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The present work deals with the biotechnological valorization of the vinasse through a sustainable biorefinery. The aim was to design and analyze, through process simulation, a biorefinery for the valorization of vinasse in products such as organo-mineral fertilizers, supplements for animal feed, and biogas. For this purpose, the SuperPro Designer software was used, and a modified hierarchical decomposition method was applied. One base case and three scenarios were techno-economically assessed, and the net present value was used as a selection criterion. The technological configuration with the best techno-economic criterion was analyzed socially and environmentally. The results suggest that the best configuration of the biorefinery corresponded to scenario 3, with a net present value of USD$73,364,000, a number of direct employees of 38, and a blue water footprint of 16.16 m3/h. These findings highlight the potential of the design of biorefineries to address the valorization of vinasses in Colombia and worldwide.
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Alimentación Animal , Biocombustibles , Fertilizantes , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Simulación por Computador , Residuos Industriales , Biotecnología/métodosRESUMEN
Mycobacterium tuberculosis (Mtb) can adopt a non-growing dormant state during infection that may be critical to both active and latent tuberculosis. During dormancy, Mtb is widely tolerant toward antibiotics, a significant obstacle in current anti-tubercular drug regimens, and retains the ability to persist in its environment. We aimed to identify novel mechanisms that permit Mtb to survive dormancy in an in vitro carbon starvation model using transposon insertion sequencing and gene expression analysis. We identified a previously uncharacterized component of the lipid transport machinery, omamC, which was upregulated and required for survival during carbon starvation. We show that OmamC plays a role both in increasing fatty acid stores during growth in rich media and enhancing fatty acid utilization during starvation. Besides its involvement in lipid metabolism, OmamC levels affected the expression of the anti-anti-sigma factor rv0516c and other genes to improve Mtb survival during carbon starvation and increase its tolerance toward rifampicin, a first-line drug effective against non-growing Mtb. Importantly, we show that Mtb can be eradicated during carbon starvation, in an OmamC-dependent manner, by inhibiting lipid metabolism with the lipase inhibitor tetrahydrolipstatin. This work casts new light into the survival processes of non-replicating, drug-tolerant Mtb by identifying new proteins involved in lipid metabolism required for the survival of dormant bacteria and exposing a potential vulnerability that could be exploited for antibiotic discovery.IMPORTANCETuberculosis is a global threat, with ~10 million yearly active cases. Many more people, however, live with "latent" infection, where Mycobacterium tuberculosis survives in a non-replicative form. When latent bacteria activate and regrow, they elicit immune responses and result in significant host damage. Replicating and non-growing bacilli can co-exist; however, non-growing bacteria are considerably less sensitive to antibiotics, thus complicating treatment by necessitating long treatment durations. Here, we sought to identify genes important for bacterial survival in this non-growing state using a carbon starvation model. We found that a previously uncharacterized gene, omamC, is involved in storing and utilizing fatty acids as bacteria transition between these two states. Importantly, inhibiting lipid metabolism using a lipase inhibitor eradicates non-growing bacteria. Thus, targeting lipid metabolism may be a viable strategy for treating the non-growing population in strategies to shorten treatment durations of tuberculosis.
