kDa Band Research Articles

Isolation of lectin from Musa acuminata for its antibiofilm potential against Methicillin-resistant Staphylococcus aureus and its synergistic effect with Enterococcus species.

Methicillin resistant Staphylococcus aureus (MRSA) is an emerging pathogen posing a considerable burden on the healthcare system due to its involvement in skin and soft tissue infections (SSTIs). Lectins are carbohydrate binding proteins found ubiquitously in animals, plants and microorganisms. Extraction and isolation of proteins from Musa acuminata were performed by using Affinity chromatography with Sephadex G 75 to determine antibiofilm activity against MRSA. Enterococcus strains obtained from dairy products, beans and vegetables were also screened for its potential to inhibit growth and biofilm formation of MRSA by using 96 well microtiter plates. Synergistic effect of cell free supernatant of Enterococcus with proteins from ripe banana were also tested. BanLec was successfully isolated and appeared as 15 KDa band after SDS-PAGE (15%) while multiple bands of unbound protein fractions were observed. The unbound fractions showed inhibition of planktonic cells and biofilm but BanLec exhibited no significant effect. The CFS of Enterococcus faecium (LCM002), Enterococcus lactis (LCM003) and Enterococcus durans (LCM004 and LCM005) displayed antagonistic effects against pathogen. The synergistic effect of CFS from E. lactis (LCM003) and unbound proteins showed inhibition of biofilm and pathogenic growth. This study demonstrates the use of Enterococcus species and plant proteins against pathogens and results suggested that it can inhibit the growth of resistant strains of Staphylococcus aureus and their synergistic effect has opened new ways to tackle emerging resistance. Furthermore, after assessment of Enterococcus as probiotics, this could be used in food industries as well as in treatment of severe skin infections.

Ligilactobacillus murinus Strains Isolated from Mice Intestinal Tract: Molecular Characterization and Antagonistic Activity against Food-Borne Pathogens.

Considering the objectives of "One Health" and the Sustainable development Goals "Good health and well-being" for the development of effective strategies to apply against bacterial resistance, food safety dangers, and zoonosis risks, this project explored the isolation and identification of Lactobacillus strains from the intestinal tract of recently weaned mice; as well as the assessment of antibacterial activity against clinical and zoonotic pathogens. For molecular identification, 16S rRNA gene-specific primers were used and, via BLAST-NCBI, 16 Ligilactobacillus murinus, one Ligilactobacillus animalis, and one Streptococcus salivarius strains were identified and registered in GenBank after the confirmation of their identity percentage and the phylogenetic analysis of the 16 Ligilactobacillus murinus strains and their association with Ligilactobacillus animalis. The 18 isolated strains showed antibacterial activity during agar diffusion tests against Listeria monocytogenes ATCC 15313, enteropathogenic Escherichia coli O103, and Campylobacter jejuni ATCC 49943. Electrophoretic and zymographic techniques confirmed the presence of bacteriolytic bands with a relative molecular mass of 107 kDa and another of 24 kDa in Ligilactobacillus murinus strains. UPLC-MS analysis allowed the identification of a 107 kDa lytic protein as an N-acetylmuramoyl-L-amidase involved in cytolysis and considered a bacteriolytic enzyme with antimicrobial activity. The 24 kDa band displayed similarity with a portion of protein with aminopeptidase function. It is expected that these findings will impact the search for new strains and their metabolites with antibacterial activity as an alternative strategy to inhibit pathogens associated with major health risks that help your solution.

A Calcium- and GTP-Dependent Transglutaminase in Leishmania infantum

While human and animal leishmaniasis affect several millions of people worldwide, L. infantum is the species responsible for visceral leishmaniasis in Europe, Middle East, and America. Antileishmanial drugs present issues associated with drug toxicity and increasing parasite resistance. Therefore, the study of this parasite with a focus on new potential drug targets is extremely useful. Accordingly, we purified and characterized a transglutaminase (TGase) from L. infantum promastigotes. While Tgases are known to be involved in cell death and autophagy, it appears that these functions are very important for parasites' virulence. For the first time, we showed a Ca2+- and GTP-dependent TGase in Leishmania corresponding to a 54 kDa protein, which was purified by two chromatographic steps: DEAE-Sepharose and Heparin-Sepharose. Using polyclonal antibodies against a 50-amino-acid conserved region of the catalytic core of human TGase 2, we revealed two other bands of 66 and 75 kDa. The 54 kDa band appears to be different from the previously reported TGase, which was shown to be Ca2+- independent. Future research should address the identification of the purified enzyme sequence and, subsequently, its cloning to more comprehensively investigate its pathophysiological function and possible differences from mammal enzymes.

