BioTech, Volume 11, Issue 2 (June 2022) – 13 articles

This research aimed to investigate the effects of the intramuscular injection of vitamins AD3E and C in combination immediately before the estrus synchronization program (the Ovsynch program) on conception and pregnancy rates, blood parameters, serum biochemical properties, immune systems, antioxidant parameters, and proteomic and transcriptomic analyses during early gestation in dairy cows. Forty nonlactating multiparous cows were randomly assigned to one of four treatments: (1) C: control with normal saline injection; (2) VAD3E: a single intramuscular injection (I/M) of vitamin AD3E; (3) VAD3EC: injection of both vitamins AD3E and C; (4) VC: a single dose of vitamin C. Blood and serum samples were taken immediately at day 0 (before AI), day 7, and day 14 (after AI for 5 days) from the coccygeal vein. Generally, injections of AD3E and C in combination had no effect on the rate of conception or pregnancy. However, they improved hematological parameters and immune and antioxidant activities. Serum samples were analyzed using LC-MS/MS, and 8190 proteins were identified. Five proteins were successfully validated using the quantitative real-time reverse transcription PCR (qRT-PCR) method. This study found that lymphocyte-specific protein 1 (LSP1, A0A3Q1M894) could be used as a protein biomarker for cows administrated with vitamins AD3E and C. Full article

Post-translational modifications (“PTMs”) in monoclonal antibodies (mAbs) contribute to charge variant distribution, which will affect biological efficacy and safety. For the characterization of mAbs, charge variants are used as a critical quality attributes for product quality, stability consistency and effectiveness. Charge variants in mAbs are characterized by a time-consuming and a multistep process starting from cation/anion exchange chromatography, acidic/basic fractions collection and subsequent reverse phase (RP) liquid chromatography, coupled with mass spectrometry (MS) analysis. Hence, an alternative characterization approach that would be highly selective for ion exchange chromatography-based charge variant analysis, which is compatible with on-line MS detection, is needed in the biopharma industry. Against this backdrop, multiple studies are being conducted to develop a simple straight on-line charge variant analysis method. In this regard, we apply the current study, which aims to develop a charge variant analytical method, based on volatile buffers with low ionic strength that can be used for on-line MS detection of charge variants of mAbs. This would enable the detection on “PTMs” using low ionic strength mobile phase compatible with MS. Hence, fruitful data can be obtained with a single chromatography run without any test sample preparation, eliminating the need for multiple steps of analysis, time-consuming process and multiple sample preparation steps. Thus, Charge Variant Analysis-MS technique will allow the characterization of charge-related PTMs on the intact protein stage. In this regard, this study is about development of a method having combination of chromatography and volatile mobile phase for mass spectrometry detection of mAbs being analyzed in native form. The method is qualified considering pharmacopeia guidelines because the ultimate aim is to transfer this method for Quality Control (QC) release testing of a monoclonal antibody, which is critical for batch release and the regulatory point of view. Acidic and basic variants have been separated with high resolution peak profile. Furthermore, there was no matrix interference and good separation selectivity in terms of specificity was obtained using this method. The experimental data suggested for the linearity of the method are 2.4 mg/mL to 3.6 mg/mL with % RSD below 2.0%. Additionally, Limit of Quantitation is found to be 0.15 mg/mL, which is 5% of loading amount. Consistently, the data show that the method is precise under the same operating conditions with a short time interval. Overall a simple, accurate, robust and precise pH gradient cation exchange chromatography method was developed and qualified for the characterization of a therapeutic native mAb. Additionally, this method can be used to claim a biosimilar product profile of an in-house product compare to an innovator. Full article

Burn injuries are among the most common peacetime injuries, with mortality ranging from 2.3% to 3.6%. At the same time, 85–90% of patients with burns are people of working age and children. Burn injury leads to metabolic disorders and systemic inflammatory response, inefficient energy consumption, and other physiological changes that can lead to dysfunction of organs and systems. The most formidable complication of burn injuries is sepsis mediated by multiple organ failure, the most common cause of poor prognosis in patients and has specific differences in these injuries. The purpose of this article was to dwell in detail on the most promising immunobiochemical markers of sepsis in the format of a mini-review, based on the main aspects of the immunopathogenesis of this complication. The pathogenesis of a burn injury and any general pathological process is based on an inflammatory reaction and large-scale changes in the skin and mucous membranes. This review is devoted to the progress in understanding the main aspects of the immunopathogenesis of burn lesions and the features of post-burn immune dysfunction, manifested by disorders in the innate and adaptive immunity systems. Attention is focused on the role in the immunopathogenesis of the development of systemic and local disorders in burn injury. Characterization of primary immunobiochemical markers of burn injury (cytokines, growth factors, C-reactive protein, procalcitonin, presepsin, matrix metalloproteinases, reactive oxygen species, nitric oxide, hemostasis parameters) is presented. The problem of treating burn lesions is associated with constant monitoring of the condition of patients and regular monitoring of specific immunobiochemical markers predicting sepsis for the timely initiation of a specific therapy. Full article

