The peptide fraction of <i>Bothrops jararaca</i> snake venom induces toxicological effects on the male reproductive system after local envenomation in mice

Bothrops envenomation is complex and provokes prominent local tissue damage and systemic disturbances, but little is known about their effects on the male reproductive system. After intratesticular injection, the bioactive peptide fraction (Bj-PF) obtained from Bothrops jararaca snake venom changes the structure of different stages of the seminiferous epithelium cycle in adult mice. For the first time, we investigated whether local envenomation of Bj-PF induces toxicological effects on the male reproductive system, particularly on the seminiferous epithelium and Sertoli cells. Male adult mice were treated with 0.24 mg.kg⁻¹ by intramuscular (i.m.) injection for 24 h. The testes samples were collected for morphological and morphometric evaluation. The toxicological effects of Bj-PF were also analyzed on mitochondrial metabolism and nitrite (NO₂) production in 15P-1 Sertoli cell culture. Bj-PF changed the structure and function of the seminiferous epithelium, particularly the disruption of the epithelium and the presence of degenerated germ cells in the adluminal compartment, but there were no alterations in the basal compartment. Bj-PF increased the thickness of the seminiferous epithelium and decreased the lumen diameter of the tubule. Semiquantitative histological assessment of the degree of tubule degeneration revealed that Bj-PF also increased the number of hypospermatogenic tubules compared to control. Bj-PF reduced NO₂ levels in 15P-1 Sertoli cells without changing the mitochondrial metabolism. Overall, the fact that Bj-PF alters the structure and function of the seminiferous epithelium suggests that bioactive peptides found in B. jararaca snake venom can have toxicological effects on the reproductive systems of affected male mice, providing new insight into the biological characteristics of snake venom and therapeutic strategies for envenomation inflammation.     

Isolation and molecular characterization of a <i>cax</i> gene from <i>Capsella bursa-pastoris</i>

A new cation exchangers (CAXs) gene was cloned and characterized from Capsella bursapastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence of cax from C. bursa-pastoris (designated as Cbcax51) was 1754 bp containing a 1398 bp open reading frame encoding a polypeptide of 466 amino-acid residues with a calculated molecular mass of 50.5 kDa and an isoelectric point of 5.69. The predicted CbCAX51 contained an IMP dehydrogenase/GMP reductase domain, two Na⁺/Ca²⁺ exchanger protein domains. Comparative and bioinformatics analyses revealed that CbCAX51 showed extensive homology with CAX from other plant species. The expression analysis by different treatments indicated that Cbcax51 could be activated by cold triggering and was related to the cold acclimation process, but its expression is regulated negatively by drought and not affected by ABA or salt.     

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.     

Isolation of epithelial cells,villi and crypts from small intestine of pigeons (<i>Columba livia</i>)

The isolation of viable enterocytes, villi and crypts from the small intestine of a feral bird (Columba livia) is important for performing physiological experiments in ecologically relevant processes of membrane transport. The effectiveness of mechanical disruption, enzymatic digestion and chelating agents were compared. The objectives were to isolate enterocytes, villi and crypts from the small intestine of young pigeons; to evaluate the viability of the isolated intestinal epithelial cells isolated; and to verify the integrity of enterocytes by biochemical features. Enzymatic and mechanical methods yielded both elongated columnar and spherical cells. With the chelating method villi and crypts were obtained. All methods produced a high yield of intestinal epithelial cells with about 50 % viability. Brush border enzymes (sucrase-isomaltase and alkaline phosphatase) activities were high and, as reported in chickens, they did not differ along the intestinal villus-crypt axis. Although the three methods have good viabilities, the enzymatic technique gives the best yield in cell number, while the chelating method provides the highest populations of morphologically distinctive villi and crypts.     

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.     

Cloning and characterization of 5-enopyruvylshikimate-3-phosphate synthase from <i>Phragmites australis</i>

Glyphosate is a non-selective broad-spectrum herbicide that blocks plant growth by inhibiting 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme of the shikimate pathway in microorganisms andplants. The full-length epsps cDNA sequence (paepsps, Genebank: KY860582.1) was cloned and characterized for thefirst time from Phragmites australis. The full-length cDNA of paepsps was 1308 bp encoding a polypeptide of 435amino acids. The bioinformatic analyses showed that PaEPSPS has highly homologous with EPSPS from other plants.RT-PCR analysis of paepsps expression indicated that the gene expressed in leaves, stems, and roots, with higherexpression in leaves. The expression of the paepsps gene increased with glyphosate application. In addition, thetransgenic tobacco containing the paepsps gene showed glyphosate resistance in comparison with control. The novelpaepsps is a good candidate gene in transgenic crops with glyphosate tolerance in the future.     

