaction. By employing MTT assay, the cytotoxicity activity of the aforesaid compound was examined on K562 cell line, which revealed a proper activity compared to cisplatin. To ascertain the lipophilicity of the newly made compound, the partition coefficient measurement was accomplished, which follows the order of cisplatin < Pd(II) complex. Next, investigation of binding properties of the studied compound with DNA of calf thymus and BSA were done by spectroscopic (CD, fluorescence emission and electronic adsorption) and non-spectroscopic (viscosity measurements, DNA gel electrophoresis, molecular docking and molecular dynamics simulation) methods. The outcomes of CD and UV-Vis spectroscopy demonstrated that the title compound refolded the protein via increasing the alpha helix percentage. The data obtained from UV-Vis studies indicated the non-intercalative mutual action between Pd(II) complex with DNA. It also revealed that the Kapp magnitude of CT-DNA (7.43 × 104 M- 1) is higher than the BSA (5.17 × 103 M- 1), and L1/2 (midpoint of transition) of CT-DNA (5 µM) is lower than the BSA (5.7 µM), indicating that the complex has a greater binding affinity to CT-DNA than BSA. Fluorescence quenching mechanism of the two biomolecules by the metal complex is static and the calculated thermodynamic parameters (ΔS° < 0 and ΔH° < 0) suggested the hydrogen bonding and/ or van der Waals forces with DNA and BSA. Further, molecular docking indicated that the studied compound fits into the groove of DNA and the site I of BSA. The stability of metal compound-DNA/-BSA in the presence of H2O solvent and over the time were validated via molecular dynamics simulation.

Leptin-mediated suppression of lipoprotein lipase cleavage enhances lipid uptake and facilitates lymph node metastasis in gastric cancer.

Lymph node metastasis (LNM) is the primary mode of metastasis in gastric cancer (GC). However, the precise mechanisms underlying this process remain elusive. Tumor cells necessitate lipid metabolic reprogramming to facilitate metastasis, yet the role of lipoprotein lipase (LPL), a pivotal enzyme involved in exogenous lipid uptake, remains uncertain in tumor metastasis. Therefore, the aim of this study was to investigate the presence of lipid metabolic reprogramming during LNM of GC as well as the role of LPL in this process. Intracellular lipid levels were quantified using oil red O staining, BODIPY 493/503 staining, and flow cytometry. Lipidomics analysis was employed to identify alterations in intracellular lipid composition following LPL knockdown. Protein expression levels were assessed through immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays. The mouse popliteal LNM model was utilized to investigate differences in LNM. Immunoprecipitation and mass spectrometry were employed to examine protein associations. In vitro phosphorylation assays and Phos-tag sodium dodecyl-sulfate polyacrylamide gel electrophoresis assays were conducted to detect angiopoietin-like protein 4 (ANGPTL4) phosphorylation. We identified that an elevated intracellular lipid level represents a crucial characteristic of node-positive (N+) GC and further demonstrated that a high-fat diet can expedite LNM. LPL was found to be significantly overexpressed in N+ GC tissues and shown to facilitate LNM by mediating dietary lipid uptake within GC cells. Leptin, an obesity-related hormone, intercepted the effect exerted by ANGPTL4/Furin on LPL cleavage. Circulating leptin binding to the leptin receptor could induce the activation of inositol-requiring enzyme-1 (IRE1) kinase, leading to the phosphorylation of ANGPTL4 at the serine 30 residue and subsequently reducing its binding affinity with LPL. Moreover, our research revealed that LPL disrupted lipid homeostasis by elevating intracellular levels of arachidonic acid, which then triggered the cyclooxygenase-2/prostaglandin E2 (PGE2) pathway, thereby promoting tumor lymphangiogenesis. Leptin-induced phosphorylation of ANGPTL4 facilitates LPL-mediated lipid uptake and consequently stimulates the production of PGE2, ultimately facilitating LNM in GC.

Gas Cluster Ion Beam-Assisted Deposition for Fabricating Dry Multilayers Containing Enzyme and Substrate with On-Demand Release.

