Search

Human health and the human microbiome are inevitably intertwined, increasing their relevance in clinical research. However, the collection, transportation and storage of faecal samples may introduce bias due to methodological differences, especially since postal shipping is a common practise in large-scale clinical cohort studies. Using four different Omics layer, we determined the structural (16S rRNA sequencing, cytometric microbiota profiling) and functional integrity (SCFAs, global metabolome) of the microbiota in relation to different easy-to-handle conditions. These conditions were storage at −20 °C, −20 °C as glycerol stock, 4 °C and room temperature with and without oxygen exposure for a maximum of one week. Storage time affected the microbiota on all Omics levels. However, the magnitude was donor-dependent, highlighting the need for purpose-optimized sample collection in clinical multi-donor studies. The effects of oxygen exposure were negligible for all analyses. At ambient temperature, SCFA and compositional profiles were stable for 24 h and 48 h, respectively, while at 4 °C, SCFA profiles were maintained for 48 h. The global metabolome was highly susceptible, already changing at 24 h in non-frozen conditions. Thus, faecal microbiota was best preserved on all levels when transported as a native sample frozen within 24 h, leading to the least biased outcomes in the analysis. We conclude that the immediate freezing of native stool samples for transportation to the lab is best suited for planned multi-Omics analyses that include metabolomics to extend standard sequencing approaches.

Abstract Tooth classes are an innovation that has contributed to the evolutionary success of mammals. However, our understanding of the mechanisms by which tooth classes diversified remain limited. We use the evolutionary radiation of noctilionoid bats to show how the tooth developmental program evolved during the adaptation to new diet types. Combining morphological, developmental and mathematical modeling approaches, we demonstrate that tooth classes develop through independent developmental cascades that deviate from classical models. We show that the diversification of tooth number and size is driven by jaw growth rate modulation, explaining the rapid gain/loss of teeth in this clade. Finally, we mathematically model the successive appearance of tooth buds, supporting the hypothesis that growth acts as a key driver of the evolution of tooth number and size. Our work reveal how growth, by tinkering with reaction/diffusion processes, drives the diversification of tooth classes and other repeated structure during adaptive radiations.

Urinary incontinence (UI) or involuntary loss of urine is a common chronic medical condition among women. It is estimated that 5%−70% of the population experiences incontinence with most studies suggesting 25%−45% of the population. Varying definitions of UI (e.g., stress, urgency, mixed) exist, and inconsistent symptom assessment tools, age, and gender can affect the estimate of incidence. Disposable Adult Incontinence products were first introduced into the market in the late 1970s and initially were used mostly in nursing homes and hospitals. However, during the 1980s, the market for incontinence products via retail outlets dramatically increased as awareness of the benefits of the products grew and stigma about their use declined. Today's products that manage urine loss have an extensive history and have evolved with time. Always products were introduced into the market in 2014 and are designed to meet the needs of women of all ages. Considered medical devices in some countries, regional regulations and global guidelines require clear planning, thorough assessment, and concise documentation of clinical safety. This manuscript will briefly review the regulatory landscape with a specific focus on European Union regulations. As previously published, the iterative, risk assessment framework used to assess the safety of Always incontinence products confirms that these products are compatible with skin and can be used safely. This manuscript will expand on the current literature highlighting additional steps that help assure the safety and compliance of the products from quality assurance programs through comprehensive post-market safety surveillance. Recommendations to help ensure several of the key regulatory requirements are met are outlined in the context of a risk assessment framework used to assure safety.

Ribosome-targeting antibiotics comprise over half of antibiotics used in medicine, but our fundamental knowledge of their binding sites is derived primarily from ribosome structures of non-pathogenic species. These include Thermus thermophilus, Deinococcus radiodurans and the archaean Haloarcula marismortui, as well as the commensal and sometimes pathogenic organism, Escherichia coli. Advancements in electron cryomicroscopy have allowed for the determination of more ribosome structures from pathogenic bacteria, with each study highlighting species-specific differences that had not been observed in the non-pathogenic structures. These observed differences suggest that more novel ribosome structures, particularly from pathogens, are required for a more accurate understanding of the level of diversity of the entire bacterial ribosome, with the potential of leading to innovative advancements in antibiotic research. In this study, high accuracy covariance and hidden Markov models were used to annotate ribosomal RNA and protein sequences respectively from genomic sequence, allowing us to determine the underlying ribosomal sequence diversity using phylogenetic methods. This analysis provided evidence that the current non-pathogenic ribosome structures are not sufficient representatives of some pathogenic bacteria, such as Campylobacter pylori, or of whole phyla such as Bacteroidota (Bacteroidetes).

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

M. tuberculosis cytochrome bd oxidase is of interest as a TB drug target. Here, the authors present the 2.5 Å cryo-EM structure of M. tuberculosis cytochrome bd oxidase and identify a disulfide bond within the canonical quinol binding and oxidation domain (Q-loop) and a menaquinone-9 binding site at heme b 595.

