Microbiota of pest insect Nezara viridula mediate detoxification and plant defense repression.

The Southern green shield bug, Nezara viridula, is an invasive piercing and sucking pest insect that feeds on crop plants and poses a threat to global food production. Given that insects are known to live in a close relationship with microorganisms, our study provides insights into the community composition and function of the N. viridula-associated microbiota and its effect on host-plant interactions. We discovered that N. viridula hosts both vertically and horizontally transmitted microbiota throughout different developmental stages and their salivary glands harbor a thriving microbial community that is transmitted to the plant while feeding. The N. viridula microbiota was shown to aid its host with the detoxification of a plant metabolite, namely 3-nitropropionic acid, and repression of host plant defenses. Our results demonstrate that the N. viridula-associated microbiota plays an important role in interactions between insects and plants and could therefore be considered a valuable target for the development of sustainable pest control strategies.

On the importance of sequence alignment inspections in plastid phylogenomics - an example from revisiting the relationships of the water-lilies.

The water-lily clade represents the second earliest-diverging branch of angiosperms. Most of its species belong to Nymphaeaceae, of which the "core Nymphaeaceae"-comprising the genera Euryale, Nymphaea and Victoria-is the most diverse clade. Despite previous molecular phylogenetic studies on the core Nymphaeaceae, various aspects of their evolutionary relationships have remained unresolved. The length-variable introns and intergenic spacers are known to contain most of the sequence variability within the water-lily plastomes. Despite the challenges with multiple sequence alignment, any new molecular phylogenetic investigation on the core Nymphaeaceae should focus on these noncoding plastome regions. For example, a new plastid phylogenomic study on the core Nymphaeaceae should generate DNA sequence alignments of all plastid introns and intergenic spacers based on the principle of conserved sequence motifs. In this investigation, we revisit the phylogenetic history of the core Nymphaeaceae by employing such an approach. Specifically, we use a plastid phylogenomic analysis strategy in which all coding and noncoding partitions are separated and then undergo software-driven DNA sequence alignment, followed by a motif-based alignment inspection and adjustment. This approach allows us to increase the reliability of the character base compared to the default practice of aligning complete plastomes through software algorithms alone. Our approach produces significantly different phylogenetic tree reconstructions for several of the plastome regions under study. The results of these reconstructions underscore that Nymphaea is paraphyletic in its current circumscription, that each of the five subgenera of Nymphaea is monophyletic, and that the subgenus Nymphaea is sister to all other subgenera of Nymphaea. Our results also clarify many evolutionary relationships within the Nymphaea subgenera Brachyceras, Hydrocallis and Nymphaea. In closing, we discuss whether the phylogenetic reconstructions obtained through our motif-based alignment adjustments are in line with morphological evidence on water-lily evolution.

Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA.

Nuclear and organellar genomes can evolve at vastly different rates despite occupying the same cell. In most bilaterian animals, mitochondrial DNA (mtDNA) evolves faster than nuclear DNA, whereas this trend is generally reversed in plants. However, in some exceptional angiosperm clades, mtDNA substitution rates have increased up to 5,000-fold compared with closely related lineages. The mechanisms responsible for this acceleration are generally unknown. Because plants rely on homologous recombination to repair mtDNA damage, we hypothesized that mtDNA copy numbers may predict evolutionary rates, as lower copy numbers may provide fewer templates for such repair mechanisms. In support of this hypothesis, we found that copy number explains 47% of the variation in synonymous substitution rates of mtDNA across 60 diverse seed plant species representing ~300 million years of evolution. Copy number was also negatively correlated with mitogenome size, which may be a cause or consequence of mutation rate variation. Both relationships were unique to mtDNA and not observed in plastid DNA. These results suggest that homologous recombinational repair plays a role in driving mtDNA substitution rates in plants and may explain variation in mtDNA evolution more broadly across eukaryotes. Our findings also contribute to broader questions about the relationships between mutation rates, genome size, selection efficiency, and the drift-barrier hypothesis.

Out of Place: Phylogenomics resolves the placement of Eurasian taxa and sheds light on origin of Thermopsideae in North America.