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Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Ácidos Grasos/metabolismo , Antibacterianos/farmacología , Carbono/metabolismo , Lipasa/metabolismoRESUMEN
New drugs to treat tuberculosis which target intractable bacterial populations are required to develop shorter and more effective treatment regimens. The benzene amide ether scaffold has activity against intracellular Mycobacterium tuberculosis, but low activity against extracellular, actively replicating M. tuberculosis. We determined that these molecules have bactericidal activity against non-replicating M. tuberculosis but not actively replicating bacteria. Exposure to compounds depleted ATP levels in non-replicating bacteria and increased the oxygen consumption rate; a subset of molecules led to the accumulation of intrabacterial reactive oxygen species. A comprehensive screen of M. tuberculosis strains identified a number of under-expressing strains as more sensitive to compounds under replicating conditions including QcrA and QcrB hypomorphs. We determined the global gene expression profile after compound treatment for both replicating and nutrient-starved M. tuberculosis. We saw compound-dependent changes in the expression of genes involved in energy metabolism under both conditions. Taken together, our data suggest that the scaffold targets respiration in M. tuberculosis.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Antituberculosos/metabolismo , Benceno/farmacología , Éter/metabolismo , Éter/farmacología , Éter/uso terapéutico , Amidas/farmacología , Pruebas de Sensibilidad Microbiana , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Éteres de Etila/metabolismo , Éteres de Etila/farmacología , Éteres de Etila/uso terapéutico , Éteres/metabolismo , Éteres/farmacología , Éteres/uso terapéuticoRESUMEN
Purpose: This study aimed to compare the success and complication rates among patients implanted with Ahmed Glaucoma Valve (AGV) using the short and long tunnel technique through retrospective chart review. Patients and Methods: We reviewed 54 charts of adult patients who underwent AGV implantation using a Short-Needle Track (SNT) or Long-Needle Track (LNT) technique. Intraocular pressures (IOP), Best Corrected Visual Acuity (BCVA) and number of medications were recorded pre-operatively, and at Day 1, 3, 7, Month 1, 3, 6 post-operatively. Treatment success, occurrence of Hypertensive Phase (HP), complication and procedures done after AGV implantation were compared between the two groups using one-tailed Z-test of proportions. Results: A total of 20 (LNT) and 21 (SNT) charts were included in the study. There was no significant difference between the median postoperative IOP, BCVA, and number of anti-glaucoma medications between the two groups at each time interval. The comparison between the occurrence of HP (P = 0.435) and success rates (P = 0.476) between the two groups yielded no significant difference. Flat/shallow anterior chamber (AC) was seen exclusively in three eyes (14%) in the SNT group (P = 0.039). There was one occurrence of plate exposure in the LNT group (P = 0.149). Conclusion: The LNT technique of AGV Implantation may be used as an alternative to the traditional SNT (with autologous graft). The long needle track offers the advantage reducing the risk of complications arising from shallow anterior chamber post-operatively.
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Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.
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Mycobacterium tuberculosis , Sistema Libre de Células , ADN Bacteriano/análisis , ADN Bacteriano/genética , Mycobacterium tuberculosis/genética , Genoma Bacteriano , Sistemas CRISPR-Cas , Humanos , Tuberculosis/diagnóstico , Tuberculosis/microbiologíaRESUMEN
The ability to measure neutralizing antibodies on large scale can be important for understanding features of the natural history and epidemiology of infection, as well as an aid in determining the efficacy of interventions, particularly in outbreaks such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Because of the assay's rapid scalability and high efficiency, serology measurements that quantify the presence rather than function of serum antibodies often serve as proxies of immune protection. Here, we report the development of a high-throughput, automated fluorescence-based neutralization assay using SARS-CoV-2 virus to quantify neutralizing antibody activity in patient specimens. We performed large-scale testing of over 19,000 COVID-19 convalescent plasma (CCP) samples from patients who had been infected with SARS-CoV-2 between March and August 2020 across the United States. The neutralization capacity of the samples was moderately correlated with serological measurements of anti-receptor-binding domain (RBD) IgG levels. The neutralizing antibody levels within these convalescent-phase serum samples were highly variable against the original USA-WA1/2020 strain with almost 10% of individuals who had had PCR-confirmed SARS-CoV-2 infection having no detectable antibodies either by serology or neutralization, and ~1/3 having no or low neutralizing activity. Discordance between neutralization and serology measurements was mainly due to the presence of non-IgG RBD isotypes. Meanwhile, natural infection with the earliest SARS-CoV-2 strain USA-WA1/2020 resulted in weaker neutralization of subsequent B.1.1.7 (alpha) and the B.1.351 (beta) variants, with 88% of samples having no activity against the BA.1 (omicron) variant. IMPORTANCE The ability to directly measure neutralizing antibodies on live SARS-CoV-2 virus in individuals can play an important role in understanding the efficacy of therapeutic interventions or vaccines. In contrast to functional neutralization assays, serological assays only quantify the presence of antibodies as a proxy of immune protection. Here, we have developed a high-throughput, automated neutralization assay for SARS-CoV-2 and measured the neutralizing activity of ~19,000 COVID-19 convalescent plasma (CCP) samples collected across the United States between March and August of 2020. These data were used to support the FDA's interpretation of CCP efficacy in patients with SARS-CoV-2 infection and their issuance of emergency use authorization of CCP in 2020.
