A novel prognostic gene signature,nomogram and immune landscape based on tanshinone IIA drug targets for hepatocellular carcinoma: Comprehensive bioinformatics analysis and <i>in vitro</i> experiments

Background: Tanshinone IIA, one of the main ingredients of Danshen, is used to treat hepatocellular carcinoma (HCC). However, potential targets of the molecule in the therapy of HCC are unknown. Methods: In this study, we collected the tanshinone IIA targets from public databases for investigation. We screened differentially expressed genes (DEGs) across HCC and normal tissues using mRNA expression profiles from The Cancer Genome Atlas (TCGA). Univariate Cox regression analysis and least absolute shrinkage and selection operator (LASSO) Cox regression models were used to identify and construct the prognostic gene signature. Results: Finally, we discovered common genes across tanshinone IIA targets and HCC DEGs. We reported Fatty acid binding protein-6 (FABP6), Polo-like Kinase 1 (PLK1), deoxythymidylate kinase (DTYMK), Uridine Cytidine Kinase 2 (UCK2), Enhancer of Zeste Homolog 2 (EZH2), and Cytochrome P450 2C9 (CYP2C9) as components of a gene signature. The six-gene signature’s prognostic ability was evaluated using the Kaplan-Meier curve, time-dependent receiver operating characteristic (ROC), multivariate Cox regression analysis, and the nomogram. The mRNA level and protein expression of UCK2 were experimentally validated after treatment with different concentrations of tanshinone IIA in HEPG2 cells. CIBERSORTx, TIMER2.0, and GEPIA2 tools were employed to explore the relationship between the prognostic signature and immune cell infiltration. Conclusion: We established a six-gene signature as a reliable model with significant therapeutic possibility for prognosis and overall survival estimation in HCC patients, which might also benefit medical decision-making for appropriate treatment.     

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.     

Phytohormonal and metabolism analysis of <i>Brassica rapa</i> L. ssp. <i>pekinensis</i> with different resistance during <i>Plasmodiophora brassicae</i> infection

Clubroot of Chinese cabbage (Brassica rapa L. ssp. pekinensis), caused by the obligate parasite Plasmodiophorabrassicae, accounts for serious yield losses. The aim of our study was to explore the phytohormone levels and metabolomechanges in the roots of resistant and susceptible B. rapa genotypes at a late stage of infection, i.e., 28 days post-infection.Both genotypes showed decreased auxin levels after P. brassicae infection except for indole-3-acetic acid. Overall, thesusceptible genotype had higher auxin and cytokinin levels after infection, with the exception of trans-zeatin and 3-indolebutyric acid as compared to the resistant genotype. Jasmonic acid levels declined after infection regardless of thegenotype. Resistance against clubroot was evident with the increased levels of salicylic acid in the resistant genotype.The susceptible genotype had a higher number of differentially accumulated metabolites (DAMs) (262) than theresistant genotype (238) after infection. Interestingly, 132 DAMs were commonly detected in both genotypes wheninfected with the pathogen, belonging to metabolite classes such as phenolic acids, amino acids, and derivatives,glucosinolates, organic acids, flavonoids, nucleotides and derivatives, and fatty acids. The differential metaboliteanalysis revealed that metabolites related to amino acid biosynthesis, fatty acid biosynthesis and elongation,glutathione metabolism, and glucosinolate metabolism were highly accumulated in the resistant genotype, suggestingtheir essential roles in resistance against P. brassicae infection.     

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.     

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.     

Immune strategies of phagoctytic cells stimulated <i>in vitro</i> with live and heat-inactivated <i>Streptococcus pyogenes</i>

Streptococcus pyogenes (group A Streptococcus) is frequently involved in a wide range of human diseases. Here we evaluated polymorphonuclear neutrophils and mononuclear cells from healthy subjects for their bactericidal function after stimulation with live and inactivated Streptococcus pyogenes (Streptococcus Group A). Mononuclear cells and Neutrophils were isolated from heparinized blood samples (n=18) using a Ficoll-Hypaque gradient and cultured in RPMI 1640 for 18 hours with a suspension of either live or inactivated Streptococcus pyogenes. Both the respiratory burst (flow cytometry) and nitrite, TNF and IL17 production (ELISA) were measured in the cell culture supernatants. An increased respiratory burst (expressed as R index) was induced by both live and inactivated bacteria. Also, increased nitrite, TNF and IL17 concentrations were found in cell culture supernatants in both cases. These findings may provide some explanation as to the roles played by neutrophils and mononuclear cells in Streptococcus pyogenes immunopathogenicity.     

