Extensively Current Activity of Transposable Elements in Natural Rice Accessions Revealed by Singleton Insertions

Active transposable elements (TEs) have drawn more attention as they continue to create new insertions and contribute to genetic diversity of the genome. However, only a few have been discovered in rice up to now, and their activities are mostly induced by artificial treatments (e.g., tissue culture, hybridization etc.) rather than under normal growth conditions. To systematically survey the current activity of TEs in natural rice accessions and identify rice accessions carrying highly active TEs, the transposon insertion polymorphisms (TIPs) profile was used to identify singleton insertions, which were unique to a single accession and represented the new insertion of TEs in the genome. As a result, 10,924 high-confidence singletons from 251 TE families were obtained, covering all investigated TE types. The number of singletons varied substantially among different superfamilies/families, perhaps reflecting distinct current activity. Particularly, eight TE families maintained potentially higher activity in 3,000 natural rice accessions. Sixty percent of rice accessions were detected to contain singletons, indicating the extensive activity of TEs in natural rice accessions. Thirty-five TE families exhibited potentially high activity in at least one rice accession, and the majority of them showed variable activity among different rice groups/subgroups. These naturally active TEs would be ideal candidates for elucidating the molecular mechanisms underlying the transposition and activation of TEs, as well as investigating the interactions between TEs and the host genome.

TGF-β modulates cell fate in human ES cell-derived foregut endoderm by inhibiting multiple endogenous signaling pathways

Genetic differences between pluripotent stem cell lines causes variable activity of extra-cellular signaling pathways, which limits the reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenously provided factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that TGF-β1 activates an OTX2/LHX1 gene regulatory network that promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, the effects of TGF-β1 cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors make TGF-β1 treated cells refractory to Wnt signaling. Subsequently, TGF-β1-mediated inhibition of Bmp- and Wnt-signaling suppresses liver- and promotes pancreas fate. However, pancreas differentiation is delayed by TGF-β1-induced CYP26A1 expression and inhibition of RA signaling. Our study thus identifies multiple mechanisms of crosstalk between major developmental signaling pathways during foregut patterning.

Specificity and Mechanism of Coronavirus, Rotavirus and Mammalian Two-Histidine-Phosphoesterases That Antagonize Antiviral Innate Immunity

2′,5′-oligoadenylate(2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2′,5′-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses and rotaviruses encode 2-5A degrading enzymes thereby antagonizing RNase L and its antiviral effects. A-kinase anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self double-stranded-RNAs that activate OAS. We show these enzymes, members of the two-histidine-phosphoesterase (2H-PE) superfamily, constitute a sub-family referred here as 2′,5′-PEs. 2′,5′-PEs from mouse coronavirus (CoV) MHV (NS2), MERS-CoV (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3′,5′-oligoribonucleotides, all cleaved 2′,5′-oligoadenylates efficiently, but with variable activity against other 2′,5′-oligonucleotides. The 2′,5′-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2′,5′-p3A3) producing mono- or di-adenylates with 2′,3′-cyclic phosphate termini. Our results suggest that elimination of 2-5A might be the sole function of viral 2′,5′-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L.

Leishmanicidal Activity and Ultrastructural Changes of Maslinic Acid Isolated from Hyptidendron canum

The therapeutic arsenal for the treatment of leishmaniasis is limited and has serious obstacles, such as variable activity, high toxicity, and costs. To overcome such limitations, it becomes urgent to characterize new bioactive molecules. Plants produce and accumulate different classes of bioactive compounds, and these molecules can be studied as a strategy to combat leishmaniasis. The study presented herein evaluated the leishmanicidal effect of maslinic acid isolated from the leaves of Hyptidendron canum (Lamiaceae) and investigated the morphological that occurred on Leishmania (Leishmania) infantum upon treatment. Maslinic acid was active and selective against promastigote and amastigote forms in a dose-dependent manner. Additionally, it was not toxic to peritoneal macrophages isolated from golden hamsters, while miltefosine and amphotericin B showed mild toxicity for macrophages. Morphological changes in promastigotes of L. (L.) infantum treated with maslinic acid were related to cytoplasmic degeneration, intense exocytic activity, and blebbing in the kDNA; disruption of mitochondrial cristae was observed in some parasites. The nucleus of promastigote forms seems to be degraded and the chromatin fragmented, suggesting that maslinic acid triggers programmed cell death. These results indicate that maslinic acid may be an interesting molecule to develop new classes of drugs against leishmaniasis.

Calprotectin-mediated zinc chelation inhibits Pseudomonas aeruginosa protease activity in cystic fibrosis sputum

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. Importance The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc-starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

Calprotectin-mediated zinc chelation inhibits Pseudomonas aeruginosa protease activity in cystic fibrosis sputum

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment.

