Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

Peptidyl tRNA Hydrolase Is Required for Robust Prolyl-tRNA Turnover in Mycobacterium tuberculosis.

Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacteria. We developed a sequencing-based strategy called copper sulfate-based tRNA sequencing (Cu-tRNAseq) to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtb's susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB. IMPORTANCE Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why. We have used genetic and novel biochemical approaches to show that when Pth levels decline in Mtb, peptidyl tRNA accumulates to such an extent that usable tRNA pools drop. Thus, Pth is needed to maintain normal tRNA levels, most strikingly for prolyl-tRNAs. Many antibiotics act on protein synthesis and could be affected by altering the availability of tRNA. This is certainly true for tRNA synthetase inhibitors, several of which are drug candidates for tuberculosis. We find that their action is potentiated by Pth depletion. Furthermore, Pth depletion results in hypersensitivity to macrolides, drugs that are not active enough under ordinary circumstances to be useful for tuberculosis.

Effects of intensity and training on cardiovascular responses to the Valsalva maneouvre.

OBJECTIVES: To establish the effect of intensity of effort during Valsalva's manoeuvre (VM) on blood pressure and heart rate responses. METHODS: Fourteen participants consisting of eight powerlifters and six controls completed four 15-s VMs at 30 and 50% maximum expiratory pressure. Expiratory pressure (EP), systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were recorded continuously during manoeuvres and response amplitudes were related to well-established phases (I-IV). RESULTS: For all participants, SBP and DBP responses during Phase I were significantly higher at 50% EPmax than 30% EPmax. Phase II responses were significantly higher at 50% EPmax than 30% EPmax for HR (39.9 ± 24.3 vs. 24.5 ± 12.5 bpm), SBP (43.6 ± 24.6 vs. 35.3 ± 18.3 mm Hg) and DBP (26.7 ± 7.1 vs. 21.1 ± 7.4 mm Hg). For Phase IV, only the HR response was affected significantly by intensity. These intensity-dependent effects were similar between powerlifters and controls. Powerlifters had significantly greater Phase II responses (HR, SBP and DBP), but in the whole group these cardiovascular responses were also significantly correlated with expiratory pressure (mm Hg) at 30% EPmax (r=0.59-0.84) and 50% EPmax (r=0.53-0.82) which was higher in powerlifters than controls. CONCLUSIONS: This study demonstrates that heart rate and blood pressure responses during Phase II are affected by the relative intensity of VM, whereas during Phase IV only heart rate is affected. The higher Phase II responses in powerlifters can be attributed to the higher expiratory pressures they developed.

Priming exercise accelerates oxygen uptake kinetics during high-intensity cycle exercise in middle-aged individuals with type 2 diabetes.

Background: The primary phase time constant of pulmonary oxygen uptake kinetics ( V · O 2 τ p) during submaximal efforts is longer in middle-aged people with type 2 diabetes (T2D), partly due to limitations in oxygen supply to active muscles. This study examined if a high-intensity "priming" exercise (PE) would speed V · O 2 τ p during a subsequent high-intensity cycling exercise in T2D due to enhanced oxygen delivery. Methods: Eleven (4 women) middle-aged individuals with type 2 diabetes and 11 (4 women) non-diabetic controls completed four separate cycling bouts each starting at an 'unloaded' baseline of 10 W and transitioning to a high-intensity constant-load. Two of the four cycling bouts were preceded by priming exercise. The dynamics of pulmonary V · O 2 and muscle deoxygenation (i.e. deoxygenated haemoglobin and myoglobin concentration [HHb + Mb]), were calculated from breath-by-breath and near-infrared spectroscopy data at the vastus lateralis, respectively. Results: At baseline V · O 2 τ p, was slower (p < 0.001) in the type 2 diabetes group (48 ± 6 s) compared to the control group (34 ± 2 s) but priming exercise significantly reduced V · O 2 τ p (p < 0.001) in type 2 diabetes (32 ± 6 s) so that post priming exercise it was not different compared with controls (34 ± 3 s). Priming exercise reduced the amplitude of the V · O 2 slow component (As) in both groups (type 2 diabetes: 0.26 ± 0.11 to 0.16 ± 0.07 L/min; control: 0.33 ± 0.13 to 0.25 ± 0.14 L/min, p < 0.001), while [HHb + Mb] kinetics remained unchanged. Conclusion: These results suggest that in middle-aged men and women with T2D, PE speeds V · O 2 τ p likely by a better matching of O2 delivery to utilisation and reduces the V · O 2 As during a subsequent high-intensity exercise.

Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

Low-volume HIIT and MICT speed V̇o2 kinetics during high-intensity "work-to-work" cycling with a similar time-course in type 2 diabetes.

We assessed the rates of adjustment in oxygen uptake (V̇o2) and muscle deoxygenation [i.e., deoxygenated hemoglobin and myoglobin, (HHb + Mb)] during the on-transition to high-intensity cycling initiated from an elevated baseline (work-to-work, w-to-w) before training and at weeks 3, 6, 9, and 12 of low-volume high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) in type 2 diabetes (T2D). Participants were randomly assigned to MICT (n = 11, 50 min of moderate-intensity cycling), HIIT (n = 8, 10 × 1 min of high-intensity cycling separated by 1 min of light cycling) or nonexercising control (n = 9) groups. Exercising groups trained three times per week. Participants completed two w-to-w transitions at each time point consisting of sequential step increments to moderate- and high-intensity work-rates. [HHb + Mb] kinetics were measured by near-infrared spectroscopy at the vastus lateralis muscle. The pretraining time constant of the primary phase of V̇o2 (V̇o2 τp) and the amplitude of the V̇o2 slow component (V̇o2As) of the high-intensity w-to-w bout decreased (P < 0.05) by a similar magnitude at week 3 of training in both MICT (from 56 ± 9 to 43 ± 6 s, and from 0.17 ± 0.07 to 0.09 ± 0.05 L/min, respectively) and HIIT (from 56 ± 8 to 42 ± 6 s, and from 0.18 ± 0.05 to 0.09 ± 0.08 L/min, respectively) with no further changes thereafter. No changes were reported in controls. The parameter estimates of Δ[HHb + Mb] remained unchanged in all groups. MICT and HIIT elicited comparable improvements in V̇o2 kinetics without changes in muscle deoxygenation kinetics during high-intensity exercise initiated from an elevated baseline in T2D despite training volume and time commitment being ∼50% lower in the HIIT group.NEW & NOTEWORTHY Three weeks of high-intensity interval training and moderate-intensity continuous training decreased the time constant of the primary phase of oxygen uptake (V̇o2) and amplitude of the V̇o2 slow component during a high-intensity exercise initiated from an elevated baseline, a protocol that mimics the abrupt metabolic transitions akin to those in daily life, in type 2 diabetes. These V̇o2 kinetics improvements were maintained until the end of the 12-wk intervention without changes in muscle deoxygenation kinetics.

Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα, and cell-cycle signaling in a breast cancer model.

Aim: A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation. Methods: The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17ß-estradiol (E2)-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9). Results: In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis. Conclusions: Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.

Differential effects of sex on adaptive responses of skeletal muscle vasodilation to exercise training in type 2 diabetes.

AIMS: We tested the hypotheses that exercise training improves the peak and dynamic responses of leg vascular conductance (LVC) in males and females with type 2 diabetes (T2DM). METHODS: Forty-one males and females with T2DM were assigned to two training groups and two control groups. Twelve weeks of aerobic/resistance training was performed three times per week, 60-90 min per session. Responses of calf muscle blood flow and systemic arterial pressure during incremental and constant-load (30% maximal voluntary contraction) intermittent plantar-flexion protocols in the supine position were recorded. RESULTS: Training significantly increased peak LVC in males (4.86 ± 1.88 to 6.06 ± 2.06 ml·min-1·mm Hg-1) and females (3.91 ± 1.13 to 5.40 ± 1.38 ml·min-1·mm Hg-1) with no changes in control groups. For dynamic responses, training significantly increased the amplitude of the fast growth phase of LVC (1.81 ± 1.12 to 2.68 ± 1.01 ml·min-1·mm Hg-1) and decreased the time constant of the slow growth phase (43.6 ± 46.4 s to 16.1 14.0 s) in females, but no improvements were observed in control females or in any of the two male groups. CONCLUSIONS: These data suggest that training increases the peak vasodilatory response in males and females, whereas the speed of the dynamic response of vasodilation is improved in females but not males.

Optimization of TAM16, a Benzofuran That Inhibits the Thioesterase Activity of Pks13; Evaluation toward a Preclinical Candidate for a Novel Antituberculosis Clinical Target.

