Expression profiles and polymorphism analysis of CDIPT gene on Qinchuan cattle

BackgroundCDIPT (CDP-diacylglycerol–inositol 3-phosphatidyltransferase, EC 2.7.8.11) was found on the cytoplasmic side of the Golgi apparatus and the endoplasmic reticulum. It was an integral membrane protein performing the last step in the de novo biosynthesis of phosphatidylinositol (PtdIns). In recent years, PtdIns has been considered to play an essential role in energy metabolism, fatty acid metabolic pathway and intracellular signal transduction in eukaryotic cells.ResultsIn this study, the results of real-time polymerase chain reaction (PCR) showed that the expression of CDIPT gene was remarkably different in diverse tissues. We also detected the polymorphism of bovine CDIPT gene and analyzed its association with body measurement and meat quality traits of Qinchuan cattle. Blood samples were obtained from 638 Qinchuan cattle aged from 18 to 24 months. DNA sequencing and PCR-restriction fragment length polymorphism (RFLP) were used to find CDIPT gene single nucleotide polymorphism (SNP). Three SNPs g.244T>C (NCBI: rs42069760), g.1496G>A and g.1514G>A were found in this study. g.244T>C located at 5′untranslated region (5′UTR) of exon 1 showed three genotypes: TT, TC and CC. g.1496G>A and g.1514G>A detected the first time were located in intron 3 and showed the same genotypes: GG, GA and AA.ConclusionsAnalysis results showed that these three SNPs were significantly associated with body measurement traits (BMTs) and meat quality traits (MQTs). We suggested that CDIPT gene may have potential effects on BMTs and MQTs and can be used for marker-assisted selection.     

Bovine TMEM95 gene: Polymorphisms detecting in five Chinese indigenous cattle breeds and their association with growth traits

BackgroundTransmembrane protein 95 (TMEM95) plays a role in male fertility. Previous studies showed that genes with a significant impact on reproductive traits can also affect the growth traits of livestock. Thus, we speculated that the genetic variation of TMEM95 gene may have effects on growth traits of cattle.ResultsTwo SNPs were genotyped. The rs136174626 and rs41904693 were in the intron 4 and 3′-untranslated region, respectively. The linkage disequilibrium analysis illustrated that these two loci were not linked. The rs136174626 was associated with six growth traits of Nanyang cattle, four traits of Luxi cattle, and three traits of Ji’an cattle. For rs41904693 locus, the GG individuals had greater body height and abdominal girth in Ji’ an cattle than TT and TG individuals. In Jinnan cattle, GG and TT individuals had greater body height, height at hip cross, body length, and heart girth than TG individuals. The potential splice site prediction results suggest that the rs136174626 may influence the splicing efficiency of TMEM95, and the miRNA binding site prediction results showed that the rs41904693 may influence the expression of TMEM95 by affecting the binding efficiency of Bta-miR-1584 and TMEM95 3′-UTR.ConclusionsThe findings of the study suggested that the two SNPs in TMEM95 could be a reliable basis for molecular breeding in cattle.How to cite: Guo X, Zhang S, Yang H, et al. Bovine TMEM95 gene: Polymorphisms detecting in five Chinese indigenous cattle breeds and their association with growth traits. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.004     

Characteristics of faecal bacterial flora and volatile fatty acids in Min pig,Landrace pig,and Yorkshire pig

BackgroundIn order to study the influence of long-term growth process and evolution environment on intestinal bacteria of different breeds, the intestinal bacteria and volatile fatty acids among the faeces of Min, Landrace and Yorkshire pigs were analysed by Illumina high-throughput sequencing of the 16S-rDNA and gas chromatography.ResultsThe shared core microbiota of Landrace, Yorkshire and Min pig were 1273, accounting for 69.56% of total abundance of organisms. The proportion of Firmicutes in Min pig faeces (57.89%) was significantly higher than that in Landrace and Yorkshire pig faeces (47.01% and 46.40%, respectively) (P < 0.05), but that of Bacteroidetes was exactly opposite. Moreover, Min pig presented more highly efficient membrane transport, environmental adaptation, carbohydrate transport, and metabolism than Yorkshire pig (P < 0.05). The acetic acid/total volatile fatty acid ratio in Min pig was significantly higher than that in Landrace pig (P < 0.05), and the isobutyric acid/ total volatile fatty acid ratio in Min pig was significantly larger than that in Yorkshire pig (P < 0.05). Furthermore, the content of branched chain volatile fatty acids in Min pig was significantly higher than that in Yorkshire pig (P < 0.05).ConclusionsThis study demonstrated that Min pig, as an excellent breed in the cold area of China, possessed special intestinal floral structure compared to the imported pigs in order to satisfy their physiological and metabolic demands, which may influence their characteristics such as resistance to cold, diseases, and crude feeding, and the ability to deposit intramuscular fat.How to cite: Yang Y, Sun C, Li F, et al. Characteristics of faecal bacterial flora and volatile fatty acids in Min pig, Landrace pig, and Yorkshire pig. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.05.002     

