Search Results (95)

This study aimed to determine the prevalence of antibodies against equine brucellosis in three distinct equid groups (farm animal, urban carthorse, and sport horse) in Western Pará, Brazil, and to identify associated risk factors. A cross-sectional study, involving 75 farms across 14 municipalities, sampled 1069 equids composed of urban carthorses and sport horses. Serum samples were tested for antibodies against Brucella spp. using the buffered acidified plate antigen (BAPA) test for screening, followed by the serum agglutination in tubes with 2-mercaptoethanol (2-ME) for confirmation. Univariate and multivariate analyses assessed associations between brucellosis and potential risk factors. Out of the 1059 equids sampled, 4.05% (95% CI: 3–5.5%) tested positive in the BAPA test. Of the 44 BAPA-positive samples, 11 were confirmed positive by 2-ME, 15 were inconclusive, and 18 were negative, yielding an animal-level prevalence of 1.03% (95% CI: 0.5–1.8%) for equine brucellosis in western Pará. Prevalence was higher in the Southwest Pará Mesoregion (p = 0.047) compared to the Lower Amazon Mesoregion. No significant associations were found with animal type, reproductive problems, animal sex, species, breed, and age group. Out of the 75 farms, 8 (10.66%) had at least one seropositive equid. Confirmed cases were identified in five municipalities (Brasil Novo, Uruará, Altamira, Rurópolis, and Itaituba). This first report of equine brucellosis in Western Pará underscores the need for enhanced surveillance and control measures to mitigate its zoonotic risk to humans and transmission to other animals. Full article

This study aims to develop a robust and reproducible method for fabricating efficient ultrathin antifouling coatings on gold surfaces by leveraging hydroxylation-based surface modifications. An ultrathin antifouling coating of a monoethylene glycol silane derivative, known to reduce fouling by at least 90% on flat hydroxylated surfaces, was successfully replicated on flat gold (reducing fouling by ~75%) by hydroxylating its surface with β-mercaptoethanol. This tandem coating contains the monoethylene glycol silane layer on top of the β-mercaptoethanol on the gold. Characterization was performed using contact angle goniometry, atomic force microscopy, x-ray photoelectron spectroscopy, and antifouling measurements. The results from these techniques, consistent with the literature, confirmed the successful and reproducible application of the tandem coating. Through heterogeneities, including defects and incomplete coverage, the AFM data revealed distinct visible layers of the tandem coating. The direct application of monoethylene glycol silane onto gold resulted in superior antifouling performance (88% reduction), demonstrating that direct silylation exploits pre-existing oxygen-containing species on the gold surface for a more effective antifouling layer. These findings offer a scalable approach for engineering antifouling coatings on gold substrates, with potential applications in biosensing and implantable device antifouling technologies. Full article

Designed hen eggs enriched in DHA and EPA are an alternative source of essential phospholipids. This study assessed the effects of ovophospholipids on lymphocyte subsets in non-immunized mice and on the humoral immune response in sheep red blood cells (SRBC)-immunized mice. Ovophospholipids were administered orally for 14 days (once a day) at doses of 100, 10, and 1 mg/kg. Ovophospholipids increased the total lymphocyte count of in the lymphoid organs. At 10 and 1 mg/kg, ovophospholipids increased the subsets of CD4⁻CD8⁻ and CD4⁺CD8⁺ thymocytes but decreased the percentage of CD4⁺ and CD8⁺ thymocytes. A stimulating effect on splenocytes was particularly evident 24 h after administration of the 10 and 1 mg/kg doses. Ovophospholipids also elevated the absolute counts of CD3⁺ and CD19⁺ splenocytes. An increase in the absolute count of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ lymphocytes of the mesenteric lymph nodes was observed 24 h after administration of the lowest dose. The increase in the percentage and absolute count of CD19⁺ cells and in the absolute count of CD3⁺ cells was still observed after 72 h. At all doses, ovophospholipids elevated the number of plaque-forming cells on day 4 and increased 2-mercaptoethanol-resistant antibody titer on day 7 after priming. In conclusion, ovophospholipids can modulate the immune response in mice. Full article