The North American Thermopsideae (Fabaceae: Papilionoideae), a monophyletic group comprising the North American endemic genus Baptisia, and the paraphyletic Eurasian-North American disjunct Thermopsis, is nested within the tribe Sophoreae. Previous phylogenetic studies have identified two East Asian taxa within the North American Thermopsideae, suggesting two independent dispersal events between North America-East Asia. More recent studies have also placed a third taxon, Vuralia turcica, an endemic species from Turkey, among the North American Thermopsideae. The presence of three geographically distant Eurasian taxa within a relatively young clade of North American origin is unprecedented among papilionoid legumes, and the biogeographic implications of this observation are not clear. To investigate this matter, 1540 low-copy nuclear genes and complete plastomes were obtained from 36 taxa across the core genistoids, including 26 newly sequenced taxa. Nuclear and plastome based maximum likelihood (ML) and ASTRAL analyses were conducted based on varying degrees of taxon coverage and read mapping consensus threshold values. Additional analyses were performed to estimate divergence times and to reconstruct biogeographic history. The results strongly support a previously undetected Old World clade, presently composed of V. turcica and T. chinensis, which diverged from the ancestor of the North American lineage during the mid to late Miocene. A single and recent North America-East Asia dispersal involving T. lupinoides is reported. Furthermore, the traditional inclusion of the genus Ammopiptanthus among Thermopsideae is not supported, and the monotypic generic status of Vuralia is called into question. A relatively high degree of cytonuclear discordance is reported within each sub-clade of the North American Thermopsideae. This finding is likely attributable to the high degree of interspecific hybridization reported within these groups and raises the need for more rigorous genome-scale testing to better delimit species within each of the reticulating subclades. Subjects: Biodiversity, Biogeography, Evolutionary Studies, Genetics, Plant Science.

Microbial degradation of plant toxins.

Plants produce a variety of secondary metabolites in response to biotic and abiotic stresses. Although they have many functions, a subclass of toxic secondary metabolites mainly serve plants as deterring agents against herbivores, insects, or pathogens. Microorganisms present in divergent ecological niches, such as soil, water, or insect and rumen gut systems have been found capable of detoxifying these metabolites. As a result of detoxification, microbes gain growth nutrients and benefit their herbivory host via detoxifying symbiosis. Here, we review current knowledge on microbial degradation of toxic alkaloids, glucosinolates, terpenes, and polyphenols with an emphasis on the genes and enzymes involved in breakdown pathways. We highlight that the insect-associated microbes might find application in biotechnology and become targets for an alternative microbial pest control strategy.

IMPACT OF THE COVID-19 PANDEMIC ON PUBLIC HOSPITALS IN THE UNITED STATES.

The country's public hospitals, guided by the principles established by the first such hospital in 1736 and codified through the policies of the Surgeon General in 1936, have played an outsized role as safety net institutions for disadvantaged populations. Public hospitals are predominantly located in urban, under-resourced neighborhoods and treat a larger percentage of low-income individuals who are uninsured or enrolled in Medicaid. In assessing the status of public hospitals and urban communities in the twenty-first century, the impact of the COVID-19 pandemic was evaluated at two high-performing public hospitals, Grady Memorial Hospital and Rush University Medical Center, and a network of safety hospitals affiliated with the Missouri Hospital Association. COVID-19 infections and death rates stratified by race and ethnicity were examined. The results suggest a trend toward lower mortality in African American patients in the first year of the pandemic and possible adverse outcomes in a subset of rural hospitals in Missouri. This study highlights the need to expand funding and support for the nation's essential hospitals.

The potential role of drug transporters and amikacin modifying enzymes in M. avium.

OBJECTIVES: Mycobacterium avium (M. avium) complex bacteria cause opportunistic infections in humans. Treatment yields cure rates of 60% and consists of a macrolide, a rifamycin, and ethambutol, and in severe cases, amikacin. Mechanisms of antibiotic tolerance remain mostly unknown. Therefore, we studied the contribution of efflux and amikacin modification to antibiotic susceptibility. METHODS: We characterised M. avium ABC transporters and studied their expression together with other transporters following exposure to clarithromycin, amikacin, ethambutol, and rifampicin. We determined the effect of combining the efflux pump inhibitors berberine, verapamil and CCCP (carbonyl cyanide m-chlorophenyl hydrazone), to study the role of efflux on susceptibility. Finally, we studied the modification of amikacin by M. avium using metabolomic analysis. RESULTS: Clustering shows conservation between M. avium and M. tuberculosis and transporters from most bacterial subfamilies (2-6, 7a/b, 10-12) were found. The largest number of transporter encoding genes was up-regulated after clarithromycin exposure, and the least following amikacin exposure. Only berberine increased the susceptibility to clarithromycin. Finally, because of the limited effect of amikacin on transporter expression, we studied amikacin modification and showed that M. avium, in contrast to M. abscessus, is not able to modify amikacin. CONCLUSION: We show that M. avium carries ABC transporters from all major families important for antibiotic efflux, including homologues shown to have affinity for drugs included in treatment. Efflux inhibition in M. avium can increase susceptibility, but this effect is efflux pump inhibitor- and antibiotic-specific. Finally, the lack of amikacin modifying activity in M. avium is important for its activity.

