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A Papi,1 D Dokic,2 W Tzimas,3 I Mészáros,4 A Olech-Cudzik,5 Z Koroknai,6 K McAulay,7 S Mersmann,8 PS Dalvi,9 T Overend9 1Department of Internal and CardioRespiratory Medicine, Reseach Center on Asthma and COPD, University of Ferrara, Ferrara, Italy; 2Clinic of Pulmology and Allergy, Clinical Centre, Medical Faculty, Ss. Cyril and Methodius University, Skopje, Macedonia; 3Pneumologische Praxis, München, Germany; 4Coral Szakorvosi Centrum, Budapest, Hungary; 5Ostrowieckie Centrum Medyczne Spólka, Ostrowiec Swietokrzyski, Poland; 6PAREXEL International, Global Medical Services, Budapest, Hungary; 7Medical Operations, Mundipharma Research Limited, Cambridge, UK; 8Biostatistics and Clinical Data Science, Mundipharma Research GmbH & Co. KG, Limburg, Germany; 9Medical Science - Respiratory, Mundipharma Research Limited, Cambridge, UK Purpose: To evaluate fluticasone propionate/formoterol (FP/FORM) in COPD. Patients and methods: COPD patients with forced expiratory volume in 1 s (FEV1) ≤50% predicted and ≥1 moderate/severe COPD exacerbation in the last 12 months were randomized to FP/FORM 500/20 or 250/10 µg bid, or formoterol (FORM) 12 µg bid for 52 weeks. The primary outcome was the annualized rate of moderate/severe COPD exacerbations. Results: In total, 1,765 patients were randomized. There were fewer discontinuations with FP/FORM 500/20 µg (20.6%) and 250/10 µg (24.0%) compared with FORM (26.1%). None of the two FP/FORM doses reduced the moderate/severe exacerbation rate versus FORM (rate ratios [RR]: 0.93; P≤0.402). There was a trend toward a lower moderate/severe exacerbation rate with FP/FORM 500/20 µg versus FORM in patients with ≤2 exacerbations in the preceding year (RR: 0.79; P=0.084). Pre- and post-dose FEV1 and forced vital capacity were greater with FP/FORM 500/20 µg versus FORM (P≤0.039). There was a trend toward a lower EXAcerbations of Chronic pulmonary disease Tool (EXACT) exacerbation rate with FP/FORM 500/20 µg versus FORM (RR: 0.87; P=0.077). There were more St George’s Respiratory Questionnaire for COPD (SGRQ-C) responders with FP/FORM 500/20 µg than FORM (odds ratios [OR] at weeks 6, 23 and 52 ≥1.28; P≤0.054). EXACT-respiratory symptoms total and breathlessness scores were lower with both FP/FORM 500/20 µg and 250/10 µg versus FORM (P≤0.066). Acute β2-agonist-induced effects and 24-hour Holter findings were similar for all treatments. Mean 24-hour urinary cortisol was similarly reduced with both FP/FORM doses. Radiologically confirmed pneumonia was seen in 2.4%, 3.2% and 1.5% of FP/FORM 500/20 µg, FP/FORM 250/10 µg and FORM-treated patients, respectively. Adverse events were otherwise similar across treatment groups. Conclusion: FP/FORM did not reduce exacerbation rates versus FORM. Numerical benefits were observed with FP/FORM 500/20 µg versus FORM for secondary variables, including lung function, EXACT exacerbations, SGRQ-C and EXACT-respiratory symptoms total and breathlessness scores. Few efficacy differences were evident between FP/FORM 250/10 µg and FORM. Pneumonia was more frequent in FP/FORM-treated patients, although the absolute difference was low. Adverse events were otherwise similar between treatments. Keywords: flutiform, chronic bronchitis, emphysema, exacerbations, eosinophils

