A Genetic Lab-on-Phone Test for Point-of-Care Diagnostic of Lactose Intolerance near Patient and in less than 90 Minutes.

BACKGROUND: The -13910 C/T single nucleotide polymorphism located within the MCM6 gene, an enhancer region located upstream of the lactase-phlorizin hydrolase gene, is associated with lactase persistence/non-persistence traits among the Caucasian population. The performance of a new point-of-care CE-IVD (In Vitro Diagnostic) marked isothermal lab-on-phone lactose intolerance assay, using crude samples, was assessed in comparison with Sanger sequencing using purified DNA, as reference method. METHODS: The study was conducted following a non-probability sampling using direct buccal swab (n = 63) and capillary blood (n = 43) clinical samples from a total of 63 volunteers. A 3 × 3 confusion matrix/contingency table was used to evaluate the performance of the isothermal lab-on-phone lactose intolerance assay. RESULTS: The isothermal lab-on-phone lactose intolerance assay successfully detected the -13910 C/T variant with a limit of detection of 5 cells/assay and demonstrated an overall accuracy of 98.41% (95% CI, 91.47%-99.96%) for buccal swab samples and 100% (95% CI, 91.19%-100%) for capillary blood, taking just 90 min from sample to result, with only 2 min hands-on. CONCLUSIONS: The lab-on-phone pocket-sized assay displayed good performance when using direct buccal swab and capillary blood samples, enabling a low-cost, real-time, and accurate genotyping of the -13910 C/T region for the rapid diagnosis of primary lactose intolerance at point-of-care, which enables a prompt implementation of appropriate diet habits and/or intolerance therapies. To our knowledge, this is the first point-of-care genetic test for lactose intolerance to be made available on the market.

Trans(gender) journeys: rights and the (non-)recognition of "human".

A human right paradigm has been challenging the biomedical perspectives that tend to be normalized in the Western context concerning the lives of trans people. The aim of this study is to understand how trans people in Portugal and Brazil perceive the (non-)recognition of their socio-cultural, economic and political rights. Specifically, the study intends to know in what extent these perceptions influence the processes of identity (de)construction. For this purpose, 35 semi-structured interviews were conducted with people self-identified as trans, transsexuals and transvestites in Brazil and Portugal. The narratives of the participants were analyzed according to the thematic analysis method and the following six main themes emerged: (i) Who are the rights for; (ii) Types of rights; (iii) Paradigm of distribution of rights; (iv) Local or global rights; (v) Non-recognition of the "human"; and, (vi) Transphobias (and cissexism). The results allowed the knowledge of rights and the non-recognition of the "human" which is the central organizer of the analysis. Among the main conclusions of this study, we emphasize the circumscription of rights to certain international, regional and/or national contexts; the existence of local instead of global rights, since they are influenced by regional and international law, but they depend on the legislation in force in each country; and the way human rights can also be understood as a platform of invisibility and exclusion of other people. Based on a commitment to social transformation, this article also contributes to rethinking the violence that is exercised on trans people as a continuum, whether through 'normalizing devices' by medical contexts, family contexts, public space, or even through internalized transphobia. Social structures produce and sustain transphobias and, simultaneously, are responsible for fighting them by changing the paradigm about the conception of transsexualities.

Drug Repurposing for COVID-19: A Review and a Novel Strategy to Identify New Targets and Potential Drug Candidates.

In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) was first identified in the province of Wuhan, China. Since then, there have been over 400 million confirmed cases and 5.8 million deaths by COVID-19 reported worldwide. The urgent need for therapies against SARS-CoV-2 led researchers to use drug repurposing approaches. This strategy allows the reduction in risks, time, and costs associated with drug development. In many cases, a repurposed drug can enter directly to preclinical testing and clinical trials, thus accelerating the whole drug discovery process. In this work, we will give a general overview of the main developments in COVID-19 treatment, focusing on the contribution of the drug repurposing paradigm to find effective drugs against this disease. Finally, we will present our findings using a new drug repurposing strategy that identified 11 compounds that may be potentially effective against COVID-19. To our knowledge, seven of these drugs have never been tested against SARS-CoV-2 and are potential candidates for in vitro and in vivo studies to evaluate their effectiveness in COVID-19 treatment.

