Morphological study of the spermatogenesis in the teleost <i>Piaractus mesopotamicus</i>

The spermatogenesis of Piaractus mesopotamicus was investigated under light and transmission electron microscopy. The specimens were captured from their natural environment (Rio Miranda and Rio Aquidauana, Pantanal Matogrossense, Brazil) during April and September. The results were compared with the spermatogenic data of specimens under captivity condition. In both conditions, P. mesopotamicus presented the typical spermatogenesis pattern of the teleost fishes, showing no significative differences. The spermatozoon was classified as type I, which has a globular head without acrosome, a short middle piece and a long tail constituted only by the flagellum. This type of spermatozoon is considered the basic type in fishes.     

<i>Agrobacterium rhizogenes</i> vs auxinic induction for <i>in vitro</i> rhizogenesis of <i>Prosopis chilensis</i> and <i>Nothofagus alpina</i>

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg.L^-1 benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg.L^-1 of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg.L^-1 IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg.L^-1 IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.     

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.     

MicroRNA-382 inhibits the proliferation of mouse spermatogonia by targeting <i>Kmt5a</i>

Spermatogenesis is a highly efficient and intricate process in the testis by which mature spermatozoa are produced daily to maintain lifelong male fertility. Essential to this process are spermatogonia capable of both proliferation and differentiation. Nevertheless, the underlying mechanisms for spermatogonial proliferation and differentiation remain poorly understood. MicroRNAs (miRNAs) are a category of non-coding small RNAs with regulatory functions by binding to the 3’ untranslated region (UTR) of the target mRNA. Previous studies have demonstrated that miRNAs are capable of modulating cell proliferation, differentiation and apoptosis, but the roles of individual miRNAs in spermatogonial fate determination remain largely elusive. Here, by using a mouse spermatogonial cell line (GC-1), we investigated the role for miRNA-382 in spermatogonial proliferation. We found that pre-miRNA-382 was expressed in spermatogonia. The luciferase reporter assay demonstrated Kmt5a but not Top1 as a target gene of miRNA-382. Overexpression of miRNA-382 by transfecting a miRNA mimic downregulated Kmt5a at both RNA and protein levels, and further reduced the proliferation and viability of spermatogonia. Knockdown of Kmt5a by RNA interference (RNAi) resulted in a uniform phenotype in spermatogonia. We therefore conclude that miRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a. Our finding extends the knowledge about the regulatory roles of miRNAs in spermatogonia and lays the groundwork for diagnosis and treatment of male infertility.     

Ultrastructural analysis of sperm from the genus <i>Idarnes</i> (Hymenoptera: Chalcidoidea,Sycophaginae)

In this study, the sperm ultrastructure of three species of Idarnes genus was investigated using light and transmission electron microscopy. Spermatozoon morphology of the three species was similar to that of most Chalcidoidea, with helicoidally twisted nucleus and flagellum. The head region consists of an acrosome and a nucleus; the nucleus-flagellum transition region characterized by the presence of mitochondrial derivatives and the centriolar adjunct; a flagellum region, which includes the axoneme with microtubular arrangement 9 + 9 + 2 and two mitochondrial derivatives. However, the sperm of these three species exhibit features that discriminate one species from each other: (1) only one species, Idarnes sp. 2 (carme group) exhibited an extracellular sheath surrounding the anterior portion of the nucleus, which extends to the anterior region of the flagellum, but it did not present filaments; (2) the acrosome in the three species was quite different, Idarnes sp. 1 and Idarnes sp. 2 (carme group) has two compartments (acrosomal and subacrosomal vesicles) while Idarnes sp. 3 (flavicollis group) has a third compartment (perforatorium); (3) the centriolar adjunct elongated and its location among the mitochondrial derivatives is similar for the three species analyzed; (4) mitochondrial derivatives differ between the species, with triangular (Idarnes sp. 1 and sp. 3) and elongated or flat shaped (Idarnes sp. 2) appearance. These data shows that sperm structure may differ within the same genus and confirms the potential of these cells in phylogenetic and taxonomic analyses in the Chalcidoidea superfamily, as well as in Hymenoptera in general.     