Electron microscopy of squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck carries a bad prognosis. In order to achieve cure, the most important thing to attain is local tumour control. The main therapy available is external radiotherapy, which can be supplemented when necessary, with interstitial radiotherapy, chemotherapy and surgery. In this paper we have evaluated specimens, taken before therapy, from 35 patients with squamous cell carcinoma of the head and neck. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were made. With SEM, the parameters analysed were the amount and appearance of microvilli, filaments, and blood vessels. From TEM, scoring was made of the filaments, desmosomes, nuclei, nucleoli, mitochondria, and blood vessels. Scoring of the samples showed a difference between the group with recurrent disease (n = 10, Group 1) and the group with local tumor control (n = 25, Group 2) in regard to both blood vessels and intracellular filaments. No differences of the nuclei, nucleoli, or the mitochondria were observed.     

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.     

<i>In vitro</i> effects of 2-methoxyestradiol-bis-sulphamate on cell growth,morphology and cell cycle dynamics in the MCF-7 breast adenocarcinoma cell line

In the search for new and improved anticancer therapies, researchers have identified several potentially useful compounds. One of these agents is 2-methoxyestradiol-bis-sulphamate (2ME-BM), a sulphamoylated derivative of 2-methoxyestradiol. The objective of this study was to evaluate 2ME-BM’s in vitro efficacy as antiproliferative agent in the MCF-7 breast adenocarcinoma cell line. Light- and fluorescent microscopy showed decreased cell density, increased apoptotic characteristics and significant ultrastructural aberrations indicative of autophagic cell death after 24 hours of exposure at a concentration of 0.4μM. In addition, mitotic indices revealed that 2ME-BM induces a G₂M block. The latter was confirmed by flow cytometric analyses where increased sub-G₁ and G₂ /M fractions, as well as an increase in cyclin B1 levels were observed. Further in vitro research into the mechanism of this potentially useful anticancer compound is thus warranted.     

Differential metabolome landscape of <i>Kadsura coccinea</i> fruit tissues and potential valorization of the peel and seed tissues

Kadsura coccinea (Lem.) is a woody wine plant with a peculiar fruit enriched in important health-promotingcompounds. The non-editable part of the fruit, i.e., the seed and peel, represents more than 60% of the fruit and isconsidered a biowaste. This significantly restricts the development of the K. coccinea fruit industry. Clarifying themetabolic components of the different fruit parts can help to improve the utilization rate and valorization ofK. coccinea. Herein, we evaluated K. coccinea fruit peel, pulp, and seed using widely-targeted metabolomics andquantified a set of 736 bioactive compounds from 11 major metabolite classes. The most prominent metabolite classesincluded lipids, amino acids, flavonoids, and lignans. Furthermore, our results emphasized a significant accumulationof flavonoids in pulp tissues, while alkaloids and lignans were abundant in peel and seed tissues, respectively. A totalof 183 metabolites were differentially accumulated among the three tissues. Procyanidin C2, rutinoside,2-hydroxyoleanolic acid, 5-hydroxymethyluracil, nootkatol, isoquercitrin, isohyperoside, quercetin-7-O-glucoside,hyperin, and rutin showed elevated accumulation in the peel. In the seed, kadsuralignan G, kadcoccilactone A,kadsuralignan H, lysoPE 20:5, iso-schisandrin ethyl alcohol, and kadangustin were significantly enriched. Our resultshighlight the diverse metabolome composition of K. coccinea fruit parts, which can be further exploited for itsvalorization in various industries.     

Glucocorticoid reduces the efficacy of afatinib on the head and neck squamous cell carcinoma