Low-Phytotoxic Deep Eutectic Systems as Alternative Extraction Media for the Recovery of Chitin from Brown Crab Shells.

The versatility of chitin and its derivatives has allowed their utilization in a wide range of applications, from wastewater treatment to pharmaceutical or biomedical industries. However, even though the extraction method used industrially is extremely efficient, it involves the use of strong acids and bases and results in the disposal of large quantities of toxic effluents. Deep eutectic systems (DESs) have emerged as a promising new class of alternative solvents, including for chitin recovery. Yet, the assessment of their toxicity has often been neglected. Therefore, in this work, the phytotoxicity of choline chloride (ChCl)/organic acid-based DESs toward wheat seeds was evaluated by measuring different growth parameters and stress biomarkers. DESs were then explored for the efficient recovery of chitin contained in brown crab shell residues at varying conditions of temperature and processing time as well as with and without water addition. The obtained chitin was then characterized through different analytical techniques and compared to a standard as well as to chitin obtained by a conventional acid/alkaline hydrolysis. Results have shown that by applying a ChCl/lactic acid-based DES (which was the system that showed the least phytotoxic effects on wheat; EC50 ≥ 1.6 mg/mL) at 130 °C, it was possible to obtain pure chitin (up to 98%) with characteristics similar to those presented by commercial chitin or chitin recovered by conventional hydrolysis in a shorter time (more than 8-fold faster), thus suggesting that ChCl/organic acid-based DESs can truly represent a low-phytotoxic alternative extraction media for the recovery of chitin from the crab shell biomass.

Priority effects of stream eutrophication and assembly history on beta diversity across aquatic consumers, decomposers and producers.

Priority effects are stochastic processes that consider the influence of the order of arrival of species on community dynamics and structure. We evaluated the short-term effects of stream eutrophication and colonization time in freshwater benthic communities (primary producers - periphytic algae, decomposers - fungi, and consumers - macroinvertebrates) to test whether (i) beta diversity is higher in eutrophic streams due to priority effects driven by stochastic community formation processes (ecological drift or random dispersal), and (ii) in the early stages of colonization, priority effects drive the history of the formation and the initial establishment of the community in the stream, resulting in higher beta diversity. The present study was conducted in situ over 28 days in temperate streams along a trophic gradient, with colonization being evaluated every seven days. The study identified 84 species of alga, 43 families of macroinvertebrates, and 44 species of aquatic fungi. Our results demonstrated that deterministic processes were responsible for the formation of aquatic producers, while priority effects (stochasticity) were more important for the aquatic decomposers and consumers. In the case of the producers, beta diversity was highest in the hypertrophic stream, but did not vary significantly over colonization time. The beta diversity of the decomposers was highest in the hypertrophic stream and in the later stages of succession, due primarily to mechanisms of facilitation. The beta diversity of the consumers was lowest in the hypertrophic stream due primarily to the priority and inhibitory effects of the predominant groups, and highest at seven and 21 days of colonization. As these three taxonomic groups differ in their intrinsic biological characteristics, and in their functional role in the ecosystem, our short-term field study demonstrated that both stochastic and deterministic processes combine to influence the configuration of the community, and that the relative importance of the two processes varies systematically along a trophic gradient.

Violence in the affective-sexual trajectories of young gay men: "new" settings and "old" challenges. / Violências nas trajetórias afetivo-sexuais de jovens gays: "novas" configurações e "velhos" desafios.

This paper discusses the phenomenon of violence in the affective-sexual trajectories of young, cisgender gay men, from popular strata, in the metropolitan region of Rio de Janeiro, Brazil. Brazilian literature on violence against gay men generally focuses on the discrimination suffered by this population. However, the violence they are submitted to among family relationships due to their sexual orientation, or even their relationships known as "dating" or "hookup", is hardly discussed. This qualitative study used in-depth interviews based on a semi-structured guide to discuss violence during the young gay men's affective-sexual trajectory. The results evidenced multiple faces of violence during childhood and adolescence in family relationships, spanning their affective-sexual relationships in adolescence and youth, including sexual, physical, psychological, and institutional violence. Support networks are limited, for example, to a few friends and access to blogs on the Internet. No health professional was cited as a helping source. There is a need to discuss the prevention of violence and discuss health promotion of this social group, expanding the perspective on the various contemporary ways of relating intimately.

