Molecular Biotechnology最新文献

This study focused on identifying potential key lncRNAs associated with gout under the mechanisms of copper death and iron death through ceRNA network analysis and Random Forest (RF) algorithm, which aimed to provide new insights into the molecular mechanisms of gout, and potential molecular targets for future therapeutic strategies of gout. Initially, we conducted an in-depth bioinformatics analysis of gout microarray chips to screen the key cuproptosis-related genes (CRGs) and key ferroptosis-related genes (FRGs). Using these data, we constructed a key ceRNA network for gout. Finally, key lncRNAs associated with gout were identified through the RF algorithm combined with ROC curves, and validated using the Comparative Toxicogenomics Database (CTD). We successfully identified NLRP3, LIPT1, and DBT as key CRGs associated with gout, and G6PD, PRKAA1, LIG3, PHF21A, KLF2, PGRMC1, JUN, PANX2, and AR as key FRGs associated with gout. The key ceRNA network identified four downregulated key lncRNAs (SEPSECS-AS1, LINC01054, REV3L-IT1, and ZNF883) along with three downregulated mRNAs (DBT, AR, and PRKAA1) based on the ceRNA theory. According to CTD validation inference scores and biological functions of target mRNAs, we identified a potential gout-associated lncRNA ZNF883/hsa-miR-539-5p/PRKAA1 regulatory axis. This study identified the key lncRNA ZNF883 in the context of copper death and iron death mechanisms related to gout for the first time through the application of ceRNA network analysis and the RF algorithm, thereby filling a research gap in this field and providing new insights into the molecular mechanisms of gout. We further found that lncRNA ZNF883 might function in gout patients by regulating PRKAA1, the mechanism of which was potentially related to uric acid reabsorption in the proximal renal tubules and inflammation regulation. The proposed lncRNA ZNF883/hsa-miR-539-5p/PRKAA1 regulatory axis might represent a potential RNA regulatory pathway for controlling the progression of gout disease. This discovery offered new molecular targets for the treatment of gout, and had significant implications for future therapeutic strategies in managing the gout.

Potato (Solanum tuberosum L.), an important horticultural crop is a member of the family Solanaceae and is mainly grown for consumption at global level. Starch, the principal component of tubers, is one of the significant elements for food and non-food-based applications. The genes associated with biosynthesis of starch have been investigated extensively over the last few decades. However, a complete regulation pathway of constituent of amylose and amylopectin are still not deeply explored. The current in-silico study of genes related to amylose and amylopectin synthesis and their genomic organization in potato is still lacking. In the current study, the nucleotide and amino acid arrangement in genome and twenty-two genes linked to starch biosynthesis pathway in potato were analysed. The genomic structure analysis was also performed to find out the structural pattern and phylogenetic relationship of genes. The genome mining and structure analysis identified ten specific motifs and phylogenetic analysis of starch biosynthesis genes divided them into three different clades on the basis of their functioning and phylogeny. Quantitative real-time PCR (qRT-PCR) of amylose biosynthesis pathway genes in three contrast genotypes revealed the down-gene expression that leads to identify potential cultivar for functional genomic approaches. These potential lines may help to achieve higher content of resistant starch.

Invasive plants are known to cause biodiversity loss and pose a major risk to human health and environment. Identification of invasive plants and distinguishing them from native species has been relied on morphological examination. Stringent requirement of floral characters and decreasing number of expert taxonomists are making conventional morphology-based identification system tedious and resource-intensive. DNA barcoding may help in quick identification of invasive species if distinct sequence divergence pattern at various taxonomic levels is observed. The present work evaluates the utility of four molecular markers; rbcL, matK, their combination (rbcL + matK), and psbA-trnH for identification of 37 invasive plant species from India and also in distinguishing them from 97 native species. A psbA-trnH locus was found to be of restricted utility in this work as it was represented by the members of a single family. A hierarchical increase in K2P mean divergence across different taxonomic levels was found to be the maximum for matK alone followed by rbcL + matK and rbcL alone, respectively. NJ clustering analysis, however, confirmed the suitability of combined locus (rbcL + matK) over individual rbcL and matK as the DNA barcode. RbcL showed the lowest resolution power among the three markers studied. MatK exhibited much better performance compared to rbcL alone in identifying most of the species accurately although it failed to show monophyly of genus Dinebra. Two families; Asteraceae and Poaceae, remained polyphyletic in the trees constructed by all three markers. Combined locus (rbcL + matK) was found to be the most suitable marker as it raised the resolution power of both the markers and could identify more than 90% of genera correctly. Phylogenetic tree constructed by Maximum-Parsimony method using combined locus as a molecular marker exhibited the best resolution, thus, supporting the significance of two-locus combination of rbcL + matK for barcoding invasive plant species from India. Present study contributes to the global barcode data of invasive plant species by adding fifty-one new sequences to it. Effective barcoding of additional number of native as well as invasive plant species from India is possible using this dual locus if it is combined with one or more new molecular plastid markers. Expansion of barcode database with a focus on barcode performance optimisation to improve discrimination ability at species level can be undertaken in future.