Clinical Characteristics and Sensitization Profile of Patients Allergic to Cow Epithelium

Introduction: Cow epithelium allergy (CEA) has been described in workers highly exposed to cattle, such as farmers and veterinarians, being a health problem in this population since it is their main livelihood. This study aimed to characterize the main clinical manifestations and define the sensitization profile of the cow epithelium-allergic population treated in our health area. Methods: This is a retrospective study including a total of 34 patients with a clinical diagnosis of CEA, confirmed by skin tests, bovine epithelium-specific IgE levels and allergen-specific conjunctival challenge test in some cases. They were distributed by age, sex, profession, clinical symptoms, specific IgE levels to other mammalian epithelia, pollens, mites, and foods. Immunoblotting was performed with extracts from cow dander, cow body fluids (urine and saliva), bull urine, and 17 sera from immunotherapy-untreated CEA patients. Results: The mean age of the patients was 44 years, with a higher incidence in cattle farmers. Rhinoconjunctivitis occurred in 100% of cases, with 35% having monosensitization to cow epithelium. Sera from most patients detected a 20-kDa IgE-binding band in cow dander, cow saliva, cow urine, and bull urine, corresponding to the major allergen Bos d 2 (bovine lipocalin). In 70% of the patients, a 25-kDa band was detected in cow and bull urine extracts, whose identification by mass spectrometry and investigation with protein databases led to the identification of a Bos taurus lipocalin (UniProt protein ID: A0A3Q1LGU7_BOVIN). Conclusion: CEA should be considered in patients exposed to cattle and as a cause of occupational disease. The IgE immunodetection revealed sensitization to a protein present in cow and bull urine (odorant-binding protein) not previously described.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Trypsin/α-Amylase Inhibitors from Capsicum chinense Seeds: Characterization and Antifungal Activity against Fungi of Agronomic Importance.

Protease inhibitors (PIs) have attracted attention due to their important roles in plant defense. The objective of this work was to characterize and evaluate the antimicrobial activity of the peptides of a family of serine PIs from Capsicum chinense Jacq. seeds. Initially, PIs were extracted from the seeds and subjected to purification by chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Subsequently, the PEF3 was subjected to trypsin inhibition assays, α-amylase activity assays, antimicrobial activity assays on phytopathogenic fungi, and assays to determine the likely mechanisms of action. The PEF3 was composed of three protein bands with molecular masses ranging between 6 and 14 kDa. The amino acid residues of the ~6 kDa band showed high similarity with serine PIs. PEF3 inhibited the activity of the enzymes trypsin, human salivary α-amylase, and Tenebrio molitor larval α-amylase and inhibited the growth of phytopathogenic fungi, showing 83.7% loss of viability in Fusarium oxysporum. PEF3 induced reactive oxygen species in Colletotrichum lindemuthianum and F. oxysporum to dissipate their mitochondrial membrane potential and activated caspases in C. lindemuthianum. Our results reinforce the importance of PIs in plant defense mechanisms against phytopathogenic fungi as well as in their biotechnological applications for the control of plant pathogens.

Bread Biopreservation through the Addition of Lactic Acid Bacteria in Sourdough.

Nowadays, the consumer seeks to replace synthetic preservatives with biopreservation methods, such as sourdough in bread. Lactic acid bacteria (LAB) are used as starter cultures in many food products. In this work, commercial yeast bread and sourdough breads were prepared as controls, as well as sourdough breads with L. plantarum 5L1 lyophilized. The impact of L. plantarum 5L1 on the properties of bread was studied. Antifungal compounds and the impact on the protein fraction by the different treatments in doughs and breads were also analyzed. In addition, the biopreservation capacity of the treatments in breads contaminated with fungi was studied and the mycotoxin content was analyzed. The results showed significant differences with respect to the controls in the properties of the bread and a higher total phenolic and lactic acid content in breads with higher amounts of L. plantarum 5L1. In addition, there was a higher content of alcohol and esters. Furthermore, adding this starter culture produced hydrolysis of the 50 kDa band proteins. Finally, the higher concentration of L. plantarum 5L1 delayed fungal growth and reduced the content of AFB1 and AFB2 compared to the control.