Alberto Papi,1 Paul W Jones,2 Prashant S Dalvi,3 Kirsten McAulay,4 Tammy McIver,5 Sanjeeva Dissanayake3 1Department of Internal and CardioRespiratory Medicine, Research Centre on Asthma and COPD, University of Ferrara, Ferrara, Italy; 2Institute for Infection and Immunity, St George’s, University of London, London, UK; 3Medical Science – Respiratory, 4Medical Operations, 5Data Management and Statistics, Mundipharma Research Ltd, Cambridge, UK Abstract: Inhaled corticosteroid/long-acting β2-agonist combination therapy is recommended in chronic obstructive pulmonary disease (COPD) patients at high risk of exacerbations. The EFFECT (Efficacy of Fluticasone propionate/FormotErol in COPD Treatment) trial is a Phase III, 52-week, randomized, double-blind study to evaluate the efficacy and safety of two doses of fluticasone propionate/formoterol compared to formoterol monotherapy in COPD patients with FEV1 ≤50% predicted and a history of exacerbations. The primary endpoint is the annualized rate of moderate and severe exacerbations. Secondary endpoints include pre-dose FEV1, EXACT-PRO (EXAcerbations of Chronic pulmonary disease Tool – Patient-Reported Outcome)-defined exacerbations, St George’s Respiratory Questionnaire for COPD, COPD Assessment Test, and EXACT-Respiratory Symptoms total score. Lung-specific biomarkers (surfactant protein D and CC chemokine ligand-18) will be measured in a subset of patients to explore their relationship to other clinical indices in COPD and their predictive utility. Pneumonia will be diagnosed per criteria defined by the British Thoracic Society community acquired pneumonia guideline, primarily by radiological confirmation and, additionally, using clinical criteria when a chest radiograph cannot be obtained. Serial measurements of serum potassium, vital signs and electrocardiograms, 24-hour Holter monitoring, and 24-hour urinary cortisol measurement will be performed in a subset of patients in addition to conventional safety assessments. Keywords: chronic obstructive pulmonary disease, flutiform, inhaled corticosteroids, long-acting β2-agonist

This study was conducted in treatment-naive adults with drug-susceptible pulmonary tuberculosis in Port-au-Prince, Haiti, to assess the safety, bactericidal activity, and pharmacokinetics of nitazoxanide (NTZ). This was a prospective phase II clinical trial in 30 adults with pulmonary tuberculosis. Twenty participants received 1 g of NTZ orally twice daily for 14 days. A control group of 10 participants received standard therapy over 14 days. The primary outcome was the change in time to culture positivity (TTP) in an automated liquid culture system. The most common adverse events seen in the NTZ group were gastrointestinal complaints and headache. The mean change in TTP in sputum over 14 days in the NTZ group was 3.2 h ± 22.6 h and was not statistically significant (P = 0.56). The mean change in TTP in the standard therapy group was significantly increased, at 134 h ± 45.2 h (P

Abstract Background The inhaled corticosteroid (ICS) fluticasone propionate (fluticasone) and the long-acting β2-agonist (LABA) formoterol fumarate (formoterol) are being made available as a combination product (fluticasone/formoterol, flutiform®) in a single aerosol inhaler. This 12-week, open-label, randomized, active-controlled, parallel-group, multicentre, phase 3 study compared the efficacy and safety of fluticasone/formoterol with the commercially available combination product fluticasone/salmeterol. Methods Patients aged ≥ 18 years (N = 202) with mild-to-moderate–severe, persistent asthma for ≥ 6 months prior to screening were included in the study. After a screening phase (4–10 days), eligible patients were randomized 1:1 to receive fluticasone/formoterol or fluticasone/salmeterol during the 12-week treatment period. The primary objective was to demonstrate non-inferiority of fluticasone/formoterol versus fluticasone/salmeterol, measured by pre-dose forced expiratory volume in the first second (FEV1), at week 12. Results Fluticasone/formoterol was comparable to fluticasone/salmeterol for the primary efficacy endpoint, mean pre-dose FEV1 at week 12. The new combination was also comparable to fluticasone/salmeterol for change from baseline to week 12 in pre-dose FEV1, change from pre-dose FEV1 at baseline to 2-hour post-dose FEV1 at week 12 and discontinuations due to lack of efficacy. Importantly, fluticasone/formoterol was superior to fluticasone/salmeterol in time to onset of action throughout the duration of the study. The two treatments demonstrated similar results for various other secondary efficacy parameters, including other lung function tests, patient-reported outcomes, rescue medication use, asthma exacerbations and Asthma Quality of Life Questionnaire scores. Fluticasone/formoterol was well tolerated and had a good safety profile that was similar to fluticasone/salmeterol. Conclusions The results of this study indicate that fluticasone/formoterol is as effective as fluticasone/salmeterol, and has a more rapid onset of action, reflecting the faster bronchodilatory effects of formoterol compared with those of salmeterol. If patients perceive the benefits of therapy with fluticasone/formoterol more rapidly than with fluticasone/salmeterol, this could have a positive impact on preference and adherence. Trial Registration ClinicalTrials.gov: NCT00476073

Competing Interests: Competing interests: The authors declare no competing interests.

