AOX1 Research Articles

Detoxification of ars genotypes by arsenite-oxidizing bacteria through arsenic biotransformation.

The detoxification process of transforming arsenite (As(III)) to arsenate (As(V)) through bacterial oxidation presents a potent approach for bioremediation of arsenic-polluted soils in abandoned mines. In this study, twelve indigenous arsenic-oxidizing bacteria (AOB) were isolated from arsenic-contaminated soils. Among these, Paenibacillus xylanexedens EBC-SK As2 (MF928871) and Ochrobactrum anthropi EBC-SK As11 (MF928880) were identified as the most effective arsenic-oxidizing isolates. Evaluations for bacterial arsenic resistance demonstrated that P. xylanexedens EBC-SK As2 (MF928871) could resist As(III) up to 40 mM, while O. anthropi EBC-SK As11 (MF928880) could resist As(III) up to 25 mM. From these bacterial strains, genotypes of arsenic resistance system (ars) were detected, encompassing ars leader genes (arsR and arsD), membrane genes (arsB and arsJ), and aox genes known to be crucial for arsenic detoxification. These ars genotypes in the isolated AOBs might play an instrumental role in arsenic-contaminated soils with potential to reduce arsenic contamination.

Effect of Partial Elimination of Mitochondrial DNA on Genome-Wide Identified AOX Gene Family in Chlamydomonas reinhardtii

Using Chlamydomonas as a model organism, we attempted to eliminate mitochondrial DNA (mtDNA) similar to rho0 or rho− cells (completely or partially mtDNA-eliminated cells) in yeast. We successfully generated partially mtDNA-eliminated cells named as crm- cells, causing the inactivation of mitochondrial activity. We used three different chemicals to eliminate mtDNA including acriflavine (AF), ethidium bromide (EB) and dideoxycytidine (ddC) which prevents replication, inhibits POLG (DNA polymerase gamma) and terminates the mtDNA chain, respectively. The qPCR method was used to detect the mtDNA copy number and the selected rrnL6 gene for the detection of mitochondria, as well as the selected Chlamydomonas CC-124 strain. A reduction in the mitochondrial copy number led to a higher expression of AOX1, UCP1, PGRL1 and ICL1, which indicates the disturbance of the mitochondria–chloroplast ATP and NADPH balance. We selected AOX genes to further study this family and carried out a genome-wide search to identify AOX genes in green algae (C. reinhardtii). Our results revealed that C. reinhardtii contains four AOX genes, i.e., CrAOX1, CrAOX2, CrAOX3 and CrAOX4, which are distributed on Chr 3, Chr7 and Chr9. All CrAOX genes were predicted to localize in mitochondria using bioinformatics tools. Phylogenetic analysis suggests that these CrAOXs are subdivided into four groups and genes existing in the same group could perform identical functions. Collinearity analysis describes the strong evolutionary relationships of AOXs between the unicellular green algae Chlamydomonas reinhardtii and the multicellular green algae Volvox carteri. GO (gene ontology) annotation analysis predicted that CrAOXs played an integral part in carrying out alternate oxidative and respirative activities. Three putative miRNAs, cre-miR1162-3p, cre-miR1171 and cre-miR914, targeting the CrAOX2 gene were identified. Our studies have laid a foundation for the further use of partially mtDNA-eliminated cells and elucidating the functional characteristics of the AOX gene family.

Overexpression of the transcriptional activators Mxr1 and Mit1 enhances lactic acid production on methanol in Komagataellaphaffii

A bio-based production of chemical building blocks from renewable, sustainable and non-food substrates is one key element to fight climate crisis. Lactic acid, one such chemical building block is currently produced from first generation feedstocks such as glucose and sucrose, both requiring land and water resources. In this study we aimed for lactic acid production from methanol by utilizing Komagataella phaffii as a production platform. Methanol, a single carbon source has potential as a sustainable substrate as technology allows (electro)chemical hydrogenation of CO2 for methanol production. Here we show that expression of the Lactiplantibacillus plantarum derived lactate dehydrogenase leads to L-lactic acid production in Komagataella phaffii, however, production resulted in low titers and cells subsequently consumed lactic acid again. Gene expression analysis of the methanol-utilizing genes AOX1, FDH1 and DAS2 showed that the presence of lactic acid downregulates transcription of the aforementioned genes, thereby repressing the methanol-utilizing pathway. For activation of the methanol-utilizing pathway in the presence of lactic acid, we constructed strains deficient in transcriptional repressors Nrg1, Mig1-1, and Mig1-2 as well as strains with overrepresentation of transcriptional activators Mxr1 and Mit1. While loss of transcriptional repressors had no significant impact on lactic acid production, overexpression of both transcriptional activators, MXR1 and MIT1, increased lactic acid titers from 4 g L−1 to 17 g L−1 in bioreactor cultivations.