To address the growing consumer demands for improved fish meat quality, desirable morphological traits, and sustainable production practices, researchers have intensified efforts in the selective breeding and genetic improvement of carp (Cyprinus carpio) varieties. However, traditional breeding methods are often time-consuming and inefficient, which poses challenges to the sustainable development of the carp aquaculture industry. The establishment of germ stem cell lines offers a crucial tool for the study of germ cells, genetic improvement, and species conservation. In this study, we successfully established a spermatogonial stem cell line (YRSSCs) from Yellow River carp (Cyprinus carpio haematopterus) that can be cultured in vitro for the long term. We optimized the culture conditions to maintain their self-renewal and differentiation capabilities. The results demonstrated that YRSSCs have a diploid karyotype and can stably proliferate for over a year in L-15 medium supplemented with 5 mmol/L HEPES, 50 μmol/L β-mercaptoethanol, 15% FBS, 2 ng/mL bFGF, 2 ng/mL LIF, 1% carp serum, 800 IU/mL penicillin, 0.8 mg/mL streptomycin, 2 μg/mL amphotericin B, 1% zebrafish embryo extract, and 1% glutamine at 30 °C in the absence of CO₂. The cells exhibited a typical germ stem cell gene expression profile, with strong expression of the vasa, plzf-a, and Oct4-a genes. Additionally, this study found that YRSSCs possess the ability to differentiate in vitro and functionally colonize in vivo within recipient bodies. This research explored the establishment of YRSSCs and their differentiation potential both in vitro and in vivo, providing a novel strategy for the genetic improvement of aquaculture fish species through germ stem cell-based gene editing and transplantation technologies. Full article

Endogenous α-amylase activity is crucial for determining the end-use value of glutinous rice flour (GRF), and controlling it is a key goal in the milling process. Although the structure and properties of starch and protein in GRF have been extensively studied, there is little information on endogenous α-amylase in GRF. In this study, endogenous α-amylase isolated from GRF was purified and characterized. It was found to have a molecular weight of about 32 kDa, with the highest specific activity at 60 °C and a pH of 6.0. The enzyme is stable below 50 °C and in the pH range of 4.0–7.0. Its activity is Ca²⁺-independent but inhibited by Cu²⁺, Zn²⁺, Mg²⁺, Mn²⁺, and Ba²⁺. Its activity is also reduced by β-mercaptoethanol. The enzyme hydrolyzes amylopectin most efficiently. Circular dichroism spectroscopy showed that the enzyme contains 7.9% α-helix, 35.4% β-folding, 21.1% β-turning, and 35.9% random coils, with a T_m value of 63.68 °C. These results suggest that temperature control may be the best strategy for reducing amylase activity in dry-milled GRF, providing a new approach for the development of GRF dry-milling techniques. Full article

Keratin-made biomaterials, including feathers, are considered a protein-rich bioresource due to their intrinsic properties, including biocompatibility, biodegradability, mechanical resistance, and biological abundance. Beta-keratin exists as an insoluble stringy protein due to the high presence of disulfide cross-links, and as a result, it is mechanically stable and resistant to enzymatic digestion. Because of this, it is not easily decomposed, and this has made the application of feathers difficult. In this study, after dissolving feathers in NaOH, sodium sulfide, and 2-Mercaptoethanol (2-ME), the relative molecular mass of beta-keratin was calculated. Thin-layer chromatography was also used to display proteins with lower molecular weights. The antioxidant activities of the samples were evaluated by Fe-chelating and free radical scavenging tests with 2,2-diphenyl-1-picrylhydrazyl (DPPH). To investigate the effect of blocking thiol groups on the antioxidant activity of dissolved keratin, iodoacetamide and H₂O₂ were used. According to the three methods—(A) sodium hydroxide, (B) sodium sulfide, and (C) urea and 2-ME—used to extract and dissolve the feathers, method C caused the least change in the chemical structure of keratin molecules. Method A destroyed the primary structure of keratin and drastically reduced its molecular mass, but method B caused a drastic increase in the molecular mass from 9.6 kDa to higher masses, due to intermolecular bonds. For the keratin molecules dissolved by method C, the Fe-chelating activity was 93.18% and free radical scavenging was 77.45%. Blocking the thiol group with iodoacetamide initially reduced the free radical scavenging activity with DPPH by 42%, but blocking it with H₂O₂ did not affect this activity. Also, blocking of the thiol group did not initially affect Fe-chelating activity and free radical scavenging activity. After a kinetic study of the activities, an interesting observation was that both blocking agents had negative effects on radical scavenging activity, but had positive effects on Fe-chelating activity. This indicates the complexity of the role of disulfide bonds in keratin’s antioxidant behavior types. According to the observed antioxidant activities, it can be expected that beta-keratin extracted from chicken feathers is a suitable candidate for application in industrial, pharmaceutical, and health applications. Full article

