Ongoing evolution of the Mycobacterium tuberculosis lactate dehydrogenase reveals the pleiotropic effects of bacterial adaption to host pressure.

The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacterium in vivo by assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome is lldD2, which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection and lldD2 is essential for Mtb growth in macrophages. We determined the extent of lldD2 variation across a set of global clinical isolates and defined how prevalent mutations modulate Mtb fitness. We show the stepwise nature of lldD2 evolution that occurs as a result of ongoing lldD2 selection in the background of ancestral lineage-defining mutations and demonstrate that the genetic evolution of lldD2 additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolved lldD2 mutations functionally increase the quinone-dependent activity of LldD2. Using 13C-lactate metabolic flux tracing, we find that lldD2 is necessary for robust incorporation of lactate into central carbon metabolism. In the absence of lldD2, label preferentially accumulates in dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accrued lactate toxicity via the deleterious buildup of sugar phosphates. The evolved lldD2 variants increase lactate incorporation to pyruvate while altering triose phosphate flux, suggesting both an anaplerotic and detoxification benefit to lldD2 evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with altered lldD2 activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX- 1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provides context for the selective advantage of lldD2 mutations in adapting to host stress.

Mitochondrial GPAT-derived LPA controls auxin-dependent embryonic and postembryonic development.

Membrane properties are emerging as important cues for the spatiotemporal regulation of hormone signaling. Lysophosphatidic acid (LPA) evokes multiple biological responses by activating G protein-coupled receptors in mammals. In this study, we demonstrated that LPA derived from the mitochondrial glycerol-3-phosphate acyltransferases GPAT1 and GPAT2 is a critical lipid-based cue for auxin-controlled embryogenesis and plant growth in Arabidopsis thaliana. LPA levels decreased, and the polarity of the auxin efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane (PM) was defective in the gpat1 gpat2 mutant. As a consequence of distribution defects, instructive auxin gradients and embryonic and postembryonic development are severely compromised. Further cellular and genetic analyses revealed that LPA binds directly to PIN1, facilitating the vesicular trafficking of PIN1 and polar auxin transport. Our data support a model in which LPA provides a lipid landmark that specifies membrane identity and cell polarity, revealing an unrecognized aspect of phospholipid patterns connecting hormone signaling with development.

A Bacterial Responsive Microneedle Dressing with Hydrogel Backing Layer for Chronic Wound Treatment.

The treatment of chronic wounds still presents great challenges due to being infected by biofilms and the damaged healing process. The current treatments do not address the needs of chronic wounds. In this study, a highly effective dressing (Dox-DFO@MN Hy) for the treatment of chronic wounds is described. This dressing combines the advantages of microneedles (MNs) and hydrogels in the treatment of chronic wounds. MNs is employed to debride the biofilms and break down the wound barrier, providing rapid access to therapeutic drugs from hydrogel backing layer. Importantly, to kill the pathogenic bacteria in the biofilms specifically, Doxycycline hydrochloride (Dox) is wrapped into the polycaprolactone (PCL) microspheres that have lipase-responsive properties and loaded into the tips of MNs. At the same time, hydrogel backing layer is used to seal the wound and accelerate wound healing. Benefiting from the combination of two advantages of MNs and hydrogel, the dressing significantly reduces the bacteria in the biofilms and effectively promotes angiogenesis and cell migration in vitro. Overall, Dox-DFO@MN Hy can effectively treat chronic wounds infected with biofilms, providing a new idea for the treatment of chronic wounds.

SAM-TB: a whole genome sequencing data analysis website for detection of Mycobacterium tuberculosis drug resistance and transmission.

Whole genome sequencing (WGS) can provide insight into drug-resistance, transmission chains and the identification of outbreaks, but data analysis remains an obstacle to its routine clinical use. Although several drug-resistance prediction tools have appeared, until now no website integrates drug-resistance prediction with strain genetic relationships and species identification of nontuberculous mycobacteria (NTM). We have established a free, function-rich, user-friendly online platform for MTB WGS data analysis (SAM-TB, http://samtb.szmbzx.com) that integrates drug-resistance prediction for 17 antituberculosis drugs, detection of variants, analysis of genetic relationships and NTM species identification. The accuracy of SAM-TB in predicting drug-resistance was assessed using 3177 sequenced clinical isolates with results of phenotypic drug-susceptibility tests (pDST). Compared to pDST, the sensitivity of SAM-TB for detecting multidrug-resistant tuberculosis was 93.9% [95% confidence interval (CI) 92.6-95.1%] with specificity of 96.2% (95% CI 95.2-97.1%). SAM-TB also analyzes the genetic relationships between multiple strains by reconstructing phylogenetic trees and calculating pairwise single nucleotide polymorphism (SNP) distances to identify genomic clusters. The incorporated mlstverse software identifies NTM species with an accuracy of 98.2% and Kraken2 software can detect mixed MTB and NTM samples. SAM-TB also has the capacity to share both sequence data and analysis between users. SAM-TB is a multifunctional integrated website that uses WGS raw data to accurately predict antituberculosis drug-resistance profiles, analyze genetic relationships between multiple strains and identify NTM species and mixed samples containing both NTM and MTB. SAM-TB is a useful tool for guiding both treatment and epidemiological investigation.