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

We examined the correlation between three different methods of Mycobacterium tuberculosis quantification: time to positivity (TTP), log 10 CFU, and an assay to detect differentially detectable M. tuberculosis (DD Mtb) from three different prospective studies. Participants with DD Mtb have significantly more variation in the CFU/TTP correlation than participants with no DD Mtb ( P < 0.001). This may impact the design of early bactericidal activity studies that use TTP as the primary outcome., Competing Interests: The authors declare no conflict of interest.

As the focus tilts toward online detection methodologies for transformer oil aging, bypassing challenges associated with traditional offline methods, such as sample contamination and misinterpretation, fiber optic sensors are gaining traction due to their compact nature, cost-effectiveness, and resilience to electromagnetic disturbances that are typical in high-voltage environments. This study delves into the sensitivity analysis of intensity-modulated plastic optical fiber sensors. The investigation encompasses key determinants such as the influence of optical source wavelengths, noise response dynamics, ramifications of varying sensing lengths, and repeatability assessments. Our findings highlight that elongating sensing length detrimentally affects both linearity response and repeatability, largely attributed to a diminished resistance to noise. Additionally, the choice of the optical source wavelength proved to be a critical variable in assessing sensor sensitivity.

Eradicating HIV/AIDS by 2030 is a central goal of the World Health Organization. Patient adherence to complicated dosage regimens remains a key barrier. There is a need for convenient long-acting formulations that deliver drugs over sustained periods. This paper presents an alternative platform, an injectable in situ forming hydrogel implant to deliver a model antiretroviral drug (zidovudine [AZT]) over 28 days. The formulation is a self-assembling ultrashort d or l-α peptide hydrogelator, namely phosphorylated (naphthalene-2-ly)-acetyl-diphenylalanine-lysine-tyrosine-OH (NapFFKY[p]-OH), covalently conjugated to zidovudine via an ester linkage. Rheological analysis demonstrates phosphatase enzyme instructed self-assembly, with hydrogels forming within minutes. Small angle neutron scattering data suggest hydrogels form narrow radius (≈2 nm), large length fibers closely fitting the flexible cylinder elliptical model. d-Peptides are particularly promising for long-acting delivery, displaying protease resistance for 28 days. Drug release, via hydrolysis of the ester linkage, progress under physiological conditions (37 °C, pH 7.4, H 2 O). Subcutaneous administration of Napffk(AZT)Y[p]G-OH in Sprague Dawley rats demonstrate zidovudine blood plasma concentrations within the half maximal inhibitory concentration (IC 50 ) range (30-130 ng mL -1 ) for 35 days. This work is a proof-of-concept for the development of a long-acting combined injectable in situ forming peptide hydrogel implant. These products are imperative given their potential impact on society., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Background: Point-of-care diagnostic devices, such as lateral-flow assays, are becoming widely used by the public. However, efforts to ensure correct assay operation and result interpretation rely on hardware that cannot be easily scaled or image processing approaches requiring large training datasets, necessitating large numbers of tests and expert labeling with validated specimens for every new test kit format., Methods: We developed a software architecture called AutoAdapt POC that integrates automated membrane extraction, self-supervised learning, and few-shot learning to automate the interpretation of POC diagnostic tests using smartphone cameras in a scalable manner. A base model pre-trained on a single LFA kit is adapted to five different COVID-19 tests (three antigen, two antibody) using just 20 labeled images., Results: Here we show AutoAdapt POC to yield 99% to 100% accuracy over 726 tests (350 positive, 376 negative). In a COVID-19 drive-through study with 74 untrained users self-testing, 98% found image collection easy, and the rapidly adapted models achieved classification accuracies of 100% on both COVID-19 antigen and antibody test kits. Compared with traditional visual interpretation on 105 test kit results, the algorithm correctly identified 100% of images; without a false negative as interpreted by experts. Finally, compared to a traditional convolutional neural network trained on an HIV test kit, the algorithm showed high accuracy while requiring only 1/50th of the training images., Conclusions: The study demonstrates how rapid domain adaptation in machine learning can provide quality assurance, linkage to care, and public health tracking for untrained users across diverse POC diagnostic tests., (© 2023. The Author(s).)

