RESUMEN

Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 µg/ml) and (14.80 ± 0.13 µg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.

Asunto(s)

Gonadotropina Coriónica , Cricetulus , Proteínas Recombinantes , Células CHO , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Humanos , Gonadotropina Coriónica/genética , Gonadotropina Coriónica/biosíntesis , Gonadotropina Coriónica/farmacología , Cricetinae , Señales de Clasificación de Proteína/genética , Expresión Génica , Transfección

RESUMEN

Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some ß-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.

Asunto(s)

Celulasa , Cryptococcus gattii , Escherichia coli , Cryptococcus gattii/genética , Cryptococcus gattii/enzimología , Cryptococcus gattii/química , Celulasa/genética , Celulasa/química , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Celulasa/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/biosíntesis , Expresión Génica , Clonación Molecular , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Concentración de Iones de Hidrógeno , Temperatura

RESUMEN

This study aimed to examine whether dietary supplementation of broiler chickens with turmeric essential could mitigate the effects of cyclic heat stress conditions. Intestinal and immunological parameters and gene expression were evaluated during the grower phase. A total of 320 21-day-old male Cobb 500 broilers were distributed according to a completely randomized design with a 4 (diet) × 2 (environment) factorial arrangement and eight replications of five birds each. Dietary treatments consisted of a basal diet without essential oil (EO, negative control) and three diets containing low (100 mg kg-1), intermediate (200 mg kg-1), or high (300 mg kg-1) levels of turmeric EO. In the heat stress group, dietary supplementation with turmeric EO at 100 and 200 mg kg-1 improved body weight, feed conversion, breast yield, and relative liver weight. These supplementation levels reduced villus width, increased villus/crypt ratio, reduced the H/L ratio, and improved hepatic (HSP70 and SREBP1) and intestinal (OCLN) gene expression in birds under heat stress. These findings support the hypothesis that turmeric EO can be used to improve or restore intestinal integrity, modulate inflammation parameters, and, consequently, enhance the performance of broilers challenged by cyclic heat stress.

Asunto(s)

Pollos , Curcuma , Dieta , Suplementos Dietéticos , Expresión Génica , Respuesta al Choque Térmico , Intestinos , Aceites Volátiles , Animales , Pollos/inmunología , Pollos/crecimiento & desarrollo , Aceites Volátiles/farmacología , Aceites Volátiles/administración & dosificación , Masculino , Intestinos/efectos de los fármacos , Respuesta al Choque Térmico/efectos de los fármacos , Dieta/veterinaria , Expresión Génica/efectos de los fármacos , Alimentación Animal , Calor , Hígado/metabolismo , Trastornos de Estrés por Calor/veterinaria , Trastornos de Estrés por Calor/prevención & control , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética

RESUMEN

CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2-12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r2 by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site's position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.

Asunto(s)

Sistemas CRISPR-Cas , Uso de Codones , Edición Génica , Uso de Codones/genética , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Biología Computacional/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Codón/genética , Expresión Génica/genética

RESUMEN

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: • Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells • The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly • The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.

Asunto(s)

Dependovirus , Retículo Endoplásmico , Vectores Genéticos , Proteína 1 de Unión a la X-Box , Humanos , Células HEK293 , Dependovirus/genética , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismo , Retículo Endoplásmico/metabolismo , Vectores Genéticos/genética , Expresión Génica , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Cápside/metabolismo

RESUMEN

Episomal AMA1-based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, the gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next-generation AMA1-based plasmids that ensure homogeneous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5-fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10-fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi.