Pharmacogenomic Assessment of Genes Implicated in Thiopurine Metabolism and Toxicity in a UK Cohort of Pediatric Patients With Inflammatory Bowel Disease.

Thiopurine drugs are effective treatment options in inflammatory bowel disease and other conditions but discontinued in some patients due to toxicity. We investigated thiopurine-induced toxicity in a pediatric inflammatory bowel disease cohort by utilizing exome sequencing data across a panel of 46 genes, including TPMT and NUDT15. The cohort included 487 patients with a median age of 13.1 years. Of the 396 patients exposed to thiopurines, myelosuppression was observed in 11%, gastroenterological intolerance in 11%, hepatotoxicity in 4.5%, pancreatitis in 1.8%, and "other" adverse effects in 2.8%. TPMT (thiopurine S-methyltransferase) enzyme activity was normal in 87.4%, intermediate 12.3%, and deficient in 0.2%; 26% of patients with intermediate activity developed toxicity to thiopurines. Routinely genotyped TPMT alleles associated with defective enzyme activity were identified in 28 (7%) patients: TPMT*3A in 4.5%, *3B in 1%, and *3C in 1.5%. Of these, only 6 (21%) patients developed toxic responses. Three rare TPMT alleles (*3D, *39, and *40) not assessed on routine genotyping were identified in 3 patients, who all developed toxic responses. The missense variant p.R139C (NUDT15*3 allele) was identified in 4 patients (azathioprine 1.6mg/kg/d), but only 1 developed toxicity. One patient with an in-frame deletion variant p.G13del in NUDT15 developed myelosuppression at low doses. Per-gene deleteriousness score GenePy identified a significant association for toxicity in the AOX1 and DHFR genes. A significant association for toxicity was observed in the AOX1 and DHFR genes in individuals negative for the TPMT and NUDT15 variants. Patients harboring the NUDT15*3 allele, which is associated with myelosuppression, did not show an increased risk of toxicity.

Ars Genotype of Arsenic Oxidizing Bacteria and Detoxification

Objectives:The objectives of this study is bioremediation and detoxification of arsenite using arsenic resistance system (ars) genotypes of Arsenic Oxidizing Bacteria (AOB) isolated from highly As-contaminated mine.Methods:Bacterial strains that are resistant to arsenic were isolated from the Samkwang mine. The identification of AOB was conducted by analyzing the 16S rRNA gene using universal primers. To determine the genotypes of the arsenic resistance system (ars), specific primers were used for each gene. The extent of arsenic resistance was measured, and the efficiency of arsenite oxidation was assessed through a batch test. Arsenic concentration was measured using ICP-MS.Results and Discussion:The arsenic concentrations at site 1 of the Samkwang mine were found to be 1,322 mg/kg. This concentration is 26.4 times higher than the standard for soil pollution concerns (50 mg/kg) and 8.8 times higher than the standard for soil pollution measures (150 mg/kg). The appropriate remediation is studied such as bacterial remediation. The three efficient AOBs were identified as Agrobacterium tumefaciens EBC-SK1 (MF928870), Ochrobactrum anthrophi EBC-SK4 (MF928873), Ochrobactrum anthrophi EBC-SK12 (MF928881), respectively. The arsenic resistance system (ars) genotype were detected, which is the leader genes of the arsenic oxidation system (arsR and arsD), and the membrane gene (arsB). The arsB is involved in the encoding of the efflux/influx pumping system and moves arsenite into the bacterial cells. Arsenite-oxidizing (aox) genes are activated to oxidize arsenite into arsenate. The AOBs biotransform arsenite to arsenate with the regulation of ars genes, which detoxify highly As-contaminated mine.Conclusion:The AOBs from Samkwang mine are known for their resistance to highly toxic arsenic environments. They play a crucial role in the bioremediation of abandoned mines by transforming As(III) into As(V) through biotransformation.

Unraveling the dynamics of salt stress response in wheat cultivars: insights into growth inhibition, antioxidant enzyme activity, and AOX gene expression

Unraveling the dynamics of salt stress response in wheat cultivars: insights into growth inhibition, antioxidant enzyme activity, and AOX gene expression

Generation, Transfer, and Loss of Alternative Oxidase Paralogues in the Aspergillaceae Family.

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance-Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens-paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins.

