In vitro activity of new combinations of ß-lactam and ß-lactamase inhibitors against the Mycobacterium tuberculosis complex.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a ß-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as ß-lactams in combination with ß-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

Effect of high-energy mechanical milling on the physicochemical and rheological properties of chayotextle (Sechium edule Sw.) starch.

This work evaluates the effect of high-energy mechanical milling time (7 levels, 20-80 min) on amylose content, crystallinity pattern, temperature and gelatinization enthalpy, morphology, and rheological properties of chayotextle (Sechium edule Sw.) starch. After 30 min of milling, granular structure was affected, and amylose values were the highest while crystallinity and gelatinization enthalpy decreased significantly. These changes allowed to obtain gels with viscoelastic properties where the elastic character (Ç´) prevailed upon the viscous modulus (Ǵ́). Native starch showed Tan δ values of 0.6, increased significantly (0.9) after 30 min of milling due to the surge in linear chains (amylose) and loss of granular structure. Native and modified starches showed high dependence on cutting or shear speed, presenting a non-Newtonian behavior (reofluidizers). These results indicate that mechanical grinding is an alternative to obtain modified starches with applications in the food industry.

Premature termination of transcription is shaped by Rho and translated uORFS in Mycobacterium tuberculosis.

Little is known about the decisions behind transcription elongation versus termination in the human pathogen Mycobacterium tuberculosis (M.TB). By applying Term-seq to M.TB we found that the majority of transcription termination is premature and associated with translated regions, i.e., within previously annotated or newly identified open reading frames. Computational predictions and Term-seq analysis, upon depletion of termination factor Rho, suggests that Rho-dependent transcription termination dominates all transcription termination sites (TTS), including those associated with regulatory 5' leaders. Moreover, our results suggest that tightly coupled translation, in the form of overlapping stop and start codons, may suppress Rho-dependent termination. This study provides detailed insights into novel M.TB cis-regulatory elements, where Rho-dependent, conditional termination of transcription and translational coupling together play major roles in gene expression control. Our findings contribute to a deeper understanding of the fundamental regulatory mechanisms that enable M.TB adaptation to the host environment offering novel potential points of intervention.

A novel kinetic model for a cocoa waste fermentation to ethanol reaction and its experimental validation.

A non-segregated kinetic model is proposed to describe a fermentation process of agro-industrial residues derived via cocoa (mucilage juice) by Pichia kudriavzevii. The novel proposed hybrid model is based on a multiple coupling reaction mechanisms (structured) to describe the kinetics of substrate consumption, biomass, carbon dioxide, and ethanol, coupled to an unstructured model for the activity enzyme. The parameters of the kinetic model are estimated by non-linear least-squares curve fitting using the Marquardt-Levenberg algorithm. In addition, numerical simulations were compared with the experimental data via residual graphs. The effectiveness of the model was statistically evaluated using dimensionless efficiency coefficients under different initial conditions. A global sensitivity analysis was applied (Fisher's information matrix). The experimental results of the batch reactor showed a maximum ethanol concentration of 29 g/L, with a yield of 0.48 g-ethanol/g-glucose and a productivity of 0.30 g/L h. The method determined that the cell formation coefficient and the specific substrate consumption rate (θ1 and θ2) directly influence most of the states of our system. The proposed scheme is particularly suitable to assist in the rational design of cell factory properties or fermentation processes because it can represent the complex biochemistry in more detail and under different initial experimental conditions; the above reveals that the generated model is robust and can be considered for control and optimization purposes.

Ribosome profiling enhances understanding of mycobacterial translation.

A recent addition to the -omics toolkit, ribosome profiling, enables researchers to gain insight into the process and regulation of translation by mapping fragments of mRNA protected from nuclease digestion by ribosome binding. In this review, we discuss how ribosome profiling applied to mycobacteria has led to discoveries about translational regulation. Using case studies, we show that the traditional view of "canonical" translation mechanisms needs expanding to encompass features of mycobacterial translation that are more widespread than previously recognized. We also discuss the limitations of the method and potential future developments that could yield further insight into the fundamental biology of this important human pathogen.

Growth and Yield of Purple Kculli Corn Plants under Different Fertilization Schemes.

Globally, corn is the most economically important crop, surpassing other cereals of economic importance. However, the tillage methods, monoculture and the abuse of synthetic agrochemicals used in Mexico have led to the loss of fertility and soil yield. In this sense, the application of alternative fertilization methods based on chemical fertilizer, organic matter and biofertilizer, applied alone or in combination, can stimulate the defense systems of corn plants and increase their yield. Therefore, in this research, some fertilization schemes were tested on purple corn plants of the Kculli race through the evaluation of some growth and yield variables, as well as the subsequent evaluation of the chemical characteristics of the corn grain produced in each fertilization scheme. The results indicate highly significant differences (p ≤ 0.05) between treatments, for the different growth and yield variables studied. Of all the fertilization schemes evaluated, treatment T7 obtained the best grain yield of 6.19 ± 0.07 t ha-1, with respect to treatment T1 of 1.02 ± 0.01 t ha-1, as well as the highest protein content and starch quality. Being clear the positive effect of the adequate contribution of the macro and micronutrients used exerts on the corn crop in each of the fertilization schemes studied. On the other hand, the analysis carried out on the grains was found within the values reported by other authors.