Background. The aim of this retrospective study with short, differently dispersed follow-up is to record the relationships between the pathologies of the individual foveal layers, measured by spectral domain optical coherence tomography (SD-OCT), and to investigate their influence on pre- and postoperative best-corrected decimal far visual acuity (BCVA) by phacovitrectomy for epiretinal membrane (ERM) in comorbidity with cataract. Patients and Methods. 208 eyes of 173 patients with symptomatic idiopathic ERM and moderate cataract were included. Results. In all OCT morphological stages of ERM, as well as in their combination with macular lamellar hole (MLH) and vitreomacular traction (VMT), a significant difference in the thickness of the individual fovea layers was found. In addition, the entire fovea thickening led to the proportional thickening of the individual fovea layers (p

Background: The Northern Territory (NT) has the highest tuberculosis (TB) rate of all Australian jurisdictions. We combined TB public health surveillance data with genomic sequencing of Mycobacterium tuberculosis isolates in the tropical ‘Top End’ of the NT to investigate trends in TB incidence and transmission. Methods: This retrospective observational study included all 741 culture-confirmed cases of TB in the Top End over three decades from 1989–2020. All 497 available M. tuberculosis isolates were sequenced. We used contact tracing data to define a threshold pairwise SNP distance for hierarchical single linkage clustering, and examined putative transmission clusters in the context of epidemiologic information. Findings: There were 359 (48%) cases born overseas, 329 (44%) cases among Australian First Nations peoples, and 52 (7%) cases were Australian-born and non-Indigenous. The annual incidence in First Nations peoples from 1989-2019 fell from average 50.4 to 11.0 per 100,000 (P

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.

The Northern Territory (NT) is a geographically remote region of northern and central Australia. Approximately a third of the population are First Nations Australians, many of whom live in remote regions. Due to the physical environment and climate, and scale of social inequity, the rates of many infectious diseases are the highest nationally. Molecular typing and genomic sequencing in research and public health have provided considerable new knowledge on the epidemiology of infectious diseases in the NT. We review the applications of genomic sequencing technology for molecular typing, identification of transmission clusters, phylogenomics, antimicrobial resistance prediction, and pathogen detection. We provide examples where these methodologies have been applied to infectious diseases in the NT and discuss the next steps in public health implementation of this technology.

BackgroundScabies is listed as a neglected tropical disease by the World Health Organization. Crusted scabies affects vulnerable and immunosuppressed individuals and is highly contagious because of the enormous number of Sarcoptes scabiei mites present in the hyperkeratotic skin. Undiagnosed and untreated crusted scabies cases can result in outbreaks of scabies in residential facilities and can also undermine the success of scabies mass drug administration programs.Methods and principal findingsCrusted scabies became a formally notifiable disease in the Northern Territory of Australia in 2016. We conducted a 2-year prospective study of crusted scabies cases notified between March 2016 and February 2018, with subsequent follow up for 22 months. Demographics, clinical and laboratory data, treatment and outcomes were analysed, with cases classified by severity of disease. Over the 2-year study period, 80 patients had 92 episodes of crusted scabies; 35 (38%) were Grade 1 crusted scabies, 36 (39%) Grade 2 and 21 (23%) Grade 3. Median age was 47 years, 47 (59%) were female, 76 (95%) Indigenous Australians and 57 (71%) from remote Indigenous communities. Half the patients were diabetic and 18 (23%) were on dialysis for end-stage kidney failure. Thirteen (16%) patients had no comorbidities, and these were more likely to have Grade 3 disease. Eosinophilia was present in 60% and high immunoglobulin E in 94%. Bacteremia occurred in 11 episodes resulting in one fatality with methicillin-susceptible Staphylococcus aureus bacteremia. Two other deaths occurred during admission and 10 others died subsequent to discharge consequent to comorbidities. Treatment generally followed the recommended guidelines, with 3, 5 or 7 doses of oral ivermectin depending on the documented grade of crusted scabies, together with daily alternating topical scabicides and topical keratolytic cream. While response to this therapy was usually excellent, there were 33 episodes of recurrent crusted scabies with the majority attributed to new infection subsequent to return to a scabies-endemic community.ConclusionsCrusted scabies can be successfully treated with aggressive guideline-based therapy, but high mortality remains from underlying comorbidities. Reinfection on return to community is common while scabies remains endemic.