Gas cluster ion beam (GCIB)-assisted deposition is used to build multilayered protein-based structures. In this process, Ar3000-5000+ clusters bombard and sputter molecules from a reservoir (target) to a collector, an operation that can be sequentially repeated with multiple targets. The process occurs under a vacuum, making it adequate for further sample conservation in the dry state, since many proteins do not have long-term storage stability in the aqueous state. First of all, the stability in time and versatility in terms of molecule selection are demonstrated with the fabrication of peptide multilayers featuring a clear separation. Then, lysozyme and trypsin are used as protein models to show that the activity remaining on the collector after deposition is linearly proportional to the argon ion dose. The energy per atom (E/n) of the Ar clusters is a parameter that was also changed for lysozyme deposition, and its increase negatively affects activity. The intact detection of larger protein molecules by SDS-PAGE gel electrophoresis and a bioassay (trypsin at ≈25 kDa and glucose oxidase (GOx) at ≈80 kDa) is demonstrated. Finally, GOx and horseradish peroxidase, two proteins involved in the same enzymatic cascade, are successively deposited on β-d-glucose to build an on-demand release material in which the enzymes and the substrate (β-d-glucose) are combined in a dry trilayer, and the reaction occurs only upon reintroduction in aqueous medium.

Laccase/caffeic acid-catalyzed crosslinking coupled with galactomannan alters the conformational structure of ovalbumin and alleviates Th2-mediated allergic asthma

Ovalbumin (OVA) is the major allergenic protein that can induce T helper 2 (Th2)-allergic reactions, for which current treatment options are inadequate. In this study, we developed a polymerized hypoallergenic OVA product via laccase/caffeic acid (Lac/CA)-catalyzed crosslinking in conjunction with galactomannan (Man). The formation of high molecular weight crosslinked polymers and the IgG-binding were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The study indicated that Lac/CA-catalyzed crosslinking plus Man conjugation substantially altered secondary and tertiary structures of OVA along with the variation in surface hydrophobicity. Gastrointestinal digestion stability assay indicated that crosslinked OVA exhibited less resistance in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Mouse model study indicated that Lac-Man/OVA ameliorated eosinophilic airway inflammatory response and efficiently downregulated the expression of Th2-related cytokines (interleukin (IL)-4, IL-5, and IL-13), and upregulated IFN-γ and IL-10 expression. Stimulation of bone marrow-derived dendritic cells with Lac-Man/OVA suppressed the expression of phenotypic maturation markers (CD80 and CD86) and MHC class II molecules, and suppressed the expression levels of proinflammatory cytokines. The knowledge obtained in the present study offers an effective way to acquire a hypoallergenic OVA product that can have a therapeutic effect in alleviating OVA-induced allergic asthma.

Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Nested PCR optimization for detecting hepatitis E virus

The occurrence of Hepatitis E virus (HEV) infections is on the rise in developing countries, frequently linked to the consumption of undercooked meats and exposure to animal feces. Typically, HEV diagnosis relies on the immunodetection of anti-HEV antibodies and reverse transcriptase polymerase chain reaction. In this study, nested polymerase chain reaction (Nested PCR) for detecting HEV in domesticated pig and farmed wild boar samples was optimized to provide an alternative method for reliable and precise detection of HEV, particularly in animal samples. All samples were collected in Ho Chi Minh City, Vietnam. Total RNA was extracted from liver tissues of domestic pigs (n=48), rectal swabs samples (n=60), and feces samples (n=25) of farmed wild boars. Complementary DNA (cDNA) was synthesized using random hexamers. Nested PCR was performed under four different conditions: protocol 1–4, with variations in reaction components and concentrations. Two PCR programs, designated as A and B, were examined, featuring distinct cycling times and annealing temperatures for the outer and inner amplification stages. The resulting amplification products (306 bp) were visualized through gel electrophoresis. Protocol 1 and 2, when employed in conjunction with both program A and B, failed to identify any positive samples, including the positive control (0%). In contrast, protocol 3, in combination with program A, and protocol 4, paired with program B, successfully identified 28 positive results out of 133 tested samples (21%). It is noteworthy that protocol 4, when used with program B, yielded clearer and more specific bands. The study successfully optimized a nested-PCR protocol for detecting HEV in animal samples, comprising tissues, rectal swabs, and fecal samples.

