Molecular Biotechnology最新文献

Whether it involves human subjects or non-human animals, basic, translational, or clinical sleep research poses significant ethical challenges for researchers and ethical committees alike. Sleep research greatly benefits from using diverse animal models, each offering unique insights into sleep control mechanisms. The fruit fly (Drosophila melanogaster) is a superior genetic model due to its quick generation period, large progenies, and rich genetic tools. Its well-characterized genome and ability to respond to hypnotics and stimulants make it an effective tool for studying sleep genetics and physiological foundations. The nematode (Caenorhabditis elegans) has a simpler neural organization and transparent body, allowing researchers to explore molecular underpinnings of sleep control. Vertebrate models, like zebrafish (Danio rerio), provide insights into circadian rhythm regulation, memory consolidation, and drug effects on sleep. Invertebrate models, like California sea hare (Aplysia californica) and Upside-down jellyfish (Cassiopea xamachana), have simpler nervous systems and behave similarly to humans, allowing for the examination of sleep principles without logistical and ethical challenges. Combining vertebrate and invertebrate animal models offers a comprehensive approach to studying sleep, improving our understanding of sleep regulation and potentially leading to new drug discovery processes for sleep disorders and related illnesses.

One of the recent emerging global health issues is long COVID. Among long COVID patients, long COVID-associated brain fog is an important area. We noted an immense gap in understanding the genes and associated pathways involved in long COVID-associated brain fog. Therefore, the study has been selected to understand the genes and pathways involved in patients with long COVID-associated brain fog. A GEO dataset, which was developed through the RNA-seq, was used for the analysis. The dataset encompasses 22 human samples of PBMC. The dataset (human samples of PBMC) was grouped into four cohorts for this study: healthy cohort, COVID convalescent, long COVID, and long COVID brain fog. Therefore, the selection criteria for the 22 PBMC samples were based on the individual infection type (COVID convalescent, long COVID, and long COVID brain fog) and the healthy cohort. Using DEG profile evaluation, we revealed 250 top-ranked DEGs with P values, Padj, baseMean, etc. From the top-ranked DEGs, we listed 24 significant DEGs and some significant DEGs are SMAD3 (P value = 6.34e-07), PF4 (P value = 1.88e-05), TNFAIP3 (P value = 3.70e-06), CXCL5 (P value = 1.22e-08), etc. Among the top-ranked DEGs, we found some genes linked with different biological functions, such as inflammatory cytokine secretion, inflammation, microclot formation, and BBB disruption. From our investigation, we found some genes that are associated with this condition, namely PF4, SMAD3, CXCL5, TNFAIP3, etc. From the literature survey and functional pathway enrichment analysis, we noted the function of the genes such as PF4, SMAD3, and CXCL5. We found that PF4 assists in clot formation, and SMAD3 is associated with neuroinflammation. Similarly, CXCL5 is an inflammatory marker associated with neuroinflammation and BBB damage. At the same time, the study with functional pathway enrichment analysis reflects that DEGs of long COVID-related brain fog might be associated with several biological pathways and processes, cell signatures, and gene-disease associations. It reflects that the disease is a highly complex one. Our study will provide an understanding of the genes and associated pathways in long COVID-related brain fog, which will assist in the next-generation biomarker discovery and therapeutics for these patients.

Diabetic osteoporosis (DOP), a complication associated with diabetes mellitus (DM), is a metabolic bone disorder characterized by a reduction in bone mass per unit volume, impaired bone tissue microarchitecture, heightened bone fragility, and increased susceptibility to fractures. Individuals with diabetes exhibit a significantly greater incidence of osteoporosis and related fractures than those without diabetes. These fractures present a significant challenge in terms of the healing process and can result in severe consequences, including fatalities. MicroRNAs (miRNAs), a class of noncoding RNAs, play a pivotal role in numerous human diseases and are implicated in the pathogenesis of DOP. This review initially elucidates the essential role of miRNAs in the pathogenesis of DOP. Next, we emphasize the potential significance of miRNAs as valuable biomarkers for diagnosing DOP and predicting DOP-related fractures. Furthermore, we explore the involvement of miRNAs in managing DOP through various pathways, including conventional pharmaceutical interventions and exercise therapy. Importantly, miRNAs exhibit potential as targeted therapeutic agents for effectively treating DOP. Finally, we highlight the use of novel materials and exosomes for miRNA delivery, which has significant advantages in the treatment of DOP and overcomes the limitations associated with miRNA delivery.

