Babesia microti Causing Intravascular Hemolysis in Immunocompetent Child, China.

We report a case of Babesia microti infection in an immunocompetent child <5 years of age that caused fever and severe intravascular hemolysis. Physicians in China should be aware of babesiosis, especially in the differential diagnosis of immune hemolytic anemia with negative results for antiglobulin tests.

A Novel Oral GyrB/ParE Dual Binding Inhibitor Effective against Multidrug-Resistant Neisseria gonorrhoeae and Other High-Threat Pathogens.

Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances. Here, we describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6-fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methanol], which was developed to target both ATP-binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae, including drug-resistant strains. A phosphate pro-drug, JSF-2659, was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram-positive organisms, and JSF-2659 was shown in a deep tissue model of vancomycin-resistant Staphylococcus aureus (VRSA) and a model of Clostridioides difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel preclinical drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

Comorbid early esophageal cancer and Gongylonema pulchrum infection: a case report.

BACKGROUND: Gongylonema pulchrum is a zoonotic parasite rarely found in humans. To date, there have been no reports on the carcinogenic properties of G. pulchrum, and there are few reports overall on the relationship between esophageal cancer and parasites. CASE PRESENTATION: This report describes the first case of esophageal gongylonemiasis coexisting with early esophageal cancer. The patient had no high-risk factors for esophageal cancer, such as smoking, flushing after drinking, or tumor history. We speculate the existence of unknown links between esophageal cancer and parasitic infection in this patient. DISCUSSION AND CONCLUSIONS: We report the first case of a human presenting both esophageal G. pulchrum infection and esophageal squamous cell carcinoma with the hope that it may provide evidence for a new hypothesis of tumorigenesis.

De novo transcriptome sequencing and analysis of the cuttlefish (Sepiella japonica) with different embryonic developmental stages.

In this study, embryos of Sepiella japonica from eye primordium formation to the larval growing stage were collected and used for RNA-Seq analysis. A total of 183,542,186 clean reads were assembled de novo into 58,054 unigenes consisting of 54,118,228 bp, with the average length at 932 bp and the N50 at 1667 bp. 21,469 (36.98%) unigenes were annotated at least in one of four databases including non-redundant protein (NR), Swiss-Prot, clusters of orthologous groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 4460 (7.68%) unigenes were annotated in all databases. Analysis of differentially expressed genes (DEGs) was carried out on embryos at Eye primordium formation stage (SJ1), organ differentiation stage (SJ2), and hatching stage (SJ3). Overall, the current study provided the de novo assembly of S. japonica transcriptome and identified the DEGs and pathways during embryonic development, which will provide a fundamental genetic resource for further functional research.

Metagenome-Guided Analogue Synthesis Yields Improved Gram-Negative-Active Albicidin- and Cystobactamid-Type Antibiotics.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics. Our search for PABA-specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio-informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

Dynamic compression locking system versus multiple cannulated compression screw for the treatment of femoral neck fractures: a comparative study.

BACKGROUND: Femoral neck fractures are one of the problems in clinical treatment. The prognosis is uncertain. Currently, No internal fixation method is superior to other internal fixation methods in the treatment of femoral neck fractures. Therefore, the internal fixation system needs to be further explored. The aim of this study was to compare clinical outcomes of femoral neck dynamic compression locking system (DCLS) and multiple cannulated compression screws(MCCS) in the treatment of femoral neck fractures. METHODS: A prospective analysis of 54 cases of femoral neck fractures treated with either a DCLS (n = 28) or MCCS (n = 26) was conducted between December 2015 and November 2017 in authors' hospitals. The perioperative and postoperative parameters of the two groups were recorded and evaluated. RESULTS: Fifty-four patients were followed up for 24-47 months. The etiology was caused by a fall. There was no significant difference in follow-up time, operation time, incision length, surgical blood loss, the incidence of perioperative and postoperative healing complications, and mobility in the two groups (all P > 0.05). The Harris score, fracture healing time, femoral neck shortening, partial weight-bearing time and complete weight-bearing time were significantly better in the DCLS group than in the MCCS group (all P < 0.05). The fracture healing rate in the DCLS group was higher than that in the MCCS group. CONCLUSIONS: The DCLS and MCCS might be equally effective in terms of operation time, incision length, surgical blood loss, the incidence of perioperative and postoperative healing complications, and mobility in the treatment of femoral neck fractures. However, the DCLS is superior to the MCCS in Harris score, fracture healing time, femoral neck shortening, weight-bearing time and fracture healing rate. So, DCLS deserves further study.

