Molecular docking, molecular dynamics simulations and binding free energy studies of interactions between Mycobacterium tuberculosis Pks13, PknG and bioactive constituents of extremophilic bacteria.

Mycobacterial pathogens present a significant challenge to disease control efforts globally due to their inherent resistance to multiple antibiotics. The rise of drug-resistant strains of Mycobacterium tuberculosis has prompted an urgent need for innovative therapeutic solutions. One promising way to discover new tuberculosis drugs is by utilizing natural products from the vast biochemical space. Multidisciplinary methods can used to harness the bioactivity of these natural products. This study aimed to evaluate the antimycobacterial efficacy of functional crude extracts from bacteria isolated from gold mine tailings in South Africa. Bacterial strains were identified using 16S rRNA sequencing. The crude extracts obtained from the bacteria were tested against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. Untargeted HPLC-qTOF and molecular networking were used to identify the functional constituents present in extracts that exhibited inhibitory activity. A virtual screening workflow (VSW) was used to filter compounds that were strong binders to Mycobacterium tuberculosis Pks13 and PknG. The ligands returned from the VSW were subjected to optimization using density functional theory (DFT) at M06-2X/6-311++ (d,p) level of theory and basis set implemented in Gaussian16 Rev.C01. The optimized ligands were re-docked against Mycobacterium tuberculosis Pks13 and PknG. Molecular dynamics simulation and molecular mechanics generalized born surface area were used to evaluate the stability of the protein-ligand complexes formed by the identified hits. The hit that showed promising binding characteristics was virtually modified through multiple synthetic routes using reaction-driven enumeration. Three bacterial isolates showed significant activity against the two strains of Mycobacterium, while only two, Bacillus subtilis and Bacillus licheniformis, exhibited activity against both Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. The tentatively identified compounds from the bacterial crude extracts belonged to various classes of natural compounds associated with antimicrobial activity. Two compounds, cyclo-(L-Pro-4-OH-L-Leu) and vazabitide A, showed strong binding against PknG and Pks13, with pre-MD MM-GBSA values of - 42.8 kcal/mol and - 47.6 kcal/mol, respectively. The DFT-optimized compounds exhibited the same docking scores as the ligands optimized using the OPSL-4 force field. After modifying vazabitide A, its affinity to the Pks13 binding site increased to - 85.8 kcal/mol, as revealed by the post-MD MM-GBSA analysis. This study highlights the potential of bacteria isolates from gold mine tailings as a source of new scaffolds for designing and optimizing anti-Mycobacterium agents. These agents synthesized in-silico can be further tested in-vitro to evaluate their efficacy.

Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7-151.6 nM against M. tuberculosis H37RvTin vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Efficacies and ADME properties of redox active methylene blue and phenoxazine analogues for use in new antimalarial triple drug combinations with amino-artemisinins.

Efforts to develop new artemisinin triple combination therapies effective against artemisinin-tolerant strains of Plasmodium falciparum based on rational combinations comprising artemisone or other amino-artemisinins, a redox active drug and a third drug with a different mode of action have now been extended to evaluation of three potential redox partners. These are the diethyl analogue AD01 of methylene blue (MB), the benzo [α]phenoxazine PhX6, and the thiosemicarbazone DpNEt. IC50 values in vitro against CQ-sensitive and resistant P. falciparum strains ranged from 11.9 nM for AD01-41.8 nM for PhX6. PhX6 possessed the most favourable pharmacokinetic (PK) profile: intrinsic clearance rate CLint was 21.47 ± 1.76 mL/min/kg, bioavailability was 60% and half-life was 7.96 h. AD01 presented weaker, but manageable pharmacokinetic properties with a rapid CLint of 74.41 ± 6.68 mL/min/kg leading to a half-life of 2.51 ± 0.07 h and bioavailability of 15%. DpNEt exhibited a half-life of 1.12 h and bioavailability of 8%, data which discourage its further examination, despite a low CLint of 10.20 mL/min/kg and a high Cmax of 6.32 µM. Efficacies of AD01 and PhX6 were enhanced synergistically when each was paired with artemisone against asexual blood stages of P. falciparum NF54 in vitro. The favourable pharmacokinetics of PhX6 indicate this is the best partner among the compounds examined thus far for artemisone. Future work will focus on extending the drug combination studies to artemiside in vitro, and conducting efficacy studies in vivo for artemisone with each of PhX6 and the related benzo[α]phenoxazine SSJ-183.