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COVID-19 , SARS-CoV-2 , Humanos , Inmunidad Humoral , Sueroterapia para COVID-19 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus , Prueba de COVID-19RESUMEN
The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.
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Background: Unbiased assessment of the risks associated with acquisition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to informing mitigation efforts during pandemics. The objective of our study was to understand the risk factors for acquiring coronavirus disease 2019 (COVID-19) in a large prospective cohort of adult residents in a large US metropolitan area. Methods: We designed a fully remote longitudinal cohort study involving monthly at-home SARS-CoV-2 polymerase chain reaction (PCR) and serology self-testing and monthly surveys. Results: Between October 2020 and January 2021, we enrolled 10 289 adults reflective of the Boston metropolitan area census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared with Whites, Black non-Hispanic participants had a 2.2-fold greater risk of acquiring COVID-19 (hazard ratio [HR], 2.19; 95% CI, 1.91-2.50; P < .001), and Hispanics had a 1.5-fold greater risk (HR, 1.52; 95% CI, 1.32-1.71; P < .016). Individuals aged 18-29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities were associated with an increased risk of acquiring COVID-19. Individuals with medical risk factors for severe disease were at a decreased risk of SARS-CoV-2 acquisition. Conclusions: These results demonstrate that race/ethnicity and socioeconomic status are the biggest determinants of acquisition of infection. This disparity is significantly underestimated if based on PCR data alone, as noted by the discrepancy in serology vs PCR detection for non-White participants, and points to persistent disparity in access to testing. Medical conditions and advanced age, which increase the risk for severity of SARS-CoV-2 disease, were associated with a lower risk of COVID-19 acquisition, suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics.
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Purpose: This study aimed to determine if near visual acuity measurements using a Philippine peso bill are comparable to a standard Jaeger chart. Study Design: This is a cross-sectional study comparing the near visual acuity measurements of a Philippine peso bill and a Jaeger chart among sixty subjects. Methods: LogMAR scores from the two methods were analyzed using Wilcoxon Mann-Whitney test. The relationship of the logMAR scores between these methods was determined using Spearman rank order correlation. The Bland-Altman plot was used to determine the comparability between quantitative measurements for near visual acuity using the standard Jaeger chart (reference) and the Philippine peso bill. Results: There were no significant differences in the proportion of Jaeger scores and mean logMAR equivalents between the two methods (p > 0.05). The scatter plot diagram shows a positive upward trend with a very strong and significant correlation between logMAR scores of Jaeger chart and Philippine peso bill methods (r = 0.9258, p < 0.0001). With a concordance correlation coefficient of 0.9505, there is a high agreement between these two measures. The Philippine peso bill overestimates the Jaeger chart visual acuity by 0.04 logMAR units. We suspect that the contrast between the background color and the serial numbers of the peso bill may have brought about this outcome. Conclusion: The Philippine peso bill may be used as an alternative measuring tool for near visual acuity. However, there is a tendency to overestimate the scores obtained using the Philippine peso bill. Future studies are recommended to validate the results in a tele-ophthalmology setting.