Cloning and Expression of 3 Chymotrypsin-like Protease of SARS-CoV-2 in E. coli using pET28a Vector

The 3 chymotrypsin-like protease (3CLpro) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of the primary targets for the development of antiviral drug therapies as it plays a critical role in viral replication. In this study, the gene encoding for SARS-CoV-2 3CLpro (918 bp) was amplified from the cDNA of the virus by polymerase chain reaction (PCR) and cloned into the pGEM-T vector. 3CLpro was then inserted into the expression vector pET28a at the end of the 6 histidine residue encoding sequence to form a fusion protein (6xHis-3CLpro). The 6xHis-3Clpro construct was successfully expressed in E. coli. The expression of 3CLpro was highest when E. coli BL21(DE3) RIL harboring pET28a-3CLpro vector was cultured in LB medium at 20 oC, induced by 1.0 mM Isopropyl thiogalactopyranosie (IPTG) when cell density measured by optical density at 600 nm (OD600) reached 0.7-0.8 and harvested after 24 hours of induction. The recombinant 3CLpro was purified by Ni-sepharose affinity chromatography under denaturation conditions. The purified 3CLpro showed to have a 41 kDa band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using polyclonal anti-3CLpro antibody and hydrolyzed a fluorescent specific substrate of 3CLpro after renaturation.

Pru p 10: the polygalacturonase described as a new peach tree pollen allergen

Peach tree pollen (PTP) is responsible for the induction of respiratory and ocular symptoms in a population exposed to peach cultivars, with great relevance in occupational allergy. In its allergenic profile, several high molecular mass proteins with significant IgE recognition were described. In order to deepen into these allergens, the main objective was the purification, identification and verification of the allergenic capacity of the 46 kDa band protein.

Anti-LGI4 Antibody Is a Novel Juxtaparanodal Autoantibody for Chronic Inflammatory Demyelinating Polyneuropathy.

The objective of this study was to discover novel nodal autoantibodies in chronic inflammatory demyelinating polyneuropathy (CIDP). We screened for autoantibodies that bind to mouse sciatic nerves and dorsal root ganglia (DRG) using indirect immunofluorescence (IFA) assays with sera from 113 patients with CIDP seronegative for anti-neurofascin 155 and anticontactin-1 antibodies and 127 controls. Western blotting, IFA assays using HEK293T cells transfected with relevant antigen expression plasmids, and cell-based RNA interference assays were used to identify target antigens. Krox20 and Periaxin expression, both of which independently control peripheral nerve myelination, was assessed by quantitative real-time PCR after application of patient and control sera to Schwann cells. Sera from 4 patients with CIDP, but not control sera, selectively bound to the nodal regions of sciatic nerves and DRG satellite glia (p = 0.048). The main immunoglobulin G (IgG) subtype was IgG4. IgG from these 4 patients stained a 60-kDa band on Western blots of mouse DRG and sciatic nerve lysates. These features indicated leucine-rich repeat LGI family member 4 (LGI4) as a candidate antigen. A commercial anti-LGI4 antibody and IgG from all 4 seropositive patients with CIDP showed the same immunostaining patterns of DRG and cultured rat Schwann cells and bound to the 60-kDa protein in Western blots of LGI4 overexpression lysates. IgG from 3 seropositive patients, but none from controls, bound to cells cotransfected with plasmids containing LGI4 and a disintegrin and metalloprotease domain-containing protein 22 (ADAM22), an LGI4 receptor. In cultured rat Schwann and human melanoma cells constitutively expressing LGI4, LGI4 siRNA effectively downregulated LGI4 and reduced patients' IgG binding compared with scrambled siRNA. Application of serum from a positive patient to Schwann cells expressing ADAM22 significantly reduced the expression of Krox20, but not Periaxin. Anti-LGI4 antibody-positive patients had a relatively old age at onset (mean age 58 years), motor weakness, deep and superficial sensory impairment with Romberg sign, and extremely high levels of CSF protein. Three patients showed subacute CIDP onset resembling Guillain-Barré syndrome. IgG4 anti-LGI4 antibodies are found in some elderly patients with CIDP who present subacute sensory impairment and motor weakness and are worth measuring, particularly in patients with symptoms resembling Guillain-Barré syndrome.

