Genome-wide identification,characterization, and expression analysis of aluminum-activated malate transporter genes (ALMTs) in <i>Gossypium hirsutum</i> L.

Aluminum-activated malate transporters (ALMT) are widely involved in plant growth and metabolic processes, including adaptation to acid soils, guard cell regulation, anion homeostasis, and seed development. Although ALMT genes have been identified in Arabidopsis, wheat, barley, and Lotus japonicus, little is known about its presence in Gossypium hirsutum L. In this study, ALMT gene recognition in diploid and tetraploid cotton were done using bioinformatics analysis that examined correlation between homology and evolution. Differentially regulated ALMT genetic profile in G. hirsutum was examined, using RNA sequencing and qRT-PCR, during six fiber developmental time-points, namely 5 d, 7 d, 10 d, 15 d, 20 d, and 25 d. We detected 36 ALMT genes in G. hirsutum, which were subsequently annotated and divided into seven sub-categories. Among these ALMT genes, 34 had uneven distribution across 14/26 chromosomes. Conserved domains and gene structure analysis indicated that ALMT genes were highly conserved and composed of exons and introns. The GhALMT gene expression profile at different DPA (days post anthesis) in different varieties of G. hirsutum is indicative of a crucial role of ALMT genes in fiber development in G. hirsutum. This study provides basis for advancements in the cloning and functional enhancements of ALMT genes in enhancing fiber development and augmenting high quality crop production.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

Targeting the “undruggable” cancer driver genes: <i>Ras</i>, <i>myc</i>, and <i>tp53</i>

The term “undruggable” is to describe molecules that are not targetable or at least hard to target pharmacologically. Unfortunately, some targets with potent oncogenic activity fall into this category, and currently little is known about how to solve this problem, which largely hampered drug research on human cancers. Ras, as one of the most common oncogenes, was previously considered “undruggable”, but in recent years, a few small molecules like Sotorasib (AMG-510) have emerged and proved their targeted anti-cancer effects. Further, myc, as one of the most studied oncogenes, and tp53, being the most common tumor suppressor genes, are both considered “undruggable”. Many attempts have been made to target these “undruggable” targets, but little progress has been made yet. This article summarizes the current progress of direct and indirect targeting approaches for ras, myc, two oncogenes, and tp53, a tumor suppressor gene. These are potential therapeutic targets but are considered “undruggable”. We conclude with some emerging research approaches like proteolysis targeting chimeras (PROTACs), cancer vaccines, and artificial intelligence (AI)-based drug discovery, which might provide new cues for cancer intervention. Therefore, this review sets out to clarify the current status of targeted anti-cancer drug research, and the insights gained from this review may be of assistance to learn from experience and find new ideas in developing new chemicals that directly target such “undruggable” molecules.     

Genome-wide identification of <i>U-box</i> gene family and expression analysis under abiotic stresses in <i>Salvia miltiorrhiza</i>

Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and in response to different abiotic stresses, but little is known about U-box genes in Danshen (root of Salvia miltiorrhiza Bunge). Here, we identified and characterized 70 SmPUB genes based on its genome sequence. Phylogenetic analysis of U-box genes from S. miltiorrhiza and Arabidopsis suggested that they can be clustered into seven subgroups (I–VII). Typical U-box domains were found in all identified SmPUB genes through the analysis of conserved motifs. Moreover, qRT-PCR was applied to analyze the relative expression levels of U-box genes in S. miltiorrhiza roots and leaves under PEG-induced water deficit and salt stresses. Results revealed that the SmPUB genes exhibited stronger response to drought than to salt stress. To the best of our knowledge, this report is the first to perform genome-wide identification and analysis of the U-box gene family in S. miltiorrhiza, and the results provide valuable information for better understanding of the function of U-box in S. miltiorrhiza.     

The chloroplast genome comparative characteristic of artificial breeding tree,a case about <i>Broussonetia kazinoki</i> × <i>Broussonetia papyrifera</i>

Broussonetia kazinoki × Broussonetia papyrifera (ZJGS) is a hybrid species in Moraceae family, which has a very complicated hybrid origin. The excellent characteristics of fast growth, strong soil and water conservation ability, high leaf protein content and stem fiber content in ZJGS make it both ecological benefits in the mining area and economically valuable. This study aims to further understand ZJGS and other Moraceae taxa through the ZJGS chloroplast (cp) genome structure and the comparison with 12 closely related Moraceae species. Among the 13 Moraceae species, the cp genome length of seven Broussonetia species (ranges from 160,239 bp to 162,594 bp) is larger than that of six Morus species (ranges from 158,459 bp to 159,265 bp). Among the 77 shared protein-coding genes (PCGs) in Moraceae species, the obvious positive selection of Ka/Ks ratios acted on petD and rpl16 genes of B. kazinoki and B. papyrifera, respectively. Phylogenetic analysis based on shared PCGs from 28 species shows that ZJGS is closely related to maternal B. kazinoki. These findings provide data support for the origin of ZJGS hybridization and provide genomic resources for future ZJGS resource development and molecular breeding.     