Transcriptomic Characterization of Tuberculous Sputum Reveals a Host Warburg Effect and Microbial Cholesterol Catabolism.

The crucial transmission phase of tuberculosis (TB) relies on infectious sputum and yet cannot easily be modeled. We applied one-step RNA sequencing (RNA-Seq) to sputum from infectious TB patients to investigate the host and microbial environments underlying transmission of Mycobacterium tuberculosis. In such TB sputa, compared to non-TB controls, transcriptional upregulation of inflammatory responses, including an interferon-driven proinflammatory response and a metabolic shift toward glycolysis, was observed in the host. Among all bacterial sequences in the sputum, approximately 1.5% originated from M. tuberculosis, and its transcript abundance was lower in HIV-1-coinfected patients. Commensal bacterial abundance was reduced in the presence of M. tuberculosis infection. Direct alignment to the genomes of the predominant microbiota species also reveals differential adaptation, whereby firmicutes (e.g., streptococci) displayed a nonreplicating phenotype with reduced transcription of ribosomal proteins and reduced activities of ATP synthases, while Neisseria and Prevotella spp. were less affected. The transcriptome of sputum M. tuberculosis more closely resembled aerobic replication and shared similarity in carbon metabolism to in vitro and in vivo models with significant upregulation of genes associated with cholesterol metabolism and downstream propionate detoxification pathways. In addition, and counter to previous reports on intracellular M. tuberculosis infection in vitro, M. tuberculosis in sputum was zinc, but not iron, deprived, and the phoP loci were also significantly downregulated, suggesting that the pathogen is likely extracellular in location. IMPORTANCE Although a few studies have described the microbiome composition of TB sputa based on 16S ribosomal DNA, these studies did not compare to non-TB samples and the nature of the method does not allow any functional inference. This is the first study to apply such technology using clinical specimens and obtained functional transcriptional data on all three aspects simultaneously. We anticipate that an improved understanding on the biological interactions in the respiratory tract may also allow novel interventions, such as those involving microbiome manipulation or inhibitor targeting disease-specific metabolic pathways.

Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5' leader region, which usually contains the Shine-Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin-antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine-Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine-Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine-Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine-Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine-Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

Evolutionary Implications of a Peroxidase with High Affinity for Cinnamyl Alcohols from Physcomitrium patens, a Non-Vascular Plant.

Physcomitrium (Physcomitrella) patens is a bryophyte highly tolerant to different stresses, allowing survival when water supply is a limiting factor. This moss lacks a true vascular system, but it has evolved a primitive water-conducting system that contains lignin-like polyphenols. By means of a three-step protocol, including ammonium sulfate precipitation, adsorption chromatography on phenyl Sepharose and cationic exchange chromatography on SP Sepharose, we were able to purify and further characterize a novel class III peroxidase, PpaPrx19, upregulated upon salt and H2O2 treatments. This peroxidase, of a strongly basic nature, shows surprising homology to angiosperm peroxidases related to lignification, despite the lack of true lignins in P. patens cell walls. Moreover, PpaPrx19 shows catalytic and kinetic properties typical of angiosperm peroxidases involved in oxidation of monolignols, being able to efficiently use hydroxycinnamyl alcohols as substrates. Our results pinpoint the presence in P. patens of peroxidases that fulfill the requirements to be involved in the last step of lignin biosynthesis, predating the appearance of true lignin.

Subfunctionalization of Paralog Transcription Factors Contributes to Regulation of Alkaloid Pathway Branch Choice in Catharanthus roseus.

Catharanthus roseus produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available C. roseus transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the C. roseus flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.

Evidencia molecular de la infección por Trypanosoma cruzi TcI en Meccus pallidipennis capturados en el municipio de Tepecuacuilco, Guerrero.

No disponible.

A snapshot of translation in Mycobacterium tuberculosis during exponential growth and nutrient starvation revealed by ribosome profiling.