"Candidatus Hydrogenisulfobacillus filiaventi" strain R50 gen. nov. sp. nov., a highly efficient producer of extracellular organic compounds from H2 and CO2.

Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe "Candidatus Hydrogenisulfobacillus filiaventi" R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h-1, doubling time of 20 h) in combination with O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.

Plastid Genome Assembly Using Long-read data.

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and two single-copy regions, and a few dispersed repeats; however, the plastomes of some taxa harbour long repeat sequences (>300 bp). These long repeats make it challenging to assemble complete plastomes using short-read data, leading to misassemblies and consensus sequences with spurious rearrangements. Single-molecule, long-read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long-read data. We generated a pipeline, plastid Genome Assembly Using Long-read data (ptGAUL), to address the problem of plastome assembly using long-read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long-read data sets. We showed that ptGAUL quickly produces accurate and unbiased assemblies using only ~50× coverage of plastome data. Additionally, we deployed ptGAUL to assemble four new Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales. The ptGAUL pipeline is available on GitHub: https://github.com/Bean061/ptgaul.

MS2Query: reliable and scalable MS2 mass spectra-based analogue search.

Metabolomics-driven discoveries of biological samples remain hampered by the grand challenge of metabolite annotation and identification. Only few metabolites have an annotated spectrum in spectral libraries; hence, searching only for exact library matches generally returns a few hits. An attractive alternative is searching for so-called analogues as a starting point for structural annotations; analogues are library molecules which are not exact matches but display a high chemical similarity. However, current analogue search implementations are not yet very reliable and relatively slow. Here, we present MS2Query, a machine learning-based tool that integrates mass spectral embedding-based chemical similarity predictors (Spec2Vec and MS2Deepscore) as well as detected precursor masses to rank potential analogues and exact matches. Benchmarking MS2Query on reference mass spectra and experimental case studies demonstrate improved reliability and scalability. Thereby, MS2Query offers exciting opportunities to further increase the annotation rate of metabolomics profiles of complex metabolite mixtures and to discover new biology.

Early Fruit Development Regulation-Related Genes Concordantly Expressed with TCP Transcription Factors in Tomato (Solanum lycopersicum).

The tomato (Solanum lycopersicum L.) is considered one of the most important vegetable crops globally, both agronomically and economically; however, its fruit development regulation network is still unclear. The transcription factors serve as master regulators, activating many genes and/or metabolic pathways throughout the entire plant life cycle. In this study, we identified the transcription factors that are coordinated with TCP gene family regulation in early fruit development by making use of the high-throughput sequencing of RNA (RNAseq) technique. A total of 23 TCP-encoding genes were found to be regulated at various stages during the growth of the fruit. The expression patterns of five TCPs were consistent with those of other transcription factors and genes. There are two unique subgroups of this larger family: class I and class II TCPs. Others were directly associated with the growth and/or ripening of fruit, while others were involved in the production of the hormone auxin. Moreover, it was discovered that TCP18 had an expression pattern that was similar to that of the ethylene-responsive transcription factor 4 (ERF4). Tomato fruit set and overall development are under the direction of a gene called auxin response factor 5 (ARF5). TCP15 revealed an expression that was in sync with this gene. This study provides insight into the potential processes that help in acquiring superior fruit qualities by accelerating fruit growth and ripening.

Rate accelerations in plastid and mitochondrial genomes of Cyperaceae occur in the same clades.

