Design, Synthesis, and Biological Evaluation of Pyrrole-2-carboxamide Derivatives as Mycobacterial Membrane Protein Large 3 Inhibitors for Treating Drug-Resistant Tuberculosis.

In this work, pyrrole-2-carboxamides were designed with a structure-guided strategy based on the crystal structure of MmpL3 and a pharmacophore model. The structure-activity relationship studies revealed that attaching phenyl and pyridyl groups with electron-withdrawing substituents to the pyrrole ring and attaching bulky substituents to the carboxamide greatly improved anti-TB activity. Most compounds showed potent anti-TB activity (MIC < 0.016 µg/mL) and low cytotoxicity (IC50 > 64 µg/mL). Compound 32 displayed excellent activity against drug-resistant tuberculosis, good microsomal stability, almost no inhibition of the hERG K+ channel, and good in vivo efficacy. Furthermore, the target of the pyrrole-2-carboxamides was identified by measuring their potency against M. smegmatis expressing wild-type and mutated variants of the mmpL3 gene from M. tuberculosis (mmpL3tb) and determining their effect on mycolic acid biosynthesis using a [14C] acetate metabolic labeling assay. The present study provides new MmpL3 inhibitors that are promising anti-TB agents.

Carpenter Bees (Xylocopa) Harbor a Distinctive Gut Microbiome Related to That of Honey Bees and Bumble Bees.

Eusocial corbiculate bees, including bumble bees and honey bees, maintain a socially transmitted core gut microbiome that contributes to digestion and pathogen defense. In contrast, solitary bees, which have fewer opportunities for direct interhost transmission, typically have less consistent microbiomes dominated by bacteria associated with pollen and food reserves. Carpenter bees (genus Xylocopa) are long-lived bees that are not eusocial but that often live in shared nesting sites. We characterized gut microbiomes for Xylocopa micans, X. mexicanorum, X. tabaniformis parkinsoniae, and X. virginica and for five solitary bee species from other genera (Andrena, Habropoda, Megachile, and Svastra), sampled in the same localities in central Texas. Unexpectedly, all four Xylocopa species had microbiomes dominated by bacterial lineages previously known only from social bees or other insect groups. Microbiomes were similar across three Xylocopa species and included lineages in the families Bifidobacteriaceae, Orbaceae, Lactobacillaceae, Pseudomonadaceae, and Enterobacteriaceae. In contrast, X. virginica had a distinct microbiome dominated by the genus Bombilactobacillus, a group abundant in guts of eusocial bees. Phylogenetic analyses support a past transfer of bacterial lineages into Xylocopa from bumble bees or honey bees. Gut microbiome compositions of Xylocopa species were distinct from those of other co-occurring solitary bees that had variable gut microbiomes dominated by bacteria from environmental sources. IMPORTANCE Gut microbiomes from social bees, such as honey bees and bumble bees, are conserved and consist of host-restricted bacteria that are transmitted among sterile female workers within a colony and that are important to the health of these key insect pollinators. In contrast, solitary bee species typically have more erratic, environmentally acquired microbiomes. Carpenter bees (genus Xylocopa) can be solitary as they lack a worker caste, and each female can excavate nests and raise offspring alone, although females are often social share nests at least in some species. This study showed that the gut microbiomes of four Xylocopa species have distinctive and consistent compositions and are dominated by bacterial lineages previously known from honey bees and bumble bees. Thus, eusociality is not required for bees to maintain a specialized, host-restricted gut microbiome. These findings suggest that gut bacteria are transmitted at shared nesting sites and that they play a role in host ecology.

Therapeutic efficacy of antimalarial drugs targeting DosRS signaling in Mycobacterium abscessus.

A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

Indole-2-Carboxamides Are Active against Mycobacterium abscessus in a Mouse Model of Acute Infection.

Nontuberculous mycobacteria (NTM) pathogens particularly infect patients with structural lung disorders. We previously reported novel indole-2-carboxamides (ICs) that are active against a wide panel of NTM pathogens. This study discloses in vivo data for two lead molecules (compounds 5 and 25) that were advanced for efficacy studies in Mycobacterium abscessus-infected mouse models. Oral administration of the lead molecules showed a statistically significant reduction in the bacterial loads in lung and spleen of M. abscessus-infected mice.

Impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections.