Phosphatidic acid regulates ammonium uptake by interacting with AMMONIUM TRANSPORTER 1;1 in Arabidopsis.

Ammonium (NH4+) is a key inorganic nitrogen source in cellular amino acid biosynthesis. The coupling of transcriptional and posttranslational regulation of AMMONIUM TRANSPORTER (AMT) ensures that NH4+ acquisition by plant roots is properly balanced, which allows for rapid adaptation to a variety of nitrogen conditions. Here, we report that phospholipase D (PLD)-derived phosphatidic acid (PA) interacts with AMT1;1 to mediate NH4+ uptake in Arabidopsis (Arabidopsis thaliana). We examined pldα1 pldδ-knockout mutants and found that a reduced PA level increased seedling growth under nitrogen deficiency and inhibited root growth upon NH4+ stress, which was consistent with the enhanced accumulation of cellular NH4+. PA directly bound to AMT1;1 and inhibited its transport activity. Mutation of AMT1;1 R487 to Gly (R487G) resulted in abolition of PA suppression and, subsequently, enhancement of ammonium transport activity in vitro and in vivo. Observations of AMT1;1-GFP showed suppressed endocytosis under PLD deficiency or by mutation of the PA-binding site in AMT1;1. Endocytosis was rescued by PA in the pldα1 pldδ mutant but not in the mutant AMT1;1R487G-GFP line. Together, these findings demonstrated PA-based shutoff control of plant NH4+ transport and point to a broader paradigm of lipid-transporter function.

In situ Hg2+ improved the peroxidase-like activity and triggered "ON" the oxidase-like activity of yolk-shell Co3S4 microspheres for the detection of Hg2.

Exploring highly active nanozymes is an important task to realize the real-time detection of some heavy metal ions in water. In this work, yolk-shell Co3S4 microspheres have been verified to possess excellent peroxidase-like activity, which can be further improved by adding Hg2+. Very interestingly, Hg2+ can trigger "ON" the oxidase-like activity of Co3S4 microspheres. The dual peroxidase-/oxidase-like activity of the yolk-shell Co3S4 microspheres is evaluated by using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). Furthermore, comprehensive studies verify that the enhanced peroxidase-like activity, together with the "ON" oxidase-like activity of the yolk-shell Co3S4 microspheres, is attributed to the in situ generation of HgS on the surface of Co3S4 microspheres and then the release of more active sites. Importantly, the in situ generated HgS on the surface of Co3S4 microspheres can form a heterojunction, which also accelerates the catalytic process. During the catalytic reaction, some active species (O2- and h+) can be detected by ESR. Thus, a colorimetric sensing platform based on Hg2+-triggered signal amplification has been successfully constructed, which can be validated by the detection of Hg2+ residue in environmental water.

Local adaptation of Mycobacterium tuberculosis on the Tibetan Plateau.

During its global dispersal, Mycobacterium tuberculosis (Mtb) has encountered varied geographic environments and host populations. Although local adaptation seems to be a plausible model for describing long-term host-pathogen interactions, genetic evidence for this model is lacking. Here, we analyzed 576 whole-genome sequences of Mtb strains sampled from different regions of high-altitude Tibet. Our results show that, after sequential introduction of a few ancestral strains, the Tibetan Mtb population diversified locally while maintaining strict separation from the Mtb populations on the lower altitude plain regions of China. The current population structure and estimated past population dynamics suggest that the modern Beijing sublineage strains, which expanded over most of China and other global regions, did not show an expansion advantage in Tibet. The mutations in the Tibetan strains showed a higher proportion of A > G/T > C transitions than strains from the plain regions, and genes encoding DNA repair enzymes showed evidence of positive selection. Moreover, the long-term Tibetan exclusive selection for truncating mutations in the thiol-oxidoreductase encoding sseA gene suggests that Mtb was subjected to local selective pressures associated with oxidative stress. Collectively, the population genomics of Mtb strains in the relatively isolated population of Tibet provides genetic evidence that Mtb has adapted to local environments.

Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence.

Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from ß-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.

An antibody-drug conjugate targeting GPR56 demonstrates efficacy in preclinical models of colorectal cancer.

BACKGROUND: Long-term prognosis remains poor for colorectal cancer (CRC) patients with advanced disease due to treatment resistance. The identification of novel targets is essential for the development of new therapeutic approaches. GPR56, an adhesion GPCR, is highly expressed in CRC tumours and correlates with poor survival. Here, we describe the generation and preclinical evaluation of a novel ADC consisting of an anti-GPR56 antibody (10C7) conjugated with the DNA-damaging payload duocarmycin. METHODS: RNA-seq dataset analysis was performed to determine GPR56 expression in CRC subtypes. The specificity of binding, epitope mapping, and internalisation of 10C7 was examined. 10C7 was conjugated to payload and ADC cytotoxicity was assessed against a panel of CRC cell lines and tumour organoids. Antitumour efficacy was evaluated in xenograft models of CRC cell lines and patient-derived tumours. RESULTS: High GPR56 was shown to be associated with the microsatellite stable (MSS) subtype that accounts for 80-85% of CRC. GPR56 ADC selectively induced cytotoxicity in CRC cells and tumour organoids at low nanomolar potency in a GPR56-dependent manner and showed significant antitumour efficacy against GPR56-expressing xenograft models. CONCLUSIONS: This study provides the rationale for the future development of a GPR56-targeted ADC approach to potentially treat a large fraction of MSS CRC patients.

Ablation of myeloid discoidin domain receptor 2 exacerbates arthritis and high fat diet induced inflammation.

Chronic systemic inflammation leads to sever disorders and diseases. It is of great importance to explore novel target for effective treatment. Discoidin domain receptor 2 (Ddr2) is a member of receptor tyrosine kinase (RTK) family and is implicated in skeletal and fat hemostasis. However, the role of Ddr2 in myeloid cells remains obscure. In this study, we conditionally deleted Ddr2 in myeloid lineage cells to generate cKO mice to investigate the role of Ddr2 in myeloid lineage cells. We found that cKO mice exhibited more severe inflammation both in collagen antibody-induced arthritis (CAIA) and high-fat diet (HFD)-induced obesity, indicating the protective role of Ddr2 against inflammation. Mechanistically, Ddr2 promotes macrophage repolarization from the M1 to M2 phenotype, and protect against systemic inflammation. Our study reveals for the first time that Ddr2 modulates macrophage repolarization and plays critical roles in macrophage-mediated inflammation, providing potential target for the intervention of inflammation and related diseases.

Enhanced Performance of Porphyrin-Modified CuO-Co3O4 Heterojunction as a Photoelectrocatalyst for Methanol Oxidation.

It is desirable to improve the methanol oxidation ability of heterojunction catalysts because of their potential in the field of electrocatalysis. In this article, we have integrated CuO/Co3O4 heterojunction with porphyrin (TCPP) for TCPP/Cu2Co1. The results demonstrate that the specific surface area and electrochemical active surface area (ECSA) are enhanced, and the unblocked separation and transfer of photogenerated charges are guaranteed by the matched band energy of CuO, Co3O4, and TCPP. As such, the photocatalytic methanol oxidation reaction (MOR) performance and stability of TCPP/Cu2Co1 are significantly enhanced compared with those of Cu2Co1. This study provides a promising pathway for the design of MOR catalysts with high MOR activity.

Colorimetric sensor arrays for antioxidant recognition based on Co3O4 dual-enzyme activities.

Antioxidants are considered as essential compounds for monitoring human health. In this work, a colorimetric sensor array was developed using oxidase-like (OXD) and peroxidase-like (POD) activities of Co3O4 nanoflowers as sensing elements, together with a substrate, 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB), as a signal reader to effectively identify different antioxidants. In the presence of Co3O4, colorless TMB can be oxidized into blue oxTMB to different degrees in the presence and absence of H2O2. Interestingly, after the addition of antioxidants, the sensor array showed cross-reactions, and different changes in color and absorbance were observed, as TMB and antioxidants competed for binding. Different colorimetric responses were obtained on the sensor array and identified by linear discriminant analysis (LDA). The LDA result indicated that the sensor array can be used to distinguish 4 antioxidants, namely, dopamine (DA), glutathione (GSH), ascorbic acid (AA), and cysteine (Cys) at seven different concentrations, namely, 10, 20, 30, 50, 100, 200, and 250 nM. Different concentrations of antioxidants and proportions of mixed antioxidants were determined. This demonstrates the potential application of sensor arrays in diagnosis and food monitoring.