Mycobacterium tuberculosis, which causes tuberculosis, can undergo prolonged periods of non-replicating persistence in the host. The mechanisms underlying this are not fully understood, but translational regulation is thought to play a role. A large proportion of mRNA transcripts expressed in M. tuberculosis lack canonical bacterial translation initiation signals, but little is known about the implications of this for fine-tuning of translation. Here, we perform ribosome profiling to characterize the translational landscape of M. tuberculosis under conditions of exponential growth and nutrient starvation. Our data reveal robust, widespread translation of non-canonical transcripts and point toward different translation initiation mechanisms compared to canonical Shine-Dalgarno transcripts. During nutrient starvation, patterns of ribosome recruitment vary, suggesting that regulation of translation in this pathogen is more complex than originally thought. Our data represent a rich resource for others seeking to understand translational regulation in bacterial pathogens.

Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations.

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

Membrane-Bound Class III Peroxidases: Unexpected Enzymes with Exciting Functions.

Class III peroxidases are heme-containing proteins of the secretory pathway with a high redundance and versatile functions. Many soluble peroxidases have been characterized in great detail, whereas only a few studies exist on membrane-bound isoenzymes. Membrane localization of class III peroxidases has been demonstrated for tonoplast, plasma membrane and detergent resistant membrane fractions of different plant species. In silico analysis revealed transmembrane domains for about half of the class III peroxidases that are encoded by the maize (Zea mays) genome. Similar results have been found for other species like thale-cress (Arabidopsis thaliana), barrel medic (Medicago truncatula) and rice (Oryza sativa). Besides this, soluble peroxidases interact with tonoplast and plasma membranes by protein⁻protein interaction. The topology, spatiotemporal organization, molecular and biological functions of membrane-bound class III peroxidases are discussed. Besides a function in membrane protection and/or membrane repair, additional functions have been supported by experimental data and phylogenetics.

Translational regulation in mycobacteria and its implications for pathogenicity.

Protein synthesis is a fundamental requirement of all cells for survival and replication. To date, vast numbers of genetic and biochemical studies have been performed to address the mechanisms of translation and its regulation in Escherichia coli, but only a limited number of studies have investigated these processes in other bacteria, particularly in slow growing bacteria like Mycobacterium tuberculosis, the causative agent of human tuberculosis. In this Review, we highlight important differences in the translational machinery of M. tuberculosis compared with E. coli, specifically the presence of two additional proteins and subunit stabilizing elements such as the B9 bridge. We also consider the role of leaderless translation in the ability of M. tuberculosis to establish latent infection and look at the experimental evidence that translational regulatory mechanisms operate in mycobacteria during stress adaptation, particularly focussing on differences in toxin-antitoxin systems between E. coli and M. tuberculosis and on the role of tuneable translational fidelity in conferring phenotypic antibiotic resistance. Finally, we consider the implications of these differences in the context of the biological adaptation of M. tuberculosis and discuss how these regulatory mechanisms could aid in the development of novel therapeutics for tuberculosis.

Delayed effects of transcriptional responses in Mycobacterium tuberculosis exposed to nitric oxide suggest other mechanisms involved in survival.

Mycobacterium tuberculosis has succeeded as a human pathogen for tens of thousands of years thanks to its ability to resist and adapt to the adverse conditions it encounters upon infection. Bacterial adaptation to stress is commonly viewed in the context of transcriptional regulation, with the implicit expectation that an initial transcriptomic response is tightly coupled to an ensuing proteomic response. However, after challenging M. tuberculosis with nitric oxide we found that the rapid transcriptional responses, detectable within minutes of nitric oxide exposure, typically took several hours to manifest on the protein level. Furthermore, early proteomic responses were dominated by the degradation of a set of proteins, specifically those containing damaged iron-sulphur clusters. Overall, our findings are consistent with transcriptional responses participating mostly in late-stage recovery rather than in generating an immediate resistance to nitric oxide stress, suggesting that survival of M. tuberculosis under acute stress is contingent on mechanisms other than transcriptional regulation. These findings provide a revised molecular understanding of an important human pathogen.

Deciphering the role of the phenylpropanoid metabolism in the tolerance of Capsicum annuum L. to Verticillium dahliae Kleb.

Verticillium dahliae is an economically relevant soilborne pathogen that causes vascular wilt in several crops, including pepper (Capsicum annuum). Fungal infection is usually visualized as a vascular browning, likely due to the onset of phenylpropanoid metabolism, which also seems to play a crucial role in the tolerance of some pepper varieties. In the current work, the potential function of distinct phenylpropanoid derivatives (suberin, lignin and phenolic compounds) in the pepper tolerance response against V. dahliae, was investigated. Histochemical and biochemical analyses ruled out suberin as a key player in the pepper-fungus interaction. However, changes observed in lignin composition and higher deposition of bound phenolics in infected stems seemed to contribute to the reinforcement of cell walls and the impairment of V. dahliae colonization. Most importantly, this is the first time that the accumulation of the hydroxycinnamic acid amide N-feruloyltyramine was reported in pepper stems in response to a vascular fungus. Fungitoxic activity for that hydroxycinnamate-tyramine conjugate was demonstrated as well.

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.