Non-classical transpeptidases yield insight into new antibacterials.

Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the ß-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of ß-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for ß-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

Allele-specific real-time PCR testing for minor macrolide-resistant Mycoplasma Pneumoniae.

BACKGROUND: The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. METHODS: We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of < 0.1% for A2063G and A2064G. We also compared the findings of ASPCR with the results of nested PCR with sequencing. RESULTS: Of 178 samples, 164 were found to have M. pneumoniae including 90.85% (149/164) samples with macrolide-resistant M. pneumoniae (MRMP) quasispecies by ASPCR, while 153 were found to be M. pneumoniae-positive including 71.90% (110/153) samples with MRMP quasispecies by nested PCR with sequencing. Of the 164 M. pneumoniae-positive samples, 61.59% (101/164) had the mixed population of wild-type and mutant M. pneumoniae, and 56.44% (57/101) of the latter contained the mutations at low frequency (≤50%). CONCLUSION: ASPCR indicated that sensitive and resistant quasispecies coexisted in most of the M. pneumoniae positive samples. The ASPCR was a highly sensitive, accurate and rapid method for detecting the macrolide resistance-associated mutations and it could provide earlier and more drug-resistant information for M. pneumoniae research and the clinical therapy.

Curcumin Inhibits Cell Viability and Increases Apoptosis of SW620 Human Colon Adenocarcinoma Cells via the Caudal Type Homeobox-2 (CDX2)/Wnt/ß-Catenin Pathway.

BACKGROUND Curcumin is a polyphenol compound extracted from the root of the herb Curcuma longa, which is used in traditional Chinese medicine (TCM). Worldwide, colorectal carcinoma (CRC) is an increasing cause of morbidity and mortality. This study aimed to investigate the effects of increasing concentrations of curcumin on cell viability, proliferation, and apoptosis of SW620 human colonic adenocarcinoma cells cultured in vitro, and the signaling pathways involved. MATERIAL AND METHODS SW620 human colonic adenocarcinoma cells were cultured in curcumin at concentrations of 0, 4, 8, 16, and 32 µmol/l for 48 hours. Specific small interfering RNA (siRNA) was transfected into SW620 cells to silence the expression of caudal type homeobox-2 (CDX2). Cell viability was measured using the MTT assay. Flow cytometry evaluated cell apoptosis. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess the nuclear translocation of b-catenin and the activation of Wnt signaling. RESULTS Curcumin reduced cell viability and increased apoptosis of SW620 human colonic adenocarcinoma cells in a dose-dependent way, and increased the expression of CDX2 but decreased ß-catenin nuclear translocation and the expression of Wnt3a, c-Myc, survivin, and cyclin D1. CDX2 silencing significantly reduced the effects of curcumin on SW620 human colonic adenocarcinoma cells. The nuclear translocation of ß-catenin, and expression levels of Wnt3a, c-Myc, survivin, and cyclin D1 were significantly higher in CDX2-silenced SW620 cells. CONCLUSIONS Curcumin reduced cell viability and increased apoptosis in SW620 human colonic adenocarcinoma cells by restoring CDX2, which inhibited the Wnt/ß-catenin signaling pathway.

Polyparasitism of Rhabditis Axei and Enterobius Vermicularis in a Child from Beijing, China.