Asunto(s)

Aspergillus nidulans , Sistemas CRISPR-Cas , Plásmidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Plásmidos/genética , Expresión Génica , Ingeniería Metabólica/métodos , Vías Biosintéticas/genética , Productos Biológicos/metabolismo

RESUMEN

Herbicides are the main class of pesticides applied in crops and are capable of polluting the surrounding freshwater system; thus, understanding their impact on non-target species, whose mechanism of action is not described, helps to elucidate the real risks of these pollutants to the environment. 2,4-dichlorophenoxyacetic acid (2,4-D) is frequently detected in water and, due to its persistence, poses a risk to wildlife. In this way, the present work aimed to describe the implication of exposure to concentrations of 2,4-D already reported in aquatic environments in several physiological mechanisms of C. riparius at molecular and biochemical levels. To achieve this, bioassays were conducted with fourth instar larvae exposed to three concentrations of 2,4-D (0.1, 1.0, and 7.5 µg L-1). Larvae were collected after 24 and 96 h of exposure, and the expression of 42 genes, related to six subcellular mechanisms, was assessed by Real-Time PCR (RT-PCR). Besides, the activity of the enzymes catalase (CAT), glutathione S-transferase (GST), and acetylcholinesterase (AChE) was determined. The main metabolic route altered after exposure to 2,4-D was the endocrine system (mainly related to 20-hydroxyecdysone and juvenile hormone), confirming its endocrine disruptor potential. Four of the eleven stress response genes studied were down-regulated, and later exposure modulated DNA-repair genes suggesting genotoxic capacity. Moreover, only one gene from each detoxification phase was modulated at short exposure to 1.0 µg L-1. The molecular responses were not dose-dependent, and some early responses were not preserved after 96 h, indicating a transient response to the herbicide. Exposure to 2,4-D did not alter the activity of CAT, GST, and AChE enzymes. The responses described in this study reveal new mechanistic pathways of toxicity for 2,4-D in non-target organisms and highlight potential ecological consequences for chironomids in aquatic systems at the edges of agricultural fields.

Asunto(s)

Ácido 2,4-Diclorofenoxiacético , Chironomidae , Glutatión Transferasa , Herbicidas , Ácido 2,4-Diclorofenoxiacético/toxicidad , Animales , Chironomidae/efectos de los fármacos , Chironomidae/genética , Herbicidas/toxicidad , Glutatión Transferasa/metabolismo , Glutatión Transferasa/genética , Acetilcolinesterasa/metabolismo , Acetilcolinesterasa/genética , Larva/efectos de los fármacos , Larva/genética , Larva/metabolismo , Contaminantes Químicos del Agua/toxicidad , Catalasa/metabolismo , Catalasa/genética , Expresión Génica/efectos de los fármacos

RESUMEN

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.

Asunto(s)

Glucosiltransferasas , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/biosíntesis , Glicosilación , Especificidad por Sustrato , Expresión Génica

RESUMEN

OBJECTIVE: Glucagon-like peptide-1 (GLP1) and leptin (Lep) are afferent signals that regulate energy metabolism. Lactational hypernutrition results in hyperphagia and adiposity in adult life, and these events can be prevented by exercise. We evaluated the effects of swimming training on hypothalamic (GLP1-R) and Lep receptor (Lep-R) gene expressions in lactational hypernutrition-induced obesity. METHODS: On the 3rd postnatal day, the litter sizes of lactating dams were adjusted to small litters (SL; 3 pups/dams) or normal litters (NL; 9 pups/dams). After weaning (21 days), NL and SL male rats were randomly distributed to sedentary (Sed) and exercised (Exe) groups. Exercised mice swam (30 min/3 times/week) for 68 days. Food intake and body weight gain were registered. At 92 days, intraperitoneal glucose and insulin tolerance tests were performed and rats were euthanized at 93 days; adipose tissue depots were weighed, and blood counts and plasma biochemical analyses performed. Hypothalamus were isolated to evaluate Lep-R and GLP1-R gene expressions. RESULTS: Small litters sedentary rats presented increased body weight gain, adiposity, insulin sensibility and higher fasting values of glucose and triglycerides, besides higher hypothalamic gene expressions of Lep-R and GLP1-R, compared to NLSed animals. SLExe rats did not develop obesity or metabolic abnormalities and Lep-R and GLP1-R hypothalamic gene expressions were normalized. CONCLUSION: Lactational hypernutrition induces obesity and metabolic dysfunction in adult life, in association with higher hypothalamic expressions of the Lep-R and GLP1-R genes. Exercise prevented obesity and improved metabolic state in SL overnourished rats, and normalized their hypothalamic Lep-R and GLP1-R gene expressions.