The evaluation of ADME and pharmacokinetic properties of decoquinate derivatives for the treatment of malaria.

The emergence of Plasmodium falciparum (Pf) parasite strains tolerant of the artemisinin component and resistant to the other drug component in artemisinin combination therapies (ACTs) used for treatment now markedly complicates malaria control. Thus, development of new combination therapies are urgently required. For the non-artemisinin component, the quinolone ester decoquinate (DQ) that possesses potent activities against blood stage Pf and acts on a distinct target, namely the Pf cytochrome bc 1 complex, was first considered. However, DQ has poor drug properties including high lipophilicity and exceedingly poor aqueous solubility (0.06 µg/ml), rendering it difficult to administer. Thus, DQ was chemically modified to provide the secondary amide derivative RMB005 and the quinoline O-carbamate derivatives RMB059 and RMB060. The last possesses sub-nanomolar activities against multidrug resistant blood stages of Pf, and P. berghei sporozoite liver stages. Here we present the results of ADME analyses in vitro and pharmacokinetic analyses using C57BL/6 mice. The amide RMB005 had a maximum mean whole blood concentration of 0.49 ± 0.02 µM following oral administration; however, the area under the curve (AUC), elimination half-life (t1/2) and bioavailability (BA) were not significantly better than those of DQ. Surprisingly, the quinoline O-carbamates which can be recrystallized without decomposition were rapidly converted into DQ in human plasma and blood samples. The maximum concentrations of DQ reached after oral administration of RMB059 and RMB060 were 0.23 ± 0.05 and 0.11 ± 0.01 µM, the DQ elimination half-lives were 4.79 ± 1.66 and 4.66 ± 1.16 h, and the DQ clearance were 19.40 ± 3.14 and 21.50 ± 3.38 respectively. Under these assay conditions, the BA of DQ could not be calculated Overall although RMB059 and -060 are labile in physiological medium with respect to the DQ parent, the potential to apply these as prodrugs is apparent from the current data coupled with their ease of preparation.

The Critical Importance of Spatial and Temporal Scales in Designing and Interpreting Immune Cell Migration Assays.

Intravital microscopy and other direct-imaging techniques have allowed for a characterisation of leukocyte migration that has revolutionised the field of immunology, resulting in an unprecedented understanding of the mechanisms of immune response and adaptive immunity. However, there is an assumption within the field that modern imaging techniques permit imaging parameters where the resulting cell track accurately captures a cell's motion. This notion is almost entirely untested, and the relationship between what could be observed at a given scale and the underlying cell behaviour is undefined. Insufficient spatial and temporal resolutions within migration assays can result in misrepresentation of important physiologic processes or cause subtle changes in critical cell behaviour to be missed. In this review, we contextualise how scale can affect the perceived migratory behaviour of cells, summarise the limited approaches to mitigate this effect, and establish the need for a widely implemented framework to account for scale and correct observations of cell motion. We then extend the concept of scale to new approaches that seek to bridge the current "black box" between single-cell behaviour and systemic response.

Discovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin.

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4-5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57-65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

Toward New Transmission-Blocking Combination Therapies: Pharmacokinetics of 10-Amino-Artemisinins and 11-Aza-Artemisinin and Comparison with Dihydroartemisinin and Artemether.

As artemisinin combination therapies (ACTs) are compromised by resistance, we are evaluating triple combination therapies (TACTs) comprising an amino-artemisinin, a redox drug, and a third drug with a different mode of action. Thus, here we briefly review efficacy data on artemisone, artemiside, other amino-artemisinins, and 11-aza-artemisinin and conduct absorption, distribution, and metabolism and excretion (ADME) profiling in vitro and pharmacokinetic (PK) profiling in vivo via intravenous (i.v.) and oral (p.o.) administration to mice. The sulfamide derivative has a notably long murine microsomal half-life (t1/2 > 150 min), low intrinsic liver clearance and total plasma clearance rates (CLint 189.4, CLtot 32.2 ml/min/kg), and high relative bioavailability (F = 59%). Kinetics are somewhat similar for 11-aza-artemisinin (t1/2 > 150 min, CLint = 576.9, CLtot = 75.0 ml/min/kg), although bioavailability is lower (F = 14%). In contrast, artemether is rapidly metabolized to dihydroartemisinin (DHA) (t1/2 = 17.4 min) and eliminated (CLint = 855.0, CLtot = 119.7 ml/min/kg) and has low oral bioavailability (F) of 2%. While artemisone displays low t1/2 of <10 min and high CLint of 302.1, it displays a low CLtot of 42.3 ml/min/kg and moderate bioavailability (F) of 32%. Its active metabolite M1 displays a much-improved t1/2 of >150 min and a reduced CLint of 37.4 ml/min/kg. Artemiside has t1/2 of 12.4 min, CLint of 673.9, and CLtot of 129.7 ml/kg/min, likely a reflection of its surprisingly rapid metabolism to artemisone, reported here for the first time. DHA is not formed from any amino-artemisinin. Overall, the efficacy and PK data strongly support the development of selected amino-artemisinins as components of new TACTs.