BACKGROUND: Rhabditis (Rhabditellae) axei is a common species in soil, which has been reported repeatedly in human urine and the digestive system. Humans exposed to sewage or mistakenly polluted sewage is the cause of larvae infecting the digestive tract or via the urethra. We reported a patient infected with Rhabditis axei and Enterobius Vermicularis. The migration of the nematodes caused true signs of hematuria, diarrhea, and high eosinophilia. METHODS: Stool and urine are collected to detect parasite eggs and genotype. Specimens are sent for polymerase chain reaction (PCR)-based species identification. Amplification of the 18S ribosomal RNA gene was performed by PCR as described [1]. RESULTS: Morphological features and PCR amplification of the 18S ribosomal RNA gene confirmed Rhabditis axei and Enterobius vermicularis as the pathogen of infection. CONCLUSIONS: Herein, we presented a case that confirmed Rhabditis axei and Enterobius vermicularis infection in humans can be associated with high eosinophilia.

Mycobacterium abscessus l,d-Transpeptidases Are Susceptible to Inactivation by Carbapenems and Cephalosporins but Not Penicillins.

As a growing number of clinical isolates of Mycobacterium abscessus are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of M. abscessus are synthesized by nonclassical transpeptidases, namely, the l,d-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of ß-lactams determines their activity against Mycobacterium tuberculosis Here, we studied the interactions of ß-lactams with two l,d-transpeptidases in M. abscessus, namely, LdtMab1 and LdtMab2, and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of ß-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with ß-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible M. abscessus and clinical isolates that are resistant to most antibiotics, which suggests that dual-ß-lactam therapy has potential for the treatment of M. abscessus Finally, we solved the first crystal structure of an M. abscessus l,d-transpeptidase, LdtMab2, and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and ß-lactams, which provide an insight into the molecular basis for the relative efficacy of different ß-lactams against M. abscessus.

A virtual screen discovers novel, fragment-sized inhibitors of Mycobacterium tuberculosis InhA.

Isoniazid (INH) is usually administered to treat latent Mycobacterium tuberculosis (Mtb) infections and is used in combination therapy to treat active tuberculosis (TB). Unfortunately, resistance to this drug is hampering its clinical effectiveness. INH is a prodrug that must be activated by Mtb catalase-peroxidase (KatG) before it can inhibit InhA (Mtb enoyl-acyl-carrier-protein reductase). Isoniazid-resistant cases of TB found in clinical settings usually involve mutations in or deletion of katG, which abrogate INH activation. Compounds that inhibit InhA without requiring prior activation by KatG would not be affected by this resistance mechanism and hence would display continued potency against these drug-resistant isolates of Mtb. Virtual screening experiments versus InhA in the GO Fight Against Malaria (GO FAM) project were designed to discover new scaffolds that display base-stacking interactions with the NAD cofactor. GO FAM experiments included targets from other pathogens, including Mtb, when they had structural similarity to a malaria target. Eight of the 16 soluble compounds identified by docking against InhA plus visual inspection were modest inhibitors and did not require prior activation by KatG. The best two inhibitors discovered are both fragment-sized compounds and displayed Ki values of 54 and 59 µM, respectively. Importantly, the novel inhibitors discovered have low structural similarity to known InhA inhibitors and thus help expand the number of chemotypes on which future medicinal chemistry efforts can be focused. These new fragment hits could eventually help advance the fight against INH-resistant Mtb strains, which pose a significant global health threat.

Evolution of a thienopyrimidine antitubercular relying on medicinal chemistry and metabolomics insights.

The metabolic instability of an antitubercular small molecule CD117 was addressed through iterative alteration of a key sulfide substituent and interrogation of the effect on growth inhibition of cultured Mycobacterium tuberculosis. This process was informed by studies of the intramycobacterial metabolism of CD117 and its inactive carboxylic acid derivative. Isoxazole 4e and thiazole 4m demonstrated significant gains in mouse liver microsomal stability with slight losses in whole-cell activity. This work illustrates the challenges of antitubercular hit evolution, requiring a balance of chemical and biological insights.

Lactobacillus melliventris promotes hive productivity and immune functionality in Bombus terrestris performance in the greenhouse.