Self-sorting in functionalized dipeptide systems can be driven by the chirality of a single amino acid, both at a high pH in the micellar state and at a low pH in the gel state. The structures formed are affected to some degree by the relative concentrations of each component showing the complexity of such an approach. The structures underpinning the gel network are predefined by the micellar structures at a high pH. Here, we describe the systems prepared from two dipeptide-based gelators that differ only by the chirality of one of the amino acids. We provide firm evidence for self-sorting in the micellar and gel phases using small-angle neutron scattering and cryo-transmission electron microscopy (cryo-TEM), showing that complete self-sorting occurs across a range of relative concentrations.

Fragment based drug discovery has long been used for the identification of new ligands and interest in targeted covalent inhibitors has continued to grow in recent years, with high profile drugs such as osimertinib and sotorasib gaining FDA approval. It is therefore unsurprising that covalent fragment-based approaches have become popular and have recently led to the identification of novel targets and binding sites, as well as ligands for targets previously thought to be 'undruggable'. Understanding the properties of such covalent fragments is important, and characterizing and/or predicting reactivity can be highly useful. This review aims to discuss the requirements for an electrophilic fragment library and the importance of differing warhead reactivity. Successful case studies from the world of drug discovery are then be examined.

Fragment-based drug discovery (FBDD) is now established as a complementary approach to high-throughput screening (HTS). Contrary to HTS, where large libraries of drug-like molecules are screened, FBDD screens involve smaller and less complex molecules which, despite a low affinity to protein targets, display more 'atom-efficient' binding interactions than larger molecules. Fragment hits can, therefore, serve as a more efficient start point for subsequent optimisation, particularly for hard-to-drug targets. Since the number of possible molecules increases exponentially with molecular size, small fragment libraries allow for a proportionately greater coverage of their respective 'chemical space' compared with larger HTS libraries comprising larger molecules. However, good library design is essential to ensure optimal chemical and pharmacophore diversity, molecular complexity, and physicochemical characteristics. In this review, we describe our views on fragment library design, and on what constitutes a good fragment from a medicinal and computational chemistry perspective. We highlight emerging chemical and computational technologies in FBDD and discuss strategies for optimising fragment hits. The impact of novel FBDD approaches is already being felt, with the recent approval of the covalent KRAS G12C inhibitor sotorasib highlighting the utility of FBDD against targets that were long considered undruggable., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: After receiving a COVID-19 vaccine, most recipients want to know if they are protected from infection and for how long. Since neutralizing antibodies are a correlate of protection, we developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies from a drop of blood. The LFA is based on the principle that neutralizing antibodies block binding of the receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2)., Methods: The ability of the LFA was assessed to correctly measure neutralization of sera, plasma or whole blood from patients with COVID-19 using SARS-CoV-2 microneutralization assays. We also determined if the LFA distinguished patients with seasonal respiratory viruses from patients with COVID-19. To demonstrate the usefulness of the LFA, we tested previously infected and non-infected COVID-19 vaccine recipients at baseline and after first and second vaccine doses., Results: The LFA compared favorably with SARS-CoV-2 microneutralization assays with an area under the ROC curve of 98%. Sera obtained from patients with seasonal coronaviruses did not show neutralizing activity in the LFA. After a single mRNA vaccine dose, 87% of previously infected individuals demonstrated high levels of neutralizing antibodies. However, if individuals were not previously infected, only 24% demonstrated high levels of neutralizing antibodies after one vaccine dose. A second dose boosted neutralizing antibody levels just 8% higher in previously infected individuals, but over 63% higher in non-infected individuals., Conclusions: A rapid, semi-quantitative, highly portable and inexpensive neutralizing antibody test might be useful for monitoring rise and fall in vaccine-induced neutralizing antibodies to COVID-19., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.