Characterization of sclerostin's response within white adipose tissue to an obesogenic diet at rest and in response to acute exercise in male mice.

Introduction: It is well established that sclerostin antagonizes the anabolic Wnt signalling pathway in bone, however, its physiological role in other tissues remains less clear. This study examined the effect of a high-fat diet (HFD) on sclerostin content and downstream markers of the Wnt signaling pathway (GSK3β and β-catenin) within subcutaneous inguinal white adipose tissue (iWAT), and visceral epididymal WAT (eWAT) depots at rest and in response to acute aerobic exercise. Methods: Male C57BL/6 mice (n = 40, 18weeks of age) underwent 10 weeks of either a low-fat diet (LFD) or HFD. Within each diet group, mice were assigned to either remain sedentary (SED) or perform 2h of endurance treadmill exercise at 15mmin-1 with 5° incline (EX), creating four groups: LFD + SED (N = 10), LFD + EX (N = 10), HFD + SED (N = 10), and HFD + EX (N = 10). Serum and WAT depots were collected 2h post-exercise. Results: Serum sclerostin showed a diet-by-exercise interaction, reflecting HFD + EX mice having higher concentration than HFD + SED (+31%, p = 0.03), and LFD mice being unresponsive to exercise. iWAT sclerostin content decreased post-exercise in both 28kDa (-31%, p = 0.04) and 30kDa bands (-36%, main effect for exercise, p = 0.02). iWAT β-catenin (+44%, p = 0.03) and GSK3β content were higher in HFD mice compared to LFD (+128%, main effect for diet, p = 0.005). Monomeric sclerostin content was abolished in eWAT of HFD mice (-96%, main effect for diet, p < 0.0001), was only detectable as a 30kDa band in LFD mice and was unresponsive to exercise. β-catenin and GSK3β were both unresponsive to diet and exercise within eWAT. Conclusion: These results characterized sclerostin's content to WAT depots in response to acute exercise, which appears to be specific to a reduction in iWAT and identified a differential regulation of sclerostin's form/post-translational modifications depending on diet and WAT depot.

Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29cells with the chimeric protein was significantly higher than the stimulated Ht29cells with CNF-1 alone and un-stimulated Ht29cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients.

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

In Vitro Activity, Stability and Molecular Characterization of Eight Potent Bacteriophages Infecting Carbapenem-Resistant Klebsiella pneumoniae.

Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment, including bacteriophage therapy. Bacteriophages are considered very safe and effective in treating bacterial infections. In this study, we characterize eight lytic bacteriophages that were previously isolated by our team against carbapenem-resistant Klebsiella pneumoniae. The one-step-growth curves, stability and lytic ability of eight bacteriophages were characterized. Restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) typing analysis and protein profiling were used to characterize the microbes at the molecular level. Phylogenetic trees of four important proteins were constructed for the two selected bacteriophages. All eight bacteriophages showed high efficiency for reducing bacterial concentration with high stability under different physical and chemical conditions. We found four major protein bands out of at least ten 15-190 KDa bands that were clearly separated by SDS-PAGE, which were assumed to be the major head and tail proteins. The genomes were found to be dsDNA, with sizes of approximately 36-87 Kb. All bacteriophages reduced the optical density of the planktonic K. pneumoniae abruptly, indicating great potential to reduce K. pneumoniae infection. In this study, we have found that tail fiber protein can further distinguished closely related bacteriophages. The characterised bacteriophages showed promising potential as candidates against carbapenem-resistant Klebsiella pneumoniae via bacteriophage therapy.

Short-Term Exposure of Dactylis glomerata Pollen to Atmospheric Gaseous Pollutants Is Related to an Increase in IgE Binding in Patients with Grass Pollen Allergies.

