Disparity of gene expression in coronary artery disease: insights from MEIS1, HIRA, and Myocardin.

INTRODUCTION: Coronary artery disease (CAD) in young adults can have devastating consequences. The cardiac developmental gene MEIS1 plays important roles in vascular networks and heart development. This gene effects on the regeneration capacity of the heart. Considering role of MEIS1 in cardiac tissue development and the progression of myocardial infarction this study investigated the expression levels of the MEIS1, HIRA, and Myocardin genes in premature CAD patients compared to healthy subjects and evaluated the relationships between these genes and possible inflammatory factors. METHODS AND RESULTS: The study conducted a case-control design involving 35 CAD patients and 35 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were collected, and gene expression analysis was performed using real-time PCR. Compared with control group, the number of PBMCs in the CAD group exhibited greater MEIS1 and HIRA gene expression, with fold changes of 2.45 and 3.6. The expression of MEIS1 exhibited a negative correlation with IL-10 (r= -0.312) expression and positive correlation with Interleukin (IL)-6 (r = 0.415) and tumor necrosis factor (TNF)-α (r = 0.534) gene expression. Moreover, there was an inverse correlation between the gene expression of HIRA and that of IL-10 (r= -0.326), and a positive correlation was revealed between the expression of this gene and that of the IL-6 (r = 0.453) and TNF-α (r = 0.572) genes. CONCLUSION: This research demonstrated a disparity in expression levels of MEIS1, HIRA, and Myocardin, between CAD and healthy subjects. The results showed that, MEIS1 and HIRA play significant roles in regulating the synthesis of proinflammatory cytokines, namely, TNF-α and IL-6.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Plasticity of Gene Expression and Thermal Tolerance: Implications for Climate Change Vulnerability in a Tropical Forest Lizard.

AbstractTropical ectotherms are thought to be especially vulnerable to climate change because they have evolved in temporally stable thermal environments and therefore have decreased tolerance for thermal variability. Thus, they are expected to have narrow thermal tolerance ranges, live close to their upper thermal tolerance limits, and have decreased thermal acclimation capacity. Although models often predict that tropical forest ectotherms are especially vulnerable to rapid environmental shifts, these models rarely include the potential for plasticity of relevant traits. We measured phenotypic plasticity of thermal tolerance and thermal preference as well as multitissue transcriptome plasticity in response to warmer temperatures in a species that previous work has suggested is highly vulnerable to climate warming, the Panamanian slender anole lizard (Anolis apletophallus). We found that many genes, including heat shock proteins, were differentially expressed across tissues in response to short-term warming. Under long-term warming, the voluntary thermal maxima of lizards also increased, although thermal preference exhibited only limited plasticity. Using these data, we modeled changes in the activity time of slender anoles through the end of the century under climate change and found that plasticity should delay declines in activity time by at least two decades. Our results suggest that slender anoles, and possibly other tropical ectotherms, can alter the expression of genes and phenotypes when responding to shifting environmental temperatures and that plasticity should be considered when predicting the future of organisms under a changing climate.

Generation of Eroded Nanoplastics from Domestic Wastes and Their Impact on Macrophage Cell Viability and Gene Expression.

This study reports an innovative approach for producing nanoplastics (NP) from various types of domestic waste plastics without the use of chemicals. The plastic materials used included water bottles, styrofoam plates, milk bottles, centrifuge tubes, to-go food boxes, and plastic bags, comprising polyethylene terephthalate (PET), polystyrene (PS), polypropylene (PP), high-density polyethylene (HDPE), and Poly (Ethylene-co-Methacrylic Acid) (PEMA). The chemical composition of these plastics was confirmed using Raman and FTIR spectroscopy, and they were found to have irregular shapes. The resulting NP particles ranged from 50 to 400 nm in size and demonstrated relative stability when suspended in water. To assess their impact, the study investigated the effects of these NP particulates on cell viability and the expression of genes involved in inflammation and oxidative stress using a macrophage cell line. The findings revealed that all types of NP reduced cell viability in a concentration-dependent manner. Notably, PS, HDPE, and PP induced significant reductions in cell viability at lower concentrations, compared to PEMA and PET. Moreover, exposure to NP led to differential alterations in the expression of inflammatory genes in the macrophage cell line. Overall, this study presents a viable method for producing NP from waste materials that closely resemble real-world NP. Furthermore, the toxicity studies demonstrated distinct cellular responses based on the composition of the NP, shedding light on the potential environmental and health impacts of these particles.

Extremely low-frequency electromagnetic field (ELF-EMF) induced alterations in gene expression and cytokine secretion in clear cell renal carcinoma cells.