Cyperaceae, the second largest family in the monocot order Poales, comprises >5500 species and includes the genus Eleocharis with âˆ¼ 250 species. A previous study of complete plastomes of two Eleocharis species documented extensive structural heteroplasmy, gene order changes, high frequency of dispersed repeats along with gene losses and duplications. To better understand the phylogenetic distribution of gene and intron content as well as rates and patterns of sequence evolution within and between mitochondrial and plastid genomes of Eleocharis and Cyperaceae, an additional 29 Eleocharis organelle genomes were sequenced and analyzed. Eleocharis experienced extensive gene loss in both genomes while loss of introns was mitochondria-specific. Eleocharis has higher rates of synonymous (dS) and nonsynonymous (dN) substitutions in the plastid and mitochondrion than most sampled angiosperms, and the pattern was distinct from other eudicot lineages with accelerated rates. Several clades showed higher dS and dN in mitochondrial genes than in plastid genes. Furthermore, nucleotide substitution rates of mitochondrial genes were significantly accelerated on the branch leading to Cyperaceae compared to most angiosperms. Mitochondrial genes of Cyperaceae exhibited dramatic loss of RNA editing sites and a negative correlation between RNA editing and dS values was detected among angiosperms. Mutagenic retroprocessing and dysfunction of DNA replication, repair and recombination genes are the most likely cause of striking rate accelerations and loss of edit sites and introns in Eleocharis and Cyperaceae organelle genomes.

Peering down the sink: A review of isoprene metabolism by bacteria.

Isoprene (2-methyl-1,3-butadiene) is emitted to the atmosphere each year in sufficient quantities to rival methane (>500 Tg C yr-1 ), primarily due to emission by trees and other plants. Chemical reactions of isoprene with other atmospheric compounds, such as hydroxyl radicals and inorganic nitrogen species (NOx ), have implications for global warming and local air quality, respectively. For many years, it has been estimated that soil-dwelling bacteria consume a significant amount of isoprene (~20 Tg C yr-1 ), but the mechanisms underlying the biological sink for isoprene have been poorly understood. Studies have indicated or confirmed the ability of diverse bacterial genera to degrade isoprene, whether by the canonical iso-type isoprene degradation pathway or through other less well-characterized mechanisms. Here, we review current knowledge of isoprene metabolism and highlight key areas for further research. In particular, examples of isoprene-degraders that do not utilize the isoprene monooxygenase have been identified in recent years. This has fascinating implications both for the mechanism of isoprene uptake by bacteria, and also for the ecology of isoprene-degraders in the environments.

A new enzymatic assay to quantify inorganic pyrophosphate in plasma.

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

Plastid phylogenomics uncovers multiple species in Medicago truncatula (Fabaceae) germplasm accessions.

Medicago truncatula is a model legume that has been extensively investigated in diverse subdisciplines of plant science. Medicago littoralis can interbreed with M. truncatula and M. italica; these three closely related species form a clade, i.e. TLI clade. Genetic studies have indicated that M. truncatula accessions are heterogeneous but their taxonomic identities have not been verified. To elucidate the phylogenetic position of diverse M. truncatula accessions within the genus, we assembled 54 plastid genomes (plastomes) using publicly available next-generation sequencing data and conducted phylogenetic analyses using maximum likelihood. Five accessions showed high levels of plastid DNA polymorphism. Three of these highly polymorphic accessions contained sequences from both M. truncatula and M. littoralis. Phylogenetic analyses of sequences placed some accessions closer to distantly related species suggesting misidentification of source material. Most accessions were placed within the TLI clade and maximally supported the interrelationships of three subclades. Two Medicago accessions were placed within a M. italica subclade of the TLI clade. Plastomes with a 45-kb (rpl20-ycf1) inversion were placed within the M. littoralis subclade. Our results suggest that the M. truncatula accession genome pool represents more than one species due to possible mistaken identities and gene flow among closely related species.

Insect Gut Isolate Pseudomonas sp. Strain Nvir Degrades the Toxic Plant Metabolite Nitropropionic Acid.