Fragment-based hit identification (FBHI) allows proportionately greater coverage of chemical space using fewer molecules than traditional high-throughput screening approaches. However, effectively exploiting this advantage is highly dependent on the library design. Solubility, stability, chemical complexity, chemical/shape diversity, and synthetic tractability for fragment elaboration are all critical aspects, and molecule design remains a time-consuming task for computational and medicinal chemists. Artificial neural networks have attracted considerable attention in automated de novo design applications and could also prove useful for fragment library design. Chemical autoencoders are neural networks consisting of encoder and decoder parts, which respectively compress and decompress molecular representations. The decoder is applied to samples drawn from the space of compressed representations to generate novel molecules that can be scored for properties of interest. Here, we report an autoencoder model using a recurrent neural network architecture, which was trained using 486,565 fragments curated from commercial sources, to simultaneously reconstruct both SMILES and chemical fingerprints. To explore its utility in fragment design, we applied transfer learning to the fingerprint decoder layers to train a classifier using 66 frequent hitter fragments identified from our screening campaigns. Using a particle swarm optimization sampling approach, we compare the performance of this "dual" model to an architecture encoding SMILES only. The dual model produced valid SMILES with improved features, considering a range of properties including aromatic ring counts, heavy atom count, synthetic accessibility, and a new fragment complexity score we term Feature Complexity (FeCo). Additionally, we demonstrate that generative performance is further enhanced by use of a simple syntax-correction procedure during training, in which invalid and undesirable SMILES are spiked into the training set. Finally, we used the syntax-corrected model to generate a library of novel candidate privileged fragments.

Objectives: Pseudomonas aeruginosa is a leading cause of opportunistic infections worldwide, particularly in healthcare settings, and frequently demonstrates resistance to commonly prescribed antimicrobials. Carbapenem resistance is prevalent worldwide, however there are currently limited data available from Haiti. The aim of this study was to characterise and document this phenotype in Port-au-Prince, Haiti, to further inform the need for appropriate infection control, empirical treatment guidelines and laboratory screening measures, both in Haiti and globally., Methods: A total of 50 P. aeruginosa isolates were characterised by multilocus sequence typing (MLST) and antimicrobial susceptibility testing, of which 8 isolates were also subjected to whole-genome sequencing (WGS) to identify potential genetic correlations of phenotypic resistance., Results: By MLST, 23 sequence types (STs) were identified, including 13 new STs. Nineteen isolates belonged to a single, previously characterised ST (ST654), all of which demonstrated a multidrug-resistant phenotype, including resistance to meropenem, imipenem and ceftazidime; two isolates were also resistant to colistin. WGS revealed the presence of genes encoding several previously characterised resistance determinants in ST654; notably ACC(6')-Ib3-cr and GES-7. Metallo-β-lactamase genes (bla VIM-5 ) were also detected in three isolates., Conclusion: These findings confirm that drug-resistant clones of P. aeruginosa are present in Haiti, supporting the need for appropriate screening and control measures and confirming that drug-resistant micro-organisms pose a global threat. Further investigations are required to guide appropriate antimicrobial prescribing in this region., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The composition of the gastrointestinal microbiota influences systemic immune responses, but how this affects infectious disease pathogenesis and antibiotic therapy outcome is poorly understood. This question is rarely examined in humans due to the difficulty in dissociating the immunologic effects of antibiotic-induced pathogen clearance and microbiome alteration. Here, we analyze data from two longitudinal studies of tuberculosis (TB) therapy (35 and 20 individuals) and a cross sectional study from 55 healthy controls, in which we collected fecal samples (for microbiome analysis), sputum (for determination of Mycobacterium tuberculosis (Mtb) bacterial load), and peripheral blood (for transcriptomic analysis). We decouple microbiome effects from pathogen sterilization by comparing standard TB therapy with an experimental TB treatment that did not reduce Mtb bacterial load. Random forest regression to the microbiome-transcriptome-sputum data from the two longitudinal datasets reveals that renormalization of the TB inflammatory state is associated with Mtb pathogen clearance, increased abundance of Clusters IV and XIVa Clostridia, and decreased abundance of Bacilli and Proteobacteria. We find similar associations when applying machine learning to peripheral gene expression and microbiota profiling in the independent cohort of healthy individuals. Our findings indicate that antibiotic-induced reduction in pathogen burden and changes in the microbiome are independently associated with treatment-induced changes of the inflammatory response of active TB, and the response to antibiotic therapy may be a combined effect of pathogen killing and microbiome driven immunomodulation.