Wearable Technology May Assist in Retraining Foot Strike Patterns in Previously Injured Military Service Members: A Prospective Case Series.

A rearfoot strike (RFS) pattern with increased average vertical loading rates (AVLR) while running has been associated with injury. This study evaluated the ability of an instrumented sock, which provides real-time foot strike and cadence audio biofeedback, to transition previously injured military service members from a RFS to a non-rearfoot strike (NRFS) running pattern. Nineteen RFS runners (10 males, 9 females) were instructed to wear the instrumented socks to facilitate a change in foot strike while completing an independent walk-to-run progression and lower extremity exercise program. Kinetic data were collected during treadmill running while foot strike was determined using video analysis at initial (T1), post-intervention (T2), and follow-up (T3) data collections. Nearly all runners (18/19) transitioned to a NRFS pattern following intervention (8 ± 2.4 weeks after the initial visit). Most participants (16/18) maintained the transition at follow-up (5 ± 0.8 weeks after the post-intervention visit). AVLR of the involved and uninvolved limb decreased 29% from initial [54.7 ± 13.2 bodyweights per sec (BW/s) and 55.1 ± 12.7 BW/s] to post-intervention (38.7 ± 10.1 BW/s and 38.9 ± 10.0 BW/s), respectively. This effect persisted 5-weeks later at follow-up, representing an overall 30% reduction on the involved limb and 24% reduction on the uninvolved limb. Cadence increased from the initial to the post-intervention time-point (p = 0.045); however, this effect did not persist at follow-up (p = 0.08). With technology provided feedback from instrumented socks, approximately 90% of participants transitioned to a NRFS pattern, decreased AVLR, reduced stance time and maintained these running adaptations 5-weeks later.

A Novel Ventilator Design for COVID-19 and Resource-Limited Settings.

There has existed a severe ventilator deficit in much of the world for many years, due in part to the high cost and complexity of traditional ICU ventilators. This was highlighted and exacerbated by the emergence of the COVID-19 pandemic, during which the increase in ventilator production rapidly overran the global supply chains for components. In response, we propose a new approach to ventilator design that meets the performance requirements for COVID-19 patients, while using components that minimise interference with the existing ventilator supply chains. The majority of current ventilator designs use proportional valves and flow sensors, which remain in short supply over a year into the pandemic. In the proposed design, the core components are on-off valves. Unlike proportional valves, on-off valves are widely available, but accurate control of ventilation using on-off valves is not straightforward. Our proposed solution combines four on-off valves, a two-litre reservoir, an oxygen sensor and two pressure sensors. Benchtop testing of a prototype was performed with a commercially available flow analyser and test lungs. We investigated the accuracy and precision of the prototype using both compressed gas supplies and a portable oxygen concentrator, and demonstrated the long-term durability over 15 days. The precision and accuracy of ventilation parameters were within the ranges specified in international guidelines in all tests. A numerical model of the system was developed and validated against experimental data. The model was used to determine usable ranges of valve flow coefficients to increase supply chain flexibility. This new design provides the performance necessary for the majority of patients that require ventilation. Applications include COVID-19 as well as pneumonia, influenza, and tuberculosis, which remain major causes of mortality in low and middle income countries. The robustness, energy efficiency, ease of maintenance, price and availability of on-off valves are all advantageous over proportional valves. As a result, the proposed ventilator design will cost significantly less to manufacture and maintain than current market designs and has the potential to increase global ventilator availability.

Comparison of chest X-ray interpretation by Emergency Department clinicians and radiologists in suspected COVID-19 infection: a retrospective cohort study.