Recent research has demonstrated that MAIT cells are activated by individual bacterial or yeasts species that possess the riboflavin biosynthesis pathway. However, little is known about the MAIT cell activating potential of microbial communities and the contribution of individual community members. Here, we analyze the MAIT cell activating potential of a human intestinal model community (SIHUMIx) as well as intestinal microbiota after bioreactor cultivation. We determined the contribution of individual SIHUMIx community members to the MAIT cell activating potential and investigated whether microbial stress can influence their MAIT cell activating potential. The MAIT cell activating potential of SIHUMIx was directly related to the relative species abundances in the community. We therefore suggest an additive relationship between the species abundances and their MAIT cell activating potential. In diverse microbial communities, we found that a low MAIT cell activating potential was associated with high microbial diversity and a high level of riboflavin demand and vice versa. We suggest that microbial diversity might affect MAIT cell activation via riboflavin utilization within the community. Microbial acid stress significantly reduced the MAIT cell activating potential of SIHUMIx by impairing riboflavin availability through increasing the riboflavin demand. We show that MAIT cells can perceive microbial stress due to changes in riboflavin utilization and that riboflavin availability might also play a central role for the MAIT cell activating potential of diverse microbiota.

Abstract The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the firefly. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1H NMR spectroscopy. The purified luciferase enzyme is in the same protein family as firefly luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely different to that of the firefly and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confirmed by chemical synthesis and bioluminescence assays. These findings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally different luciferins.

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

At the end of 2019 a newly emerged betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of an outbreak of severe pneumonia, subsequently termed COVID-19, in a number of patients in Wuhan, China. Subsequently, SARS-CoV-2 rapidly spread globally, resulting in a pandemic that has to date infected over 200 million individuals and resulted in more than 4.3 million deaths. While SARS-CoV-2 results in severe disease in 13.8%, with increasing frequency of severe disease with age, over 80% of infections are asymptomatic or mild. The immune response is an important determinant of outcome following SARS-CoV-2 infection. While B cell and T cell responses are associated with control of infection and protection against subsequent challenge with SARS-CoV-2, failure to control viral replication and the resulting hyperinflammation are associated with severe COVID-19. Towards the end of 2020, several variants of concern emerged that demonstrate increased transmissibility and/or evasion of immune responses from prior SARS-CoV-2 infection. This article reviews what is known about the humoral and cellular immune responses to SARS-CoV-2 and how mutation and structural/functional changes in the emerging variants of concern impact upon the immune protection from prior infection or vaccination.

Abstract Background As part of the second wave of the German Health Interview and Examination Survey for Children and Adolescents (KiGGS Wave 2), food and nutrient intake of children and adolescents aged 6–17 years living in Germany is assessed in EsKiMo II – the Eating Study as a KiGGS Module. Methods EsKiMo II is a cross-sectional study, conducted from June 2015 until September 2017. The study population comprises 6 to 17-year-old study participants from the cross-sectional sample of KiGGS Wave 2 in 167 KiGGS sample points, which are revisited by trained nutritionists. Dietary intake is assessed by weighted food records during three consecutive days plus one randomly selected day within the following 3 months for children aged 6–11 years. Dietary intake for adolescents aged 12–17 years is assessed by computer-assisted dietary history interviews, reflecting the past four weeks, using the software DISHES. Further information, for example, about specific diets and dietary supplement intake, is reported during a standardised computer assisted interview for all participants. Food items are coded by the German Food Code and Nutrient Database (BLS 3.02). Discussion EsKiMo II provides actual data on the dietary behaviour of children and adolescents living in Germany and their determinants. Results of EsKiMo II will be relevant for decision-making, measures, and evaluations within nutrition, consumer and health policy.

Rheumatic heart disease (RHD) poses a major disease burden among disadvantaged populations globally. It results from acute rheumatic fever (ARF), a complication of Group A Streptococcal (GAS) infection. These conditions are acknowledged as diseases of poverty, however the role of specific social and environmental factors in GAS infection and progression to ARF/RHD is not well understood. The aim of this systematic review was to determine the association between social determinants of health and GAS infection, ARF and RHD, and the effect of interventions targeting these.We conducted a systematic literature review using PubMed, the Cochrane Library and Embase. Observational and experimental studies that measured: crowding, dwelling characteristics, education, employment, income, nutrition, or socioeconomic status and the relationship with GAS infection, ARF or RHD were included. Findings for each factor were assessed against the Bradford Hill criteria for evidence of causation. Study quality was assessed using a standardised tool.1,164 publications were identified. 90 met inclusion criteria, comprising 91 individual studies. 49 (50.5%) were poor quality in relation to the specific study question. The proportion of studies reporting significant associations between socioeconomic determinants and risk of GAS infection was 57.1%, and with ARF/RHD was 50%. Crowding was the most assessed factor (14 studies with GAS infection, 36 studies with ARF/RHD) followed by socioeconomic status (6 and 36 respectively). The majority of studies assessing crowding, dwelling characteristics, education and employment status of parents or cases, and nutrition, reported a positive association with risk of GAS infection, ARF or RHD. Crowding and socioeconomic status satisfactorily met the criteria of a causal association. There was substantial heterogeneity across all key study aspects.The extensive literature examining the role of social determinants in GAS infection, ARF and RHD risk lacks quality. Most were observational, not interventional. Crowding as a cause of GAS infection and ARF/RHD presents a practical target for prevention actions.