Este artigo aborda o fenômeno da violência nas trajetórias afetivo-sexuais de jovens gays cisgênero, pertencentes a camadas populares da região metropolitana do Rio de Janeiro, Brasil. A literatura brasileira sobre violência contra homens gays, geralmente, tem como foco a discriminação sofrida por esta população, mas pouco se problematiza a violência que eles sofrem nas relações familiares devido à sua orientação sexual, ou ainda, em suas relações ditas como "namoro" ou "ficar". Com o objetivo de discutir a presença de violências durante a trajetória afetivo-sexual dos jovens, este estudo qualitativo realizou entrevistas em profundidade a partir de um roteiro semiestruturado. Os resultados mostraram que há múltiplas faces de violência que ocorrem durante a infância e adolescência nas relações familiares, perpassando nos seus relacionamentos afetivo-sexuais na adolescência e juventude, incluindo violências sexuais, físicas, psicológicas e institucionais. As redes de apoio dos jovens são limitadas, como exemplo, a poucos amigos e ao acesso de blogs na internet. Nenhum profissional de saúde foi citado pelos jovens como fonte de ajuda. Destaca-se também a necessidade de debater a prevenção da violência e promoção da saúde destes jovens, ampliando o olhar para as várias formas contemporâneas de se relacionar intimamente.

Measuring Efflux and Permeability in Mycobacteria.

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.

Violências nas trajetórias afetivo-sexuais de jovens gays: "novas" configurações e "velhos" desafios / Violence in the affective-sexual trajectories of young gay men: "new" settings and "old" challenges

Resumo Este artigo aborda o fenômeno da violência nas trajetórias afetivo-sexuais de jovens gays cisgênero, pertencentes a camadas populares da região metropolitana do Rio de Janeiro, Brasil. A literatura brasileira sobre violência contra homens gays, geralmente, tem como foco a discriminação sofrida por esta população, mas pouco se problematiza a violência que eles sofrem nas relações familiares devido à sua orientação sexual, ou ainda, em suas relações ditas como "namoro" ou "ficar". Com o objetivo de discutir a presença de violências durante a trajetória afetivo-sexual dos jovens, este estudo qualitativo realizou entrevistas em profundidade a partir de um roteiro semiestruturado. Os resultados mostraram que há múltiplas faces de violência que ocorrem durante a infância e adolescência nas relações familiares, perpassando nos seus relacionamentos afetivo-sexuais na adolescência e juventude, incluindo violências sexuais, físicas, psicológicas e institucionais. As redes de apoio dos jovens são limitadas, como exemplo, a poucos amigos e ao acesso de blogs na internet. Nenhum profissional de saúde foi citado pelos jovens como fonte de ajuda. Destaca-se também a necessidade de debater a prevenção da violência e promoção da saúde destes jovens, ampliando o olhar para as várias formas contemporâneas de se relacionar intimamente.

Abstract This paper discusses the phenomenon of violence in the affective-sexual trajectories of young, cisgender gay men, from popular strata, in the metropolitan region of Rio de Janeiro, Brazil. Brazilian literature on violence against gay men generally focuses on the discrimination suffered by this population. However, the violence they are submitted to among family relationships due to their sexual orientation, or even their relationships known as "dating" or "hookup", is hardly discussed. This qualitative study used in-depth interviews based on a semi-structured guide to discuss violence during the young gay men's affective-sexual trajectory. The results evidenced multiple faces of violence during childhood and adolescence in family relationships, spanning their affective-sexual relationships in adolescence and youth, including sexual, physical, psychological, and institutional violence. Support networks are limited, for example, to a few friends and access to blogs on the Internet. No health professional was cited as a helping source. There is a need to discuss the prevention of violence and discuss health promotion of this social group, expanding the perspective on the various contemporary ways of relating intimately.