Asunto(s)

Hipotálamo , Obesidad , Condicionamiento Físico Animal , Ratas Wistar , Receptores de Leptina , Natación , Animales , Hipotálamo/metabolismo , Obesidad/metabolismo , Obesidad/genética , Obesidad/prevención & control , Condicionamiento Físico Animal/fisiología , Condicionamiento Físico Animal/métodos , Masculino , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Femenino , Natación/fisiología , Tamaño de la Camada , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Ratas , Lactancia/metabolismo , Lactancia/fisiología , Péptido 1 Similar al Glucagón/metabolismo , Leptina/sangre , Leptina/metabolismo , Distribución Aleatoria , Expresión Génica , Ingestión de Alimentos/fisiología , Adiposidad/fisiología

RESUMEN

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Asunto(s)

Clostridium butyricum , Plásmidos , Clostridium butyricum/genética , Plásmidos/genética , Humanos , Expresión Génica , Biotecnología/métodos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Celulasa/genética , Celulasa/metabolismo , Genes Reporteros , Microbiología Industrial/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos

RESUMEN

Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.

Asunto(s)

Baculoviridae , Glicoproteínas Hemaglutininas del Virus de la Influenza , Subtipo H9N2 del Virus de la Influenza A , Virus de la Enfermedad de Newcastle , Spodoptera , Animales , Baculoviridae/genética , Células Sf9 , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H9N2 del Virus de la Influenza A/genética , Virus de la Enfermedad de Newcastle/genética , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Expresión Génica , Clonación Molecular/métodos , Vectores Genéticos/genética

RESUMEN

Cumulative evidence suggests that zebrafish is a useful model in psychiatric research. Weighted Gene Co-expression Network Analysis (WGCNA) enables the reduction of genome-wide expression data to modules of highly co-expressed genes, which are hypothesized to interact within molecular networks. In this study, we first applied WGCNA to zebrafish brain expression data across different experimental conditions. Then, we characterized the different co-expression modules by gene-set enrichment analysis and hub gene-phenotype association. Finally, we analyzed association of polygenic risk scores (PRSs) based on genes of some interesting co-expression modules with alcohol dependence in 524 patients and 729 controls from Galicia, using competitive tests. Our approach revealed 34 co-expression modules in the zebrafish brain, with some showing enrichment in human synaptic genes, brain tissues, or brain developmental stages. Moreover, certain co-expression modules were enriched in psychiatry-related GWAS and comprised hub genes associated with psychiatry-related traits in both human GWAS and zebrafish models. Expression patterns of some co-expression modules were associated with the tested experimental conditions, mainly with substance withdrawal and cold stress. Notably, a PRS based on genes from co-expression modules exclusively associated with substance withdrawal in zebrafish showed a stronger association with human alcohol dependence than PRSs based on randomly selected brain-expressed genes. In conclusion, our analysis led to the identification of co-expressed gene modules that may model human brain gene networks involved in psychiatry-related traits. Specifically, we detected a cluster of co-expressed genes whose expression was exclusively associated with substance withdrawal in zebrafish, which significantly contributed to alcohol dependence susceptibility in humans.

Asunto(s)

Alcoholismo , Encéfalo , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Pez Cebra , Animales , Encéfalo/metabolismo , Alcoholismo/genética , Humanos , Herencia Multifactorial , Modelos Animales de Enfermedad , Expresión Génica/genética

