Molecular Biotechnology最新文献

Chinese hamster ovary (CHO) cells play a crucial role in biopharmaceutical production due to their ability to produce complex proteins. Enhancing the productivity of CHO cells is essential for meeting the growing demand for biologics. Caspase 8-Associated Protein 2 (CASP8AP2), a key regulator of apoptosis and cell survival, has been identified as a potential target to increase CHO cell productivity. To this end, CRISPR-mediated homology-independent targeted integration (HITI) was used to silence CASP8AP2. Results of the cell viability assay revealed that CASP8AP2-deficient clones (C2, C3, and C4) were more resistant to sodium butyrate (NaBu) compared to native cells, with IC50 values of 11.83, 12.77, 10.25, and 8.55 mM, respectively. Protein production assays showed a significant increase in JRed and luciferase expression in silenced clones (C2 and C4) compared to wild-type cells, with up to 1.2- and 1.9-fold increases for JRed, and 1.4- and 1.7-fold increases for luciferase, respectively. These findings could be attributed to the clones experiencing cell cycle arrest specifically during the S phase. While these results demonstrate proof-of-principle using reporter proteins, future validation with therapeutic biologics such as implementing monoclonal antibodies in bioreactor settings could confirm scalability for industrial bioprocessing. Transcriptomic analyses would further elucidate downstream effects on apoptosis and metabolism pathways. The results suggest that targeting CASP8AP2 could be a promising strategy for improving bioprocess efficiency and yield in CHO cell-based production systems.

Bacterial lipases and esterases are central to lipid metabolism and biotechnology, yet they are underexplored in purple non-sulfur photosynthetic bacteria. To address this gap, we mined the genome of Cereibacter sphaeroides 2.4.1 by retrieving the sequences annotated by alpha beta hydrolase, esterase, and lipase from NCBI database. All sequences aligned to well-known of bacterial esterase or lipase and the higher identity was cloned and expressed in Escherichi coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed soluble expression of four proteins, with LipCs2 (26.4 kDa), LipCs3 (39.4 kDa), and LipCs9 (24.1 kDa) showing activity on tributyrin agar and preference for short-chain para-nitrophenyl esters. Although LipCs9 (314.11 ± 2.44 mM h^-1 mg^-1) showed the highest activity on pNP-butyrate, LipCs3 (25.57 ± 2.22 mM h^-1 mg^-1) was prioritized due to its novelty and strong performance rather than LipCs2 (17.00 ± 0.62 mM h^-1 mg^-1). Optimization of IPTG induction (0.25, 0.5, and 1 mM) for LipCs3 expression in E. coli revealed severe cytotoxicity in both cytoplasmic and periplasmic systems, prompting its heterologous expression in Bacillus subtilis WB800. Despite successful secretion, the intracellular (10.29 U) and extracellular (11.24 U) levels remained nearly equal, indicating inefficient signal peptide function. Phylogenetic analysis classified LipCs3 within bacterial family IV lipolytic enzymes, closely related to Brefeldin A esterase. Docking analysis revealed a close proximity between the ligand carbonyl group of pNP-butyrate and the hydroxyl of Ser153 within the binding pocket. Genomic analysis revealed a GntR binding motif overlapping the -10 promoter region of lipCs3 gene, suggesting transcriptional regulation. These findings broaden the current understanding of family IV bacterial lipolytic by establishing LipCs3 as a novel representative of this group.

CRISPR-Cas9 technology is revolutionizing genetic engineering by enabling precise genome modifications across a wide range of organisms, particularly in livestock. This review focuses on livestock improvement and the most recent transformative developments in the application of CRISPR-based genome editing techniques. We analyzed productivity improvements achieved by editing growth factor genes, immunogenic resistance enhancement through the editing of immune loci, and ecological footprint reduction for sustainability. This review also addresses the intricate ethical and regulatory issues posed by the application of CRISPR technology in animal breeding, including concerns about animal welfare violations, unintended off-target effects, and impacts on public perception. Furthermore, we discuss key untapped CRISPR targets in livestock genetics enabled by precision gene editing, the emerging integration of CRISPR with artificial intelligence, and the need for collaboration to address complex ethical and regulatory challenges related to applying CRISPR in animal breeding.

Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer, representing around 97% of cases in humans. Mitochondria play a crucial role in slowing the progression of HCC and are utilized in developing precision medicine strategies targeting various mitochondria-related pathways. Recent developments in nanomedicine exploit the unique features of nanoparticles for improving drug delivery and efficacy specifically to mitochondria, bypassing the problems related to conventional chemotherapy. Mitochondria-targeted nanomedicines are developed for damaging mitochondrial DNA in liver cancer tissues by increasing reactive oxygen species perturbing redox balance and respiratory chain. This review emphasizes the future of mitochondrial-targeted nanomedicine in transforming liver cancer management and encourages an attempt to continuously improve these strategies to provide patients with better survival chances and quality of life.

Inflammation-related disorders constitute a major global health challenge, with annual incidence exceeding tens of millions of cases and economic losses surpassing hundreds of billions USD. These burdens significantly impact both healthcare systems and socioeconomic development. Current clinical therapies are limited by suboptimal efficacy, which poses health risks to patients and underscores the demand for innovative therapeutic strategies. Emerging evidence demonstrates that extracellular vesicles (EVs) exhibit significant therapeutic advantages over conventional pharmacological interventions in managing inflammatory disorders. Native EVs possess inherent advantages including superior biocompatibility, structural integrity, and unique biological barrier penetration capacities, positioning them as promising therapeutic agents for inflammatory conditions. Engineered EVs can be functionalized with targeted delivery systems to transport bioactive components, such as miRNAs and proteins, enabling precise modulation of inflammatory signaling pathways. This approach enhances therapeutic efficacy, shortens treatment duration, reduces morbidity rates, and decreases healthcare costs. This review examines the therapeutic potential of EVs in inflammatory diseases and summarizes recent advances in their application to specific inflammatory disorders. We discuss the clinical translation challenges of EVs and highlight their prospects for inflammatory disease therapeutics.

Molecular diagnostics have revolutionized veterinary and clinical medicine by enabling rapid, sensitive, and specific detection of pathogens and genetic markers. This comprehensive review traces the evolution of diagnostic techniques from traditional culture and serology methods to advanced molecular technologies such as polymerase chain reaction (PCR), digital PCR (dPCR), real-time PCR, isothermal amplification, and hybridization-based approaches including DNA arrays and padlock probes. The review highlights the benefits and limitations of each technology, emphasizing the enhanced sensitivity and specificity molecular methods offer over conventional diagnostics. Emerging technologies such as microfluidic lab-on-a-chip devices and nanotechnology-based sensors are discussed for their potential to provide rapid, cost-effective, and multiplexed diagnostics at the point of care. The critical importance of assay validation and laboratory quality standards is also explored, underscoring their role in ensuring reliable diagnostic outcomes in veterinary clinical and regulatory settings. Collectively, these advances have significantly improved disease detection, surveillance, and management in animal health, with ongoing innovations promising further breakthroughs in diagnostic capabilities.

The whitefly Bemisia tabaci is an invasive, polyphagous pest that transmits pathogenic plant viruses in several important crops worldwide. Whitefly management is challenging because of their rapid reproduction, polyphagy and development of resistance against conventional insecticides. RNA interference (RNAi) is a next-generation biopesticide technology with high species-specificity and environmentally friendly. Clay nanosheets are used in this study as a carrier for dsRNAs delivery targeting two B. tabaci insecticide receptors, ryanodine receptor (BtRyR) and nicotinic acetylcholine receptor subunit β1 (BtnAChR-β1), via foliar spray and root dip method. Clay nanoparticles are synthesised by the hydrothermal method and characterised to confirm an average diameter of 42 nm. Synthesised nanoclay loaded with dsRNA, and the complete loading ratio achieved was 1:10 (dsRNA: Nanoclay). Three different concentrations of dsRNA-nanoclay complex were taken (20 µg/mL, 40 µg/mL, and 60 µg/mL). A Real-Time qPCR analysis indicated a significant reduction in the expression of both targeted genes (~ 30-70%). Silencing of both target genes led to decreased survival (60-100% for different treatments), reduced egg laying (48.19-10.81% as compared to control) and delayed adult emergence (34.88-7.26% as compared to control). Gene silencing using the root dip method shows higher efficiency than the foliar spray method. LC₅₀ for the dsRNA-nanoclay complex of BtRyR and BtnAChR-β1 for the root dip method was 16.08 µg/mL and 14.13 µg/mL, respectively, lower than those of dsRNA alone for both target genes. These findings emphasize the practical applicability of clay nanosheet-based RNAi as an eco-friendly and scalable strategy for effective management of B. tabaci sustainable alternative to chemical insecticides.