Polymorphism of bovine lipocalin-2 gene and its impact on milk production traits and mastitis in Holstein Friesian cattle

BackgroundMastitis is one of the most serious diseases of dairy cattle, causing substantial financial losses. While predisposition to reduced somatic cell count in milk has been considered for in cattle breeding programs as the key indicator of udder health status, scientists are seeking genetic markers of innate immune response, which could be helpful in selecting cows with improved immunity to mastitis. Lipocalin-2 (LCN2) is a protein involved in the response of the immune system by eliminating iron ions which are necessary for the growth of pathogenic bacteria, so LCN2 may be considered as a natural bacteriostatic agent and could become a marker of infection.ResultsA total of five SNPs were identified in LCN2 gene (one in the promoter, three in exon 1, and one in intron 1). A single haplotype block was identified. The locus g.98793763G > C was found to have a significant impact on protein levels in milk, and alleles of this locus were identified to have a significant positive dominance effect on this trait. None of the four analysed loci had a statistically significant impact on the milk yield, fat levels in milk or the somatic cell score. LCN-2 gene had no significant impact on the incidence of mastitis in the cows.ConclusionsAlthough the identified SNPs were not found to have any impact on the somatic cell count or the incidence of mastitis in cows, it seems that further research is necessary, covering a larger population of cattle, to confirm the association between lipocalin-2 and milk production traits and mastitis.How to cite: Pokorska J, Piestrzyńska-Kajtoch A, Kułaj D, et al. Polymorphism of bovine lipocalin-2 gene and its impact on milk production traits and mastitis in Holstein Friesian cattle. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.04.004     

Identification of genetic variants the CCKAR gene and based on body measurement and carcass quality characteristics in Qinchuan beef cattle (Bos taurus)

BackgroundThis study aimed to explore genetic polymorphisms of the CCKAR gene and their relationship with the growth and development of Qinchuan cattle which could be used as molecular markers for the improvement of the breeding of Qinchuan cattle.ResultsHere, we have identified seven single nucleotide polymorphisms (SNPs) at loci g. 1463 C>G; g. 1532 T>A; g. 1570 G>A; g. 1594 C>A; g. 1640 T>C; g. 1677 G>C; and g. 1735 C>T in the coding region of the bovine CCKAR gene. The frequencies identified on allelic and genotypic characteristics have shown that all seven SNPs diverged from the Hardy-Weinberg-Equilibrium. The SNP2, SNP3, SNP6 and SNP7 had the lowest polymorphism information content values, and remaining SNPs were found to be moderate (0.25 < PIC < 0.50). The genotype CG in SNP1 at loci g.1463 C>G had the greatest association with WH, HW, CD and CCF, while the genotype TA at the very same loci was associated with BFT, ULA and IMF content in Qinchuan cattle. The CCKAR gene expression level in adipose tissue, small intestine, liver and skeleton muscle was found to be higher, whereas, the expression level of mRNA in organs of other digestive system including reticulum, abomasum and omasum was moderate. Some expression of CCKAR mRNA was found in the large intestine, kidney and rumen.ConclusionsIn summary, our finding suggested that the CCKAR gene could be used as a potential candidate for the improvement of carcass quality and body measurements of Qinchuan cattle.How to citeNurgulsim K, Raza SHA, Khan R, et al. Identification of genetic variants the CCKAR gene and based on body measurement and carcass quality characteristics in Qinchuan beef cattle (Bos taurus). Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.02.001     

Changes in freshwater sediment microbial populations during fermentation of crude glycerol