Zinc pyrovanadate nanorods with excellent peroxidase-like activity at physiological pH for the colorimetric assay of H2O2 and epinephrine.

Exploring highly active peroxidase mimics at physiological pH is important for the construction of efficient and convenient colorimetric sensing platforms for detecting small biomolecules. In this work, prepared zinc pyrovanadate (Zn3V2O7(OH)2·2H2O) nanorods exhibit excellent peroxidase-like activity, which is verified by the fast oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (oxTMB) by H2O2 at physiological pH (pH = 7) in 2 min. In addition, the catalytic behaviors of Zn3V2O7(OH)2·2H2O as a peroxidase-like nanozyme conform to the Michaelis-Menten equation. Scavenger experiments prove that the catalytic activity of Zn3V2O7(OH)2·2H2O is ascribed to ˙O2- radicals generated in the process of catalysis. Based on the peroxidase-like activity of the Zn3V2O7(OH)2·2H2O nanozyme, a fast and convenient colorimetric sensor has been constructed to detect H2O2 and epinephrine (EP) under physiological pH. The detection limit of EP is as low as 0.26 µM. In addition, the feasibility of the proposed sensor has been validated to detect H2O2 in milk and EP in serum.

Engineering Lattice Planes of NiCo-LDH Ultrathin Sheets for Boosting Methanol/Ethanol Oxidation Performance.

Alcohol oxidation reactions are known to be significant in the advancement of sustainable, renewable energy sources. Searching for catalytic materials with powerful, reliable, and economic performance is of great importance. Due to their excellent intrinsic performance, outstanding stability, and inexpensiveness, ultrathin layered double hydroxides (LDHs) are considered to be competitive electrocatalysts. However, the electrocatalytic property of ultrathin LDHs is still confined by the predominant exposure of the (003) basal plane. Hence, we have engineered active edge facets in ultrathin NiCo-LDHs, which possess abundant oxygen vacancies (VO), by a facile one-step strategy. Experimental results show that NiCo-LDH-E synthesized in ethanol demonstrates an ultrathin structure, rich oxygen vacancies, and more active facets, exhibiting a higher electrochemical active area of 3.25 cm2, which is 1.18 times that of NiCo-LDH-W (2.75 cm2). In addition, the current density of NiCo-LDH-E in methanol and ethanol oxidation reactions could reach 159.5 and 136.3 mA cm-2, which are 2.8 and 1.7 times that of NiCo-LDH-W, respectively.

Enhanced peroxidase-like activity of 2(3), 9(10), 16(17), 23(24)-octamethoxyphthalocyanine modified CoFe LDH for a sensor array for reducing substances with catechol structure.

Improving the catalytic activity of artificial nanozymes to realize the real-time detection of small molecules becomes an important task. Herein, a highly active nanozyme, 2(3), 9(10), 16(17), 23(24)-octamethoxyphthalocyanine (Pc(OH)8) modified CoFe LDH microspheres (Pc(OH)8-CoFe LDH) have been prepared by the two-step hydrothermal method. The 3,3',5,5'-tetramylbenzidine (TMB), a chromogenic substrate, was fast oxidized into blue oxTMB by H2O2 in the presence of Pc(OH)8-CoFe LDH, indicating that Pc(OH)8-CoFe LDH possesses high peroxidase-like activity rather than pure CoFe LDH. The enhancement peroxidase-like activity of the Pc(OH)8-CoFe LDH is ascribed to the synergistic action between Pc(OH)8 and CoFe LDH. Experimental results of radical scavenger and fluorescence probe verify that superoxide radical (•O2-) plays an important role during the catalytic reaction. Interestingly, the absorption intensity of reaction system has been enhanced largely, due to adding of the reducing substances containing catechol structure. Based on this, the three reducing substances (dopamine, procyanidin B2, catechins) containing catechol structure were distinguished from other reducing substances without catechol structure. Thus, a colorimetric array has been constructed using reaction time as the sensing element to realize the sensitive and selective recognition of catechol structures at a certain concentration.

ATP synthase subunit e is a shrimp growth-associated breeding marker.