BACKGROUND: The study aimed to investigate the influence of extremely low-frequency electromagnetic fields (ELF-EMF) on clear cell renal cell carcinoma (ccRCC) by assessing alterations in gene expression and the secretion of cytokines and chemokines. MATERIAL AND METHODS: Three ccRCC cell lines (786-O, 769-P, and CAKI-1) and a healthy HEK293 cell line were subjected to ELF-EMF exposure (frequency 50 Hz, magnetic field strength 4.5 mT) for 30 min daily for 5 days. The study examined the expression of ADAM28, NCAM1, and VEGFC genes, along with the secretion of 30 cytokines and chemokines. RESULTS: Notably, primary tumor-derived cell lines, but not those from metastatic sites, exhibited ADAM28 gene expression, which increased following ELF-EMF exposure. A statistically significant reduction in VEGFC gene expression was observed in 769-P cells after ELF-EMF exposure. Additionally, NCAM1 gene expression was upregulated in HEK293, 769-P, and 786-O cells, representing normal embryonic kidney cells and primary tumor cells, but not in CAKI-1 cells, which model metastatic sites. After EMF exposure, there was a statistically significant decrease in transforming growth factor ß1 (TGF-ß1) concentration in the cell culture supernatants of HEK293 and CAKI-1 cell lines, with no other significant changes in the secretion of tested cytokines. CONCLUSIONS: Given the study's findings and available research, caution is warranted when drawing conclusions about the potential inhibitory effect of ELF-EMF on ccRCC progression. Standardization of experimental models is imperative when assessing the effects of EMF in a human context. Med Pr Work Health Saf. 2024;75(2):133-141.

TA-Cloning for Diabetes Treatment: Expressing Corynebacterium Malic Enzyme Gene in E. coli.

Diabetes mellitus represents a persistent metabolic condition marked by heightened levels of blood glucose, presenting a considerable worldwide health concern, and finding targeted treatment for it is a crucial priority for global health. Gram-positive aerobic bacteria, predominantly inhabiting water and soil, are known carriers of various enzyme-encoding genetic material, which includes the malic enzyme gene that plays a role in insulin secretion. Corynebacterium glutamicum bacteria (ATCC 21799) were acquired from the Pasteur Institute and confirmed using microbiological and molecular tests, including DNA extraction. After identification, gene purification and cloning of the maeB gene were performed using the TA Cloning method. Additionally, the enhancement of enzyme expression was assessed using the expression vector pET-28a, and validation of simulation results was monitored through a real-time PCR analysis. Based on previous studies, the malic enzyme plays a pivotal role in maintaining glucose homeostasis, and increased expression of this enzyme has been associated with enhanced insulin sensitivity. However, the production of malic enzyme has encountered numerous challenges and difficulties. This study successfully isolated the malic enzyme genes via Corynebacterium glutamicum and introduced them into Escherichia coli for high-yield production. According to the results, the optimum temperature for the activity of enzymes has been identified as 39 °C.

Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

OBJECTIVE: To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. MATERIALS AND METHODS: A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. RESULTS: Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. CONCLUSION: The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. WHAT DOES THIS STUDY ADDS TO THE CLINICAL WORK: Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

The role of lncFABP4 in modulating adipogenic differentiation in buffalo intramuscular preadipocytes.

Intramuscular fat (IMF) is a crucial determinant of meat quality and is influenced by various regulatory factors. Despite the growing recognition of the important role of long noncoding RNAs (lncRNAs) in IMF deposition, the mechanisms underlying buffalo IMF deposition remain poorly understood. In this study, we identified and characterized a lncRNA, lncFABP4, which is transcribed from the antisense strand of fatty acid-binding protein 4 (FABP4). lncFABP4 inhibited cell proliferation in buffalo intramuscular preadipocytes. Moreover, lncFABP4 significantly increased intramuscular preadipocyte differentiation, as indicated by an increase in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG), CCAAT enhancer binding protein alpha (C/EBPα), and FABP4. Mechanistically, lncFABP4 was found to have the potential to regulate downstream gene expression by participating in protein-protein interaction pathways. These findings contribute to further understanding of the intricate mechanisms through which lncRNAs modulate intramuscular adipogenesis in buffaloes.

Disproportionate neuroanatomical effects of DCC haploinsufficiency in adolescence compared with adulthood: links to dopamine, connectivity, covariance, and gene expression brain maps in mice.