Psychomedical Interventions with Transgender People in Portugal and Brazil: A Critical Approach.

This study aims to analyze biopsychomedical interventions with transgender people. For this purpose, we carried out 35 semi-structured interviews with people who self-identify as transsexuals and transvestites in Brazil and Portugal. The responses of the study participants were systematized according to a thematic analysis, which led to the emergence of the following three main themes: "institutional power", "expectations of trans-bodies", and "experiences in health services". This study demonstrates how some trans people perform bodily modifications to fight the transphobia they experience throughout their lives. In addition, they believe that, by making their bodies conform to each other, they may become more attractive and desirable. The process of cisnormativity is, furthermore, conveyed by the idea present in the answers of some respondents: that having "integrated" bodies means facing less discrimination and that they will, therefore, obtain more satisfactory ways of personally and socially experiencing their identities. This study contributes to a deepening critical reflection on the experiences/exclusions of trans people, especially in the psychomedical context of "normalization" devices. Hence, just as social structures produce and sustain transphobia, the same structures are responsible for combating it.

História das abordagens científicas, médicas e psicológicas sobre as transexualidades e suas aproximações críticas / A history of scientific, medical and psychological approaches to transsexualities and their critical approach

Resumo Neste artigo, apresenta-se um breve enquadramento histórico das abordagens científicas, médicas e psicológicas sobre as transexualidades, tecendo um conjunto de considerações sobre a forma como tal enquadramento foi fundacional da noção de patologia associada às pessoas trans e como tem sido responsável pela manutenção da patologização destes indivíduos no imaginário coletivo. Para atingir tal objetivo, é desenhado um mapa cronológico dos acontecimentos que têm vindo a marcar, ao longo da história, o estudo e a intervenção com as pessoas trans a partir do modelo biomédico, referenciando algumas das personalidades que, no contexto ocidental, tiveram responsabilidade nesta visão biomédica das pessoas trans. No final deste trabalho, apresenta-se a discussão em torno da (des)patologização das transexualidades a partir do surgimento do paradigma centrado nos direitos humanos das pessoas trans e em propostas de autodeterminação dos seus corpos e identidades.

Abstract This study provides a brief historical background of scientific, medical and psychological approaches to transsexualities. It also makes considerations on how such approaches based the concept of pathology that is associated with trans people and how it has been responsible for maintaining the pathologization in the collective imaginary. More specifically, we will establish a chronology of events that, throughout history, have affected the study on and the intervention with trans people, based on the biomedical model. We will also refer to some Western figures responsible for creating this biomedical vision of trans people. Finally, the discussion about the (de)pathologization of transsexualities is presented, based on the emergence of the paradigm centered on the human rights of trans people and proposals for self-determination of their bodies and identities.

Efflux pump inhibitors as a promising adjunct therapy against drug resistant tuberculosis: a new strategy to revisit mycobacterial targets and repurpose old drugs.

INTRODUCTION: In 2018, an estimated 377,000 people developed multidrug-resistant tuberculosis (MDR-TB), urging for new effective treatments. In the last years, it has been accepted that efflux pumps play an important role in the evolution of drug resistance. Strategies are required to mitigate the consequences of the activity of efflux pumps. AREAS COVERED: Based upon the literature available in PubMed, up to February 2020, on the diversity of efflux pumps in Mycobacterium tuberculosis and their association with drug resistance, studies that identified efflux inhibitors and their effect on restoring the activity of antimicrobials subjected to efflux are reviewed. These support a new strategy for the development of anti-TB drugs, including efflux inhibitors, using in silico drug repurposing. EXPERT OPINION: The current literature highlights the contribution of efflux pumps in drug resistance in M. tuberculosis and that efflux inhibitors may help to ensure the effectiveness of anti-TB drugs. However, despite the usefulness of efflux inhibitors in in vitro studies, in most cases their application in vivo is restricted due to toxicity. In a time when new drugs are needed to fight MDR-TB and extensively drug-resistant TB, cost-effective strategies to identify safer efflux inhibitors should be implemented in drug discovery programs.

