<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.     

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 Plasmodiophora brassicae, accounts for serious yield losses. The aim of our study was to explore the phytohormone levels and metabolome changes 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, the susceptible 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 the genotype. 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 the resistant genotype (238) after infection. Interestingly, 132 DAMs were commonly detected in both genotypes when infected 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 metabolite analysis 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, suggesting their essential roles in resistance against P. brassicae infection.     

Intracellular life of protozoan <i>Toxoplasma gondii</i>: Parasitophorous vacuole establishment and survival strategies

Toxoplasma gondii is a protozoan of worldwide distribution and the agent of toxoplasmosis. It is estimated that 30%–50% of the world population could be infected with this parasite. Although the infection in immunocompetent individuals is mostly asymptomatic, the disease in immunosuppressed and pregnant is a risk condition. As a member of the phylum Apicomplexa, T. gondii has an obligatory intracellular lifestyle; therefore, invading a host cell and establishing it inside a parasitophorous vacuole (PV) are mandatories for the survival of this parasite. The construction of a perfect intracellular niche for T. gondii requires the secretion of an arsenal of proteins from unique secretory organelles. These proteins will remodel the vacuolar environment and the host cell organization and functions, allowing the parasite to access essential nutrients and stay “invisible” inside a host cell. In the present review, we will discuss the main steps involved in the PV formation and its differentiation to tissue cyst, focusing mainly on the strategies employed in the acquisition of nutrients and proteins involved in host cell modification.     

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.     

<i>In vitro</i> propagation of <i>Opuntia ellisiana</i> Griff. and acclimatization to field conditions

The genus Opuntia is a valuable forage resource in arid and semiarid lands during periods of drought and shortage of herbaceous plants. However, absolute minimum temperatures in the plains of Mendoza represent a limiting factor to cultivate several species.
Opuntia ellisiana is a cold hardy species, so the goals of this study were to massively propagate it using in vitro culture techniques, and then to acclimatize plantlets obtained to field conditions.
Different sterilization protocols were tested. Areoles were isolated in laminar airflow cabinet, and cultured on Murashige-Skoog medium, supplemented with sucrose and different BAP and IBA combinations. Explants were grown at 27±2ºC, under a 16-h photoperiod. The shoots produced were used in the rooting assay using different auxin combinations. In the most efficient growth treatment, plantlets reached 100% shooting after 35 days of culture, and a mean length of 10.2 mm after 49 days of culture. A 100% rooted plantlets was obtained on a medium containing 5 mg L^-1 IBA, after 12 days of culture. Acclimatization was achieved under greenhouse conditions, showing 100% plantlet survival.
This study suggests that O. ellisiana can be successfully micropropagated by areoles, and easily acclimatizated to field conditions.     

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.     

Identification of potential inhibitors for Sterol C-24 reductase of <i>Leishmania donovani</i> through virtual screening of natural compounds

Leishmaniasis is a vector-borne parasitic neglected tropical disease caused by a group of about 30 different species of the genus Leishmania. It is transmitted by the bite of female phlebotomies sand fly. Three main clinical manifestations of leishmaniasis include cutaneous, visceral, and mucocutaneous leishmaniasis. Visceral leishmaniasis (VL) caused by Leishmania donovani, is an infection of reticuloendothelial system and fatal if untreated. Cholesterol, a sterol that is prominent in the mammalian cell membranes whereas stigmasterol and ergosterol are more prevalent in plants, yeast, and protozoa, respectively. Ergosterols which is absent in human being, is an important constituent of parasite membrane. Sterol C-24 reductase (LdSR) enzyme catalyzes the final step in the ergosterol biosynthesis pathway. The inhibition of biosynthesis of ergosterol may lead to decreased cell viability and growth. Here, we performed the molecular docking-based virtual screening of a library of natural ligands against LdSR to identify a potential inhibitor to fight leishmaniasis. Capsaicin, prenyletin, flavan-3-ol, resveratrol, and gingerol showed the top binding affinity towards LdSR. Based upon ADME properties and bioactivity score, gingerol showed the best lead-likeness and drug-likeness properties. Hence, we further annotated its leishmanicidal properties. We found that gingerol inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes. Gingerol exerted its antileishmanial action through the induction of reactive oxygen species (ROS) in concentration-dependent manner. Gingerol induced ROS led to apoptosis. Overall, this study described that gingerol would act as possible inhibitor to LdSR.     

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.     

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.     

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.     

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.     

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.     

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.     

Secondary zoospores in the algal endoparasite <i>Maullinia ectocarpii</i> (Plasmodiophoromycota)

The present paper deals with the ultrastructure of zoospores produced by the plasmodiophorid Maullinia ectocarpii, living in the marine algal host Ectocarpus siliculosus. The zoospores described here are very similar to secondary zoospores of Polymyxa graminis and Phagomyxa sp. (the latter an algal endoparasite, also). Our results indicate that M. ectocarpii produces two types of plasmodia, and suggest that is a species with a complete life cycle, as it is known for all the Plasmodiophormycota that have been studied. Sporogenic and sporangial plasmodia produce, respectively, primary zoospores with parallel flagella within thick walled resting sporangia, and secondary zoospores with opposite flagella within thin walled sporangia.