Pharmacogenetic Markers of Favipiravir Safety in COVID-19 Treatment

The aim of the study was to evaluate the associations of different variants of AOX1 and CYP1A2 genes with safety parameters of favipiravir therapy in patients with COVID-19. Material and Methods. The study included 86 patients hospitalized at Moscow Clinical Hospital No. 15 with a COVID-19 diagnosis who received favipiravir as etiotropic therapy. Frequency of adverse reactions (bradycardia, dyspeptic disorders, increased transaminase levels) and various laboratory parameters (levels of ALT, AST, leukocytes) were compared between the carriers of «wild» and polymorphic variants of the genes studied after administration of the drug. The dynamics of these indicators before and after the therapy depending on the carriage of the variants of the genes studied were also compared. Results. There was no significant difference in the frequency of ad- verse reactions and laboratory parameters between the carriers of various variants of the studied genes. Haplotype analysis of the combination of different gene variants also did not reveal associations with therapy safety parameters. Upon the comparison of the parameters before and after treatment, an increase in the level of AST was noted in carriers of the AA genotype for both studied loci of the AOX1 gene (P=0.018 и P=0.009). At the same time, the level of AST increased in carriers of polymorphic variants of the CYP1A2*F1 gene (P=0.024). Leukocyte number increase was noted in carriers of polymorphic variants of AOX1 (rs10931910) (P=0.044), as well as «wild» genotypes AOX1 (rs55754655) (P=0.002) and CYP1A2*F1 (P=0.05). Conclusion. The associations of carriers of different AOX1 and CYP1A2 gene variants on the dynamics of AST and leukocytes in patients with COVID-19 after favipiravir therapy were revealed.

Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens).

β-Cyanoalanine synthase (β-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of β-CAS and Aox genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species. We used a natural polymorphism for cyanogenesis in white clover to examine β-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure. We identified all β-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a, and Aox2d subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue. β-CAS and Aox2a expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression. Aox2d, in contrast, appeared to be upregulated as a generalized wounding response. These findings suggest a heightened constitutive role for HCN detoxification (via elevated β-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a expression) in plants that are capable of cyanogenesis. As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.

Functional characterization of the Komagataella phaffii 1033 gene promoter and transcriptional terminator.

The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.

Mutations in the Second Alternative Oxidase Gene: A New Approach to Group Aspergillus niger Strains.

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes-A. calidoustus and A. implicatus-as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species.

Effect of Methionine on Gene Expression in Komagataella phaffii Cells.

Komagataella phaffii yeast plays a prominent role in modern biotechnology as a recombinant protein producer. For efficient use of this yeast, it is essential to study the effects of different media components on its growth and gene expression. We investigated the effect of methionine on gene expression in K. phaffii cells using RNA-seq analysis. Several gene groups exhibited altered expression when K. phaffii cells were cultured in a medium with methanol and methionine, compared to a medium without this amino acid. Methionine primarily affects the expression of genes involved in its biosynthesis, fatty acid metabolism, and methanol utilization. The AOX1 gene promoter, which is widely used for heterologous expression in K. phaffii, is downregulated in methionine-containing media. Despite great progress in the development of K. phaffii strain engineering techniques, a sensitive adjustment of cultivation conditions is required to achieve a high yield of the target product. The revealed effect of methionine on K. phaffii gene expression is important for optimizing media recipes and cultivation strategies aimed at maximizing the efficiency of recombinant product synthesis.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Prospects of biosimilar recombinant human growth hormone manufacture in the Republic of Moldova

In collaboration of SUMPh „N.Testemițanu”, the pharmaceutical company Balkan Pharmaceuticals and ICGEB the technology transfer during scale-up from the laboratory phase to the pilot phase of the processes of creating the working cell bank and growing Pichia pastoris strains in the Erlenmeyer flask were carried out. Pichia pastoris was selected for recombinant growth hormone as an expression system, which has many advantages over other organisms, including protein folding, post-translational changes in protein, and easy handling. The GH1 altered recombinant human somatropin gene was introduced into the genome of Pichia pastoris culture using restriction near the AOX1 gene promoter as a vector serving the pPIC9K plasmid. The GH1 gene was activated by the AOX1 gene promoter which can be easily induced by the presence of methanol in the culture medium. Subsequently, the „Prepro-alpha Factor Leader” sequence from Saccharomyces cerevisiae was added to the modified sequence so that the obtained somatropin could be secreted into the culture medium. In the technological transfer process the influence of the chosen technological processes on some parameters of the obtained product were examined, Technological Regulations were developed for short and long term storage processes of Pichia pastoris cells and for the process of inoculum preparation, inoculation and growth of P. pastoris strains with recombinant human growth hormone in the Erlenmeyer flask. Standard Operating Procedures were developed for the processes of creating the working cell bank, determining the in-vitro biological activity of the recombinant human growth hormone preparation, with subsequent implementation in the process of producing rhGH biosimilar at CS Balkan Pharmaceuticals Ltd.