Glucocorticoids (GC) are widely used to counter the adverse events during cancer therapy; nonetheless, previous studies pointed out that GC may reduce the efficacy of chemotherapy on cancer cells, especially in epidermal growth factor receptor (EGFR)-targeted therapy of head and neck squamous cell carcinoma (HNSCC) remaining to be elucidated. The primary aim of the present study was to probe into the GC-induced resistance of EGFR-targeted drug afatinib and the underlying mechanism. HNSCC cell lines (HSC-3, SCC-25, SCC-9, and H-400) and the human oral keratinocyte (HOK) cell lines were assessed for GC receptor (GR) expression. The promoting tumor growth effect of GC was evaluated by the CCK-8 assay and flow cytometry. Levels of signaling pathways participants GR, mTOR, and EGFR were determined by quantitative polymerase chain reaction and western blotting. GC increased the proliferation of HNSCC cells in a GR-dependent manner and promoted AKT/mTOR signaling. But GC failed in counteracting the inhibition of rapamycin in the mTOR signaling pathway. Besides, GC also induced resistance to EGFR-targeted drug afatinib through AKT/mTOR instead of the EGFR/ERK signaling pathway. Thus, GCs reduce the efficacy of afatinib on HNSCC, implicating a cautious use of glucocorticoids in clinical practice.     

Synergistic combination of colistin with imipenem,amikacine or ciprofloxacin against <i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i> carbapenem-resistant isolated in Annaba hospital Algeria

Objective: The aim of this study is to detect in vitro the synergetic activity of colistin in combination withimipenem, amikacin or ciprofloxacin, at sub-inhibitory concentrations, against carbapenems-resistant (CR) Acinetobacterbaumannii and Pseudomonas aeruginosa strains isolated from various wards in Annaba teaching hospital in eastern Algeria.
Materials and Methods: The minimal inhibitory concentrations (MIC) were determined by broth macrodilution (BMD).Carbapenemase encoding genes were screened using polymerase chain reaction (PCR). The activity of colistin incombination with second antibiotic was evaluated by the Checkerboard Technique.
Results: 39 CR P. aeruginosa and 21 CR A. baumanni strains where collected. The MIC values ranging from (0.25 to4 µg/ml) to colistin, ≥16 µg/ml for imipenem, ≥4 µg/ml to amikacin and ≥8 µg/ml ciprofloxacin. The PCR reveals thepresence of the genes bla_OXA23 (n = 12), blaOXA24 (n = 6), blaNDM1 (n = 3) in A. baumannii and blaVIM2 (n = 12) in P.aeruginosa. The combination of colistin with imipenem showed synergistic effect on 57.14% and 46.15% of A.baumannii and P. aeruginosa isolates, respectively. For colistin and amikacin, the synergistic effect is detected in 28.6%of A. baumannii and 30.8% of P. aeruginosa. While colistin and ciprofloxacin showed synergy on 14.29% and 15.38% ofA. baumannii and P. aeruginosa isolates, respectively.
Conclusion: CR A. baumannii and P. aeruginosa remain the most prevalent infection agents in patients from high-riskwards at Annaba Hospital. Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations givesvery encouraging results allowing better management of infections caused by this type of bacteria.     

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.     

WGCNA and LASSO algorithm constructed an immune infiltration-related 5-gene signature and nomogram to improve prognosis prediction of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common immunogenic malignant tumor. Although the new strategies of immunotherapy and targeted therapy have made considerable progress in the treatment of HCC, the 5-year survival rate of patients is still very low. The identification of new prognostic signatures and the exploration of the immune microenvironment are crucial to the optimization and improvement of molecular therapy strategies. We studied the potential clinical benefits of the inflammation regulator miR-93-3p and mined its target genes. Weighted gene co-expression network analysis (WGCNA), univariate and multivariate COX regression and the LASSO COX algorithm are employed to identify prognostic-related genes and construct multi-gene signature-based risk model and nomogram for survival prediction. Support vector machine (SVM) based Cibersort’s deconvolution algorithm and gene set enrichment analysis (GSEA) is used to evaluate the changes in tumor immune microenvironment and pathway differences. The study found the favorable prognostic performance of miR-93-3p and identified 389 prognostic-related target genes. The risk model based on a novel 5-gene signature (cct5, cdk4, cenpa, dtnbp1 and flvcr1) was developed and has prominent prognostic significance in the training cohort (P < 0.0001) and validation cohort (P = 0.0016). The nomogram constructed by combining the gene signature and the AJCC stage further improves the survival prediction ability of the gene signature. The infiltration level of multiple immune cells (especially T cells, B cells and macrophages) were positively correlated with the expression of prognostic signature. In addition, we found that gene markers of T cells and B cells is monitored and regulated by prognostic signature. Meanwhile, several GSEA pathways related to the immune system are enriched in the high-risk group. In general, we integrated the WGCNA, LASSO COX and SVM algorithms to develop and verify 5-gene signatures and nomograms related to immune infiltration to improve the survival prediction of patients.