Precise quantification of human cells in preclinical animal models by a sensitive and specific approach is warranted. The probe-based quantitative PCR (qPCR) assay as a sensitive and swift approach is suitable for the quantification of human cells by targeting human-specific DNA sequences. In this study, we developed an efficient qPCR assay targeting human-specific DNA in ST6GALNAC3 (termed ST6GAL-qPCR) for the quantification of human cells in preclinical animal models. ST6GAL-qPCR probe was synthesized with FAM and non-fluorescent quencher-minor groove binder conjugated to the 5' and 3' end of the probe, respectively. Genomic DNA from human, rhesus monkeys, cynomolgus monkeys, New Zealand White rabbits, SD rats, C57BL/6, and BALB/c mice were utilized for analyzing the specificity and sensitivity of the ST6GAL-qPCR assay. The ST6GAL-qPCR assay targeted human-specific DNA was cloned to pUCM-T vector and released by EcoR I/Hind III digestion for generating a calibration curve. Cell mixing experiment was performed to validate the ST6GAL-qPCR assay by analysis of 0.1%, 0.01%, and 0.001% of human leukocytes mixed with murine thymocytes. The ST6GAL-qPCR assay detected human DNA rather than DNA from the tested animal species. The amplification efficiency of the ST6GAL-qPCR assay was 93% and the linearity of calibration curve was R² = 0.999. The ST6GAL-qPCR assay detected as low as 5 copies of human-specific DNA and is efficient to specially amplify as low as 30-pg human DNA in the presence of 1 μg of DNA from the tested species, respectively. The ST6GAL-qPCR assay was able to quantify as low as 0.01% of human leukocytes within murine thymocytes. This ST6GAL-qPCR assay can be used as an efficient approach for the quantification of human cells in preclinical animal models.

Sufentanil is a common opioid anesthetic agent, which exerts anti-cancer properties in several cancer types. However, its action mechanisms in non-small cell lung cancer (NSCLC) are unclear. Therefore, the present study investigated the pharmacological effect of sufentanil on miRNAs in NSCLC treatment. In this study, after treatment with sufentanil, the proliferation, migration, invasion and apoptosis of A549 and H1299 NSCLC cell lines were measured by cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assays and flow cytometry. Quantitative real time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of miR-186-5p and high mobility group box-1 (HMGB1), and their interaction was analyzed using luciferase reporter assay. The proteins of HMGB1, and apoptosis- and Wnt/β-catenin pathway-related factors were detected by western blot. It was demonstrated that sufentanil significantly upregulated miR‑186‑5p to restrict NSCLC cell proliferation, migration, invasion, and boost apoptosis in vitro. Mechanically, miR-186-5p interacted with HMGB1 and negatively regulated HMGB1 in NSCLC cells. Furthermore, rescue assay showed that sufentanil exerted antitumor activities by upregulating miR-186-5p, which targeted HMGB1 and restrained Wnt/β-catenin signal pathway in NSCLC cells. In conclusion, these results suggested that sufentanil disrupts the oncogenicity of NSCLC cells by regulating miR-186-5p/HMGB1/β-catenin axis, providing a promising implication for the anti-oncogenic effect of sufentanil.

The environment monitoring of forest is vital for the ecosystem sustainable management, especially soil quality. Ancient Gleditsia sinensis is one of the most distributed ancient trees in Shaanxi. Comprehensive soil evaluate is important for the ancient tree protection. In this study, we selected the most distributed ancient tree Gleditsia sinensis and immature tree to compare the effect of growth stage to soil quality and soil bacteria. Most ancient tree soil nutrients were in good condition compared with immature tree. The bacterial community were composed with Proteobacteria (27.55%), Acidobacteriota (16.82%), Actinobacteriota (15.77%), Gemmatimonadota (6.82%), Crenarchaeota (4.61%), Bacteroidota (4.41%), Firmicutes (4.32%), Chloroflexi (4.28%), Planctomycetota (3.24%) and Verrucomicrobiota (3.04%). The level 2 ancient tree (300-400 years old) was different in bacterial community diversity. SOC and STN were important to level 2 (300-400 years old Gleditsia sinensis), and other levels were opposite. Our results suggested that the ancient tree management should not be lumped together.