With increasing drug resistance in tuberculosis (TB) patient populations, there is an urgent need for new drugs. Ideally, new agents should work through novel targets so that they are unencumbered by preexisting clinical resistance to current treatments. Benzofuran 1 was identified as a potential lead for TB inhibiting a novel target, the thioesterase domain of Pks13. Although, having promising activity against Mycobacterium tuberculosis, its main liability was inhibition of the hERG cardiac ion channel. This article describes the optimization of the series toward a preclinical candidate. Despite improvements in the hERG liability in vitro, when new compounds were assessed in ex vivo cardiotoxicity models, they still induced cardiac irregularities. Further series development was stopped because of concerns around an insufficient safety window. However, the demonstration of in vivo activity for multiple series members further validates Pks13 as an attractive novel target for antitubercular drugs and supports development of alternative chemotypes.

The Role of Central Command in the Increase in Muscle Sympathetic Nerve Activity to Contracting Muscle During High Intensity Isometric Exercise.

We previously demonstrated that muscle sympathetic nerve activity (MSNA) increases to contracting muscle as well as to non-contracting muscle, but this was only assessed during isometric exercise at ∼10% of maximum voluntary contraction (MVC). Given that high-intensity isometric contractions will release more metabolites, we tested the hypothesis that the metaboreflex is expressed in the contracting muscle during high-intensity but not low-intensity exercise. MSNA was recorded continuously via a tungsten microelectrode inserted percutaneously into the right common peroneal nerve in 12 participants, performing isometric dorsiflexion of the right ankle at 10, 20, 30, 40, and 50% MVC for 2 min. Contractions were immediately followed by 6 min of post-exercise ischemia (PEI); 6 min of recovery separated contractions. Cross-correlation analysis was performed between the negative-going sympathetic spikes of the raw neurogram and the ECG. MSNA increased as contraction intensity increased, reaching mean values (± SD) of 207 ± 210 spikes/min at 10% MVC (P = 0.04), 270 ± 189 spikes/min at 20% MVC (P < 0.01), 538 ± 329 spikes/min at 30% MVC (P < 0.01), 816 ± 551 spikes/min at 40% MVC (P < 0.01), and 1,097 ± 782 spikes/min at 50% MVC (P < 0.01). Mean arterial pressure also increased in an intensity-dependent manner from 76 ± 3 mmHg at rest to 90 ± 6 mmHg (P < 0.01) during contractions of 50% MVC. At all contraction intensities, blood pressure remained elevated during PEI, but MSNA returned to pre-contraction levels, indicating that the metaboreflex does not contribute to the increase in MSNA to contracting muscle even at these high contraction intensities.

UV Hyperspectral Imaging as Process Analytical Tool for the Characterization of Oxide Layers and Copper States on Direct Bonded Copper.

Hyperspectral imaging and reflectance spectroscopy in the range from 200-380 nm were used to rapidly detect and characterize copper oxidation states and their layer thicknesses on direct bonded copper in a non-destructive way. Single-point UV reflectance spectroscopy, as a well-established method, was utilized to compare the quality of the hyperspectral imaging results. For the laterally resolved measurements of the copper surfaces an UV hyperspectral imaging setup based on a pushbroom imager was used. Six different types of direct bonded copper were studied. Each type had a different oxide layer thickness and was analyzed by depth profiling using X-ray photoelectron spectroscopy. In total, 28 samples were measured to develop multivariate models to characterize and predict the oxide layer thicknesses. The principal component analysis models (PCA) enabled a general differentiation between the sample types on the first two PCs with 100.0% and 96% explained variance for UV spectroscopy and hyperspectral imaging, respectively. Partial least squares regression (PLS-R) models showed reliable performance with R2c = 0.94 and 0.94 and RMSEC = 1.64 nm and 1.76 nm, respectively. The developed in-line prototype system combined with multivariate data modeling shows high potential for further development of this technique towards real large-scale processes.

Use of Hyperspectral Imaging for the Quantification of Organic Contaminants on Copper Surfaces for Electronic Applications.

To correctly assess the cleanliness of technical surfaces in a production process, corresponding online monitoring systems must provide sufficient data. A promising method for fast, large-area, and non-contact monitoring is hyperspectral imaging (HSI), which was used in this paper for the detection and quantification of organic surface contaminations. Depending on the cleaning parameter constellation, different levels of organic residues remained on the surface. Afterwards, the cleanliness was determined by the carbon content in the atom percent on the sample surfaces, characterized by XPS and AES. The HSI data and the XPS measurements were correlated, using machine learning methods, to generate a predictive model for the carbon content of the surface. The regression algorithms elastic net, random forest regression, and support vector machine regression were used. Overall, the developed method was able to quantify organic contaminations on technical surfaces. The best regression model found was a random forest model, which achieved an R2 of 0.7 and an RMSE of 7.65 At.-% C. Due to the easy-to-use measurement and the fast evaluation by machine learning, the method seems suitable for an online monitoring system. However, the results also show that further experiments are necessary to improve the quality of the prediction models.