BACKGROUND: Empiric antibiotic treatment is common among children with acute respiratory tract infections (ARTI), despite infections being predominately viral. The use of molecular respiratory panel assays has become increasingly common for medical care of patients with ARTIs. STUDY DESIGN: This was a 6-year retrospective, single-centered study of pediatric inpatients who tested positive for an ARTI respiratory pathogen. We examined the relationship between clinical outcomes and whether the patient was tested using the Luminex Respiratory Viral Panel ([RVP]; in-use: Dec. 2009 - Jul. 2012) or Biofire Respiratory Pathogen Panel ([RP]; in-use Aug. 2012 - Jun. 2016). The prevalence and duration of pre-test empiric antibiotics, post-test oseltamivir administration to influenza patients, chest x-rays and length of stay between the two assays was compared. RESULTS: A total of 5142 patients (1264 RVP; 3878 RP) were included. The median laboratory turn-around-time for RP was significantly shorter than RVP (1.4 vs. 27.1 h, respectively; p < .001). Patients tested with RP were less likely to receive empiric antibiotics (OR: 0.45; p < .001; 95% CI: 0.39, 0.52) and had a shorter duration of empiric broad-spectrum antibiotics (6.4 h vs. 32.9 h; p < .001) compared to RVP patients. RP influenza patients had increased oseltamivir use post- test compared to RVP influenza patients (OR: 13.56; p < .001; 95% CI: 7.29, 25.20). CONCLUSIONS: Rapid molecular testing positively impacts patient management of ARTIs. Adopting assays with a shorter turn-around-time improves decision making by decreasing empirical antibiotic use and duration, decreasing chest x-rays, increasing timely oseltamivir administration, and reducing length of stay.

Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association.

BACKGROUND: Kawasaki disease is an acute vasculitis of childhood that leads to coronary artery aneurysms in ≈25% of untreated cases. It has been reported worldwide and is the leading cause of acquired heart disease in children in developed countries. METHODS AND RESULTS: To revise the previous American Heart Association guidelines, a multidisciplinary writing group of experts was convened to review and appraise available evidence and practice-based opinion, as well as to provide updated recommendations for diagnosis, treatment of the acute illness, and long-term management. Although the cause remains unknown, discussion sections highlight new insights into the epidemiology, genetics, pathogenesis, pathology, natural history, and long-term outcomes. Prompt diagnosis is essential, and an updated algorithm defines supplemental information to be used to assist the diagnosis when classic clinical criteria are incomplete. Although intravenous immune globulin is the mainstay of initial treatment, the role for additional primary therapy in selected patients is discussed. Approximately 10% to 20% of patients do not respond to initial intravenous immune globulin, and recommendations for additional therapies are provided. Careful initial management of evolving coronary artery abnormalities is essential, necessitating an increased frequency of assessments and escalation of thromboprophylaxis. Risk stratification for long-term management is based primarily on maximal coronary artery luminal dimensions, normalized as Z scores, and is calibrated to both past and current involvement. Patients with aneurysms require life-long and uninterrupted cardiology follow-up. CONCLUSIONS: These recommendations provide updated and best evidence-based guidance to healthcare providers who diagnose and manage Kawasaki disease, but clinical decision making should be individualized to specific patient circumstances.

Clinical Course of Enterovirus D68 in Hospitalized Children.

BACKGROUND: Enterovirus D68 (EV-D68) has been sporadically reported as a cause of respiratory tract infections. In 2014, an international outbreak of EV-D68 occurred and caused severe respiratory disease in the pediatric population. METHODS: A retrospective chart review was performed of children admitted to Children's Mercy Hospital from August 1, 2014, to September 15, 2014, with positive multiplex polymerase chain reaction testing for EV/rhinovirus (RV). Specimens were subsequently tested for EV-D68, and clinical data were obtained from the medical records. Patients with EV-D68 were compared with children presenting simultaneously with other EV/RV. RESULTS: Of 542 eligible specimens, children with EV-D68 were significantly older than children with other EV/RV (4.6 vs. 2.2 years, P < 0.001). Children with EV-D68 were more likely to have a history of asthma (38.6% vs. 30.0%, P = 0.04) or recurrent wheezing (22.1% vs. 14.8%, P = 0.04). EV-D68-positive children more commonly received supplemental oxygen (86.7% vs. 65.0%, P < 0.001), albuterol (91.2% vs. 65.5%, P < 0.001) and corticosteroids (82.9% vs. 58.6%, P < 0.001). Age ≥5 years was an independent risk factor for intensive care unit management in EV-D68-infected children. Children with a history of asthma or recurrent wheezing and EV-D68 received supplemental oxygen (92.7% vs. 82.4%, P = 0.007) and magnesium (42.7% vs. 29.7%, P = 0.03) at higher rates and more continuous albuterol (3 vs. 2 hours, P = 0.03) than those with other EV/RV. CONCLUSIONS: EV-D68 causes severe disease in the pediatric population, particularly in children with a history of asthma or recurrent wheezing. EV-D68-positive children are more likely to require therapy for refractory bronchospasm and may need intensive care unit- level care.

Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3.

In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.

New approaches to target the mycolic acid biosynthesis pathway for the development of tuberculosis therapeutics.

Mycolic acids are the major lipid components of the unique mycobacterial cell wall responsible for the protection of the tuberculosis bacilli from many outside threats. Mycolic acids are synthesized in the cytoplasm and transported to the outer membrane as trehalose- containing glycolipids before being esterified to the arabinogalactan portion of the cell wall and outer membrane glycolipids. The large size of these unique fatty acids is a result of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids to the mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, including fatty acid synthesis (KasA, KasB, MabA, InhA, HadABC), mycolic acid modifying enzymes (SAM-dependent methyltransferases, aNAT), fatty acid activating and condensing enzymes (FadD32, Acc, Pks13), transporters (MmpL3) and tranferases (Antigen 85A-C) all of which are excellent potential drug targets. Not surprisingly, in recent years many new compounds have been reported to inhibit specific portions of this pathway, discovered through both phenotypic screening and target enzyme screening. In this review, we analyze the new and emerging inhibitors of this pathway discovered in the post-genomic era of tuberculosis drug discovery, several of which show great promise as selective tuberculosis therapeutics.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis.

Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed.

Screening a library of 1600 adamantyl ureas for anti-Mycobacterium tuberculosis activity in vitro and for better physical chemical properties for bioavailability.

Adamantyl ureas were previously identified as a group of compounds active against Mycobacterium tuberculosis in culture with minimum inhibitor concentrations (MICs) below 0.1 µg/ml. These compounds have been shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate. They also inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases. However, active compounds to date have high cLogP's and are poorly soluble, leading to low bioavailability and thus limiting any therapeutic application. In this study, a library of 1600 ureas (mostly adamantyl ureas), which were synthesized for the purpose of increasing the bioavailability of inhibitors of hsEH, was screened for activity against M. tuberculosis. 1-Adamantyl-3-phenyl ureas with a polar para substituent were found to retain moderate activity against M. tuberculosis and one of these compounds was shown to be present in serum after oral administration to mice. However, neither it, nor a closely related analog, reduced M. tuberculosis infection in mice. No correlation between in vitro potency against M. tuberculosis and the hsEH inhibition were found supporting the concept that activity against hsEH and M. tuberculosis can be separated. Also there was a lack of correlation with cLogP and inhibition of the growth of M. tuberculosis. Finally, members of two classes of adamantyl ureas that contained polar components to increase their bioavailability, but lacked efficacy against growing M. tuberculosis, were found to taken up by the bacterium as effectively as a highly active apolar urea suggesting that these modifications to increase bioavailability affected the interaction of the urea against its target rather than making them unable to enter the bacterium.

Temporal profile of functional visual rehabilitative outcomes modulated by transcranial direct current stimulation.

OBJECTIVES: We have previously reported that transcranial direct current stimulation (tDCS) delivered to the occipital cortex enhances visual functional recovery when combined with three months of computer-based rehabilitative training in patients with hemianopia. The principal objective of this study was to evaluate the temporal sequence of effects of tDCS on visual recovery as they appear over the course of training and across different indicators of visual function. METHODS: Primary objective outcome measures were 1) shifts in visual field border and 2) stimulus detection accuracy within the affected hemifield. These were compared between patients randomized to either vision restoration therapy (VRT) combined with active tDCS or VRT paired with sham tDCS. Training comprised two half-hour sessions, three times a week for three months. Primary outcome measures were collected at baseline (pretest), monthly interim intervals, and at posttest (three months). As secondary outcome measures, contrast sensitivity and reading performance were collected at pretest and posttest time points only. RESULTS: Active tDCS combined with VRT accelerated the recovery of stimulus detection as between-group differences appeared within the first month of training. In contrast, a shift in the visual field border was only evident at posttest (after three months of training). tDCS did not affect contrast sensitivity or reading performance. CONCLUSIONS: These results suggest that tDCS may differentially affect the magnitude and sequence of visual recovery in a manner that is task specific to the type of visual rehabilitative training strategy employed.

