<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     

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.     

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.     

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.     

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.     

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.     

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.     

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 were recently reported. The potential protective roles in HP treatment were evaluated on the TRPM2-mediated cellular oxidative 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+HP group than in the wound group. In addition, the results of laser confocal microscopy analyses indicated protective roles 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 increase of early (annexin V-FITC) apoptosis and late (propidium iodide) apoptosis were also decreased in the cells by the HP treatment. In conclusion, HP treatment acted protective effects against wound-mediated oxidative cell toxicity and apoptosis through TRPM2 inhibition. These effects may be attributed to their potent antioxidant effect.     

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.     

Molecular typing of methicillin and vancomycin-resistant <i>Staphylococcus aureus</i> isolated from clinical specimens by doublelocus sequence typing (DLST) method

Methicillin-resistant Staphylococcus aureus (MRSA) strains are the essential cause of infections in communities and hospitals. The present study was conducted to determine the molecular typing of MRSA, isolated from hospitalized patients, using the double-locus sequence typing (DLST). In total, 280 S. aureus isolated from clinical specimens by phenotypic (catalase, coagulase, DNase, oxacillin, vancomycin screening agar and antibiotic disk diffusion), and molecular methods (PCR for determining the mecA, vanA and nuc genes). The DLST and sequencing was performed for MRSA containing mecA. Out of 280 specimens, confirmed as Staphylococcus aureus (S. aureus), 123 (43.9%) strains were MRSA. The highest resistance toward the erythromycin (15 μg), followed by ciprofloxacin (5 μg), clindamycin (2 μg), tetracycline (30 μg), gentamicin (10 μg) and rifampicin (5 μg), was 98.3%, 97.5%, 94.3%, 90.2%, 83.7% and 41.4%, respectively. Also, the least resistance (0%) was observed in each of teicoplanin (30 μg), linzolide (30 μg), and vancomycin (30 μg). All (100%) of MRSA strains had the mecA, and none of them have had the vanA. The results of DLST showed that the most common sequence types were BPH 2003 and 0217. The DLST type 18-32 was a significant cluster of MRSA. By sequencing MRSA and comparing the dominant types via the DLST, it is possible to establish the etiology of the disease in a much shorter time, and prevent the complications of the disease. Therefore, the combination of partial sequences of clfB and spa can serve as useful genetic markers for MRSA typing. It concluded that the MRSA in our region was relatively high, but no vancomycin resistance was found. The majority of the MRSA DLST type was 18–32.     

A <i>Brachypodium distachyon</i> calcineurin B-like protein-interacting protein kinase,BdCIPK26, enhances plant adaption to drought and high salinity stress

As sessile organisms, plants possess a complex system to cope with environmental changes. Ca²⁺ functions as a vital second messenger in the stress signaling of plants, and the CBL-interacting protein kinases (CIPKs) serve as essential elements in the plant Ca²⁺ signaling pathway. In this study, calcineurin B-like protein-interacting protein kinase 26 (BdCIPK26) from Brachypodium distachyon was characterized. Overexpression of BdCIPK26 enhanced tolerance to drought and salt stress of transgenic plants. Further investigations revealed that BdCIPK26 participated in abscisic acid (ABA) signaling, conferred hypersensitivity to exogenous ABA in transgenic plants, and promoted endogenous ABA biosynthesis. Moreover, BdCIPK26 was found to maintain ROS homeostasis in plants under stress conditions. Therefore, this study indicates that BdCIPK26 functions as a positive regulator in drought and salt stress response.     

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.     

Induction of adaptive response <i>in utero </i>by ionizing radiation: A radiation quality dependent phenomenon

Investigation on possible induction of adaptive response (AR) by high-liner energy transfer (LET) particle radiation for protection against low-LET photon radiation-induced detrimental effects has not yet been performed in utero. This study verified if an AR could be induced by high-LET particle radiation from accelerated heavy ions against low-LET X-ray radiation-induced detrimental effects on fetal mice. Total body irradiation of pregnant C57BL/6J mice were performed by delivering a priming dose ranging from 10 mGy to 320 mGy of particle radiation on gestation day 11 followed one day later by a challenge dose at 3500 mGy from X-ray radiation. The monoenergetic beams of carbon, silicon and iron with the LET values of about 15, 55, and 200 KeV/μm, respectively, were examined. Significant suppression by the priming radiation of the detrimental effects (fetal death, malformation, or low body weight) was used as the endpoints for judgment of a successful AR induction on gestation day 18. Existence of AR was not observed. On the other hand, the priming dose of high-LET particle radiation, in some cases, even increased the detrimental effects induced by the challenge dose from low-LET X-ray radiation. Although existence of AR induced by high-LET radiation in cultured mammalian cells in vitro and in certain tissues of laboratory mice in vivo was demonstrated, the present study did not suggest that low dose of high-LET particle radiation could induce an AR in fetal mice in utero under the setup of our experimental system.     

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in <i>Escherichia coli</i> to promote L-tryptophan production

In this study, phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas (EMP) pathway and pentose phosphate (PP) pathway in Escherichia coli, thus increasing the L-tryptophan production. Firstly, the effects of disrupting EMP pathway on L-tryptophan production were studied, and the results indicated that the strain with deletion of phosphofructokinase A (i.e., E. coli JW-5 ΔpfkA) produced 23.4 ± 2.1 g/L of L-tryptophan production. However, deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth, especially deletion of glucosephosphate isomerase. Next, the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd) and thus increasing the L-tryptophan production (i.e., 26.5 ± 3.2 g/L vs. 21.7 ± 1.3 g/L) without obviously changing the cell growth (i.e., 0.41 h⁻¹ vs. 0.44 h⁻¹) as compared with the original strain JW-5. Finally, the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated. It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd (i.e., E. coli JW-5 zwf243 gnd361 ΔpfkA) produced 31.9 ± 2.7 g/L of L-tryptophan, which was 47.0% higher than that of strain JW-5. In addition, the glucose consumption rate of strain JW-5 zwf243 gnd361 ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5. The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.