With this article we set out to explore the impact of migratory phenomena in an industrial district and the greater metropolitan area between Florence and Prato. The presence of migrant workers and families of Chinese origin extends across boundaries of communes, former provinces, and the metropolitan cities. In this localized encounter zone, practices of work and living trigger conflicts and processes of negotiation that unfold in the absence of a regulatory framework able to manage conditions of subalternity and self-exploitation—particularly when these occur in neighborhoods and among groups of families and migrant workers. The analysis of the construction phases of a complex control apparatus highlights the relevance of the ethnographic encounters in physical spaces, cities and neighborhoods, where economic crisis has contributed to producing conflicts between the logics and aspirations of the migrants, residents and various levels of local administrative as well as regional and national government entities.

IgA-coated fractions of the intestinal microbiota of Crohn’s disease (CD) patients have been shown to contain taxa that hallmark the compositional dysbiosis in CD microbiomes. However, the correlation between other cellular properties of intestinal bacteria and disease has not been explored further, especially for features that are not directly driven by the host immune-system, e.g. the expression of surface sugars by bacteria. By sorting and sequencing IgA-coated and lectin-stained fractions from CD patients microbiota and healthy controls, we found that lectin-stained bacteria were distinct from IgA-coated bacteria, but still displayed specific differences between CD and healthy controls. To exploit the discriminatory potential of both, immunoglobulin coated bacteria and the altered surface sugar expression of bacteria in CD, we developed a multiplexed single cell-based analysis approach for intestinal microbiota. By multi-parameter microbiota flow cytometry (mMFC) we characterized the intestinal microbiota of 55 CD patients and 44 healthy controls for 11-parameters in total, comprising host-immunoglobulin coating and the presence of distinct surface sugar moieties. The data were analyzed by machine-learning to assess disease-specific marker patterns in the microbiota phenotype. mMFC captured detailed characteristics of CD microbiota and identified patterns to classify CD patients. In addition, we identified phenotypic signatures in the CD microbiota which not only reflected remission after 6 weeks of anti-TNF treatment, but were also able to predict remission before the start of an adalimumab treatment course in a pilot study. We here present the proof-of-concept demonstrating that multi-parameter single-cell bacterial phenotyping by mMFC could be a novel tool with high translational potential to expand current microbiome investigations by phenotyping of bacteria to identify disease- and therapy-associated cellular alterations and to reveal novel target properties of bacteria for functional assays and therapeutic approaches.

In this paper, we examine the role of time in shaping decision-making processes in a town meeting, a type of legislative body common in many New England towns. Town meetings are one of the oldest and most democratic institutions of local governance in the United States, and they provide a rich arena in which to investigate how large groups of people convene and make decisions together. A mixed-methods approach enabled our team of researchers to gain insight into the processes and dynamics that played out in one town meeting. We analyze the tensions between democratic values of “taking time” vs. “being efficient.” The dynamics are particularly compelling because of an absence of the typical two parties that dominate U.S. political culture. Attitudes toward time closely aligned with voting behaviors. Our study concludes that, even in the context of a culturally and economically homogenous New England town-meeting membership, orientations to temporality are contested and meaningful. Situated historically, these orientations reflect citizens’ embrace or rejection of “time-thrift” and suggest implications for participatory democracy.

In the made in italy fast-fashion sector, the ultimate flexible workers are Chinese migrants, whose parenting practices include circulating children back to China. This paper draws on collaborative research in Prato, Italy, to grasp how Chinese families and individuals encounter the Italian state and negotiate the terms of transnational capitalism. The project innovates an encounter ethnography framework to guide research. This paper investigates the dialectic between economics and affect. Chinese immigrants’ desires to make money are situated in three structural encounters, each at a different level of scale: the Wenzhou regional model of economic development; a Central Italian small-firm environment connected to the made in italy brand; and global restructuring of the textile and clothing industry. These encounters structure migrant experiences. Underlying sentiments expose how families cope with the demands of globalization, particularly their work in a sector demanding long hours and a fast production schedule. Adjusting to garment work is expressed as a “fistful of tears”; a decision to circulate children into the care of relatives back home is said to be “the only solution”. This paper uses the concept of “global household” (Safri and Graham 2010) to examine the transnational dispersal of kin networks and migrants’ reliance on the affective circuits of those networks. They illuminate, as Marshall Sahlins (2000) observed, how “capitalist forces are realized in other forms and finalities”, as a result of local cultural practices such mediating global systems.

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1-142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein.

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.