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane.

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

Combining visual rehabilitative training and noninvasive brain stimulation to enhance visual function in patients with hemianopia: a comparative case study.

OBJECTIVE: To standardize a protocol for promoting visual rehabilitative outcomes in post-stroke hemianopia by combining occipital cortical transcranial direct current stimulation (tDCS) with Vision Restoration Therapy (VRT). DESIGN: A comparative case study assessing feasibility and safety. SETTING: A controlled laboratory setting. PATIENTS: Two patients, both with right hemianopia after occipital stroke damage. METHODS AND OUTCOME MEASUREMENTS: Both patients underwent an identical VRT protocol that lasted 3 months (30 minutes, twice a day, 3 days per week). In patient 1, anodal tDCS was delivered to the occipital cortex during VRT training, whereas in patient 2 sham tDCS with VRT was performed. The primary outcome, visual field border, was defined objectively by using high-resolution perimetry. Secondary outcomes included subjective characterization of visual deficit and functional surveys that assessed performance on activities of daily living. For patient 1, the neural correlates of visual recovery were also investigated, by using functional magnetic resonance imaging. RESULTS: Delivery of combined tDCS with VRT was feasible and safe. High-resolution perimetry revealed a greater shift in visual field border for patient 1 versus patient 2. Patient 1 also showed greater recovery of function in activities of daily living. Contrary to the expectation, patient 2 perceived greater subjective improvement in visual field despite objective high-resolution perimetry results that indicated otherwise. In patient 1, visual function recovery was associated with functional magnetic resonance imaging activity in surviving peri-lesional and bilateral higher-order visual areas. CONCLUSIONS: Results of preliminary case comparisons suggest that occipital cortical tDCS may enhance recovery of visual function associated with concurrent VRT through visual cortical reorganization. Future studies may benefit from incorporating protocol refinements such as those described here, which include global capture of function, control for potential confounds, and investigation of underlying neural substrates of recovery.

Complementary therapy used by Hispanic women during treatment for breast cancer.

The purposes of this study are to assess Hispanic women's use of complementary interventions during breast cancer treatment and delineate the association between the most burdensome side effects and the most frequently used complementary and alternative medicine (CAM). The researchers examined both descriptive statistics and correlational relationships between side effects and CAM. Data were collected from a convenience sample (N = 125). The mean age was 54, the educational average was less than 10 years, and the median income level was less than $20,000 per year. CAM was positively correlated with family income. Prayer was used by 93% of the women, humor was used by 83%, and 65% used exercise. The most frequent side effect of hair loss (70%) was the most bothersome side effect. The most burdensome and unmanageable side effects were bowel problems and nausea. Nurses play a key role in offering affordable, culturally appropriate symptom management interventions.

Disseminated nontuberculous mycobacterial infections in sickle cell anemia patients.

Nontuberculous mycobacteria (NTM) are ubiquitous in nature and have been implicated in skin/soft-tissue, pulmonary, middle ear, bone, and surgical/traumatic wound infections. Disseminated disease occurs infrequently and almost exclusively in the immunocompromised. We describe the first 2 reported cases of disseminated Mycobacterium fortuitum infection in teenagers with sickle hemoglobinopathy. Both had central venous catheters (CVCs), frequent admissions for vaso-occlusive painful episode and received hydroxyurea. Diagnosis was confirmed by multiple positive blood cultures and pulmonary dissemination occurred in both. Both had successful treatment after CVC removal and combination drug therapy. Positive cultures persisted in 1 patient due to drug resistance emphasizing the need for accurate susceptibility data. NTM infection should be added to the list of pathogens in sickle cell patients with CVCs and fever. Investigation for disseminated disease should be undertaken based on clinical signs and symptoms. Although some routine blood culture systems can identify NTM, specific mycobacterial blood culture is optimal. Removal of involved CVCs is essential and treatment of NTM must be guided by susceptibilities. As dissemination almost always occurs in those with impaired cellular immunity, human immunodeficiency virus testing should be performed. Hydroxyurea may be a risk factor for dissemination and needs further evaluation.

Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis.

The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 microg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Delta9-stearoyl desaturase as the drug target. Development of a cell-free assay for Delta9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Delta9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Delta9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Delta9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.