Bumblebees are important pollinators in agricultural ecosystems, but their abundance is declining globally. There is an urgent need to protect bumblebee health and their pollination services. Bumblebees possess specialized gut microbiota with potential to be used as probiotics to help defend at-risk bumblebee populations. However, evidence for probiotic benefits on bumblebees is lacking. Here, we evaluated how supplementation with Lactobacillus melliventris isolated from bumblebee gut affected the colony development of Bombus terrestris. This native strain colonized robustly and persisted long-term in bumblebees, leading to a significantly higher quality of offspring. Subsequently, the tyrosine pathway was upregulated in the brain and fat body, while the Wnt and mTOR pathways of the gut were downregulated. Notably, the field experiment in the greenhouse revealed the supplementation of L. melliventris led to a 2.5-fold increase in the bumblebee survival rate and a more than 10% increase in the number of flowers visited, indicating a better health condition and pollination ability in field conditions. Our study represents a first screening for the potential use of the native gut member, L. melliventris, as probiotic strains in hive supplement for bumblebee breeding, which may be a practical approach to improve immunity and hive health.

Establish an allele-specific real-time PCR for Leishmania species identification.

BACKGROUND: Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites. METHODS: A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay. RESULTS: This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients. CONCLUSIONS: Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.

Effects of axial pressure on the evolution of core-shell heterogeneous structures and magnetic properties of Fe-Si soft magnetic powder cores during hot-press sintering.

Silicon dioxide (SiO2) has attracted much attention as an ideal coating material for iron (Fe)-based soft magnetic powder cores (SMPCs). However, maintaining the integrity and uniformity of Fe-based/SiO2 core-shell heterostructures is still a challenge. The evolution mechanism of core-shell heterostructures determines the performance of Fe-based SMPCs. Herein, the evolution of the core-shell structures and heterogeneous interfaces of Fe-Si@SiO2 SMPCs with axial pressure and the influence of the evolution on the SMPCs performance were investigated. The results show that in the axial pressure range of 10-15 kN, the core-shell heterostructures were gradually integrated, whereas the SiO2 insulation coatings underwent an amorphous-to-crystalline transformation. At axial pressure above 16 kN, the Fe-Si powder melted partially, and the core-shell heterostructure collapsed due to overheating, caused by the gradient temperature field during the hot-press sintering. When the core-shell heterostructure was intact, the Fe-Si@SiO2 SMPCs showed a permeability of over 38 with a wide and stable frequency range of 100-300 kHz, a saturation magnetisation of 231.7 emu g-1, resistivity of 0.8 mΩ cm and total loss of 704.7 kW m-3 at 10 mT and 100 kHz. When the core-shell heterostructure was destroyed, the resistivity dropped dramatically and the loss increased to 765.0 and 897.4 kW m-3. These results show the relationship between the core-shell heterostructure of Fe-Si@SiO2 SMPCs, axial pressure and magnetic properties, which would be vital in achieving high power density, high efficiency and miniaturisation in SMPCs.

Detection of Pneumocystis jirovecii and Toxoplasma gondii in patients with lung infections by a duplex qPCR assay.

Pneumocystis pneumonia (PCP) and pulmonary toxoplasmosis (PT) are caused by Pneumocystis jirovecii and Toxoplasma gondii. The clinical symptoms and imaging of PCP and PT are indistinguishable. A duplex qPCR was developed to differentiate between these two pathogens. In testing 92 clinical samples to validate the performance of this method for P. jirovecii detection, it identified 31 positive samples for P. jirovecii infection, consistent with clinical diagnosis. Among the remainder of the 61 clinical samples with suspected PCP, yet showing as negative by the conventional PCR diagnosis approach, 6 of them proved positive using our new assay. Our new approach also produced similar results in identification of T. gondii infections, giving a result of 2 positive and 20 negative in clinical samples. An investigation was undertaken on the prevalence of P. jirovecii and T. gondii infections using 113 samples from lung infection patients. 9% (10/113) were shown to be positive with infections of P. jirovecii, 2% with T. gondii (2/113) and 5% (6/113) were co-infected with both pathogens. Although this duplex qPCR can detect individual P. jirovecii and T. gondii infection, and co-infection of both pathogens, further large-scale investigations are needed to validate its performance, especially in T. gondii detection. Our assay provides a rapid and accurate tool for PCP and PT diagnosis in immunocompromised population and clinical surveillance of these infections in patients with no immune defects.