Non-small cell lung cancer (NSCLC), including squamous cell carcinoma and adenocarcinoma, ranks among the top 10 cancers worldwide in terms of prevalence and mortality. NSCLC, a highly malignant tumor, exhibits distant invasion and migration as well as an unfavorable prognosis. As an innovative circular RNA, hsa _circ_0109320 (circ_0109320) has been recognized as a promising cancer modulator. However, our understanding of the influence of circ_0109320 in NSCLC remains insufficient. Our research explored the clinical significance and effects of circ_0109320 on oncogenic non-small cell lung cancer (NSCLC) phenotypes. Microarray analysis and qPCR indicated that circ_0109320 expression in NSCLC specimens increased relative to that in adjacent normal tissues and was further elevated in metastatic lymph nodes. The specimens acquired from 25 patients confirmed these findings. Additionally, circ_0109320 indicated a good score (AUC = 0.688, P = 0.013) on the ROC curves, which suggests its suitability as a promising biomarker for lung cancer. Meanwhile, circ_0109320 was noticeably upregulated in lung cancer (LC) cell lines compared to human bronchial epithelial cells. Next, we performed loss- and gain-of-function experiments to examine the role of circ_0109320 in the tumor phenotypes of the cell lines. We observed that depletion or overexpression of circ_0109320 did not alter cell viability. However, the ectopic removal of circ_0109320 repressed the migration and invasion of A549 and SK-MES-1 cells, whereas circ_0109320 overexpression promoted cell migration and invasion. Furthermore, the examination of epithelial-mesenchymal transition (EMT) markers indicated that circ_0109320 elevates cell EMT activity. In conclusion, circ_0109320 level was highly associated with increased tumor cell proliferation and metastasis. circ_0109320 could be a promising predictor of clinical outcomes and a reliable target to treat NSCLC by inhibiting metastasis.

Y-box-binding protein 1 (YBX1) is a multifunctional oncoprotein with its nuclear localization contributing to chemo-resistance in breast cancer. Through its interactions with various proteins and lncRNAs, YBX1 promotes cancer cell migration, invasion, and metastasis. Despite its significant role in cancer progression, studies on YBX1's protein-protein interactions (PPIs) remain limited. Flavonoids are natural compounds with anticancer properties that inhibit metastasis, modulate immunity, and induce apoptosis, with minimal systemic toxicity, making them strong candidates for cancer therapy. Targeting PPIs offers a promising approach for cancer therapy and flavonoids, with their anticancer properties, may modulate these interactions. Our study focused on the YBX1 PPI network, specifically targeting HSPA1A, IGF2BP1, MECP2, G3BP1, EWSR1, PURA, and SYNCRIP. We selected four flavonoids Quercetin, Fisetin, Rutin, and Myricitrin based on literature and conducted 26 docking sessions. Further ADMET analysis indicated Quercetin and Fisetin as more favorable for drug-likeness parameters than Rutin and Myricitrin, which was underscored by MD simulation data. In vitro studies showed that Quercetin and Fisetin downregulated YBX1 expression in a dose-dependent manner (50 μM to 150 μM) in MCF-7 cells. Our study provides a preliminary understanding of YBX1 PPI and the potential of flavonoids to disrupt these interactions. This study investigates the potential of flavonoids to target YBX1 PPIs, providing insights into novel therapeutic strategies for YBX1-driven cancers.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46 bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46 bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext^+/-). Off-target effect analysis did not reveal off-target mutations in Ext^+/- mice caused by the two sgRNAs used. Compared to wild-type mice, Ext^+/- mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext^+/- mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext^+/- mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext^+/- mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext^+/- mice serve as a valuable model for further biomedical investigations related to the Ext1 gene.

Horseradish peroxidase (HRP) is a chromogenic glycoenzyme widely used in research, diagnostics, and therapeutics. Due to its high demand, various eukaryotic and prokaryotic expression systems have been employed for the production of recombinant HRP. Eukaryotic systems yield properly folded, fully functional enzymes with the necessary post-translational modifications. However, these systems can be costly, time-consuming, and prone to hyperglycosylation. In contrast, prokaryotic systems are simple, inexpensive, and readily available, but achieving proper folding and subsequent modifications can be challenging. In this study, we employed a simple and cost-effective method to produce recombinant HRP in soluble form, using the E. coli expression system. The produced enzyme demonstrated substantial activity (89.75 ± 3.25 U/mg) and resistance to heat (T_1/2 = 5 min at 50 °C), pH variations (up to 8), and H₂O₂ concentrations (up to 10 mM). Additionally, we systematically compared our method with those of other researchers, highlighting methodological details and outcomes of HRP production in E. coli.