RESUMEN

BACKGROUND: Adrenal-origin and peripheral tissue-transformed 11-oxygenated androgens are recognized as significant androgens. However, our current understanding of the synthesis of 11-oxygenated androgens, including the organs and cell types involved, remains limited. METHODS: We performed comprehensive analyses on an extensive dataset of normal human tissues, which included bulk RNA data from 30 tissues, single-cell RNA sequencing (scRNA) data from 16 tissues and proteomics data from 29 tissues, to characterize the expression profiles of enzyme-encoding genes. To validate the findings, immunohistochemical and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques were employed. RESULTS: Our investigation revealed that the gene expression levels of the enzymes HSD11B2 and AKR1C3 were notably elevated in the kidney and intestines. Intriguingly, within these organs, we observed an increasing trend in enzyme expression with age in women, while a decreasing trend was apparent in men. scRNA analysis revealed that HSD11B2 was predominantly expressed in collecting duct principal cells in the kidney, while AKR1C3 was primarily expressed in the proximal tubules. Intriguingly, nearly all epithelial cells in the intestine expressed these key enzymes. Further analysis using LC-MS/MS revealed that the kidney exhibited the highest levels of 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT) among the seven tissues examined, and substantial synthesis of 11KA4 and 11KT was also observed in the intestine. Finally, we developed the TransMap website (http://gxmujyzmolab.cn:16245/TransMap/) to provide comprehensive visualization of all currently available transcriptome data. CONCLUSION: This study offers an overarching perspective on tracing the synthesis of 11-oxygenated androgens in peripheral tissues, thereby providing valuable insights into the potential role of these androgens in humans.

Asunto(s)

Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Andrógenos , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida , Masculino , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/genética , Femenino , Andrógenos/biosíntesis , Andrógenos/metabolismo , Riñón/metabolismo , Riñón/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Adulto , Persona de Mediana Edad , Expresión Génica , Cromatografía Líquida con Espectrometría de Masas

RESUMEN

C-reactive protein (CRP) plays a crucial role in the diagnosis and monitoring of the non-specific acute phase response in humans. In contrast, rat CRP (rCRP) is an atypical acute-phase protein that possesses unique features, such as a possible incapacity to trigger the complement system and markedly elevated baseline plasma concentrations. To facilitate in vitro studies on these unique characteristics, obtaining high-quality pure rCRP is essential. Here we explored various strategies for rCRP purification, including direct isolation from rat plasma and recombinant expression in both prokaryotic and eukaryotic systems. Our study optimized the recombinant expression system to enhance the secretion and purification efficiency of rCRP. Compared to traditional purification methods, we present a streamlined and effective approach for the expression and purification of rCRP in the Pichia pastoris system. This refined methodology offers significant improvements in the efficiency and effectiveness of rCRP purification, thereby facilitating further structural and functional studies on rCRP.

Asunto(s)

Proteína C-Reactiva , Proteínas Recombinantes , Animales , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Ratas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/genética , Expresión Génica , Saccharomycetales/genética , Saccharomycetales/metabolismo , Pichia/genética , Pichia/metabolismo

RESUMEN

AIMS: 5 mC methylation and hydroxymethylation (5hmC) are associated with Alzheimer's disease (AD). However, previous studies were limited by the absence of a 5hmC calculation. This study aims to find AD associated predictors and potential therapeutic chemicals using bioinformatics approach integrating 5 mC, 5hmC, and expression changes, and an AD mouse model. METHODS: Gene expression microarray and 5 mC and 5hmC sequencing datasets were downloaded from GEO repository. 142 AD and 52 normal entorhinal cortex specimens were enrolled. Data from oxidative bisulfite sequencing (oxBS)-treated samples, which represent only 5 mC, were used to calculate 5hmC level. Functional analyses, random forest supervised classification and methylation validation were applied. Potential chemicals were predicted by CMap. Morris water maze, Y maze and novel object recognition behavior tests were performed using FAD4T AD mice model. Cortex and hippocampus tissues were isolated for immunohistochemical staining. RESULTS: C1QTNF5, UBD, ZFP106, NEDD1, AKT3, and MBP genes involving 13 promoter CpG sites with 5mc, 5hmC methylation and expression difference were identified. AKT3 and MBP were down-regulated in both patients and mouse model. Three CpG sites in AKT3 and MBP showed significant methylation difference on validation. FAD4T AD mice showed recession in brain functions and lower AKT3 expression in both cortex and hippocampus. Ten chemicals were predicted as potential treatments for AD. CONCLUSIONS: AKT3 and MBP may be associated with AD pathology and could serve as biomarkers. The ten predicted chemicals might offer new therapeutic approaches. Our findings could contribute to identifying novel markers and advancing the understanding of AD mechanisms.