Taxonomy of common taxa of Chlorophyceae (Chlorophyta) and Zygnematophyceae (Streptophyta) from periphyton of a Neotropical floodplain

Some orders of algal groups such as Chlorophyceae and Zygnematophyceae have been reported as frequent taxa in periphytic communities of wetlands. The present study aimed at submitting these algal members which occurred in high abundance and frequency in the periphyton of 30 environments of the Upper Paraná River floodplain to a taxonomic survey and to present some ecological data concerning their richness. Periphyton were collected from petioles by scraping of Eichhornia azurea (Sw.) Kunth and preserved with Lugol acetic solution in the Upper Paraná River, Brazil, during high water period in March 2010. Taxa were counted in inverted microscope and those higher than 2,500 individuals in density with frequency of occurrence less than 50% simultaneously in the 30 environments sampled were subjected to a detailed taxonomical treatment under optical microscope coupled to a light camera and ocular micrometer under 1000x. Identifications and descriptions were made according to the algal literature. Some abiotic data were shown. Fifteen taxa belonging to the classes Chlorophyceae and Zygnematophyceae were abundant representing 64.6% of the total density. A Procruste analysis within a Detrended Correspondence Analysis showed that distribution pattern of richness of clorophyceans and zygnematophyceans was represented by common species of these communities, highlighting the importance of knowing about these algae taxonomy

Unveil the Anticancer Potential of Limomene Based Therapeutic Deep Eutectic Solvents.

Deep eutectic solvents have been recently reported as an interesting alternative to improve the therapeutic efficacy of conventional drugs, hence called therapeutic deep eutectic solvents (THEDES). The main objective of this work was to evaluate the potential of limonene (LIM) based THEDES as new possible systems for cancer treatment. LIM is known to have antitumor activity, however it is highly toxic and cell viability is often compromised, thus this compound is not selective towards cancer cells. Different THEDES based on LIM were developed to unravel the anticancer potential of such systems. THEDES were prepared by gently mixing saturated fatty acids menthol or ibuprofen (IBU) with LIM. Successful THEDES were obtained for Menthol:LIM (1:1), CA:LIM (1:1), IBU:LIM (1:4) and IBU:LIM(1:8). The results indicate that all the THEDES present antiproliferative properties, but IBU:LIM (1:4) was the only formulation able to inhibit HT29 proliferation without comprising cell viability. Therefore, IBU:LIM (1:4) was the formulation selected for further assessment of anticancer properties. The results suggest that the mechanism of action of LIM:IBU (1:4) is different from isolated IBU and LIM, which suggest the synergetic effect of DES. In this work, we unravel a methodology to tune the selectivity of LIM towards HT29 cell line without compromising cell viability of healthy cells. We demonstrate furthermore that coupling LIM with IBU leads also to an enhancement of the anti-inflammatory activity of IBU, which may be important in anti-cancer therapies.

Design, Synthesis, and Efficacy Testing of Nitroethylene- and 7-Nitrobenzoxadiazol-Based Flavodoxin Inhibitors against Helicobacter pylori Drug-Resistant Clinical Strains and in Helicobacter pylori-Infected Mice.

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles.

Aminoglycoside acetyltransferases are important determinants of resistance to aminoglycoside antibiotics in most bacterial genera. In mycobacteria, however, aminoglycoside acetyltransferases contribute only partially to aminoglycoside susceptibility since they are related with low level resistance to these antibiotics (while high level aminoglycoside resistance is due to mutations in the ribosome). Instead, aminoglycoside acetyltransferases contribute to other bacterial functions, and this can explain its widespread presence along species of genus Mycobacterium. This review is focused on two mycobacterial aminoglycoside acetyltransferase enzymes. First, the aminoglycoside 2'-N-acetyltransferase [AAC(2')], which was identified as a determinant of weak aminoglycoside resistance in M. fortuitum, and later found to be widespread in most mycobacterial species; AAC(2') enzymes have been associated with resistance to cell wall degradative enzymes, and bactericidal mode of action of aminoglycosides. Second, the Eis aminoglycoside acetyltransferase, which was identified originally as a virulence determinant in M. tuberculosis (enhanced intracellular survival); Eis protein in fact controls production of pro-inflammatory cytokines and other pathways. The relation of Eis with aminoglycoside susceptibility was found after the years, and reaches clinical significance only in M. tuberculosis isolates resistant to the second-line drug kanamycin. Given the role of AAC(2') and Eis proteins in mycobacterial biology, inhibitory molecules have been identified, more abundantly in case of Eis. In conclusion, AAC(2') and Eis have evolved from a marginal role as potential drug resistance mechanisms into a promising future as drug targets.