Stress and Learning in Pupils: Neuroscience Evidence and its Relevance for Teachers.

Our understanding of how stress affects primary school children's attention and learning has developed rapidly. We know that children experience differing levels of stressors (factors that cause stress) in their environments, and that this can influence how they respond to new stressors when they occur in educational contexts. Here, we review evidence showing that stress can increase children's attention and learning capacities in some circumstances but hinder them in others. We show how children differ in their attention and learning styles, dependent on stress levels: for example, more highly stressed children may be more distracted by superficial features and may find it harder to engage in planning and voluntary control. We review intervention research on stress management techniques in children, concentrating on psychological techniques (such as mindfulness and stress reappraisal), physiological techniques (such as breathing exercises) and environmental factors (such as reducing noise). At the current time, raising teachers' awareness of pupils' differing stress responses will be an important step in accommodating the differing needs of children in their classrooms.

Randomised controlled trial comparing immobilisation in above-knee plaster of Paris to controlled ankle motion boots in undisplaced paediatric spiral tibial fractures.

BACKGROUND: Management of common childhood spiral tibial fractures, known as toddler's fractures, has not significantly changed in recent times despite the availability of immobilisation devices known as controlled ankle motion (CAM) boots. We compared standard therapy with these devices on quality-of-life measures. METHODS: A prospective randomised controlled trial, comparing immobilisation with an above-knee plaster of Paris cast (AK-POP) with a CAM boot in children aged 1-5 years with proven or suspected toddler's fractures presenting to a tertiary paediatric ED in Perth, Western Australia, between March 2018 and February 2020. The primary outcome measure was ease of personal care, as assessed by a Care and Comfort Questionnaire (eight questions scored from 0, very easy, to 8, impossible) completed by the caregiver and assessed during three treatment time-points and preintervention and postintervention. Secondary outcome measures included weight-bearing status as well as complications of fracture healing and number of pressure injuries. RESULTS: 87 patients were randomised (44 CAM boot, median age 2 (IQR 1.5-2.3), 71% male; 43 AK-POP, median age 2 (IQR 1.7-2.8), 80% male), a significant difference in the care and comfort score was demonstrated at all treatment time-points; with the AK-POP group reporting greater personal care needs on assessment on day 2, day 7-10 and 4-week review (all p≤0.001). Weight-bearing status was significantly different at day 7-10 (77.5% CAM vs 53.8% AK-POP, p=0.027). There was no difference in fracture healing or pressure areas between the two treatment groups. CONCLUSIONS: Immobilisation of toddler's fractures in a CAM boot allows faster return to activities of daily living and weight-bearing without any effect on fracture healing. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry (ACTRN12618001311246).

Targeting Mycobacterium tuberculosis CoaBC through Chemical Inhibition of 4'-Phosphopantothenoyl-l-cysteine Synthetase (CoaB) Activity.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

Time-course of V̇o2 kinetics responses during moderate-intensity exercise subsequent to HIIT versus moderate-intensity continuous training in type 2 diabetes.