OBJECTIVES: We describe the inter-rater agreement between Emergency Department (ED) clinicians and reporting radiologists in the interpretation of chest X-rays (CXRs) in patients presenting to ED with suspected COVID-19. METHODS: We undertook a retrospective cohort study of patients with suspected COVID-19. We compared ED clinicians' and radiologists' interpretation of the CXRs according to British Society of Thoracic Imaging (BSTI) guidelines, using the area under the receiver operator curve (ROC area). RESULTS: CXRs of 152 cases with suspected COVID-19 infection were included. Sensitivity and specificity for 'classic' COVID-19 CXR findings reported by ED clinician was 84 and 83%, respectively, with a ROC area of 0.84 (95%CI 0.77 to 0.90). Accuracy improved with ED clinicians' experience, with ROC areas of 0.73 (95%CI 0.45 to 1.00), 0.81 (95%CI 0.73 to 0.89), 1.00 (95%CI 1.00 to 1.00) and 0.90 (95%CI 0.70 to 1.00) for foundation year doctors, senior house officers, higher speciality trainees and ED consultants, respectively (p < 0.001). CONCLUSIONS: ED clinicians demonstrated moderate inter-rater agreement with reporting radiologists according to the BSTI COVID-19 classifications. The improvement in accuracy with ED clinician experience suggests training of junior ED clinicians in the interpretation of COVID-19 related CXRs might be beneficial. Large-scale survey studies might be useful in the further evaluation of this topic. ADVANCES IN KNOWLEDGE: This is the first study to examine inter-rater agreement between ED clinicians and radiologists in regards to COVID-19 CXR interpretation.Further service configurations such as 24-hr hot reporting of CXRs can be guided by these data, as well as an ongoing, nationwide follow-up study.

Repetitive Head Impacts in Football Do Not Impair Dynamic Postural Control.

PURPOSE: The purpose of this study was to assess the effect of repetitive head impacts experienced by football players compared to noncontact athletes on dynamic postural control during both single-task (ST) and dual-task (DT) conditions. METHODS: Thirty-four football players wearing accelerometer instrumented helmets and 13 cheerleaders performed a dynamic postural control battery, consisting of ST and DT gait initiation, gait, and gait termination, both prior to and following the football season. A 2 (group) × 2 (time) repeated measures ANOVA compared performance across 32 dynamic postural outcomes. A linear regression was performed on postural control change scores with common head impact kinematics serving as the independent variables. RESULTS: The football players experienced a mean of 538.1 ± 409.1 head impacts in the season with a mean linear acceleration of 27.8g ± 3.2g. There were no significant interactions for any of the ST or DT dynamic postural control tasks. There was a significant relationship between head impact kinematics and the lateral center of pressure displacement during the anticipatory postural adjustment phase (r = 0.26, P = 0.010) and transitional phase (r = 0.511, P = 0.042) during ST gait initiation. For both measures, the number of impacts exceeding 98g was the only significant predictor of decreased center of pressure displacement. CONCLUSIONS: A single competitive football season did not adversely affect dynamic postural control when comparing football players to cheerleaders who do not experience repetitive head impacts. Furthermore, there were limited relationships with head impact kinematics suggesting that a single season of football does not adversely affect most outcome measures of instrumented dynamic postural control. These findings are consistent with most studies which fail to identify clinical differences related to repetitive head impacts.

Association of Optic Nerve Head Drusen with Best Vitelliform Macular Dystrophy: A Case Series.

PURPOSE: To report the association of optic nerve head (ONH) drusen with Best vitelliform macular dystrophy (BVMD). METHODS: Chart review. PATIENTS: Five patients from 3 families. RESULTS: Multimodal imaging and ophthalmic examination demonstrated findings consistent with ONH drusen, in association with BVMD, in 5 patients. CONCLUSION: We report the association of BVMD with ONH drusen in 5 patients. This combination has previously been reported only once. We recommend that patients with a diagnosis of BVMD undergo autofluorescence and ultrasound imaging of the optic nerve to help facilitate this diagnosis, as some ONH drusen can be buried.

Integrated geometric and mechanical analysis of an image-based lymphatic valve.