Low-molecular-weight hydrogels (LMWG) can be formed by electrochemical methods. Unique to the electrochemical method, gelation is localized on the electrode surface; therefore, thin hydrogel films can be prepared in seconds while thicker gels can be prepared in minutes. Furthermore, hydrogels are suitable for use in a range of characterization methods. Here, we describe techniques to form hydrogels using cyclic voltammetry and potentiometry.

In a Clinical Laboratory Improvement Amendments laboratory setting, we evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG detection with 4 lateral flow immunoassays [LFIAs; 2 iterations from BTNX Inc. (n = 457) and 1 each from ACON Laboratories (n = 200) and SD BIOSENSOR (n = 155)]. In a cohort of primarily hospitalized, reverse-transcription polymerase chain reaction-confirmed coronavirus disease 2019 cases, sensitivity at ≥14 days from symptom onset was: BTNX kit 1, 95%; BTNX kit 2, 91%; ACON, 95%; and SD, 92%. All assays showed good concordance with the Abbott SARS-CoV-2 IgG assay at ≥14 days from symptom onset: BTNX kit 1, 99%; BTNX kit 2, 94%; ACON, 99%; and SD, 100%. Specificity, measured using specimens collected prior to SARS-CoV-2 circulation in the United States and "cross-reactivity challenge" specimens, was 98% for BTNX kit 1 and ACON and 100% for BTNX kit 2 and SD. These results suggest that LFIAs may provide adequate results for rapid detection of SARS-CoV-2., Competing Interests: Disclosures TEG represents Mayo Clinic in a joint venture with Safe Health Systems and has shared intellectual property that may result in royalty sharing., (Copyright © 2020 Elsevier Inc. All rights reserved.)

BACKGROUNDControl of the tuberculosis (TB) pandemic remains hindered in part by a lack of simple and accurate measures of treatment efficacy, as current gold standard markers rely on sputum-based assays that are slow and challenging to implement. However, previous work identified urinary N1, N12-diacetylspermine (DiAcSpm), neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and mass-to-charge ratio (m/z) 241.0903 as potential biomarkers of active pulmonary TB (ATB). Here, we evaluated their ability to serve as biomarkers of TB treatment response and mycobacterial load.METHODSWe analyzed urine samples prospectively collected from 2 cohorts with ATB. A total of 34 study participants from African countries treated with first-line TB therapy rifampin, isoniazid, pyrazinamide, and ethambutol (HRZE) were followed for 1 year, and 35 participants from Haiti treated with either HRZE or an experimental drug were followed for 14 days. Blinded samples were analyzed by untargeted HPLC-coupled high-resolution TOF-mass spectrometry.RESULTSUrinary levels of all 7 molecules significantly decreased by week 26 of successful treatment (P = 0.01 to P < 0.0001) and positively correlated with sputum mycobacterial load (P < 0.0001). Urinary DiAcSpm levels decreased significantly in participants treated with HRZE as early as 14 days (P < 0.0001) but remained unchanged in cases of ineffective therapy (P = 0.14).CONCLUSIONUrinary DiAcSpm, neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 reductions correlated with successful anti-TB treatment and sputum mycobacterial load. Urinary DiAcSpm levels exhibited reductions capable of differentiating treatment success from failure as early as 2 weeks after the initiation of chemotherapy, advocating its further development as a potentially simple, noninvasive biomarker for assessing treatment response and bacterial load.FUNDINGThis work was supported by the Clinical and Translational Science Center at Weill Cornell College of Medicine (NIH/NCATS 1 UL1 TR002384-02 and KL2TR000458), the Department of Defense (PR170782), the National Institute of Allergy and Infectious Disease grants (NIAID T32AI007613-16, K24 AI098627, and K23 AI131913), the NIH Fogarty International Center grants (R24 TW007988 and TW010062), NIH grant (R01 GM135926), the Abby and Howard P. Milstein Program in Chemical Biology and Translational Medicine, and the Tuberculosis Research Units Networks (TBRU-N, AI111143).