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Introduction: Various barriers delay the process of patient transfer to critical care units. We implemented quality improvement methods to decrease the time required for interhospital transfer of critical care patients. As a result, we aimed to decrease the time from initial transfer call to specialized transport team arrival at the referring hospital from 150 minutes to <40 minutes over 2 years. Methods: Quality improvement initiative monitoring the length of transport time of 245 patients transferred from referral hospitals to a tertiary pediatric intensive care unit for 31 months from March 2013 to October 2015. We reviewed preexisting transport protocols and identified barriers to the timely arrival to the pediatric intensive care unit. We implemented 3 interventions: a transport information line serving as a central communication center to coordinate the transport process between all stakeholders, the formation of a specialized pediatric transport team, and a training program. We collected transport response time data and monitored the impact of interventions via statistical process control charts. Results: There was a significant decrease in the length of the time course pre- and postintervention. We noted a special cause to decrease in time from referral hospital call to arrival of our transport team by 76% from 150 minutes to 36 minutes. In addition, the statistical process chart revealed a stable and effective process without significant shifts above the process mean as early as 3 months postintervention. Conclusions: By improving our transport services with additional resources and people, we have improved the efficiency of patient transport between institutions.
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BACKGROUND: Antibody response duration following severe acute respiratory syndrome coronavirus 2 infection tends to be variable and depends on severity of disease and method of detection. STUDY DESIGN AND METHODS: COVID-19 convalescent plasma from 18 donors was collected longitudinally for a maximum of 63-129 days following resolution of symptoms. All the samples were initially screened by the Ortho total Ig test to confirm positivity and subsequently tested with seven additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, receptor binding domain, and nucleocapsid [NC]), along with two neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute [VRI] Pseudovirus reporter viral particle neutralization [RVPN]). RESULTS: The direct detection assays (Ortho total Ig total and Roche total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay. DISCUSSION: These data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag-sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1- and NC-based assays can help reduce the rate of false-positivity and discriminate between natural infection and vaccine-derived seroreactivity.
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Anticuerpos Antivirales/inmunología , Donantes de Sangre , COVID-19/epidemiología , COVID-19/inmunología , SARS-CoV-2/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , COVID-19/sangre , COVID-19/diagnóstico , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Estudios Seroepidemiológicos , Pruebas Serológicas/métodos , Pruebas Serológicas/normas , Índice de Severidad de la EnfermedadRESUMEN
COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.
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COVID-19/patología , COVID-19/virología , Riñón/patología , Hígado/patología , Pulmón/patología , Miocardio/patología , SARS-CoV-2/patogenicidad , Adulto , Anciano , Anciano de 80 o más Años , Atlas como Asunto , Autopsia , Bancos de Muestras Biológicas , COVID-19/genética , COVID-19/inmunología , Células Endoteliales , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Fibroblastos , Estudio de Asociación del Genoma Completo , Corazón/virología , Humanos , Inflamación/patología , Inflamación/virología , Riñón/virología , Hígado/virología , Pulmón/virología , Masculino , Persona de Mediana Edad , Especificidad de Órganos , Fagocitos , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , ARN Viral/análisis , Regeneración , SARS-CoV-2/inmunología , Análisis de la Célula Individual , Carga ViralRESUMEN
The increasing consumption of plantain fruits with specific quality standards generates high agricultural waste. This work aimed at valorising the rejected unripe pulp of Dominico-Hartón plantain fruits (Musa AAB Simmonds). The pulp was characterised physico-chemically, thermally and functionally. The data gathered experimentally and collected from different databases were used to design a production process of isomalto-oligosaccharides (IMO) syrup. The plantain flour contains high levels of starch (87 ± 2%) and amylose (31.2 ± 0.8%). The flour showed stability at high temperatures (pasting temperature of 79.26 ± 0.02 °C), allowing its use in high temperature processes. In vitro gastrointestinal digestion of the plantain flour showed that when cooked, the glycemic index of the flour increased from 47.7 ± 2.2 to 84.2 ± 1.8, while its resistant starch content only slightly decreased from 71.7 ± 1% to 52.6 ± 2%, suggesting that this type of flour preserves high content of dietary fibre after digestion. The conceptual process design showed that 24.48 g of IMO are theoretically obtained from 53.24 g of plantain flour maltose. These results suggest that the rejected plantain pulp holds high potential as an ingredient for the production of prebiotic compounds such as IMO.