Response of <i>MaHMA2</i> gene expression and stress tolerance to zinc stress in mulberry (<i>Morus alba</i> L.)

HMA2 (heavy metal ATPase 2) plays a crucial role in extracellular and intracellular Zn²⁺ transport across biomembranes, maintaining ion homeostasis, and playing an important role in the normal physiological metabolism, growth, and development of plants. In our study, a novel HMA2 gene, named MaHMA2, was isolated and cloned from white mulberry (Morus alba L.). The gene sequence obtained was 1,342 bp long, with an open reading frame of 1,194 bp, encoding a protein of 397 amino acids, with a predicted molecular mass of 42.852 kD and an isoelectric point of 7.53. This protein belonged to the PIB-type ATPase transport protein family. We analyzed the expression of the MaHMA2 gene by quantitative real-time PCR. The results showed that the level of MaHMA2 gene expression decreased to a Zn concentration of 800 mg/kg. Malondialdehyde and proline levels increased and responded to increasing Zn when the MaHMA2 gene was silenced, whereas the activities of peroxidase and superoxide dismutase tended to increase in response to increasing Zn²⁺ ion stress concentrations but were lower in the gene-silenced plants. These findings suggested that the MaHMA2 gene played an active role in the tolerance response of mulberry to Zn stress.     

Increased <i>MAD2L2</i> expression predicts poor clinical outcome in Colon Adenocarcinoma

Background: Colon adenocarcinoma (COAD) is the second leading cause of cancer death worldwide thus, identification of COAD biomarkers is critical. Mitotic Arrest Deficient 2 Like 2 (MAD2L2) is a key factor in mammalian DNA damage repair and is highly expressed in many malignant tumors. This is a comprehensive study of MAD2L2 expression, its diagnostic value, prognostic analysis, potential biological function, and impact on the immune system of patients with COAD. Methods: Gene expression, clinical relevance, prognostic analysis, diagnostic value, GO/KEGG cluster analysis, data obtained from TCGA, and bioinformatics statistical analysis were performed using the R package. Immune responses to MAD2L2 expression in COAD were analyzed using TIMER. The expression of MAD2L2 in HCT116 cells induced by the inflammatory factor TNF-α was detected using Western blot. Results: Our results underscore the clinical diagnostic value and potential biological significance of MAD2L2 in patients with COAD. A high level of MAD2L2 expression has been found in COAD and correlated with tumor status and colon polyps. ROC curve analysis showed that MAD2L2 expression has high diagnostic value in COAD. Analysis of immune infiltration results showed that MAD2L2 expression was positively correlated with neutrophil levels. The western blot results demonstrated that MAD2L2 was dose-dependently present with TNF-α. GO/KEGG revealed that MAD2L2 overexpressed and coexpressed genes were mostly involved in biological functions, including hypoxia response, response to reduced oxygen levels, mitochondrial translation elongation, and other processes. Conclusion: MAD2L2 as a new COAD biomarker contributes to our understanding of how alterations in gene expression and the immunological environment contribute to the development of colon cancer. Following further investigation, MAD2L2 may prove to be a viable target factor for clinical diagnosis and therapy of COAD.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

Selection and validation of reference genes for quantitative real-time polymerase chain reaction analyses of <i>Serratia ureilytica</i> DW2

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT–qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT–qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.     

Association between preterm birth risk and polymorphism and expression of the DNA repair genes <i>OGG1</i> and <i>APE1</i> in Saudi women

Genomic instability and mutations caused by increases in oxidative stress during pregnancy can damage the fetoplacental unit and can upshot preterm birth. Oxidative damage to DNA may possibly be involved in etiology of preterm birth (PTB) which can be repaired by DNA repair gene. In the present study, we assessed the association of base excision repair gene family by analyzing the association of single nucleotide polymorphisms and genes expression in 8-oxoguanine glycosylase-1 (OGG1) and apurinic-apyrimidinic endonuclease 1 (APE1) genes with risk of preterm birth in Saudi women. We analyzed genotypes of four single nucleotide polymorphisms (SNPs) (rs1052133, rs293795, rs2072668 and rs2075747) in OGG1 gene and three SNPs (rs1130409, rs3136814, and rs3136817) in APE1 gene using TaqMan Genotyping assay kits in 50 pairs of preterm cases and individually matched controls. Also, gene expression level was explored by RT-PCR in 10 pairs of preterm placental tissues and individually matched normal placental tissues. Two OGG1 SNP, rs1052133 (OR=0.497; c2=1.11; p=0.292) and rs2072668 (OR=0.408; c2=1.90; p=0.167) and one APE1 SNP rs3136817 (OR=0.458; c2=0.40; p=0.527) showed nonsignificant protective effect against PTB development. The expression of both genes under study was found lower in the PTB patients. Genotype and allele frequencies of both gene SNPs did not show any association with the risk of preterm delivery in Saudi women (P˃0.05). However, synthesis and release of OGG1 and APE1 proteins decreased in preterm placental tissues compared to term delivery reflects the probability of being one of the mechanisms leading to preterm birth.     