In this study, novel triblock copolymers, including poly(N-isopropylacrylamide)-block-poly(ε-caprolactone)-block-poly(N-isopropylacrylamide) (PNIPAM-b-PCL-b-PNIPAM), poly(N-vinyl-pyrrolidone)-block-poly(ε-caprolactone)-block-poly(N-vinyl-pyrrolidone) (PNVP-b-PCL-b-PNVP), poly(N-isopropylacrylamide-co-N,N-dimethylaminoethyl methacrylate)-block-poly(ε-caprolactone)-block-poly(N-isopropylacrylamide-co-N,N-dimethylaminoethyl methacrylate) (P(DMAEMA-co-NIPAM)-b-PCL-b-P(NIPAM-co-DMAEMA)), and poly(N,N-dimethylacrylamide)-block-poly(ε-caprolactone)-block-poly(N,N-dimethylacrylamide) (PDMA-b-PCL-b-PDMA), were synthesized via a combination of ring-opening polymerization (ROP) and reversible addition–fragmentation chain transfer (RAFT) polymerization. The synthesis was performed using novel bifunctional PCL-based RAFT macro chain transfer agents (macroCTAs; MXTPCL-X1 and MXTPCL-X2) with a m-xylene-bis(2-mercaptoethyloxy) core. Initially, m-xylene-bis(1-hydroxy-3-thia-propane) (MXTOH), which has not previously been used in lactone polymerization, was synthesized via the reaction of α,α′-dibromo-m-xylene with 2-mercaptoethanol in the presence of sodium in ethanol. Subsequently, Sn(Oct)₂-catalyzed ROP of ε-caprolactone (ε-CL) using MXTOH as an initiator yielded PCL-diol (MXTPCLOH). The resulting PCL-diol underwent further functionalization through esterification and substitution reactions, leading to the formation of PCL-based RAFT macroCTAs. Triblock copolymers were synthesized using these macroCTAs with AIBN as an initiator. The synthesized products, along with their intermediates, were characterized using FTIR and ¹H NMR spectroscopy. The number average molecular weight (M_n) and polydispersity index (Ð) of PCL-based macroCTAs were determined by using GPC analysis. The sensor capabilities of the synthesized novel triblock copolymers were investigated on the determination of syringic acid and it was determined that the most sensitive polymer was PNVP-b-PCL-b-PNVP (MXTP2). The working range was between 1.5 µg/mL and 15 µg/mL and the limit of detection (LOD) was found to be 0.44 µg/mL using DPV on MXTP2 polymer sensor. Full article