Our previous study established the effectiveness of scopoletin (SC) in protecting mice against acute pancreatitis (AP) induced by cerulein and subsequent pulmonary injury. However, the precise molecular mechanisms underlying SC protective effects have yet to be elucidated. This research suggests that SC reduces the release of pro-inflammatory cytokines and nuclear factor kappa B (NF-κB) by activating the peroxisome proliferator-activated receptor γ (PPAR-γ) in mice suffering from AP. We observed the protective role of SC against the male Swiss mice with hourly intraperitoneal injections of cerulein (50 µg/kg) for six hours, followed by the administration of SC (10 mg/kg) one hour after AP induction, with or without the PPAR-γ antagonist GW9662 (0.3 mg/kg). The study exploration into the anti-inflammatory effects of SC revealed that a concentration of 25 µM enhanced the phagocytic clearance of dying pancreatic acinar cells by triggering the PPAR-γ signaling activation. Conversely, an in vitro assessment confirmed the presence of GW9662 counteracted the beneficial impact of SC on acinar cells. Molecular docking and simulation studies have shown that SC prompts significant structural changes in PPAR-γ. The in vivo, in vitro, and in silico analyses suggest that SC has potent anti-inflammatory properties that may be mediated by the activation of PPAR-γ signaling in cerulein-induced experimental pancreatitis.

Seed samples of two types of chickpea (Cicer arietinum Linn.), including variety A (NRCGR-4452) and variety B (local varieties), with different seed colors, were collected every five days for a total of four times during the seed development period. Non-targeted metabolome and transcriptome sequencing were conducted to identify differentially expressed genes and metabolites associated with chickpea seed coat color. The results indicated that the relative quercetin, pelargonidin, luteolin, rutin, myricetin, kaempferol, glycitin, and naringin contents were higher in variety A than in variety B, and that carbohydrate and amino acid metabolites had a greater impact on flavonoid metabolites. Quercetin, luteolin, and kaempferol were most significantly associated with seed color differences, the associated enzyme genes were LOC101491583 (callose synthase 5-like), LOC101503703 (flavonoid 3',5'-hydroxylase), LOC101514158 (callose synthase 5), LOC101497872 (UDP-glycosyltransferase 74F1-like), LOC101500232 (callose synthase 7 isoform X1), LOC101511206 (UDP-glycosyltransferase 73C3-like), LOC101502065 (galactoside 2-alpha-L-fucosyltransferase), LOC101492791 (sulfoquinovosyl transferase SQD2), and LOC101509377 (flavonol synthase). Additionally, the gene transcription factor MYB44 may regulate UDP-glycosyltransferase 73C3 to affect seed color differences.

This study was designed to clarify the role of GJA1 in colorectal cancer. qPCR was adopted to detect the GJA1 and miR-20a-5p expression levels in tumor tissues and cells; and EdU, Transwell assay, Scratch test to examine the migration, invasion, and proliferation of colorectal cancer cells. The EMT-related protein expression was measured by immunofluorescence and western Blot. The binding relationship between GJA1 and miR-20a-5p was examined using dual luciferase reporting subsystem. In situ hybridization was utilized to examine the miR-20a-5p expression in tumor tissues and metastases. Rescue experiments were performed by simultaneous transfection of sh-GJA1 inhibitor and miR-20a-5p inhibitor. The miR-20a-5p expression was high and the GJA1 expression was low in colorectal cancer tissues and cells. A targeting relationship was found in GJA1 and miR-20a-5p targets. The invasion, migration, and proliferation of colorectal cancer cells can be inhibited by overexpression of GJA1. Meanwhile, overexpression of GJA1 markedly elevated the e-cadherin expression, but reduced the levels of vimentin, α-SMA and n-cadherin expression. miR-20a-5p inhibitor + sh-GJA1 promoted the invasion, migration, and proliferation of colon cancer cells and EMT process. Overall, miR-20a-5p could target GJA1 to down-regulate the GJA1 expression, thereby regulating the EMT response, and ultimately promoting the progression of colorectal cancer.