BackgroundThis work studied how the exposure to an unusual substrate forced a change in microbial populations during anaerobic fermentation of crude glycerol, a by-product of biodiesel production, with freshwater sediment used as an inoculum.ResultsThe microbial associations almost completely (99.9%) utilized the glycerol contained in crude glycerol 6 g L⁻¹ within four days, releasing gases, organic acids (acetic, butyric) and alcohols (ethanol, n-butanol) under anaerobic conditions. In comparison with control medium without glycerol, adding crude glycerol to the medium increased the amount of ethanol and n-butanol production and it was not significantly affected by incubation temperature (28 °C or 37 °C), nor incubation time (4 or 8 d), but it resulted in reduced amount of butyric acid. Higher volume of gas was produced at 37 °C despite the fact that the overall bacterial count was smaller than the one measured at 20 °C. Main microbial phyla of the inoculum were Actinobacteria, Proteobacteria and Firmicutes. During fermentation, significant changes were observed and Firmicutes, especially Clostridium spp., began to dominate, and the number of Actinobacteria and Gammaproteobacteria decreased accordingly. Concentration of Archaea decreased, especially in medium with crude glycerol. These changes were confirmed both by culturing and culture-independent (concentration of 16S rDNA) methods.ConclusionsCrude glycerol led to the adaptation of freshwater sediment microbial populations to this substrate. Changes of microbial community were a result of a community adaptation to a new source of carbon.How to cite: Paiders M, Nikolajeva V, Makarenkova G, et al. Changes in freshwater sediment microbial populations during fermentation of crude glycerol. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.10.007     

Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

BackgroundPEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating N-terminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein.ResultsSelected linear 5 kDa and 20 kDa mPEG – potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the di-PEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods.ConclusionsThis novel approach using reactive ATPS shows that it is feasible to simultaneously produce and purify PEGylated therapeutic proteins with conserved biological activity and presents another example where reactive ATPS can be successfully implemented.How to cite: Campos-García VR, Benavides J, González-Valdez J. Reactive aqueous two-phase systems for the production and purification of PEGylated proteins. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.09.003     

Synthesis of β(1 → 3) and β(1 → 6) galactooligosaccharides from lactose and whey using a recombinant β-galactosidase from Pantoea anthophila

BackgroundMilk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by β-galactosidases.ResultsB-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with β(1 → 6) and β(1 → 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD.ConclusionsB-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides.How to cite: Yañez-Ñeco CV, Cervantes FV, Amaya-Delgado L, et al. Synthesis of β(1→3) and β(1→6) galactooligosaccharides from lactose and whey using a recombinant β-galactosidase from Pantoea anthophila. Electron J Biotechnol 2021;49. https://dx.doi.org/10.1016/j.ejbt.2020.10.004     

Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

BackgroundMolecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable trans-activator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO.ResultsIn the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light.ConclusionsIn conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.How to cite: Zárate RV, Arancibia D, Fernández A, et al. Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.006     

First genome sequence of Chilean Brucella canis SCL strain provides insights on the epidemiology and virulence factors,explaining differences between geographical origins

BackgroundBrucella canis is the etiological agent of canine brucellosis, a worldwide neglected zoonosis that constitutes one of the major infectious causes of infertility and reproductive failure in dogs. Although genomic information available for this pathogen has increased in recent years, here we report the first genome sequencing of a B. canis strain in Chile, and the differences in virulence genes with other B. canis strains.ResultsGenome assembly produced a total length of 3,289,216 bp, N50 of 95,163 and GC% of 57.27, organized in 54 contigs in chromosome I, and 21 contigs in chromosome II. The genome annotation identified a total of 1981 CDS, 3 rRNA and 36 tRNA in chromosome I, and 1113 CDS and 10 tRNA in chromosome II. There is little variation between the different strains and the SCL isolate. Phylogenetic analysis showed that the Chilean SCL strain is closely related to B. canis and B. suis strains. Small differences were found when compared to the Serbian isolate, but all strains shared the same recent common ancestor. Finally, changes in the sequence of some virulence factors showed that the SCL strain is similar to other South American B. canis strains.ConclusionsThis work sequenced and characterized the complete genome of B. canis strain SCL, evidencing the complete presence of all the genes of the virB operon, and minor changes in outer membrane proteins and in the urease operon. Our data suggest that B. canis was introduced from North America and then spread throughout the South American continent.How to cite: Borie C, Bravo C, Dettleff P, et al. First genome sequence of Chilean Brucella canis SCL strain provides insights on the epidemiology and virulence factors, explaining differences between geographical origins Electron J Biotechnol 2021;49. https://dx.doi.org/10.1016/j.ejbt.2020.10.002.     

Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BackgroundLawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate.ResultsBatch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers.ConclusionsConsidering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.How to cite: Salazar S, Gutiérrez N, Sánchez O, et al. Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis. Electron J Biotechnol 2021.https://doi.org/10.1016/j.ejbt.2021.01.002     

De novo assembly of a Tibetan genome and identification of novel structural variants associated with high-altitude adaptation

Structural variants (SVs) may play important roles in human adaptation to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17 900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high-quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%–1.53%) compared to other East Asian genomes (0.70%–0.98%), reflecting a unique genomic composition of Tibetans. One such archaic hominid shared sequence—a 662-bp intronic insertion in the SCUBE2 gene—is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical studies.     