BACKGROUND: Critical adolescent neural refinement is controlled by the DCC (deleted in colorectal cancer) protein, a receptor for the netrin-1 guidance cue. We sought to describe the effects of reduced DCC on neuroanatomy in the adolescent and adult mouse brain. METHODS: We examined neuronal connectivity, structural covariance, and molecular processes in a DCC-haploinsufficient mouse model, compared with wild-type mice, using new, custom analytical tools designed to leverage publicly available databases from the Allen Institute. RESULTS: We included 11 DCC-haploinsufficient mice and 16 wild-type littermates. Neuroanatomical effects of DCC haploinsufficiency were more severe in adolescence than adulthood and were largely restricted to the mesocorticolimbic dopamine system. The latter finding was consistent whether we identified the regions of the mesocorticolimbic dopamine system a priori or used connectivity data from the Allen Brain Atlas to determine de novo where these dopamine axons terminated. Covariance analyses found that DCC haploinsufficiency disrupted the coordinated development of the brain regions that make up the mesocorticolimbic dopamine system. Gene expression maps pointed to molecular processes involving the expression of DCC, UNC5C (encoding DCC's co-receptor), and NTN1 (encoding its ligand, netrin-1) as underlying our structural findings. LIMITATIONS: Our study involved a single sex (males) at only 2 ages. CONCLUSION: The neuroanatomical phenotype of DCC haploinsufficiency described in mice parallels that observed in DCC-haploinsufficient humans. It is critical to understand the DCC-haploinsufficient mouse as a clinically relevant model system.

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Deficiency of interleukin-19 exacerbates acute lung injury induced by intratracheal treatment of hydrochloric acid.

Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.

Inferring Stochastic Rates from Heterogeneous Snapshots of Particle Positions.

Many imaging techniques for biological systems-like fixation of cells coupled with fluorescence microscopy-provide sharp spatial resolution in reporting locations of individuals at a single moment in time but also destroy the dynamics they intend to capture. These snapshot observations contain no information about individual trajectories, but still encode information about movement and demographic dynamics, especially when combined with a well-motivated biophysical model. The relationship between spatially evolving populations and single-moment representations of their collective locations is well-established with partial differential equations (PDEs) and their inverse problems. However, experimental data is commonly a set of locations whose number is insufficient to approximate a continuous-in-space PDE solution. Here, motivated by popular subcellular imaging data of gene expression, we embrace the stochastic nature of the data and investigate the mathematical foundations of parametrically inferring demographic rates from snapshots of particles undergoing birth, diffusion, and death in a nuclear or cellular domain. Toward inference, we rigorously derive a connection between individual particle paths and their presentation as a Poisson spatial process. Using this framework, we investigate the properties of the resulting inverse problem and study factors that affect quality of inference. One pervasive feature of this experimental regime is the presence of cell-to-cell heterogeneity. Rather than being a hindrance, we show that cell-to-cell geometric heterogeneity can increase the quality of inference on dynamics for certain parameter regimes. Altogether, the results serve as a basis for more detailed investigations of subcellular spatial patterns of RNA molecules and other stochastically evolving populations that can only be observed for single instants in their time evolution.

An important consideration when expressing mAbs in Escherichiacoli.

Monoclonal antibodies (mAbs) are a driving force in the biopharmaceutical industry. Therapeutic mAbs are usually produced in mammalian cells, but there has been a push towards the use of alternative production hosts, such as Escherichia coli. When the genes encoding for a mAb heavy and light chains are codon-optimized for E. coli expression, a truncated form of the heavy chain can form along with the full-length product. In this work, the role of codon optimization in the formation of a truncated product was investigated. This study used the amino acid sequences of several therapeutic mAbs and multiple optimization algorithms. It was found that several algorithms incorporate sequences that lead to a truncated product. Approaches to avoid this truncated form are discussed.

Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.

Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB. ONE-SENTENCE SUMMARY: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.

Antibiotics alter development and gene expression in the model cnidarian Nematostella vectensis.

Background: Antibiotics are commonly used for controlling microbial growth in diseased organisms. However, antibiotic treatments during early developmental stages can have negative impacts on development and physiology that could offset the positive effects of reducing or eliminating pathogens. Similarly, antibiotics can shift the microbial community due to differential effectiveness on resistant and susceptible bacteria. Though antibiotic application does not typically result in mortality of marine invertebrates, little is known about the developmental and transcriptional effects. These sublethal effects could reduce the fitness of the host organism and lead to negative changes after removal of the antibiotics. Here, we quantify the impact of antibiotic treatment on development, gene expression, and the culturable bacterial community of a model cnidarian, Nematostella vectensis. Methods: Ampicillin, streptomycin, rifampicin, and neomycin were compared individually at two concentrations, 50 and 200 µg mL-1, and in combination at 50 µg mL-1 each, to assess their impact on N. vectensis. First, we determined the impact antibiotics have on larval development. Next Amplicon 16S rDNA gene sequencing was used to compare the culturable bacteria that persist after antibiotic treatment to determine how these treatments may differentially select against the native microbiome. Lastly, we determined how acute (3-day) and chronic (8-day) antibiotic treatments impact gene expression of adult anemones. Results: Under most exposures, the time of larval settlement extended as the concentration of antibiotics increased and had the longest delay of 3 days in the combination treatment. Culturable bacteria persisted through a majority of exposures where we identified 359 amplicon sequence variants (ASVs). The largest proportion of bacteria belonged to Gammaproteobacteria, and the most common ASVs were identified as Microbacterium and Vibrio. The acute antibiotic exposure resulted in differential expression of genes related to epigenetic mechanisms and neural processes, while constant application resulted in upregulation of chaperones and downregulation of mitochondrial genes when compared to controls. Gene Ontology analyses identified overall depletion of terms related to development and metabolism in both antibiotic treatments. Discussion: Antibiotics resulted in a significant increase to settlement time of N. vectensis larvae. Culturable bacterial species after antibiotic treatments were taxonomically diverse. Additionally, the transcriptional effects of antibiotics, and after their removal result in significant differences in gene expression that may impact the physiology of the anemone, which may include removal of bacterial signaling on anemone gene expression. Our research suggests that impacts of antibiotics beyond the reduction of bacteria may be important to consider when they are applied to aquatic invertebrates including reef building corals.