A new and simple platform to produce a nanocarrier for small-peptide antigen delivery was developed. Virus-like particles (VLPs) were of interest due to their good cell-penetrating properties and ability to protect target molecules from degradation. In this study, the VLP that was entirely formed by influenza neuraminidase (NA), NA-VLPs, was employed. The platform construction includes the genetic engineering of target peptides into the NA structure immediately above its stalk, at the bottom of the NA head, by an overlap extension PCR. The resulting chimeric gene is next expressed in stably transformed insect cells. The recombinant NA protein produced by the insect cells is then naturally assembled into the NA-VLPs that display those peptides on their surfaces. For the platform demonstration, Angiotensin II (AngII) octapeptide hormones that raise blood pressure were chosen as a model peptide antigen. The NA-VLPs displaying AngII peptides were successfully produced by the stably transformed insect cells. The AngII octapeptides were successfully delivered by the NA-AngII VLPs as the anti-AngII antibodies were raised in hypertensive rats. The antibodies effectively neutralized the AngII peptide hormone in these rats, as demonstrated by the decrease in systolic blood pressure of the immunized rats. Thus, NA-VLP nanocarriers represent a promising platform for delivering small-peptide antigens to stimulate antibody production.

The subfamily Plusiinae, an economically important moth pest group, belongs to the species-rich family Noctuidae (Lepidoptera). Despite their enormous economic importance, the evolutionary history of this subfamily has not been completely resolved. In India, they are represented by a species complex, but the taxonomic delineation among these organisms is unclear. This study represents an insight into the comprehensive phylogenetic relationship among species supported by molecular approach based on mitochondrial (Cytochrome Oxidase I) and nuclear gene markers (Ribosomal Protein S5), emphasizing tribal-level classification. A total of 125 plusiinae taxa were analysed from eight biogeographical zones of India. The results revealed that Plusiinae tribes were monophyletic and considered sister groups that shared many derived characteristics. The ML/MP cladogram based on the barcoding gene successfully separates all species but not all tribes. The nuclear gene marker RPS5, separated all the species according to their tribes. The combined analysis of both genes showed tribe resolution into distinct clades. This is the first comprehensive study on phylogenetic studies of 25 species of plusiinae from India that clarifies deep divergence and gives information about species position and arrangement within taxa.

Alzheimer's disease (AD) is a neurodegenerative disease distinguished by cognitive and memory deficits. A lack of memory, cognition, and other forms of cognitive dissonance characterizes AD, which affects approximately 50 million people worldwide. This study aimed to identify the neuroprotective effects of berberine chloride (BC) against aluminium chloride (AlCl₃)-induced AD in zebrafish larvae by inhibiting oxidative stress and neuroinflammation. BC toxicity was assessed by evaluating survival rates, malformations, and heart rates in zebrafish larvae following treatment with varying concentrations of BC. This study elucidates the mechanisms of BC through an extensive range of biochemical assays, behavioral testing, and molecular docking analysis. The developmental toxicity assessment of BC indicated that doses up to 40 μM did not cause any developmental abnormalities until 96 h post fertilization. The LC₅₀ value of BC in zebrafish larvae was found to be 50.16 μM. The biochemical and behavioral changes induced by AlCl₃ in zebrafish larvae were significantly mitigated by BC treatment. Our findings demonstrate that BC can reduce total cholesterol and triglyceride levels in AlCl₃-induced AD zebrafish larvae. Our molecular docking results indicated that BC significantly interacted with the ABCA1 protein, suggesting that BC may act as an ABCA1 agonist. Based on our results, it can be concluded that BC may serve as an effective therapeutic agent for mitigating oxidative stress by altering cholesterol metabolism in AlCl₃-induced AD.

The ethylene-responsive transcription factors (ERFs) perform pivotal regulatory functions in plant growth, development, and stress responses. Nonetheless, there is limited research on the functional characterization of ERFs in the medicinal orchid, Dendrobium catenatum. Here, we identified a salt-induced ERF gene DcERF3 from a D. catenatum cultivar Tiepi. DcERF3 comprises 186 amino acids and has a confirmed molecular weight of 21 kDa. It possesses a conserved AP2/ERF domain and displays a strong affiliation with the evolutionary lineage of other characterized ERFs. Analysis of expression patterns indicated that DcERF3 exhibits predominant expression in stems and roots, with considerably higher levels than in other tissues, and it demonstrated significant upregulation in response to treatments involving salt, ETH, PEG, and SA. The DcERF3-YFP protein localizes to the nucleus, and DcERF3 displays distinct transcriptional activation characteristics. Overexpressing DcERF3 led to an increased lateral root formation and enhanced tolerance to salt stress in Arabidopsis. Furthermore, the activities of antioxidant enzymes, along with the stress-responsive genes, were significantly induced in transgenic plants when subjected to salt stress. This study aims to investigate the function and role of DcERF3 in D. catenatum to establish a foundation for examining its involvement in lateral root formation and response to salt stress.