The concentrations of nitrogen dioxide (NO2) and tropospheric ozone (O3) in urban and industrial site atmospheres are considered key factors associated with pollen-related respiratory allergies. This work studies the effects of NO2 and O3 on the protein expression profile and IgE binding in patients with grass allergies to Dactylis glomerata pollen extracts. Pollens were collected during the flowering season and were exposed to NO2 and O3 in a controlled environmental chamber. The amount of soluble protein was examined using the Bradford method, and the protein expression profile and antigenic properties were analysed using the immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Our results showed apparent inter-sera differences concerning the number and intensity of IgE reactivity, with the most prominent at bands of 55 kDa, 35, 33, and 13 kDa. In the 13 kDa band, both gases tend to induce an increase in IgE binding, the band at 33 kDa showed a tendency towards a reduction, particularly pollen exposed to O3. Reactive bands at 55 and 35 kDa presented an increase in the IgE binding pattern for all the patient sera samples exposed to NO2, but the samples exposed to O3 showed an increase in some sera and in others a decrease. Regarding the ELISA results, out of the 21 tested samples, only 9 showed a statistically significant increase in total IgE reactivity after pollen exposure to the pollutants. Our study revealed that although airborne pollen allergens might be affected by air pollution, the possible impacts on allergy symptoms might vary depending on the type of pollutant and the patient's sensitisation profile.

EFFECT OF Anti-miRNA 144 AND Anti-miRNA 150 ON THE EXPRESSION OF α GLOBIN CHAINS IN PBMC

The excess of unbound α globin chains are the basic pathophysiology of the cause of clinical symptoms in major β thalassemia. Recently there are many alternative therapies by increasing γ globin chains to reduced the effects of unbound α globin chains. Alternative therapies by decreasing α globin chains have not been much noticed. α globin expression involves complex regulatory involving transcription factors and miRNAs. It involves GATA-1, KLFD and MYB transcription factors . It also involves miRNA-144 and miRNA-150. The role of miRNA-144 and miRNA-150 to reduce the α globin chains expression in major β thalassemia patients is an alternative therapy. miRNA-144 and miRNA150 activity need to be known by using anti-miRNA 144 and anti- miRNA 150. Analyzed the effect of anti-miRNA 144 and anti-miRNA 150 on the expression of α globin chains. This study was an experimental study using PBMC of a major β thalassemia patients. PBMC divided into four groups that were not transfected, transfected by anti-miRNA 144, transfected by anti-miRNA 150 and transfected by anti-miRNA 144 and anti-miRNA 150. qPCR examinations to find out expression of miRNA-144 and miRNA-150. Western blot examination to find out the expression of α globin chains. There was significantly lower miRNA-144 expression in PBMC of major β thalassemia patients who had been transfected by anti-miRNA 144 than those not transfected. In line with the result of miRNA-144 expression which was 0.17 times lower than the control group. There was significantly lower miRNA-150 expression in PBMC of major β thalassemia patients who had been transfected by anti-miRNA 150 than those not transfected. In line with the result of miRNA-150 expression which was 0.30 times lower than the control group. There was significantly lower α globin chains expression in PBMC thalassemia patients who had been transfected by anti-miRNA 150 than those not transfected. There was significantly lower α globin chains expression in PBMC thalassemia patients who had been transfected by anti-miRNA 150 and anti-miRNA 144 than those not transfected. This is evidenced by the decreased in the area 10-25 KDa band. Based on this study, the administration of anti-miRNA 150 or anti-miRNA 144 and anti-miRNA 150 is capable of decreasing the expression α globin chains in PBMC of major β thalassemia patients.

Bona fide atypical scrapie faithfully reproduced for the first time in a rodent model

Atypical Scrapie, which is not linked to epidemics, is assumed to be an idiopathic spontaneous prion disease in small ruminants. Therefore, its occurrence is unlikely to be controlled through selective breeding or other strategies as it is done for classical scrapie outbreaks. Its spontaneous nature and its sporadic incidence worldwide is reminiscent of the incidence of idiopathic spontaneous prion diseases in humans, which account for more than 85% of the cases in humans. Hence, developing animal models that consistently reproduce this phenomenon of spontaneous PrP misfolding, is of importance to study the pathobiology of idiopathic spontaneous prion disorders. Transgenic mice overexpressing sheep PrPC with I112 polymorphism (TgShI112, 1–2 × PrP levels compared to sheep brain) manifest clinical signs of a spongiform encephalopathy spontaneously as early as 380 days of age. The brains of these animals show the neuropathological hallmarks of prion disease and biochemical analyses of the misfolded prion protein show a ladder-like PrPres pattern with a predominant 7–10 kDa band. Brain homogenates from spontaneously diseased transgenic mice were inoculated in several models to assess their transmissibility and characterize the prion strain generated: TgShI112 (ovine I112 ARQ PrPC), Tg338 (ovine VRQ PrPC), Tg501 (ovine ARQ PrPC), Tg340 (human M129 PrPC), Tg361 (human V129 PrPC), TgVole (bank vole I109 PrPC), bank vole (I109I PrPC), and sheep (AHQ/ARR and AHQ/AHQ churra-tensina breeds). Our analysis of the results of these bioassays concludes that the strain generated in this model is indistinguishable to that causing atypical scrapie (Nor98). Thus, we present the first faithful model for a bona fide, transmissible, ovine, atypical scrapie prion disease.