Environmental problems associated with marine pollution and climate warming create favorable conditions for the penetration and survival of pathogenic bacteria in marine ecosystems. These microorganisms have interspecific competitive interactions with marine bacteria. Co-culture, as an important research strategy that mimics the natural environment of bacteria, can activate silent genes or clusters through interspecies interactions. The authors used modern biotechnology of co-cultivation to dynamically study intercellular interactions between different taxa of bacteria—pathogenic enterobacteria Yersinia pseudotuberculosis and Listeria monocytogenes and saprotrophic marine bacteria Bacillus sp. and Pseudomonas japonica isolated in summer from the coastal waters of the recreational areas of the Sea of Japan. The results of the experiments showed that during the formation of polycultural biofilms, horizontal transfer of genes encoding some pathogenicity factors from Y. pseudotuberculosis and L. monocytogenes to marine saprotrophic bacteria with different secretion systems is possible. It was previously thought that this was largely prevented by the type VI secretion system (T6SS) found in marine saprotrophic bacteria. The authors showed for the first time the ability of marine bacteria Bacillus sp. and P. japonica to biofilm formation with pathogenic enterobacteria Y. pseudotuberculosis and L. monocytogenes, saprophytic bacteria with type III secretion system (T3SS). For the first time, a marine saprotrophic strain of Bacillus sp. Revealed manifestations of hyaluronidase, proteolytic and hemolytic activity after cultivation in a polycultural biofilm with listeria. Saprotrophic marine bacteria that have acquired virulence factors from pathogenic enterobacteria, including antibiotic resistance genes, could potentially play a role in altering the biological properties of other members of the marine microbial community. In addition, given the possible interdomain nature of intercellular gene translocation, acquired virulence factors can be transferred to marine unicellular and multicellular eukaryotes. The results obtained contribute to the paradigm of the epidemiological significance and potential danger of anthropogenic pollution of marine ecosystems, which creates serious problems for public health and the development of marine culture as an important area of economic activity in coastal regions. Full article

The main aim of this study was to evaluate the yield and compliance of selected Iranian garlic (Allium sativum L.) cultivars, including Tuyserkan (TSN), Heydareh (HDH), Mouien (MUN), and Taroom (TRM), during two growing seasons. The TRM cultivar germination rate is higher than the other cultivars studied. The TRM cultivars have quite remarkable values for the dry weight, fresh weight, stem diameter, and the number of leaves present. The fresh weight and dry weight of the TRM cultivar for the second year are 33.8 t/ha and 16.7 t/ha, respectively. However, on average, the HDH cultivar is the tallest plant in the experiments. Average pyruvic acid content in fresh samples of the TRM and HDH cultivars is 78 µm/gfw and 69.3 µm/gfw, respectively. It is observed that there are remarkable differences in the level of pyruvic acid between the different cultivars. The growth, development, and yield of plants are highly dependent on their genetic characteristics; in this experiment, the TRM cultivar shows a good yield (16.7 t/ha), and the evaluated characteristics improve compared to the other cultivars studied, which could be due to the high compatibility of this cultivar to the environmental conditions of the study. The excellent performance on the yield of TRM makes this cultivar more appreciable on a commercial level. Full article

In general, agroindustrial byproducts can be easily assimilated by several microorganisms due to their composition, which is rich in carbohydrates. Therefore, they could be appropriate for use as raw materials in a sustainable refinery concept, including the production of hydrolytic enzymes with industrial applicability. In this work, xylanase production by the filamentous fungi Talaromyces amestolkiae in submerged culture was evaluated using five agroindustrial byproducts, namely, wheat bran, citrus pulp, rice bran, peanut skin, and peanut shell. Firstly, the aforementioned byproducts were characterized in terms of cellulose, xylan, lignin, and extractives. Next, production studies were performed, and wheat bran generated the highest enzymatic activity (5.4 U·mL⁻¹), probably because of its large amount of xylan. Subsequently, a factorial design was performed to evaluate the independent variables yeast extract, wheat bran, K₂HPO₄, and pH, aiming to improve the variable response, xylanase activity. The condition that promoted the highest production, 13.02 U·mL⁻¹ (141% higher than the initial condition), was 20 g·L⁻¹ wheat bran, 2.5 g·L⁻¹ yeast extract, 3 g·L⁻¹ K₂HPO₄, and pH 7. Thus, industrial byproducts with a high content of xylan can be used as a culture medium to produce xylanase enzymes with a Talaromyces strain through an economical and sustainable approach. Full article