Changes in life history parameters and expression of key genes of Brachionus plicatilis exposed to a combination of organic and inorganic ultraviolet filters.

The increasing use of ultraviolet filters has become an emerging contaminant on the coast, posing potential ecological risks. Rotifers are essential components of marine ecosystems, serving as an association between primary producers and higher-level consumers. These organisms frequently encounter ultraviolet filters in coastal waters. This study aimed to assess the comprehensive effects of organic ultraviolet filters, specifically 2-ethylhexyl-4-methoxycinnamate (EHMC), and inorganic ultraviolet filters, namely, titanium dioxide nanoparticles (TiO2 NPs), on the rotifer Brachionus plicatilis. We exposed B. plicatilis to multiple combinations of different concentrations of EHMC and TiO2 NPs to observe changes in life history parameters and the expression of genes related to reproduction and antioxidant responses. Our findings indicated that increased EHMC concentrations significantly delayed the age at first reproduction, reduced the total offspring, and led to considerable alterations in the expression of genes associated with reproduction and stress. Exposure to TiO2 NPs resulted in earlier reproduction and decreased total offspring, although these changes were not synchronised in gene expression. The two ultraviolet filters had a significant interaction on the age at first reproduction and the total offspring of rotifer, with these interactions extending to the first generation. This research offers new insights into the comprehensive effects of different types of ultraviolet filters on rotifers by examining life history parameters and gene expression related to reproduction and stress, highlighting the importance of understanding the impacts of sunscreen products on zooplankton health.

Size-dependent enhancement of gene expression by Plasmodium 5'UTR introns.

BACKGROUND: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5' and 3' untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown. METHODS: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5'UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5'UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5'UTR intron were generated using the CRISPR/Cas9 system. RESULTS: We showed that promoters with 5'UTR introns had higher activities in driving gene expression than those without 5'UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5'UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5'UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages. CONCLUSIONS: Plasmodium 5'UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5'UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation.

Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Decoding cardiovascular risks: analyzing type 2 diabetes mellitus and ASCVD gene expression.

Background: ASCVD is the primary cause of mortality in individuals with T2DM. A potential link between ASCVD and T2DM has been suggested, prompting further investigation. Methods: We utilized linear and multivariate logistic regression, Wilcoxon test, and Spearman's correlation toanalyzethe interrelation between ASCVD and T2DM in NHANES data from 2001-2018.The Gene Expression Omnibus (GEO) database and Weighted Gene Co-expression Network Analysis (WGCNA) wereconducted to identify co-expression networks between ASCVD and T2DM. Hub genes were identified using LASSO regression analysis and further validated in two additional cohorts. Bioinformatics methods were employed for gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, along with the prediction of candidate small molecules. Results: Our analysis of the NHANES dataset indicated a significant impact of blood glucose on lipid levels within diabetic cohort, suggesting that abnormal lipid metabolism is a critical factor in ASCVD development. Cross-phenotyping analysis revealed two pivotal genes, ABCC5 and WDR7, associated with both T2DM and ASCVD. Enrichment analyses demonstrated the intertwining of lipid metabolism in both conditions, encompassing adipocytokine signaling pathway, fatty acid degradation and metabolism, and the regulation of adipocyte lipolysis. Immune infiltration analysis underscored the involvement of immune processes in both diseases. Notably, RITA, ON-01910, doxercalciferol, and topiramate emerged as potential therapeutic agents for both T2DM and ASCVD, indicating their possible clinical significance. Conclusion: Our findings pinpoint ABCC5 and WDR7 as new target genes between T2DM and ASCVD, with RITA, ON-01910, doxercalciferol, and topiramate highlighted as promising therapeutic agents.