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.     

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>Trypanosoma rangeli:</i> growth in mammalian cells <i>in vitro</i> and action of a repositioned drug (17-AAG) and a natural extract (<i>Artemisia sp</i>. essential oil)

Trypanosoma rangeli and T. cruzi are both parasitic unicellular species that infect humans. Unlike T. cruzi,the causative agent of Chagas disease, T. rangeli is an infective and non-pathogenic parasite for humans, but pathogenicfor vectors from the Rhodnius genus. Because both species can coexist in different hosts and overlap their infectivecycles but very little is known about the infection of T. rangeli in mammalian cells, we decided to characterize both thedevelopment of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action onit. We found that T. rangeli exhibits a cycle of infection in Vero cells similar to that for T. cruzi and that the repurposeddrug, 17-AAG, and the natural extract Artemisia sp. essential oil produce a toxic effect on epimastigotes showinga trypanocidal action from the fifth day of culture. Both treatments also affected the infection of trypomastigotesand reduced the capacity of replication of amastigotes of T. rangeli. Since T. cruzi / T. rangeli coinfection cases havebeen reported, the finding of drugs with potential activity against both species could be significant in the future.Furthermore, studies of susceptibility of both species to drugs could also help to know the different mechanisms ofpathogenicity in humans displayed by T. cruzi that are absent in T. rangeli     

Hydrothermal preparation of TiO₂-Ag nanoparticles and its antimicrobial performance against human pathogenic microbial cells in water

Water contaminated with pathogenic microbes is considered as one of the most common routes for transmitting diseases in human beings. Different methods have been applied for the decontamination of microbes in contaminated water. In the current study, an easy to do hydrothermal method has been used for the preparation of TiO₂-Ag nanoparticles. The obtained material was characterised using a scanning electron microscope (SEM) and fourier transform infra-red spectroscopy (FTIR). The morphological appearance of the obtained nanoparticles was in the shape of a sphere with a size range of 60-90 nm. The antimicrobial activity of the prepared nanoparticles was tested against several pathogenic bacteria and fungi. The obtained results proved that the nanoparticles succeeded to affect all the tested microbes in the following order: Bacillus cereus ATCC6633>Pseudomonas aeruginosa ATCC9027= Klebsiella pneumoniae ATCC13883>Vibrio cholera ATCC700=Candida albicans ATCC 700=Escherichia coli NCTC10418>Staphylococcus aureus ATCC6538. The minimum inhibitory concentration (MIC) of the prepared nanoparticles varied among the tested microbes at range of 12 mg/ml and 25 mg/ml. These results encourage the application of prepared TiO₂-Ag nanoparticles for treatment of microbe-contaminated waters.     

Overexpression of a sugarcane<i> ScCaM</i> gene negatively regulates salinity and drought stress responses in transgenic <i>Arabidopsis thaliana</i>

Calmodulin (CaM) proteins play a key role in signal transduction under various stresses. In the present study,the effects of a sugarcane ScCaM gene (NCBI accession number: GQ246454) on drought and salt stress tolerance intransgenic Arabidopsis thaliana and Escherichia coli cells were evaluated. The results demonstrated a significantnegative role of ScCaM in the drought and salt stress tolerance of transgenic lines of A. thaliana, as indicated by thephenotypes. In addition, the expression of AtP5CS and AtRD29A, two genes tightly related to stress resistance, wassignificantly lower in the overexpression lines than in the wild type. The growth of E. coli BL21 cells expressingScCaM showed weaker tolerance under mannitol and NaCl stress. Taken together, this study revealed that the ScCaMgene plays a negative regulatory role in both mannitol and NaCl stresses, and it possibly exerts protective mechanismscommon in both prokaryotes and eukaryotes under stress conditions.     