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.     

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 with imipenem, amikacin or ciprofloxacin, at sub-inhibitory concentrations, against carbapenems-resistant (CR) Acinetobacter baumannii 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 in combination 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 to 4 µg/ml) to colistin, ≥16 µg/ml for imipenem, ≥4 µg/ml to amikacin and ≥8 µg/ml ciprofloxacin. The PCR reveals the presence 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% of A. baumannii and P. aeruginosa isolates, respectively.
Conclusion: CR A. baumannii and P. aeruginosa remain the most prevalent infection agents in patients from high-risk wards at Annaba Hospital. Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations gives very encouraging results allowing better management of infections caused by this type of bacteria.     

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.     

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.     

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.     

<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 pathogenic for vectors from the Rhodnius genus. Because both species can coexist in different hosts and overlap their infective cycles but very little is known about the infection of T. rangeli in mammalian cells, we decided to characterize both the development of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action on it. We found that T. rangeli exhibits a cycle of infection in Vero cells similar to that for T. cruzi and that the repurposed drug, 17-AAG, and the natural extract Artemisia sp. essential oil produce a toxic effect on epimastigotes showing a trypanocidal action from the fifth day of culture. Both treatments also affected the infection of trypomastigotes and reduced the capacity of replication of amastigotes of T. rangeli. Since T. cruzi / T. rangeli coinfection cases have been 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 of pathogenicity in humans displayed by T. cruzi that are absent in T. rangeli     

Karyological and electrophoretic differences between <i>Pomacea flagellata</i> and <i>P. patula catemacensis</i> (Caenogastropoda: Ampullariidae)

The widespread Mexican apple snail Pomacea flagellata (Say 1827) and the strictly endemic "tegogolo" P. patula catemacensis (Baker 1922) (restricted to Lake Catemaco), are the only known American Ampullariidae that have haploid complements n=13. Pomacea patula catemacensis has suffered a critical reduction in abundance due to immoderate fishing for human consumption. Chromosome slides were obtained from colchicine-injected Pomacea snails collected from nine locations along the coastal zone of the Gulf of Mexico, including Lake Catemaco, for use in principal component analysis (PCA). Total proteins in foot homogenates were analyzed through isoelectric focusing (IEF) and native-PAGE electrophoresis on polyacrylamide gels. The chromosome number 2n=26 was confirmed for snails from all locations, with a uniform 9 m + 4 sm formula. However, P. patula catemacensis showed significantly larger chromosomes (absolute size) than any population of P. flagellata. Pomacea patula catemacensis also differed from all populations of P. flagellata in a PCA with standardized data, i.e., independently of the absolute size difference between species. Proteins with an acid isoelectric point were dominant in the foot of both species. The electrophoresis analysis showed that P. flagellata has 17 protein bands, with an upper bound at IEF=7.6, while P. patula catemacensis has only 15 bands, with an upper bound at IEF=7 and a more evenly spaced band pattern. Molecular weights ranged from 40 to approximately 130 kDa in both species. Proteins with high values (>94 kDa) were the most abundant. Pomacea patula catemacensis showed a band of 93 kDa, which was absent from all specimens of P. flagellata. Samples of P. flagellata did not cluster according to any geographical pattern in the statistical analyses, nor did they show any taxonomically useful differences in their electrophoretic patterns that merit sub-specific discrimination.     

Defense reactions of <i>Dermatobia hominis</i> (Diptera: Cuterebridae) larval hemocytes

The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis. The cellular defense performed by larval hemocytes was observed under electron microscopy. China ink particles were phagocytosed by granular cells 5 h after injection. E. coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering. H. capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation. Nylon thread was encapsulated 24 h after the introduction into the hemocoel. Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation. In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material.