Penaeus vannamei is one of the most popular aquaculture species in the world. This species is featured with its fast-growing and delicious taste, which drives people develop various strains. During this process identification of trait-associated markers could effectively increase breeding efficiency. Driven by this, we tried to screen fast-growing key regulators via a FACS-based high throughput method, in which 2-NBDG was applied as a fluorescent indicator for direct glucose uptake measurement. Totally six candidate genes were screened out followed by in vitro validation in 293T cells. After that, the correlation between these genes and shrimp growing was further verified in a hybrid lineage. The expression level of two genes including ATP synthase subunit e and inhibitor of apoptosis protein showed some correlation with shrimp growth speed. Furthermore, we tested these two candidate markers in various lineages and confirmed that ATP synthase subunit e could be a shrimp growth-associated breeding marker.

N,N-dicarboxymethyl Perylene-diimide-modified CdV2O6 Nanorods for Colorimetric Sensing of H2O2 and Pyrogallol.

The peroxidase-like activity of CdV2O6 nanorods has been considerably improved by modification with N, N-dicarboxymethyl perylene-diimide (PDI) as a photosensitizer. The peroxidase-like behaviors are evaluated by virtue of the colorless chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is fast changed into blue oxTMB in the presence of H2O2 in only 90 s. PDI-CdV2O6 exhibits high stability at elevated temperatures and PDI-CdV2O6 retains more than 70% of its catalytic activity over a wide range of 15 to 60 °C. The catalytic mechanism of PDI-CdV2O6 is ascribed to the synergistic interaction between PDI and CdV2O6 and the generation of •O2- radicals. Based on the enhanced peroxidase-like activity of PDI-CdV2O6, a selective colorimetric sensor has been constructed for H2O2 and pyrogallol (PG) with detection limits of 36.5 µM and 0.179 µM, respectively. The feasibility of the proposed sensing platform has been validated by detecting H2O2 in milk and pyrogallol in tap water.

Integrated Analysis of DNA Methylome and Transcriptome Reveals Epigenetic Regulation of Cold Tolerance in Litopenaeus vannamei.

DNA methylation is an important epigenetic modification that has been shown to be associated with responses to non-biological stressors. However, there is currently no research on DNA methylation in response to environmental signals in shrimp. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles and differentially expressed genes between two strains of Litopenaeus vannamei with significantly different cold tolerance through whole genome bisulfite sequencing (WGBS) and transcriptome sequencing. Between Lv-C and Lv-T (constant temperature of 28 °C and low temperatures of 18 °C and 10 °C) under cytosine-guanine (CG) environments, 39,100 differentially methylated regions (DMRs) were identified, corresponding to 9302 DMR-related genes (DMRGs). The DMRs were mainly located in the gene body (exons and introns). Gene Ontology (GO) analysis showed that these DMRGs were significantly enriched in cell parts, catalytic activity, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed significant enrichment of these DMRGs in pathways such as proteasome (ko03050), oxidative phosphorylation (ko00190), mTOR signaling pathway (ko04150), fatty acid metabolism (ko01212), and fatty acid degradation (ko00071). The comprehensive results suggested that L. vannamei mainly regulates gene expression in response to low temperatures through hypermethylation or demethylation of some genes involved in thermogenesis, glycolysis, the autophagy pathway, the peroxisome, and drug metabolism pathways. These results provide important clues for studying DNA methylation patterns and identifying cold tolerance genes in shrimp.

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion.

GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.

Drug Degradation Caused by mce3R Mutations Confers Contezolid (MRX-I) Resistance in Mycobacterium tuberculosis.

Contezolid (MRX-I), a safer antibiotic of the oxazolidinone class, is a promising new antibiotic with potent activity against Mycobacterium tuberculosis (MTB) both in vitro and in vivo. To identify resistance mechanisms of contezolid in MTB, we isolated several in vitro spontaneous contezolid-resistant MTB mutants, which exhibited 16-fold increases in the MIC of contezolid compared with the parent strain but were still unexpectedly susceptible to linezolid. Whole-genome sequencing revealed that most of the contezolid-resistant mutants bore mutations in the mce3R gene, which encodes a transcriptional repressor. The mutations in mce3R led to markedly increased expression of a monooxygenase encoding gene Rv1936. We then characterized Rv1936 as a putative flavin-dependent monooxygenase that catalyzes the degradation of contezolid into its inactive 2,3-dihydropyridin-4-one (DHPO) ring-opened metabolites, thereby conferring drug resistance. While contezolid is an attractive drug candidate with potent antimycobacterial activity and low toxicity, the occurrence of mutations in Mce3R should be considered when designing combination therapy using contezolid for treating tuberculosis.