Use of the Western blot technique to identify the immunogenic proteins of Borrelia burgdorferi for developing a Lyme disease vaccine

BackgroundLyme disease is a serious infectious disease having a restricted worldwide distribution for which there is no vaccine available for human use. ObjectiveThis study was designed to determine common reactive antigens involved in Borrelia burgdorferi (Bb) infection that are recognized in mammalian sera that may be useful for vaccine development. MethodsBlood samples were collected from patients with documented Lyme disease, and from rabbits and mice experimentally infected with either tick-transmitted or culture-grown Borrelia burgdorferi. All samples were then processed for sera. For performing the Western blots, sonicated Bb organisms (whole cell lysates) and protein ladders were separated by protein gel electrophoresis. Immune reactivities of the electrophoresed proteins with the serum samples were then probed with anti-HRP IgG reagent. ResultsRabbit, mouse and human sera consistently reacted with the 41 kDa band of Bb which corresponded to the flagellin protein – the major protein component of this organism’s periplasmic flagella, also known as axial filaments or fibrils. Various other Bb antigens of wide molecular weight ranges were also recognized by rabbit and human sera, and less frequently with mouse sera. ConclusionThe strong immune response to the 41 kDa flagellin protein by the different mammalian species suggests the utility of a possible vaccine targeting this protein, although other proteins may also be appropriate, for preventing Lyme disease following a bite from an infected tick.

Characterization of proteases from Irpex lacteus grown on minimally denatured soybean meal.

Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. The I.lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean β-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both β-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I.lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I.lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). Enzymatic hydrolysis of soybean proteins by the I.lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Human FSH Glycoform α-Subunit Asparagine52 Glycans: Major Glycan Structural Consistency, Minor Glycan Variation in Abundance.

Follicle-stimulating hormone (FSH), an α/β heterodimeric glycoprotein hormone, consists of functionally significant variants resulting from the presence or absence of either one of two FSHβ subunit N-glycans. The two most abundant variants are fully-glycosylated FSH24 (based on 24 kDa FSHβ band in Western blots) and hypo-glycosylated FSH21 (21 kDa band, lacks βAsn24 glycans). Due to its ability to bind more rapidly to the FSH receptor and occupy more FSH binding sites than FSH24, hypo-glycosylated FSH21 exhibits greater biological activity. Endoglycosidase F1-deglycosylated FSH bound to the complete extracellular domain of the FSH receptor crystallized as a trimeric complex. It was noted that a single biantennary glycan attached to FSHα Asn52 might preemptively fill the central pocket in this complex and prevent the other two FSH ligands from binding the remaining ligand-binding sites. As the most active FSH21 preparations possessed more rapidly migrating α-subunit bands in Western blots, we hypothesized that Asn52 glycans in these preparations were small enough to enable greater FSH21 receptor occupancy in the putative FSHR trimer model. Highly purified hFSH oligosaccharides derived from each FSH subunit, were characterized by electrospray ionization-ion mobility-collision-induced dissociation (ESI-IM-CID) mass spectrometry. FSHβ glycans typically possessed core-linked fucose and were roughly one third bi-antennary, one third tri-antennary and one third tetra-antennary. FSHα oligosaccharides largely lacked core fucose and were bi- or tri-antennary. Those αAsn52 glycans exhibiting tetra-antennary glycan m/z values were found to be tri-antennary, with lactosamine repeats accounting for the additional mass. Selective αAsn52 deglycosylation of representative pituitary hFSH glycoform Superdex 75 gel filtration fractions followed by ESI-IM-CID mass spectrometry revealed tri-antennary glycans predominated even in the lowest molecular weight FSH glycoforms. Accordingly, the differences in binding capacity of the same receptor preparation to different FSH glycoforms are likely the organization of the FSH receptor in cell membranes, rather than the αAsn52 oligosaccharide.