Structural characterization of four <i>Rhododendron</i> spp. chloroplast genomes and comparative analyses with other azaleas

Azalea is a general designation of Rhododendron in the Ericaceae family. Rhododendron not only has highornamental value but also has application value in ecological protection, medicine, and scientific research. In thisstudy, we used Illumina and PacBio sequencing to assemble and annotate the entire chloroplast genomes (cpgenomes) of four Rhododendron species. The chloroplast genomes of R. concinnum, R. henanense subsp. lingbaoense,R. micranthum, and R. simsii were assembled into 207,236, 208,015, 207,233, and 206,912 bp, respectively. Allchloroplast genomes contain eight rRNA genes, with either 88 or 89 protein-coding genes. The four cp genomes werecompared and analyzed by bioinformatics, and the phylogenetic analysis based on chloroplast genomes of 26 speciesof Ericaceae, Actinidiaceae, and Primulaceae under Ericales was conducted. A comparison of the linear structure of cpgenomes of four Rhododendron showed that there were substantial sequence similarities in coding regions, but highdifferences in non-coding regions. A phylogenetic analysis, based on chloroplast whole genome sequences, showedthat all Rhododendron species are in the clade Ericaceae. This study provides valuable genetic information for thestudy of population genetics and evolutionary relationships in Rhododendron and other azalea species.     

Micropropagation of <i>Prosopis chilensis</i> (Mol.) Stuntz from young and mature plants

Prosopis chilensis (Mol.) Stuntz (Algarrobo de Chile) is an important native tree species that can be grown in arid and semiarid regions for wood and forage production and environmental protection. Developing a simple and reliable in vitro protocol for cloning it would enable to improve it genetically. Explants of P.chilensis were taken from 4 months-old plants grown in the greenhouse or from adult trees grown in a natural environment. Nodal segments 1 – 2 cm long containing an axillary bud were selected from elongating shoots. These cuttings were aseptically cultured on two agar-solid basal media, MS or BTMm, and treated with 0.05 mg L^-1 BA and 3 mg L^-1 of either IAA, IBA or NAA. Sucrose (3% w/v) was used as carbon source. The percentage of sprouted cuttings and whole plant regeneration as well as its shoot and root length were recorded. Number, length and dry weight of shoots and roots were also measured. Rooting was successful with cuttings taken from young or adult plants, but explants from young plants showed a better response. Culturing in BTMm resulted in significantly greater shoot and root biomass than culturing in MS. Moreover, this response was higher in young explants when IBA was used as growth regulator. This paper reports a simple and effective method to micropropagate P. chilensis from young and adult plants.     

Micropropagation of <i>Ilex dumosa</i> (Aquifoliaceae) from nodal segments in a tissue culture system

Micropropagation of Ilex dumosa var. dumosa R. (“yerba señorita”) from nodal segments containing one axillary bud was investigated. Shoot regeneration from explants of six-year-old plants was readily achieved in ¹/₄ strength Murashige and Skoog medium (¹/₄ MS) plus 30 gr·L^-1 sucrose and supplemented with 4.4 µM BA. Further multiplication and elongation of the regenerated shoots were obtained by subculture in a fresh medium of similar composition with 1.5 gr·L^-1 sucrose. Rooting induction from shoots were achieved in two steps: 1) 7 days in ¹/₄ MS (30 gr·L^-1 sucrose, 0.25 % Phytagel^®) with 7.3 µM IBA and 2) 21 days in the same medium without IBA and 20 µM of cadaverine added. Regenerated plants were successfully transferred to soil. This micropropagation schedule can be implemented in breeding programs of Ilex dumosa.     

An extract of <i>Hypericum perforatum</i> induces wound healing through inhibitions of Ca²⁺ mobilizations,mitochondrial oxidative stress and cell death in epithelial cells: Involvement of TRPM2 channels

The wound is induced by several mechanical and metabolic factors. In the etiology of the wound recovery,excessive oxidative stress, calcium ion (Ca²⁺) influx, and apoptosis have important roles. Ca²⁺-permeable TRPM2 channel is activated by oxidative stress. Protective roles of Hypericum perforatum extract (HP) on the mechanical nerve injury-induced apoptosis and oxidative toxicity through regulation of TRPM2 in the experimental animals wererecently reported. The potential protective roles in HP treatment were evaluated on the TRPM2-mediated cellularoxidative toxicity in the renal epithelium (MPK) cells. The cells were divided into three groups as control, wound,and wound + HP treatment (75 µM for 72 h). Wound diameters were more importantly decreased in the wound+HPgroup than in the wound group. In addition, the results of laser confocal microscopy analyses indicated protectiveroles of HP and TRPM2 antagonists (N-(p-Amylcinnamoyl) anthranilic acid and 2-aminoethyl diphenylborinate)against the wound-induced increase of Ca²⁺ influx and mitochondrial ROS production. The wound-induced increaseof early (annexin V-FITC) apoptosis and late (propidium iodide) apoptosis were also decreased in the cells by the HPtreatment. In conclusion, HP treatment acted protective effects against wound-mediated oxidative cell toxicity andapoptosis through TRPM2 inhibition. These effects may be attributed to their potent antioxidant effect.