As a cost-effective sorbent, modified biochar has received increasing attention for the removal of heavy metal contaminants. Among several chemical modification methods, introducing thiol functional groups onto the surface of biochar has been identified as an effective enhancement approach for the heavy metal sorption and removal capacity of this porous adsorbent material. In general, chemical impregnation is a widely used method to graft thiol groups onto the surface of carbon-based materials. However, limited comparative data are available on the efficacy of the present biochar thiolation methods. In this study, the biochar of nine different organic sources was modified by two frequently used agents with distinct thiolation mechanisms: 3-Mercaptopropyltrimethoxysilane (3-MPTS) and β-mercaptoethanol. In addition to chemical impregnation, the ball milling method, a simple and environmentally friendly alternative thiolation method, was also evaluated. A comprehensive structural characterization of the biochar samples was completed before and after thiolation. A higher concentration of sulfur on the surface of the biochar was achieved through thiolation with β-mercaptoethanol, in which the thiolation mechanism is performed through an esterification reaction with the carboxylic acid functional groups of the activated biochar. Chemical impregnation was found to be a more effective thiolating method than ball milling using the same thiolating agent. Full article

This study demonstrates, for the first time, the formation of a hemiester of carbonic acid on self-assembled monolayers using voltammetric techniques and redox probes. A gold electrode (GE) was modified with 2-mercaptoethanol (ME) through self-assembly. With this modified electrode (GE-ME), a well-defined peak was observed by differential pulse voltammetry (DPV) for the negatively charged redox probe, ferricyanide/ferrocyanide, [Fe(CN)₆]³⁻/⁴⁻, in sodium acetate as an electrolyte adjusted to pH 8.2. In the presence of dissolved CO₂ in equilibrium with bicarbonate, there is a decrease in the ferrocyanide peak current with time (~30% in 60 min), attributed to the formation of hemiester 2-mercapto ethyl carbonate at the GE-ME/solution interface. Similarly, dissolved CO₂ and bicarbonate also affect the electrochemical impedance measurements by increasing resistance to the charge transfer process with time (elevation of Rct values), compatible with the formation of the hemiester. The addition of barium salt led to the displacement of the equilibrium towards BaCO₃ precipitation and consequent dissociation of the hemiester, attested by the recovery of the initial ferricyanide DPV signal. With the positively charged redox probe [Ru(NH₃)₆]²⁺, no decrease in the DPV peak was observed during the formation of the hemiester by reaction with bicarbonate. The repulsion of [Fe(CN)₆]³⁻, but not of [Ru(NH3)₆]²⁺, suggests that the formed species is the negatively charged 2-mercapto-ethyl carbonate, i.e., the hemiester with a dissociated proton. Due to the lack of a voltammetric signal from the hemiester itself, the formation of a self-assembled layer of thio-alcohol followed by the gradual formation of the corresponding carbonic acid hemiester allowed us to reach an elegant way of electrochemically demonstrating the formation of these species. Full article

A novel protease was isolated from the fruiting bodies of the straw mushroom Volvariella volvacea. The protease was purified 13.48-fold using a series of techniques, including ammonium sulfate precipitation, ultrafiltration, diethylaminoethyl fast-flow (DEAE FF) ion-exchange chromatography, and Superdex 75 gel filtration chromatography, resulting in a specific enzyme activity of 286.82 U/mg toward casein as a substrate. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the purified protease had a molecular weight of 24 kDa. The enzyme exhibited optimal activity at pH 7 and 50 °C, showing sensitivity to alkaline conditions and instability at elevated temperatures. The presence of Ca²⁺ significantly enhanced enzyme activity, whereas Ni²⁺ and Cu²⁺ exerted strong inhibitory effects, with other metal ions showing weak inhibition. β-mercaptoethanol, Tween-80, and Triton X-100 had more pronounced inhibitory effects, whereas PMSF, EDTA, and CTAB had weaker inhibitory effects. The Michaelis constant (Km) and maximum velocity (Vm) of the protease were determined to be 1.34 g/L and 3.45 μg/(mL·min), respectively. The protease exhibited a greater degree of enzymatic degradation of soybean-isolate protein (7.58%) compared to trypsin (5.24%), with the enzyme product containing a high percentage of medicinal amino acids (73.54%), particularly phenylalanine (Phe) and arginine (Arg), suggesting their presence at the enzyme’s active site. These findings suggest that the protease from V. volvacea holds promising potential for applications in the food industry, particularly in protein hydrolysate production and flavor enhancement. Full article