Assessing optimal: inequalities in codon optimization algorithms

Background Custom genes have become a common resource in recombinant biology over the last 20 years due to the plummeting cost of DNA synthesis. These genes are often “optimized” to non-native sequences for overexpression in a non-native host by substituting synonymous codons within the coding DNA sequence (CDS). A handful of studies have compared native and optimized CDSs, reporting different levels of soluble product due to the accumulation of misfolded aggregates, variable activity of enzymes, and (at least one report of) a change in substrate specificity. No study, to the best of our knowledge, has performed a practical comparison of CDSs generated from different codon optimization algorithms or reported the corresponding protein yields. Results In our efforts to understand what factors constitute an optimized CDS, we identified that there is little consensus among codon-optimization algorithms, a roughly equivalent chance that an algorithm-optimized CDS will increase or diminish recombinant yields as compared to the native DNA, a near ubiquitous use of a codon database that was last updated in 2007, and a high variability of output CDSs by some algorithms. We present a case study, using KRas4B, to demonstrate that a median codon frequency may be a better predictor of soluble yields than the more commonly utilized CAI metric. Conclusions We present a method for visualizing, analyzing, and comparing algorithm-optimized DNA sequences for recombinant protein expression. We encourage researchers to consider if DNA optimization is right for their experiments, and work towards improving the reproducibility of published recombinant work by publishing non-native CDSs.

In Vivo Activity of WCK 4282 (High-Dose Cefepime/Tazobactam) against Serine-β-lactamase-Producing Enterobacterales and Pseudomonas aeruginosa in the Neutropenic Murine Lung Infection Model

(248/250) Introduction: WCK 4282 (cefepime 2g/tazobactam 2g) maximizes systemic exposure of tazobactam and restores cefepime activity against various extended-spectrum β-lactamase (ESBL)- and cephalosporinase-producing strains in vitro. We describe clinical WCK 4282 exposure efficacy against various serine β-lactamase-producing Enterobacterales and Pseudomonas aeruginosa in a murine pneumonia model. Clinical cefepime-resistant isolates (17 Enterobacterales and 2 P. aeruginosa) were utilized. Isolates expressed ESBLs, cephalosporinases, and/or serine carbapenemases (KPC, OXA-48-like). WCK 4282 MICs were 4-32 μg/mL. For in vivo experiments, lungs of neutropenic mice were inoculated using standard inoculum (107 log10 CFU/mL). Serine-carbapenemase-producing isolates were also assessed using a low inoculum (1:5 dilution). Treatment mice received HSR of cefepime, meropenem (control for serine carbapenemase expression with low inoculum experiments), or WCK 4282 human-simulated regimens. Efficacy was assessed as change in log10 CFU/lung at 24h compared with 0h controls. Results: At standard inoculum, mean 0h bacterial burden was 6.65±0.23 log10 CFU/lung and increased at 24h by 2.48 ± 0.60 log10 CFU/lung among untreated controls. Lower inoculums initial bacterial burdens ranged from 5.81±0.12-6.39±0.13 log10 CFU/lung. At standard and/or low inoculums, cefepime and meropenem provided minimal activity. WCK 4282 produced >1-log10 reduction against 9/9 ESBL/cephalosporinase-producing strains. WCK 4282 provided variable activity among mice infected with standard or lower inoculums of OXA-48-like-producers. WCK 4282 exposures provided 0.53±1.07 log10 CFU/lung growth against KPC-producers at standard versus bacteriostasis (-0.15±0.54 change in log10 CFU/lung) at low inoculum. Conclusion: WCK 4282 produced potent in vivo activity against ESBL- and cephalosporinase-producing Enterobacterales and P. aeruginosa, and potential activity against OXA-48-like-producing Enterobacterales in a neutropenic pneumonia model.

Features of pedagogical prevention of deviant behavior among young adolescents

We consider the main prevention forms of deviant behavior of younger adolescents in general education organizations. The features of different types of prevention of deviant behavior of the individual are revealed. The model of preventive work with adolescents is possible with a number of conditions: teaching adolescents social skills, creating situations for changing the behavior of adolescents, organizing successful socially significant variable activity of a teenager. The influence of achievement motivation is important in the success of deviant behavior prevention. We reveal the features of the social environment necessary for the consolidation of positive changes in the adolescents’ behavior. Informing adolescents timely about the consequences of illegal behavior is important. We also highlight the necessity of early involvement in a healthy lifestyle of the child and the whole family. Attention is focused on the role of the state in preventing the appearance and development of deviations among young people.