The EU approved antimalarial pyronaridine shows antitubercular activity and synergy with rifampicin, targeting RNA polymerase.

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 µg/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 µg/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 µg/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.

Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis.

The spread of multidrug-resistant isolates of Mycobacterium tuberculosis requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine (SCN) and N-methyl-seconeolitsine (N-SCN), against M. tuberculosis. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of Streptococcus pneumoniae. Both SCN and N-SCN inhibited M. tuberculosis growth at 1.95-15.6 µM, depending on the strain. In M. smegmatis this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of Escherichia coli. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. In vitro inhibition of MtbTopoI activity by SCN and N-SCN was tested using a plasmid relaxation assay. Both SCN and N-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 µM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated in vivo in plasmid-containing cultures of M. tuberculosis. Plasmid supercoiling densities were -0.060 in cells untreated or treated with boldine, and -0.072 in 1 × MIC N-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of N-SCN. Altogether, these results demonstrate that the M. tuberculosis topoisomerase I enzyme is an attractive drug target, and that SCN and N-SCN are promising lead compounds for drug development.

Synergy between Circular Bacteriocin AS-48 and Ethambutol against Mycobacterium tuberculosis.

The increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains and the very few drugs available for treatment are promoting the discovery and development of new molecules that could help in the control of this disease. Bacteriocin AS-48 is an antibacterial peptide produced by Enterococcus faecalis and is active against several Gram-positive bacteria. We have found that AS-48 was active against Mycobacterium tuberculosis, including H37Rv and other reference and clinical strains, and also against some nontuberculous clinical mycobacterial species. The combination of AS-48 with either lysozyme or ethambutol (commonly used in the treatment of drug-susceptible tuberculosis) increased the antituberculosis action of AS-48, showing a synergic interaction. Under these conditions, AS-48 exhibits a MIC close to some MICs of the first-line antituberculosis agents. The inhibitory activity of AS-48 and its synergistic combination with ethambutol were also observed on M. tuberculosis-infected macrophages. Finally, AS-48 did not show any cytotoxicity against THP-1, MHS, and J774.2 macrophage cell lines at concentrations close to its MIC. In summary, bacteriocin AS-48 has interesting antimycobacterial activity in vitro and low cytotoxicity, so further studies in vivo will contribute to its development as a potential additional drug for antituberculosis therapy.

Total Synthesis of Ripostatin B and Structure-Activity Relationship Studies on Ripostatin Analogs.

Described is the total synthesis of the myxobacterial natural product ripostatin B and of a small number of analogs. Ripostatin B is a polyketide-derived 14-membered macrolide that acts as an inhibitor of bacterial RNA-polymerase, but is mechanistically distinct from rifamycin-derived RNA-polymerase inhibitors that are in use for tuberculosis treatment. The macrolactone ring of ripostatin B features two stereocenters and a synthetically challenging doubly skipped triene motif, with one of the double bonds being in conjugation with the ester carbonyl. Appended to the macrolactone core are an extended hydroxy-bearing phenylalkyl side chain at C13 and a carboxymethyl group at C3. The triene motif was established with high efficiency by ring-closing olefin metathesis, which proceeded in almost 80% yield. The side chain-bearing stereocenter α to the ester oxygen was formed in a Paterson aldol reaction between a methyl ketone and a ß-chiral ß-hydroxy aldehyde with excellent syn selectivity (dr >10:1). The total synthesis provided a blueprint for the synthesis of analogs with modifications in the C3 and C13 side chains. The C3-modified analogs showed good antibacterial activity against efflux-deficient Escherichia coli but, as ripostatin B, were inactive against Mycobacterium tuberculosis, in spite of significant in vitro inhibition of M. tuberculosis RNA-polymerase.