Binary and ternary copolymers of acrylonitrile (AN), tert-butyl acrylate (TBA), and n-butyl acrylate (BA) are synthesized through conventional radical polymerization in DMSO in the presence of 2-mercaptoethanol. The thermal behavior of binary and ternary copolymers is studied under argon atmosphere and in air. It is demonstrated that the copolymers of AN contain 1–10 mol.% of TBA split isobutylene upon heating above 160 °C, resulting in the formation of the units of acrylic acid in the chain. The carboxylic groups formed in situ are responsible for the ionic mechanism of cyclization, which starts at lower temperatures compared with pure polyacrylonitrile (PAN) or AN copolymer with BA. The activation energy of cyclization through ionic and radical mechanisms depends on copolymer composition. For the ionic mechanism, the activation energy lies in the range ca. 100–130 kJ/mole, while for the radical mechanism, it lies in the range ca. 150–190 kJ/mole. The increase in the TBA molar part in the copolymer is followed by faster consumption of nitrile groups and the evolution of a ladder structure in both binary and ternary copolymers. Thus, the incorporation of a certain amount of TBA in PAN or its copolymer with BA allows tuning the temperature range of cyclization. This feature seems attractive for applications in the production of melt-spun PAN by choosing the appropriate copolymer composition and heating mode. Full article

Aflatoxin B1 (AFB1) produced by some Aspergillus species belongs to the most dangerous contaminants of animal feeds. Development of safe and cost efficient decontamination methods saving feed quality and nutritional value are of paramount importance. The use of recombinant AFB1-detoxifying microbial enzymes represents a promising biotechnological approach meeting the aforementioned requirements. In this study, three AFB1-degrading oxidases (AFOs) from edible basidiomycetes Cantharellus cibarius, Lentinula edodes and Pleurotus eryngii as well as AFO from Armillaria tabescens were expressed in E. coli Rosetta (DE3) and purified by immobilized metal-chelate chromatography. The stabilizing effect of the addition of glycerol and β-mercaptoethanol during protein extraction is shown. The catalytic constants of the recombinant AFOs (rAFOs) and other characteristics, which might be important for their practical application (and optimal temperature and pH, thermolability, regulation of the activity by metal ions and chelating agents, storage stability) were investigated. Among the obtained enzymes, rAFO from P. eryngii (Pe-AFO), which was characterized by the highest specific activity, thermostability and pH stability (especially at acidic pH range), the lowest K_m, and relative resistance to the inhibition by phytate, showed the best AFB1-degrading efficacy. However, Pe-AFO and all other rAFOs significantly decreased the target activity during heating above 45 °C, storage frozen or lyophilization. Full article

Cell-based therapy using insulin-producing cells (IPCs) is anticipated as an alternative treatment option to insulin injection or pancreatic islet transplantation for the treatment of diabetes mellitus in both human and veterinary medicine. Several protocols were reported for the differentiation of mesenchymal stem cells (MSCs) into IPCs; to date, glucose-responsive IPCs have only been obtained from canine adipose tissue-derived MSCs (cAD-MSCs), but not from canine bone marrow-derived MSCs (cBM-MSCs). Therefore, this study aims to generate in vitro glucose-responsive IPCs from cBM-MSCs using two differentiation protocols: a two-step protocol using trichostatin (TSA) and a three-step protocol using mercaptoethanol to induce pancreatic and duodenal homeobox gene 1 (PDX-1) expression. A single experiment was carried out for each protocol. BM-MSCs from one dog were successfully cultured and expanded. Cells exposed to the two-step protocol appeared rarely grouped to form small clusters; gene expression analysis showed a slight increase in PDX-1 and insulin expression, but no insulin protein production nor secretion in the culture medium was detected either under basal conditions or following glucose stimulation. Conversely, cells exposed to the three-step protocol under a 3D culture system formed colony-like structures; insulin gene expression was upregulated compared to undifferentiated control and IPCs colonies secreted insulin in the culture medium, although insulin secretion was not enhanced by high-glucose culture conditions. The single experiment results suggest that the three-step differentiation protocol could generate IPCs from cBM-MSCs; however, further experiments are needed to confirm these data. The ability of IPCs from cBM- MSCs to produce insulin, described here for the first time, is a preliminary interesting result. Nevertheless, the IPCs’ unresponsiveness to glucose, if confirmed, would affect its clinical application. Further studies are necessary to establish a differentiation protocol in this perspective. Full article