Puerarin demonstrates therapeutic potential in alleviating chronic postoperative pain (CPSP) through multi-target mechanisms. While previous studies established its ferroptosis-inhibiting and anti-inflammatory properties via lipid peroxidation reduction and iron-mediated apoptosis regulation, its specific efficacy in CPSP remained unexplored. This study integrated network pharmacology with experimental validation using a skin/muscle incision-retraction (SMIR) rat model. Protein-protein interaction network analysis, Gene Ontology annotation, and KEGG pathway enrichment revealed puerarin's dual action pathway: modulating Th17 cell differentiation and regulating the HIF1 signaling axis. Molecular docking confirmed high-affinity binding between puerarin and five core targets: HIF1A, PTGS2, mTOR, RELA, and GSK3β. In vivo validation showed puerarin significantly elevated mechanical pain thresholds in SMIR rats while downregulating mRNA expression of these targets via qPCR. The compound's multimodal mechanism involves coordinated suppression of inflammatory signaling cascades and hypoxia-responsive pathways. These findings establish a robust methodology combining computational prediction with biological validation for herbal compound research.

In the current study, zinc hydroxide gel was prepared as an adjuvant system for adsorption of tetanus toxoid antigen. The vaccine's efficacy was established by estimating different immunological markers (IgG, IL-1α, IL-1β, IL-6) against tetanus antigen in mice (n = 48, Swiss albino Balb/C strain) at suitable intervals to develop protective antibody titers. The safety of the test formulation was confirmed by specific toxicity studies in guinea pigs (n = 7, Dunkin Hartley strain). The efficacy of the tetanus toxoid vaccine such prepared was increased by incorporating zinc that activated both humoral and cell-mediated immune responses. The test formulation demonstrated improved potency (113.8 IU/SHD) compared with the control formulation (106.4 IU/SHD). Toxicity studies were conducted on mice and guinea pigs to ensure safety. The test formulation (containing zinc hydroxide as adjuvant) showed a majority of small-sized particles (2.0-5.0 µm) compared to the control, with few larger particles (> 100 µm). Immunological markers were elevated in the test formulation compared to the control, indicating a better immune response. The low zinc concentration in the test formulation could reduce potential toxicity risks.

MicroRNAs (miRNAs) and epigenetic processes in bovine adipocytes can provide valuable new insights into controlling adipogenesis in livestock. It modulates gene expression at the posttranscriptional stage by affecting mRNA translation and stability. It plays a significant role in the regulation of a wide range of physiological and cellular functions, such as immune system processes and cellular and intramuscular fat development. Transcriptional regulators such as PPARγ and C/EBP proteins are key mediators of the critical processes governing progenitor cell differentiation into adipocytes. The differentiation of these progenitor cells into adipocytes, as well as the subsequent lipid deposition within the adipocytes, is influenced by a variety of factors. These include genetics; epigenetics; nutritional; hormonal regulation; weaning and slaughtering; aging and weight; and stress levels. Epigenetic modifications such as chromatin reorganization, histone alterations, and DNA methylation subsequently affect the activation of genes related to adipogenesis and the progression of adipocyte differentiation. By investigating how fat deposition is epigenetically regulated in beef cattle, scientists aim to unravel molecular mechanisms, identify key regulatory genes and pathways, and develop targeted strategies for modifying fat deposition to enhance desirable traits such as marbling and meat tenderness. This review paper delves into miRNAs and epigenetic factors and their role in regulating bovine adipocytes and it will identify links to transcription factors that influence diverse aspects of meat quality, encompassing flavor, juiciness, tenderness, firmness, texture, and visual appearance. Cutting-edge developments in genomics, including the identification of molecular markers and genetic variations, hold promise for enhancing animal productivity and meeting the global demand for premium-quality meat. This review establishes a foundation for future research on understanding regulatory networks linked to miRNAs and epigenetic changes, contributing to both scholarly knowledge advancement and practical applications within animal agriculture.