Evaluation of biochemical and molecular response of onion breeding lines to drought and salt stresses

Onion is the most important crop in the Alliaceae family which is diversely used all over the world. Türkiye is the 5th largest onion producer with the production of 2.24 million tons, but onion production is under threat due to climatic changes especially in the Anatolia region. Therefore, in the present study, six onion breeding lines were investigated to quantify the biochemical and gene expressional changes under drought and salt stresses. Plants were divided into control, salt stress, and drought stress to apply stress treatment under semi-controlled greenhouse conditions. Current study showed that salt stress restricted the uptake of minerals from the soil. Study also revealed that under both stresses the chlorophyll a and b contents were stable in K41, U47 and U49 breeding lines. Moreover, gene expression for stress related genes showed that K41 and U47 breeding lines showed tolerance to drought and salt stress by adequately regulating their SOD, CAT, AOX, WRKY70 and NAC29 gene expressions, moreover highest antioxidant enzymatic activity was also observed in these breeding lines under drought and salt stress. The H2O2 concentration was highest in the sensitive breeding lines suggesting the sensitivity to both stresses. PCA analysis revealed that breeding lines K41, U47 and U49 showed tolerance to both stresses. These results can further used to understand onion stress tolerance mechanism and it may enable onion breeders for the development of abiotic stress resilient onion cultivars.

Comparasion of the effectiveness of anchor proteins ScAGα1p, KpCW51p, KpCW61p for surface display in yeast <i>Komagataella phaffii</i>

BACKGROUND: Yeast display is an effective technology for exposure target proteins to the cell surface by fusing them with cell wall proteins. This technique, among other things, makes it possible to obtain vaccine preparations based on yeast by exposing antigen proteins on their cell surface. Finding and selecting proteins that allow effective exposure of target proteins on the surface of yeast cells is an urgent task.
 AIM: The aim of this work was to evaluate the efficiency of cell wall proteins ScAG1p, KpCW51p, KpCW61p for displaying the reporter protein on the Komagataella phaffii cell surface, including the study of several variants of the ScAG1 gene coding sequence.
 MATERIALS AND METHODS: The studied gene sequences were cloned under the control of the AOX1 gene promoter in the same reading frame as the eGFP reporter protein gene and integrated into the genome of the K. Phaffii yeast strain X-33.
 RESULTS: Cytoimmunochemical analysis and confocal microscopy of strains displaying the eGFP protein on their surface under conditions of induction of the AOX1 gene promoter made it possible to identify the most effective anchor protein. The best efficiency was demonstrated for the sequence of the ScAG1 gene without the native 3' non-coding region.
 CONCLUSIONS: The plasmids obtained in the work will make it possible to obtain a yeast strain K. phaffii that effectively exposure proteins, including antigens, on its surface, which can be used as a vaccine preparation.

Suppression of mitochondrial alternative oxidase can result in upregulation of the ROS scavenging network: some possible mechanisms underlying the compensation effect.

Mitochondrial alternative oxidase is an important protein involved in maintaining cellular metabolic and energy balance, especially under stress conditions. AOX genes knockout is aimed at revealing the functions of AOX genes. Under unfavourable conditions, AOX-suppressed plants (mainly based on Arabidopsis AOX1a-knockout lines) usually experience strong oxidative stress. However, a compensation effect, which consists of the absence of AOX1a leading to an increase in defence response mechanisms, concomitant with a decrease in ROS content, has also been demonstrated. This review briefly describes the possible mechanisms underlying the compensation effect upon the suppression of AOX1a. Information about mitochondrial retrograde regulation of AOX is given. The importance of ROS and mitochondrial membrane potential in triggering the signal transmission from mitochondria in the absence of AOX or disturbance of mitochondrial electron transport chain functions is indicated. The few available data on the response of the cell to the absence of AOX at the level of changes in the hormonal balance and the reactions of chloroplasts are presented. The decrease in the relative amount of reduced ascorbate at stable ROS levels as a result of compensation in AOX1a-suppressed plants is proposed as a sign of stress development. Obtaining direct evidence on the mechanisms and signalling pathways involved in AOX modulation in the genome should facilitate a deeper understanding of the role of AOX in the integration of cellular signalling pathways.