Xylanases, key enzymes for hydrolyzing xylan, have diverse industrial applications. The bioprocessing of agricultural byproducts to produce xylanase through fermentation approaches is gaining importance due to its significant potential to reduce enzyme production costs. In this work, the productivity of Streptomyces thermocarboxydus TKU045 xylanase was enhanced through liquid fermentation employing wheat bran as the sole carbon source. The maximum xylanase activity (25.314 ± 1.635 U/mL) was obtained using the following optima factors: 2% (w/v) wheat bran, 1.4% (w/v) KNO₃, an initial pH of 9.8, an incubation temperature of 37.3 °C, and an incubation time of 2.2 days. Xylanase (Xyn_TKU045) of 43 kDa molecular weight was isolated from the culture supernatant and was biochemically characterized. Analysis through liquid chromatography with tandem mass spectrometry revealed a maximum amino acid identity of 19% with an endo-1,4-β-xylanase produced by Streptomyces lividans. Xyn_TKU045 exhibited optimal activity at pH 6, with remarkable stability within the pH range of 6.0 to 8.0. The enzyme demonstrated maximum efficiency at 60 °C and considerable stability at ≤70 °C. Mg²⁺, Mn²⁺, Ba²⁺, Ca²⁺, 2-mercaptoethanol, Tween 20, Tween 40, and Triton X-100 positively influenced Xyn_TKU045, while Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, and sodium dodecyl sulfate exhibited adverse impact. The kinetic properties of Xyn_TKU045 were a K_m of 0.628 mg/mL, a k_cat of 75.075 s⁻¹ and a k_cat/K_m of 119.617 mL mg⁻¹s⁻¹. Finally, Xyn_TKU045 could effectively catalyze birchwood xylan into xylotriose and xylobiose as the major products. Full article

Chitinases are biotechnologically relevant enzymes that can be applied in such different sectors as pharmaceutical, food, environmental management, the biocontrol of pests and in the paper and cellulose industry. Microorganisms as filamentous fungi are the most important source of these biomolecules. The fungus Aspergillus niveus produces extracellular chitinase when cultured under submerged fermentation using shrimp shells, a residue generated by the fish industry, as a carbon source, for 96 h at 30 °C and 100 rpm. The particle size and concentration of the shrimp shells affected enzyme production. The chitinase was purified until electrophoretic homogeneity through the use of a Sephadex G-100 chromatographic column. It is a monomeric glycoprotein with a molecular mass of 47 kDa estimated using SDS-PAGE and 49.3 kDa determined using gel filtration. The carbohydrate content was 22.8%. The best temperature and pH for enzyme activity were 65 °C and 6.0, respectively. Approximately 80% of the enzymatic activity was preserved at pH 4.0 and 5.0 for 48 h, and the half-life (t₅₀) was maintained for 48 h at 40 °C. Salts, EDTA and β-mercaptoethanol did not affect chitinase activity significantly, but organic solvents reduced it. The kinetic parameters determined using p-NPGlycNac were K_m of 2.67 mmol L⁻¹, V_max of 12.58 U mg of protein⁻¹, Kcat of 2.47 s⁻¹ and K cat/K_m of 0.93 s⁻¹ mmol L⁻¹. The A. niveus chitinase inhibited the growth of all fungal strains used, especially Trichoderma harzianum (MIC = 22.4 μg mL⁻¹) and Penicillium purpurogenum (MIC = 11.2 μg mL⁻¹). The chitinase produced by A. niveus presented interesting characteristics that indicate its potential of application in different areas. Full article