The American cranberry, Vaccinium macrocarpon, contains fibers and (poly)phenols that could exert health-promoting effects through modulation of gut microbiota. This study aimed to investigate how a freeze-dried whole cranberry powder (FCP) modulated metabolite production and microbial composition using both a 48-h incubation strategy and a long-term human gut simulator study with the M-SHIME (Mucosal Simulator of the Human Intestinal Microbial Ecosystem). FCP was repeatedly administered over three weeks. The studies included five and three study subjects, respectively. In both models, FCP significantly increased levels of health-related short-chain fatty acids (SCFA: acetate, propionate and butyrate), while decreased levels of branched-chain fatty acids (markers of proteolytic fermentation). Interestingly, FCP consistently increased luminal Bacteroidetes abundances in the proximal colon of the M-SHIME (+17.5 ± 9.3%) at the expense of Proteobacteria (−10.2 ± 1.5%). At family level, this was due to the stimulation of Bacteroidaceae and Prevotellaceae and a decrease of Pseudomonodaceae and Enterobacteriaceae. Despite of interpersonal differences, FCP also increased the abundance of families of known butyrate producers. Overall, FCP displayed an interesting prebiotic potential in vitro given its selective utilization by host microorganisms and potential health-related effects on inhibition of pathogens and selective stimulation of beneficial metabolites. Full article

Lately, microalgae-based value-added products have been gaining market value because they moderate the dependency on fossil fuel and high-value chemical products. To this end, the purpose of this study was to develop prebiotic products from the microalgae Spirulina sp. The microalgae were isolated from the fresh water and characterized at the molecular level. The dry biomass, chlorophyll content, phycocyanin, cytotoxicity and antimicrobial and antioxidant properties of the isolated strains were analyzed. Moreover, value-added products like Spirulina cake, chocolate, tea, vermicelli and Spirulina juice were made for a vulnerable population due to high nutritive value. Full article

Several variants of SARS-CoV-2 have been identified in different parts of the world, including Gamma, detected in Brazil, Delta, detected in India, and the recent Omicron variant, detected in South Africa. The emergence of a new variant is a cause of great concern. This work considers an extended version of an SIRD model capable of incorporating the effects of vaccination, time-dependent transmissibility rates, mortality, and even potential reinfections during the pandemic. We use this model to characterise the Omicron wave in Brazil, South Africa, and Germany. During Omicron, the transmissibility increased by five for Brazil and Germany and eight for South Africa, whereas the estimated mortality was reduced by three-fold. We estimated that the reported cases accounted for less than $25\%$ of the actual cases during Omicron. The mortality among the nonvaccinated population in these countries is, on average, three to four times higher than the mortality among the fully vaccinated. Finally, we could only reproduce the observed dynamics after introducing a new parameter that accounts for the percentage of the population that can be reinfected. Reinfection was as high as $40\%$ in South Africa, which has only $29\%$ of its population fully vaccinated and as low as $13\%$ in Brazil, which has over $70\%$ and $80\%$ of its population fully vaccinated and with at least one dose, respectively. The calibrated models were able to estimate essential features of the complex virus and vaccination dynamics and stand as valuable tools for quantifying the impact of protocols and decisions in different populations. Full article

Mammary gland secretory cells produce miRNA-rich milk. In humans, these miRNAs reach infant/neonate bloodstream, playing diverse roles, like neural system development, metabolism, and immune system maturation. Notwithstanding, still few works explore human milk miRNA content, and there are no reports at the population level. Our hypothesis was that miR-146b-5p, miR148a-3p, miR155-5p, mir181a-5p, and mir200a-3p immunoregulatory miRNAs are expressed in human colostrum/milk at a higher level than infant milk formulae. The aim of this work was to evaluate the expression of the five immunoregulatory miRNAs in human milk and compare it with their expression in infant milk formula. For this purpose, miRNA relative expression was measured by qPCR in cDNA prepared from total RNA extracted from sixty human colostrum/milk samples and six different formulae. The comparative Cт method 2^−ΔCт using exogenous cel-miR-39 as internal control was employed, followed by statistical analysis. We found the relative expression levels of miRNAs are comparable among colostrum/milk samples, and these miRNAs are present in infant milk formulae but at very low concentrations. We conclude that the relative expression of the immunomodulatory miRNAs is comparable in all the human colostrum/milk samples and is higher than the expression in formulae. Full article

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing for a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using the Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions (contact time 49 min, pH 6.73, and [ASNase] 0.26 mg·mL⁻¹), the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal, and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed good thermal stability at 37 °C (RRA of 91% after 90 min), and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors. Full article

Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment–protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells. Full article

Drought is one of the most important threats to plants and agriculture. Here, the effects of four drought levels (90%, 55%, 40%, and 25% field capacity) on the relative water content (RWC), chlorophyll and carotenoids levels, and mRNA gene expression of metabolic enzymes in Thymus vulgaris (as sensitive to drought) and Thymus kotschyanus (as a drought-tolerant species) were evaluated. The physiological results showed that the treatment predominantly affected the RWC, chlorophyll, and carotenoids content. The gene expression analysis demonstrated that moderate and severe drought stress had greater effects on the expression of histone deacetylase-6 (HDA-6) and acetyl-CoA synthetase in both Thymus species. Pyruvate decarboxylase-1 (PDC-1) was upregulated in Thymus vulgaris at high drought levels. Finally, succinyl CoA ligase was not affected by drought stress in either species. Data confirmed water stress is able to alter the gene expression of specific enzymes. Furthermore, our results suggest that PDC-1 expression is independent from HDA-6 and the increased expression of ACS can be due to the activation of new pathways involved in carbohydrate production. Full article