UNVEILING THE GENETIC BASIS OF BACTERIOCIN PRODUCTION FROM Enterococcus faecium ATCC 19434

The prevalence of resistance to traditional antimicrobials is increasing rapidly, leading to a significant rise in annual deaths from antibiotic-resistant illnesses. In 2020, the global death toll from such diseases was approximately 500,000. If current trends continue, this figure could exceed 10 million by 2050, with associated economic damages surpassing 100 trillion USD. These alarming statistics highlight the urgent need for alternative antimicrobials. Bacteriocins are considered among the most promising options. Enterococcus faecium ATCC 19434 is known to produce a potent bacteriocin effective against Listeria monocytogenes and Staphylococcus aureus, though its specific bacteriocin and genetic properties have not been fully explored. This study aimed to identify the gene responsible for bacteriocin production in Enterococcus faecium ATCC 19434. DNA was extracted from the bacterium, and polymerase chain reaction (PCR) was conducted. The PCR products were analyzed through agarose gel electrophoresis, sequenced, and subjected to homology searches using the BLASTN and BLASTX tools from the National Center for Biotechnology Information (NCBI). Results revealed that Enterococcus faecium ATCC 19434 contains a gene encoding enterocin B. Additionally, it was found to harbor the gene for enterocin A immunity protein production. This discovery paves the way for future genetic modification of Enterococcus faecium ATCC 19434 to enhance enterocin B synthesis, facilitating its commercial application.

Determining the genetic diversity of some black cumin genotypes collected in different regions of Türkiye using RAPD markers

Black cumin is one of the important medicinal aromatic plants belonging to the Ranunculaceae family. It is mostly used in the Middle East and for some diseases, especially in the Iranian region. It is important to know the genetic resources of such important medicinal and aromatic plants. Characterization of genetic resources sheds light on both the conservation of genetic resources and the future breeding studies. In this study, a total of 8 black cumin plants were characterized with 17 RAPD primers. Presence (1) and absence (0) scoring of gel images was performed using the Agarose Gel Electrophoresis (AGE) method. In genetic characterization, phylogenetic dendrogram with Bayesian statistics and Principal Coordinate Analysis (PCoA) with Jaccard similarity index were performed. As a result of the findings, the Nigella damascena, one of the black cumin plant species, was 100% separated from the Nigella sativa species. Additionally, Nigella sativa species differed among Konya, Eskişehir and Çameli genotypes. It gave similar results to Bayesian statistics in PCoA. The analysis indicated that Konya, Eskişehir and Çameli genotypes of Nigella sativa species have a higher potential to be used in breeding studies compared to other genotypes.

Outbreak of Burkholderia cenocepacia in an intensive care unit of a tertiary care hospital identified by pulsed-field gel electrophoresis

Nosocomial outbreaks of Burkholderia cepacia complex, transmitted through contaminated medical surfaces or equipment have been reported. Pulsed-field Gel Electrophoresis (PFGE) is recognized as the “gold standard” for molecular subtyping, yet studies on clonal relationships in India are limited. PFGE was used to study the clonal relationships of 22 isolates of Burkholderia cenocepacia from 12 patients admitted to a critical care unit during 2 months (November and December 2021). PFGE revealed three different profiles with 15 isolates belonging to a single cluster suggesting a common source within the hospital, emphasizing the need for preventive measures to control B. cenocepacia transmission.

Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.

The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors. The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction. The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software. The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation. We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

Comprehensive Study of Thiazole‐Orange‐Based DNA Dyes

AbstractThe rapid advancement of biotechnology over the recent decades has amplified the importance of DNA detection and quantification assays. Many of these assays, such as gel electrophoresis, microscopy, flow cytometry, and the detection of amplification in quantitative polymerase chain reaction (qPCR), rely on the use of DNA‐binding fluorescent dyes. This article presents a comprehensive study of six Thiazole‐Orange‐based fluorescent DNA‐binding dyes: SYBR Safe, SYBR Green, Pico Green, SYTO‐16, SYTO‐9, and the benzothiazole‐based analogue (TOPhBu) of the latter. The selected DNA markers were synthesized at a 10‐milligram scale and characterised spectroscopically to quantify their fluorescence enhancement upon binding to double‐stranded DNA. The ability of the dyes to detect DNA at low concentrations was evaluated using two new metrics, absolute fluorescence enhancement (AFE) and relative fluorescence enhancement (RFE). Quantum chemical calculations shed new light on the mechanism of their fluorogenicity through modelling the excited state behaviour and DNA binding of the dyes. Their analytical performance was further tested in qPCR experiments. The experimental results of this work highlight some important differences in the sensitivity and qPCR efficiency of the studied DNA‐binding dyes which will facilitate the DNA marker selection for analytical purposes and the future development of novel DNA sensors.