Nitropropionic acid (NPA) is a widely distributed naturally occurring nitroaliphatic toxin produced by leguminous plants and fungi. The Southern green shield bug feeds on leguminous plants and shows no symptoms of intoxication. Likewise, its gut-associated microorganisms are subjected to high levels of this toxic compound. In this study, we isolated a bacterium from this insect's gut system, classified as Pseudomonas sp. strain Nvir, that was highly resistant to NPA and was fully degrading it to inorganic nitrogen compounds and carbon dioxide. In order to understand the metabolic fate of NPA, we traced the fate of all atoms of the NPA molecule using isotope tracing experiments with [15N]NPA and [1-13C]NPA, in addition to experiments with uniformly 13C-labeled biomass that was used to follow the incorporation of 12C atoms from [U-12C]NPA into tricarboxylic acid cycle intermediates. With the help of genomics and transcriptomics, we uncovered the isolate's NPA degradation pathway, which involves a putative propionate-3-nitronate monooxygenase responsible for the first step of NPA degradation. The discovered protein shares only 32% sequence identity with previously described propionate-3-nitronate monooxygenases. Finally, we advocate that NPA-degrading bacteria might find application in biotechnology, and their unique enzymes might be used in biosynthesis, bioremediation, and in dealing with postharvest NPA contamination in economically important products. IMPORTANCE Plants have evolved sophisticated chemical defense mechanisms, such as the production of plant toxins in order to deter herbivores. One example of such a plant toxin is nitropropionic acid (NPA), which is produced by leguminous plants and also by certain fungi. In this project, we have isolated a bacterium from the intestinal tract of a pest insect, the Southern green shield bug, that is able to degrade NPA. Through a multiomics approach, we identified the respective metabolic pathway and determined the metabolic fate of all atoms of the NPA molecule. In addition, we provide a new genetic marker that can be used for genome mining toward NPA degradation. The discovery of degradation pathways of plant toxins by environmental bacteria opens new possibilities for pretreatment of contaminated food and feed sources and characterization of understudied enzymes allows their broad application in biotechnology.

An exercise-inducible metabolite that suppresses feeding and obesity.

Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.

Effects of Salt Stress on Transcriptional and Physiological Responses in Barley Leaves with Contrasting Salt Tolerance.

Salt stress negatively impacts crop production worldwide. Genetic diversity among barley (Hordeum vulgare) landraces adapted to adverse conditions should provide a valuable reservoir of tolerance genes for breeding programs. To identify molecular and biochemical differences between barley genotypes, transcriptomic and antioxidant enzyme profiles along with several morpho-physiological features were compared between salt-tolerant (Boulifa) and salt-sensitive (Testour) genotypes subjected to salt stress. Decreases in biomass, photosynthetic parameters, and relative water content were low in Boulifa compared to Testour. Boulifa had better antioxidant protection against salt stress than Testour, with greater antioxidant enzymes activities including catalase, superoxide dismutase, and guaiacol peroxidase. Transcriptome assembly for both genotypes revealed greater accumulation of differentially expressed transcripts in Testour compared to Boulifa, emphasizing the elevated transcriptional response in Testour following salt exposure. Various salt-responsive genes, including the antioxidant catalase 3, the osmoprotectant betaine aldehyde dehydrogenase 2, and the transcription factors MYB20 and MYB41, were induced only in Boulifa. By contrast, several genes associated with photosystems I and II, and light receptor chlorophylls A and B, were more repressed in Testour. Co-expression network analysis identified specific gene modules correlating with differences in genotypes and morpho-physiological traits. Overall, salinity-induced differential transcript accumulation underlies the differential morpho-physiological response in both genotypes and could be important for breeding salt tolerance in barley.

CinA mediates multidrug tolerance in Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to resist and tolerate antibiotics complicates the development of improved tuberculosis (TB) chemotherapies. Here we define the Mtb protein CinA as a major determinant of drug tolerance and as a potential target to shorten TB chemotherapy. By reducing the fraction of drug-tolerant persisters, genetic inactivation of cinA accelerated killing of Mtb by four antibiotics in clinical use: isoniazid, ethionamide, delamanid and pretomanid. Mtb ΔcinA was killed rapidly in conditions known to impede the efficacy of isoniazid, such as during nutrient starvation, during persistence in a caseum mimetic, in activated macrophages and during chronic mouse infection. Deletion of CinA also increased in vivo killing of Mtb by BPaL, a combination of pretomanid, bedaquiline and linezolid that is used to treat highly drug-resistant TB. Genetic and drug metabolism studies suggest that CinA mediates drug tolerance via cleavage of NAD-drug adducts.

Activity-based annotation: the emergence of systems biochemistry.

Current tools to annotate protein function have failed to keep pace with the speed of DNA sequencing and exponentially growing number of proteins of unknown function (PUFs). A major contributing factor to this mismatch is the historical lack of high-throughput methods to experimentally determine biochemical activity. Activity-based methods, such as activity-based metabolite and protein profiling, are emerging as new approaches for unbiased, global, biochemical annotation of protein function. In this review, we highlight recent experimental, activity-based approaches that offer new opportunities to determine protein function in a biologically agnostic and systems-level manner.