The extracellular secretion of <i>miR-1825</i> wrapped by exosomes increases <i>CLEC5A</i> expression: A potential oncogenic mechanism in ovarian cancer

Background: Ovarian cancer (OC) is a leading cause of gynecological cancer-linked deaths worldwide. Exosomal miR-1825 and its target gene C-type lectin domain family 5 member A (CLEC5A) are associated with tumorigenesis in cancers that was further probed. Methods: Exosomal miR-1825 expression in exosomes and its impact on overall survival (OS) prediction were determined using Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data. Target genes of miR-1825 were searched in five prediction databases and prognostically significant differentially expressed genes were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out. The ability of CLEC5A to predict OS was evaluated using univariate and multivariate Cox regression analyses and Kaplan-Meier curves. The CLEC5A expression pattern in OC was validated using immunohistochemistry. The CIBERSORT algorithm was used to compare the immune cell landscape, and the results were validated in a GEO cohort. Finally, the predicted half maximal inhibitory concentration (IC50) values for five commonly used chemotherapy agents were also compared. Results: MiR-1825 level was higher in exosomes derived from OC cells and served as a tumor suppressor. The CLEC5A gene was found to be a target of miR-1825, the upregulation of which was correlated with a poor prognosis. M2 macrophage infiltration was significantly enhanced in the CLEC5A high expression group, while T follicular helper cell infiltration was reduced in it. While the predicted IC50 for cisplatin and doxorubicin was higher in the CLEC5A high expression group, that of docetaxel, gemcitabine, and paclitaxel was lower. Conclusion: MiR-1825, a promising OC biomarker, may promote OC progression by increasing CLEC5A expression via exosome-mediated efflux from tumor cells.     

Characterization of multidrug-resistant <i>Klebsiella pneumoniae</i> isolated from the Chinese cobra <i>Naja atra</i> in a Beijing suburb

The emergence and spread of antibiotic resistance genes among Bacteria are a serious threat to global health. Their occurrence in animals which are in contact with humans is also important. The Chinese cobra (Naja atra, Elapidae), though a highly venomous species, is appreciated as food and as a source of materials used in traditional Chinese medicine. We are here reporting the isolation of multidrug-resistant Klebsiella pneumoniae (Enterobacteriaceae) from the lung of Naja atra, obtained from a snake farm in a Beijing suburb. Our study analyzed, using gene sequencing, the occurrence of antibiotic resistance genes (ARGs) in three K. pneumoniae isolates from two snakes. In addition, bacterial clones were identified by biochemical tests and phylogenetic analysis. Tests of antimicrobial susceptibility showed that all K. pneumoniae isolates were resistant to a host of antibiotics (piperacillin, cefazolin, gentamicin, tetracycline, doxycyclin, ciprofloxacin, levofloxacin, lomefloxacin, ofloxacin, norfloxacin, nalidixic acid, chloramphenicol, nitrofurantoin, sulfamethoxazole, and sulfamethoxazole/trimethoprim) but were susceptible to cefotaxime, cefixime, aztreonam, bramycin, amikacin, kanamycin, netilmicin, and streptomycin. Eighteen ARGs were detected in total DNA extracted from the isolates. Results showed three quinolone resistance genes (oqxA, oqxB, qnrB), the gyrA gene that confers resistance to beta-lactam antibiotics, and the emerging aac(3)-II gene that confers resistance to aminoglycosides. K. pneumoniae is an important opportunistic human pathogen and the emergence of multidrug-resistant K. pneumoniae in N. atra suggests the increasing risk of pathogen transmission between humans, livestock, and wildlife. Given the close association between foodborne pathogenic microorganisms and humans, it is key factor to identify these antibiotic resistance genes profile thereby minimize the risk of K. pneumoniae transmission.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

Meta-analysis of the cell cycle related <i>C12orf48</i>

The cell cycle is a conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division. The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during the cycle. Although the genes involved in interphase are well studied, most of the genes which are involved in mitotic events still remain unidentified. Since, the discovery of mitosis related genes is still incomplete, we performed a co-expression and gene ontology analysis for revealing novel mitosis regulated genes. In this study, we showed that C12orf48 is co-expressed with well-known mitotic genes. Moreover, it is also co-expressed with the genes that have roles in interphase such as DNA replication. Furthermore, our results showed that C12orf48 is also differentially expressed in various cancers. Therefore, the results presented in this study suggest that C12orf48 may be an important molecule for both interphase and mitosis. Since, the molecules involved in these mechanisms are crucial for proliferation as well as in carcinogenesis, C12orf48 should be considered as a novel cell cycle and carcinogenesis related gene.