Glial cell reactivity and oxidative stress prevention in Alzheimer’s disease mice model by an optimized NMDA receptor antagonist

In Alzheimer's disease pathology, several neuronal processes are dysregulated by excitotoxicity including neuroinflammation and oxidative stress (OS). New therapeutic agents capable of modulating such processes are needed to foster neuroprotection. Here, the effect of an optimised NMDA receptor antagonist, UB-ALT-EV and memantine, as a gold standard, have been evaluated in 5XFAD mice. Following treatment with UB-ALT-EV, nor memantine, changes in the calcineurin (CaN)/NFAT pathway were detected. UB-ALT-EV increased neurotropic factors (Bdnf, Vgf and Ngf) gene expression. Treatments reduced astrocytic and microglial reactivity as revealed by glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) quantification. Interestingly, only UB-ALT-EV was able to reduce gene expression of Trem2, a marker of microglial activation and NF-κB. Pro-inflammatory cytokines Il-1β, Ifn-γ, Ccl2 and Ccl3 were down-regulated in UB-ALT-EV-treated mice but not in memantine-treated mice. Interestingly, the anti-inflammatory markers of the M2-migroglial phenotype, chitinase-like 3 (Ym1) and Arginase-1(Arg1), were up-regulated after treatment with UB-ALT-EV. Since iNOS gene expression decreased after UB-ALT-EV treatment, a qPCR array containing 84 OS-related genes was performed. We found changes in Il-19, Il-22, Gpx6, Ncf1, Aox1 and Vim gene expression after UB-ALT-EV. Hence, our results reveal a robust effect on neuroinflammation and OS processes after UB-ALT-EV treatment, surpassing the memantine effect in 5XFAD.

Heterologous synthesis of N and M fragments of capsid protein VP2 of avian infectious bursal disease virus in yeast Pichia pastoris

BACKGROUND: Infectious bursal disease is one of the most common and economically important viral diseases of birds. Vaccination is currently the most effective way to control IBD. Subunit vaccines contain only the immunogenic protein of the pathogen or its fragments, but do not contain other proteins, lipopolysaccharides, toxins, which avoids vaccination side effects.
 AIM: The aim of the work was to obtain yeast Pichia pastoris strains that synthesize and secrete the fragments of major coat protein VP2 of the infectious bursal disease virus.
 MATERIALS AND METHODS: The DNA sequences encoding the N and M fragments of VP2 protein, were cloned under the control of the AOX1 gene promoter and integrated into the genome of P. pastoris strains X-33 (mut+) and GS115 (his4).
 RESULTS: The analysis of proteins secreted by the obtained strains revealed the presence of additional proteins with a molecular weights corresponding to the target proteins.
 CONCLUSIONS: Thus, the obtained strains of P. pastoris producers of N and M fragments of VP2 protein can be used for antigen production to create a subunit vaccine against avian IBD.

Bioinformatics-Guided Analysis Uncovers AOX1 as an Osteogenic Differentiation-Relevant Gene of Human Mesenchymal Stem Cells.

Background: The available therapeutic options of bone defects, fracture nonunion, and osteoporosis remain limited, which are closely related to the osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs). Thus, there remains an urgent demand to develop a prediction method to infer osteogenic differentiation–related genes in BMSCs. Method: We performed differential expression analysis between hBMSCs and osteogenically induced samples. Association analysis, co-expression analysis, and PPI analysis are then carried out to identify potential osteogenesis-related regulators. GO enrichment analysis and GSEA are performed to identify significantly enriched pathways associated with AOX1. qRT-PCR and Western blotting were employed to investigate the expression of genes on osteogenic differentiation, and plasmid transfection was used to overexpress the gene AOX1 in hBMSCs. Result: We identified 25 upregulated genes and 17 downregulated genes. Association analysis and PPI network analysis among these differentially expressed genes show that AOX1 is a potential regulator of osteogenic differentiation. GO enrichment analysis and GSEA show that AOX1 is significantly associated with osteoblast-related pathways. The experiments revealed that AOX1 level was higher and increased gradually in differentiated BMSCs compared with undifferentiated BMSCs, and AOX1 overexpression significantly increased the expression of osteo-specific genes, thereby clearly indicating that AOX1 plays an important role in osteogenic differentiation. Moreover, our method has ability in discriminating genes with osteogenic differentiation properties and can facilitate the process of discovery of new osteogenic differentiation–related genes. Conclusion: These findings collectively demonstrate that AOX1 is an osteogenic differentiation-relevant gene and provide a novel method established with a good performance for osteogenic differentiation-relevant genes prediction.