We assessed the time-course of changes in oxygen uptake (V̇o2) and muscle deoxygenation (i.e., deoxygenated hemoglobin and myoglobin, [HHb + Mb]) kinetics during transitions to moderate-intensity cycling following 12 wk of low-volume high-intensity interval training (HIIT) vs. moderate-intensity continuous training (MICT) in adults with type 2 diabetes (T2D). Participants were randomly assigned to MICT (n = 10, 50 min of moderate-intensity cycling), HIIT (n = 9, 10 × 1 min at ∼90% maximal heart rate), or nonexercising control (n = 9) groups. Exercising groups trained three times per week, and measurements were taken every 3 wk. [HHb + Mb] kinetics were measured by near-infrared spectroscopy at the vastus lateralis muscle. The local matching of O2 delivery to O2 utilization was assessed by the Δ[HHb + Mb]/ΔV̇o2 ratio. The pretraining time constant of the primary phase of V̇o2 (τV̇o2p) decreased (P < 0.05) at wk 3 of training in both MICT (from 44 ± 12 to 32 ± 5 s) and HIIT (from 42 ± 8 to 32 ± 4 s) with no further changes thereafter, whereas no changes were reported in controls. The pretraining overall dynamic response of muscle deoxygenation (τ'[HHb + Mb]) was faster than τV̇o2p in all groups, resulting in Δ[HHb + Mb]/V̇o2p showing a transient "overshoot" relative to the subsequent steady-state level. After 3 wk, the Δ[HHb + Mb]/V̇o2p overshoot was eliminated only in the training groups, so that τ'[HHb + Mb] was not different to τV̇o2p in MICT and HIIT. The enhanced V̇o2 kinetics response consequent to both MICT and HIIT in T2D was likely attributed to a training-induced improvement in matching of O2 delivery to utilization.NEW & NOTEWORTHY High-intensity interval training and moderate-intensity continuous training elicited faster pulmonary oxygen uptake (V̇o2) kinetics during moderate-intensity cycling within 3 wk of training with no further changes thereafter in individuals with type 2 diabetes. These adaptations were accompanied by unaltered near-infrared spectroscopy-derived muscle deoxygenation (i.e. deoxygenated hemoglobin and myoglobin concentration, [HHb+Mb]) kinetics and transiently reduced Δ[HHb+Mb]-to-ΔV̇o2 ratio, suggesting an enhanced blood flow distribution within the active muscles subsequent to both training interventions.

Resistance of Mycobacterium tuberculosis to indole 4-carboxamides occurs through alterations in drug metabolism and tryptophan biosynthesis.

Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.

Oxygen and pulmonary arterial hypertension: effects, mechanisms, and therapeutic benefits.

Oxygen is a pulmonary vasodilator. Although treatment of pulmonary arterial hypertension (PAH) is focused on pulmonary vasodilation, treatment guidelines do not recommend O2 therapy for patients unless they develop hypoxaemia. These guidelines point to a lack of evidence of benefit of O2 therapy from randomized controlled trials (RCTs) and to evidence of lack of benefit in a single RCT involving patients with Eisenmenger syndrome. These guidelines did not identify major limitations with the Eisenmenger study or consider other evidence of therapeutic benefit. Recent advances in mechanistic understanding of O2 effects on pulmonary vascular tone, along with substantial evidence of acute effects of O2 in PAH patients, challenge the view that benefits of O2 arise only through correction of hypoxaemia. Evidence presented in this review shows that O2 acts as a pulmonary vasodilator in patients who are normoxaemic; that this probably involves an alveolar mechanism in addition to a blood-borne (oxyhaemoglobin) mechanism; and that therapeutic benefit of O2 does not depend on arterial O2 levels. This suggests that O2 has potential therapeutic benefit for all patients with PAH. Clinical guidelines and practice related to O2 therapy need to be reassessed, and further research is needed.

Time course of changes in V̇o2peak and O2 extraction during ramp cycle exercise following HIIT versus moderate-intensity continuous training in type 2 diabetes.

In the present study, we assessed the time course of adaptations in peak oxygen uptake (V̇o2peak) and muscle fractional oxygen (O2) extraction (using near-infrared spectroscopy) following 12 wk of low-volume high-intensity interval training (HIIT) versus moderate-intensity continuous endurance training (MICT) in adults with uncomplicated type 2 diabetes (T2D). Participants with T2D were randomly assigned to MICT (n = 12, 50 min of moderate-intensity cycling) or HIIT (n = 9, 10 × 1 min at ∼90% maximal heart rate) or to a nonexercising control group (n = 9). Exercising groups trained three times per week and measurements were taken every 3 wk. The rate of muscle deoxygenation (i.e., deoxygenated hemoglobin and myoglobin concentration, Δ[HHb + Mb]) profiles of the vastus lateralis muscle were normalized to 100% of the response, plotted against % power output (PO), and fitted with a double linear regression model. V̇o2peak increased (P < 0.05) by week 3 of MICT (+17%) and HIIT (+8%), with no further significant changes thereafter. Total increases in V̇o2peak posttraining (P < 0.05) were 27% and 14%, respectively. The %Δ[HHb + Mb] versus %PO slope of the first linear segment (slope1) was reduced (P < 0.05) beyond 3 wk of HIIT and MICT, with no further significant changes thereafter. No changes in V̇o2peak or slope1 were observed in the control group. Low-volume HIIT and MICT induced improvements in V̇o2peak following a similar time course, and these improvements were likely, at least in part, due to an improved microvascular O2 delivery.

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.