Lymphatic valves facilitate the lymphatic system's role in maintaining fluid homeostasis. Malformed valves are found in several forms of primary lymphœdema, resulting in incurable swelling of the tissues and immune dysfunction. Their experimental study is complicated by their small size and operation in low pressure and low Reynolds number environments. Mathematical models of these structures can give insight and complement experimentation. In this work, we present the first valve geometry reconstructed from confocal imagery and used in the construction of a subject-specific model in a closing mode. A framework is proposed whereby an image is converted into a valve model. An FEA study was performed to identify the significance of the shear modulus, the consequences of smoothing the leaflet surface and the effect of wall motion on valve behaviour. Smoothing is inherent to any analysis from imagery. The nature of the image, segmentation and meshing all cause attenuation of high-frequency features. Smoothing not only causes loss of surface area but also the loss of high-frequency geometric features which may reduce stiffness. This work aimed to consider these effects and inform studies by taking a manual reconstruction and through manifold harmonic analysis, attenuating higher frequency features to replicate lower resolution images or lower degree-of-freedom reconstructions. In conclusion, two metrics were considered: trans-valvular pressure required to close the valve, ΔPc, and the retrograde volume displacement after closure. The higher ΔPc, the greater the volume of lymph that will pass through the valve during closure. Retrograde volume displacement after closure gives a metric of compliance of the valve and for the quality of the valve seal. In the case of the image-specific reconstructed valve, removing features with a wavelength longer than four µm caused changes in ΔPc. Varying the shear modulus from 10 kPa to 60 kPa caused a 3.85-fold increase in the retrograde volume displaced. The inclusion of a non-rigid wall caused ΔPc to increase from 1.56 to 2.52 cmH2O.

C6 Spinous Process Fracture in a Young Adult.

A 21-year-old male military academy cadet developed acute posterior neck pain after performing multiple sets of overhead shoulder presses and resting the bar on his lower neck and shoulders. He presented to a direct-access physical therapy clinic. Due to the acute onset and focal nature of pain in the setting of repetitive weightlifting, the physical therapist ordered radiographs of the cervical spine, which revealed a C6 spinous process fracture. J Orthop Sports Phys Ther 2017;47(8):578. doi:10.2519/jospt.2017.7277.

The effect of minimalist footwear and instruction on running: an observational study.

BACKGROUND: It is not known whether the effects on altered running style which are attributed to minimalist footwear can be achieved by verbal instructions in standard running shoes (SRS). AIM: To explore the effect of Vibram FiveFingers (VFF) versus SRS plus running instruction on lower extremity spatiotemporal parameters and lower limb joint kinematics. METHODS: 35 healthy subjects (mean=30 years, 18 females) were assessed on two occasions with 3D motion analysis. At each session subjects ran on a treadmill (3.58 m/s) for 2 min in either VFF or SRS (randomised order); with and without running instruction. Differences between spatiotemporal parameters and lower limb joint kinematics between conditions were assessed using a 2x2 repeated-measures ANOVA. RESULTS: Wearing VFF significantly increased cadence (p<0.001) and reduced stride length (p<0.01). Prior to initial contact, both instruction and VFF significantly increased foot (p<0.001 and p=0.02, respectively) and ankle (p<0.001 and p=0.02, respectively) plantarflexion, while wearing VFF significantly increased knee extension (p=0.04). At initial contact, instruction significantly increased knee flexion (p=0.04), and foot (p=0.001) and ankle (p=0.03) plantarflexion. At mid-stance and toe-off, instruction significantly increased knee flexion (p=0.048 and p<0.001, respectively) and foot plantarflexion (p<0.001 and p=0.01, respectively). Instruction had a greater effect on increasing knee flexion (p=0.007) and plantarflexion angle (p<0.001) when subjects wore SRS and VFF, respectively. CONCLUSION: Alterations in spatiotemporal parameters observed when running in VFF are likely to be attributable to the minimalist footwear. However, the kinematic adaptations observed following instruction suggests that changes in joint angles previously attributed to minimalist footwear alone may be similarly achieved with instruction.

Femoral neck stress fracture in a military trainee.

The patient was a 21-year-old female who was currently enrolled in a military security forces training program. She had a 1-month history of worsening left anterior hip pain that was insidious in nature and limiting her ability to run. The patient was diagnosed as having a left hip strain, prescribed a nonsteroidal anti-inflammatory medication,given a reduced activity waiver, and referred to a physical therapist. Despite previous radiographs of the pelvis and hips that were interpreted as normal, the history and physical examination findings led the physical therapist to be concerned about the presence of a possible femoral neck stress fracture. Immediate magnetic resonance imaging of the left hip was obtained, which revealed a stress fracture of the basicervical portion of the left femoral neck. The patient was referred to an orthopaedic surgeon for an expedited consultation and underwent open reduction internal fixation of the left hip later that day. After a period of convalescence and completion of a comprehensive rehabilitation program, the patient successfully returned to full military duty without limitations.