Duplex PCR-lateral flow immunoassay for rapid and visual screening of Salmonella and Vibrio cholerae for food safety assurance and hygiene surveillance

Salmonella and Vibrio cholerae are the deadliest foodborne pathogens, requiring effective routine screening for food safety assurance. Hence, a duplex polymerase chain reaction integrated with a lateral flow immunoassay (PCR-LFIA) was developed for rapid detections of Salmonella and V. cholerae to overcome the laborious, time-consuming, complicated, and costly limitations of standard bacterial culture. The duplex PCR-LFIA could simultaneously detect Salmonella invA and V. cholerae lolB targets at lower limits of 0.1 and 1 ng, respectively. Without requiring culture enrichment, the duplex PCR-LFIA method enabled rapid and simple naked-eye identification of Salmonella and V. cholerae in spiked raw oysters and chicken meat at a limit of detection (LOD) of 106 CFU/mL. A LOD of 104 CFU/mL was achieved by visualizing the PCR amplicons by agarose gel electrophoresis (AGE). This novel duplex PCR-LFIA offers rapid and simple visual detections of pathogenic Salmonella and V. cholerae contaminations, potentially enhancing the capability to monitor and control foodborne illnesses, thereby ensuring food safety.

Antibacterial Properties of Peptide and Protein Fractions from Cornu aspersum Mucus.

The discovery and investigation of new natural compounds with antimicrobial activity are new potential strategies to reduce the spread of antimicrobial resistance. The presented study reveals, for the first time, the promising antibacterial potential of two fractions from Cornu aspersum mucus with an MW < 20 kDa and an MW > 20 kDa against five bacterial pathogens-Bacillus cereus 1085, Propionibacterium acnes 1897, Salmonella enterica 8691, Enterococcus faecalis 3915, and Enterococcus faecium 8754. Using de novo sequencing, 16 novel peptides with potential antibacterial activity were identified in a fraction with an MW < 20 kDa. Some bioactive compounds in a mucus fraction with an MW > 20 kDa were determined via a proteomic analysis on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bioinformatics. High homology with proteins and glycoproteins was found, with potential antibacterial activity in mucus proteins named aspernin, hemocyanins, H-lectins, and L-amino acid oxidase-like protein, as well as mucins (mucin-5AC, mucin-5B, mucin-2, and mucin-17). We hypothesize that the synergy between the bioactive components determined in the composition of the fraction > 20 kDa are responsible for the high antibacterial activity against the tested pathogens in concentrations between 32 and 128 µg/mL, which is comparable to vancomycin, but without cytotoxic effects on model eukaryotic cells of Saccharomyces cerevisiae. Additionally, a positive effect, by reducing the levels of intracellular oxidative damage and increasing antioxidant capacity, on S. cerevisiae cells was found for both mucus extract fractions of C. aspersum. These findings may serve as a basis for further studies to develop a new antibacterial agent preventing the development of antibiotic resistance.

Development of DNA Insertion-specific Markers Based on the Intergenic Region of Oplegnathus punctatus Cdkn1/srsf3 for Sex Identification.

Spotted knifejaw (Oplegnathus punctatus) is a marine economic fish with high food and ecological value, and its growth process has obvious male and female sexual dimorphism, with males growing significantly faster than females. However, the current sex identification technology is not yet mature, which will limit the growth rate of O. punctatus aquaculture and the efficiency of separate sex breeding, so the development of efficient sex molecular markers is imperative. This study identified a 926bp DNA insertion fragment in the cdkn1/srsf3 intergenic region of O. punctatus males through whole-genome scanning, comparative genomics, and structural variant analysis. A pair of primers was designed based on the insertion information of the Y chromosome intergenic region in male individuals. Agarose gel electrophoresis revealed the amplification of two DNA fragments, 1118bp and 192bp, in male O. punctatus individuals. The 926bp fragment was identified as the insertion in the intergenic region of cdkn1/srsf3 in males, while only a single 192bp DNA fragment was amplified in females. The biological sex of the individuals identified in this manner was consistent with their known phenotypic sex.In this study, we developed a method to detect DNA insertion variants in the intergenic region of O. punctatus. Additionally, we introduced a new DNA marker for the rapid identification of the sex of O. punctatus, which enhances detection efficiency. The text has important reference significance and application value in sex identification, all-male breeding, and lineage selection. It provides new insights into the regulation of variation in the intergenic region of cdkn1/srsf3 genes and the study of RNA shearing.