Application of Quantitative PCR in the Diagnosis and Evaluating Treatment Efficacy of Leishmaniasis.

Leishmaniasis is still a serious neglected tropical disease that may cause death in infected individuals. At present, the clinical diagnosis and treatment monitoring still rely on parasitological culture and microscopy that needs experienced technicians. The low sensitivity and inconvenience of microscopic examination could cause misdiagnosis and relapse of leishmaniasis. There is an urgent need for developing a sensitive and easily operated diagnostic method for the diagnosis and disease management of leishmaniasis. Thus, a quantitative real-time PCR (qPCR) based on the conversed regions of kinetoplast minicircle DNA (mkDNA) of Leishmania spp. was developed to detect different species of Leishmania. The designed mkDNA-based qPCR was able to detect as low as one copy of Leishmania mkDNA or DNA from single parasite. It also detected Pan-Leishmania protozoa including Leishmania donovani, Leishmania infantum and Leishmania major without cross-reaction with other pathogen DNAs available in our lab. This method was clinically applied to quantitatively detect skin lesion samples from 20 cutaneous leishmaniasis (CL) and bone marrow and/or PBMC samples from 30 current and cured visceral leishmaniasis (VL) patients, and blood samples from 11 patients with other infections and 5 normal donors as well. Total 20 skin lesion samples from current CL patients and 20 bone marrow and/or PBMC samples from current VL patients were all detected as positive with qPCR without cross-reaction with samples from patients with malaria, brucellosis and dengue or normal donors. Two VL patients with parasite converted to microscopically negative after treatment were detected positive with qPCR. The patients with bigger skin lesion in CL and higher level of immunoglobulin or splenomegaly in VL, had the higher parasite load detected by qPCR. The parasite load was significantly reduced after treatment. In conclusion, the mkDNA-based qPCR assay that we developed in this study can be used not only for diagnosis of both cutaneous and visceral leishmaniasis with high sensitivity and specificity, but also for evaluating the severity and treatment efficacy of this disease, presenting a rapid and accurate tool for clinical surveillance, treatment monitoring and the end point determination of leishmaniasis.

A Preclinical Candidate Targeting Mycobacterium tuberculosis KasA.

Published Mycobacterium tuberculosis ß-ketoacyl-ACP synthase KasA inhibitors lack sufficient potency and/or pharmacokinetic properties. A structure-based approach was used to optimize existing KasA inhibitor DG167. This afforded indazole JSF-3285 with a 30-fold increase in mouse plasma exposure. Biochemical, genetic, and X-ray studies confirmed JSF-3285 targets KasA. JSF-3285 offers substantial activity in an acute mouse model of infection and in the corresponding chronic infection model, with efficacious reductions in colony-forming units at doses as low as 5 mg/kg once daily orally and improvement of the efficacy of front-line drugs isoniazid or rifampicin. JSF-3285 is a promising preclinical candidate for tuberculosis.

Antitubercular Triazines: Optimization and Intrabacterial Metabolism.

The triazine antitubercular JSF-2019 was of interest due to its in vitro efficacy and the nitro group shared with the clinically relevant delamanid and pretomanid. JSF-2019 undergoes activation requiring F420H2 and one or more nitroreductases in addition to Ddn. An intrabacterial drug metabolism (IBDM) platform was leveraged to demonstrate the system kinetics, evidencing formation of NO⋅ and a des-nitro metabolite. Structure-activity relationship studies focused on improving the solubility and mouse pharmacokinetic profile of JSF-2019 and culminated in JSF-2513, relying on the key introduction of a morpholine. Mechanistic studies with JSF-2019, JSF-2513, and other triazines stressed the significance of achieving potent in vitro efficacy via release of intrabacterial NO⋅ along with inhibition of InhA and, more generally, the FAS-II pathway. This study highlights the importance of probing IBDM and its potential to clarify mechanism of action, which in this case is a combination of NO⋅ release and InhA inhibition.