Kernel size and weight affected by three plant bioregulators applied at bloom to Non Pareil and Carmel almond cultivars

BackgroundThe yield of almonds [Prunus dulcis (Mill.) D.A. Webb] could be low due to climatic problems and any factor improving kernel size and weight, such as the use of plant bioregulators (PBRs), should be beneficial.ResultsThree plant bioregulators: 24-epibrassinolide (BL), gibberellic acid (GA₃) and kinetin (KN) were applied at three spray concentrations to Non Pareil and Carmel cultivars, at two phenological stages during bloom, in the 2014 and 2015 seasons. The results showed significant differences (P < 0.0001). For total dry weight of Non Pareil, the best treatment was BL (30 mg·L^-1), with an average of 1.45 g, while the control was 1.30 g, at pink button during 2015. For Carmel, the best dry weight was 1.23 g, achieved with BL (30 mg·L^-1) at fallen petals in both seasons. The average dry weight of the controls varied between 1.13 and 1.18 g. The greatest almond lengths and widths in Non Pareil were 24.98 mm and 15.05 mm, achieved with BL (30 mg·L^-1) and KN (50 μL·L^-1) treatments, respectively, applied at pink button in 2015. In Carmel, the greatest length and width were 24.38 and 13.44 mm, obtained with BL (30 mg·L^-1) applied at the stages of pink button and fallen petals, respectively, in 2015. The control reached lengths between 22.33 and 23.38 mm, and widths between 11.99 and 12.93 mm.ConclusionsThe use of the bioregulators showed significant favorable effects on dry weight, length and width of kernels at harvest, in both cultivars.     

1,3-Propanediol production from crude glycerol by Clostridium butyricum DSP1 in repeated batch

BackgroundThe production of biofuels from renewable energy sources is one of the most important issues in industrial biotechnology today. The process is known to generate various by-products, for example crude glycerol, which is obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including microbial conversion to 1,3-propanediol (1,3-PD), a raw material for the synthesis of polyesters and polyurethanes.ResultsThe paper presents results of a study on the synthesis of 1,3-propanediol from crude glycerol by a repeated batch method with the use of Clostridium butyricum DSP1. Three cycles of fermentation medium replacement were carried out. The final concentration of 1,3-PD was 62 g/L and the maximum productivity, obtained during the second cycle, reached 1.68 g/L/h. Additionally, experiments conducted in parallel to the above involved using the entire quantity of the culture broth removed from the bioreactor to inoculate successive portions of fermentation media containing crude glycerol at concentrations of 80 g/L and 100 g/L. Under those conditions, the maximum 1,3-PD concentrations were 43.2 g/L and 54.2 g/L.ConclusionsThe experiments proved that by using a portion of metabolically active biomass as inoculum for another fermentation formula it is possible to eliminate the stage of inoculum growth and thereby reduce the length of the whole operation. Additionally, that strategy avoids the phase of microbial adaptation to a different source of carbon such as crude glycerol, which is more difficult to utilize, thus improving the kinetic parameters of 1,3-PD production.     

Enhancement of biodegradation potential of catechol 1,2-dioxygenase through its immobilization in calcium alginate gel

BackgroundIn biodegradation processes free enzymes often undergo deactivation. Thus, it is very important to obtain highly stable enzymes by different methods. Immobilization allows for successful stabilization of many multimeric enzymes by increasing the rigidity of the enzyme structure. This study aimed to evaluate some environmental factors that affect catechol 1,2-dioxygenase from Stenotrophomonas maltophilia KB2 immobilized in alginate hydrogel. The goal of the present work was to improve the functional stability of the enzyme by increasing its structural rigidity.ResultsImmobilization yield and expressed activity were 100% and 56%, respectively. Under the same storage conditions, the activity of the immobilized enzyme was still observed on the 28th d of incubation at 4°C, whereas the free enzyme lost its activity after 14 d. The immobilized enzyme required approximately 10°C lower temperature for its optimal activity than the free enzyme. Immobilization shifted the optimal pH from 8 for the soluble enzyme to 7 for the immobilized enzyme. The immobilized catechol 1,2-dioxygenase showed activity against 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, and 3,5-dichlorocatechol. The immobilization of the enzyme promoted its stabilization against any distorting agents: aliphatic alcohols, phenols, and chelators.ConclusionsThe entrapment of the catechol 1,2-dioxygenase from S. maltophilia KB2 has been shown to be an effective method for improving the functional properties of the enzyme. Increased resistance to inactivation by higher substrate concentration and other factors affecting enzyme activity as well as broadened substrate specificity compared to the soluble enzyme, makes the immobilized catechol 1,2-dioxygenase suitable for the bioremediation and detoxification of xenobiotic-contaminated environments.