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Digestión , Harina , Frutas , Musa , Índice Glucémico , Humanos , Valor NutritivoRESUMEN
The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.
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New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.
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Antituberculosos/clasificación , Antituberculosos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Eliminación de Gen , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Antituberculosos/farmacología , Girasa de ADN/metabolismo , Farmacorresistencia Microbiana , Ácido Fólico/biosíntesis , Terapia Molecular Dirigida , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/clasificación , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Especificidad por Sustrato , Inhibidores de Topoisomerasa II/aislamiento & purificación , Inhibidores de Topoisomerasa II/farmacología , Triptófano/biosíntesis , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Previous work established a coumarin scaffold as a starting point for inhibition of Mycobacterium tuberculosis (Mtb) FadD32 enzymatic activity. After further profiling of the coumarin inhibitor 4 revealed chemical instability, we discovered that a quinoline ring circumvented this instability and had the advantage of offering additional substitution vectors to further optimize. Ensuing SAR studies gave rise to quinoline-2-carboxamides with potent anti-tubercular activity. Further optimization of ADME/PK properties culminated in 21b that exhibited compelling in vivo efficacy in a mouse model of Mtb infection.
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Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Cumarinas/química , Animales , Antituberculosos/metabolismo , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Quinolinas/química , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.
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Farmacorresistencia Bacteriana , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Ribosomas/efectos de los fármacos , Ribosomas/genética , Proteínas Bacterianas/biosíntesis , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Proteoma/análisisRESUMEN
Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (â¼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence â¼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.
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Genoma Bacteriano/genética , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Microfluídica/métodos , Secuenciación Completa del Genoma/métodos , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Farmacorresistencia Microbiana/genética , Monitoreo del Ambiente/instrumentación , Monitoreo del Ambiente/métodos , Biblioteca de Genes , Genómica/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Dispositivos Laboratorio en un Chip , Microfluídica/instrumentación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Microbiología del Suelo , Secuenciación Completa del Genoma/instrumentaciónRESUMEN
Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.
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Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Catalasa/genética , Genómica/métodos , Humanos , Isoniazida/uso terapéutico , Mutación/genética , Mycobacterium tuberculosis/efectos de los fármacos , Polimorfismo Genético/genética , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Recent studies have renewed interest in ß-lactam antibiotics as a potential treatment for Mycobacterium tuberculosis infection. To explore the opportunities and limitations of this approach, we sought to better understand potential resistance mechanisms to ß-lactam antibiotics in M. tuberculosis. We identified mutations in the penicillin-binding protein (PBP) ponA2 that were able to confer some degree of resistance to the cephalosporin subclass of ß-lactams. Surprisingly, deletion of ponA2 also confers resistance, demonstrating that ß-lactam resistance can spontaneously arise from PBP loss of function. We show that ponA2 mutants resistant to the cephalosporin subclass of ß-lactams in fact show increased susceptibility to meropenem, a carbapenem that is known to target l,d-transpeptidases, thereby suggesting that in the absence of PonA2, an alternative mode of peptidoglycan synthesis likely becomes essential. Consistent with this hypothesis, a negative genetic selection identified the l,d-transpeptidase ldtMt2 as essential in the absence of ponA2. The mechanism of ß-lactam resistance we outline is consistent with emerging models of ß-lactam killing, while the investigation of ponA2 downstream and synthetic lethal genes sheds light on the mechanism of cell wall biosynthesis and the interaction between conventional PBPs and l,d-transpeptidases.