Asunto(s)

Enfermedad de Alzheimer , Metilación de ADN , Proteínas Proto-Oncogénicas c-akt , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Animales , Ratones , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Masculino , Modelos Animales de Enfermedad , Biomarcadores/metabolismo , Corteza Entorrinal/metabolismo , Corteza Entorrinal/patología , Femenino , Anciano , Hipocampo/metabolismo , Expresión Génica , Ratones Transgénicos

RESUMEN

Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides, an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii. The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

Asunto(s)

Proteínas Fúngicas , Edulcorantes , Edulcorantes/química , Edulcorantes/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/química , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/química , Humanos , Gusto , Expresión Génica , Simulación por Computador

RESUMEN

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (ß-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Asunto(s)

Técnicas de Transferencia de Gen , Vectores Genéticos , Penaeidae , Animales , Penaeidae/virología , Penaeidae/genética , Células Sf9 , Vectores Genéticos/genética , Baculoviridae/genética , Regiones Promotoras Genéticas , Spodoptera/virología , Densovirinae/genética , Expresión Génica , Virus del Síndrome de la Mancha Blanca 1/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo

RESUMEN

Astrocytes, a major class of glial cells, are an important element at the synapse where they engage in bidirectional crosstalk with neurons to regulate numerous aspects of neurotransmission, circuit function, and behavior. Mutations in synapse-related genes expressed in both neurons and astrocytes are central factors in a vast number of neurological disorders, making the proteins that they encode prominent targets for therapeutic intervention. Yet, while the roles of many of these synaptic proteins in neurons are well established, the functions of the same proteins in astrocytes are largely unknown. This gap in knowledge must be addressed to refine therapeutic approaches. In this chapter, we integrate multiomic meta-analysis and a comprehensive overview of current literature to show that astrocytes express an astounding number of genes that overlap with the neuronal and synaptic transcriptomes. Further, we highlight recent reports that characterize the expression patterns and potential novel roles of these genes in astrocytes in both physiological and pathological conditions, underscoring the importance of considering both cell types when investigating the function and regulation of synaptic proteins.

Asunto(s)

Astrocitos , Neuronas , Sinapsis , Astrocitos/metabolismo , Sinapsis/metabolismo , Humanos , Neuronas/metabolismo , Animales , Expresión Génica , Transmisión Sináptica , Transcriptoma

RESUMEN

Temporal ecological niche partitioning is an underappreciated driver of speciation. While insects have long been models for circadian biology, the genes and circuits that allow adaptive changes in diel-niches remain poorly understood. We compared gene expression in closely related day- and night-active non-model wild silk moths, with otherwise similar ecologies. Using an ortholog-based pipeline to compare RNA-Seq patterns across two moth species, we find over 25 pairs of gene orthologs showing differential expression. Notably, the gene disco, involved in circadian control, optic lobe and clock neuron development in Drosophila, shows robust adult circadian mRNA cycling in moth heads. Disco is highly conserved in moths and has additional zinc-finger domains with specific nocturnal and diurnal mutations. We propose disco as a candidate gene for the diversification of temporal diel-niche in moths.

Asunto(s)

Ritmo Circadiano , Mariposas Nocturnas , Animales , Mariposas Nocturnas/genética , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/fisiología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Evolución Biológica , Expresión Génica

RESUMEN

Poly-ADP-ribose polymerase-14 (PARP14) can modify proteins and nucleic acids by the reversible addition of a single ADP-ribose molecule. Aberrant PARP14 functions have been related to cancer and inflammation, and its domains are involved in processes related to viral infection. Previous research indicates that PARP14 functions might be mediated via a multitude of target proteins. In vitro studies of this large multidomain enzyme have been complicated by difficulties to obtain biochemical quantities of pure protein. Here we present a strategy that allows bacterial expression and purification of a functional multidomain construct of PARP14. We substituted an internal KH domain and its neighboring unstructured region with a SUMO domain to obtain a protein construct that encompasses three macrodomains, a WWE domain, and a PARP catalytic domain. We show that the resulting construct retains both ADP-ribosyltransferase and de-MARylase activities. This construct will be useful in structural and functional studies of PARP14.

Asunto(s)

Escherichia coli , Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Dominios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Expresión Génica , Clonación Molecular