It is common to switch between H 2 O and D 2 O when examining peptide-based systems, with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H 2 O to D 2 O in a number of low-molecular-weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little difference in the structures formed at high pH, but this is not universally true. On lowering the pH, the kinetics of gelation are affected and, in some cases, the structures underpinning the gel network are different. Where there are differences in the self-assembled structures, the resulting gel properties are different. We, therefore, show that isotopic control over gel properties is possible.

We present a method to trigger the formation of dipeptide-based hydrogels by the simple addition of dopamine. Dopamine undergoes oxidation in air, reducing the pH to induce gelation. The production of polydopamine and release of reactive oxygen species such as hydrogen peroxide confers antimicrobial activity. Gel stiffness can be controlled by modulating the initial starting pH of the gelator solution. We can use this method to tune the antimicrobial activity of the gels, with gels that are less stiff demonstrating increased bactericidal efficacy against Gram-positive bacteria.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties.

The micellar aggregates formed at high pH for dipeptide-based gelators can be varied by using different alkali metal salts to prepare the solutions. The nature of the micellar aggregates directly affects the properties of the resulting gels.

The properties of a hydrogel are controlled by the underlying network that immobilizes the solvent. For gels formed by the self-assembly of a small molecule, it is common to show the primary fibres that entangle to form the network by microscopy, but it is difficult to access information about the network. One approach to understand the network is to examine the effect of the concentration on the rheological properties, such that G'∝ c x , where G' is the storage modulus and c is the concentration. A number of reports link the exponent x to a specific type of network. Here, we discuss a small library of gels formed using functionalized dipeptides, and describe the underlying networks of these gels, using microscopy, small angle scattering and rheology. We show that apparently different networks can give very similar values of x.

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis ( Mtb ) in the presence of a β-lactamase inhibitor. However, most antimycobacterial β-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

Most low molecular weight gelators are chiral, with racemic mixtures often unable to form gels. Here, we show an example where all enantiomers, diastereomers and racemates of a single functionalized dipeptide can form gels. At high pH, different self-assembled aggregates are formed and these directly template the structures formed in the gel. Hence, solutions and gels with different properties can be accessed simply by varying the chirality. This opens up new design rules for the field., (This journal is © The Royal Society of Chemistry 2019.)

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Recent reports indicate that the sputum of 80% or more of treatment-naive subjects with tuberculosis recruited in England or South Africa contained more viable Mycobacterium tuberculosis cells detected by limiting dilution (LD) in liquid culture than detected as CFU. Efforts to generate such differentially detectable (DD) M. tuberculosis populations in vitro have been difficult to reproduce, and the LD assay is prone to artifact. Here, we applied a stringent version of the LD assay to sputum from 33 treatment-naive, HIV-negative Haitian subjects with drug-sensitive tuberculosis (TB) and to a second sputum sample after two weeks of standard treatment with isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) for 13 of these subjects. Twenty-one percent had statistically defined levels of DD M. tuberculosis in their pretreatment sputum at an average proportional excess over CFU of 3-fold. Sixty-nine percent of those who received HRZE had statistically defined levels of DD M. tuberculosis in their sputum, and of these, the mean proportionate excess over CFU was 7.9-fold. Thus, DD M. tuberculosis is detectable in pretreatment sputum from a significant proportion of subjects in the Western Hemisphere, and certain drugs or drug regimens, while reducing CFU, may at the same time increase the proportional representation of DD M. tuberculosis among the surviving bacilli. Monitoring DD M. tuberculosis may improve our ability to predict the efficacy of efforts to shorten treatment. IMPORTANCE Measurement of the reduction in CFU in sputum of patients with TB up to 2 weeks after the initiation of treatment is the gateway test for a new TB treatment. Reports have suggested that CFU assays fail to detect the majority of viable M. tuberculosis cells in sputum samples from the majority of patients when the number of M. tuberculosis is estimated by limiting dilution (LD). In an effort to avoid potential methodologic confounders, we applied a modified version of the LD assay in a study of a geographically distinct population. We confirmed that differentially detectable (DD) M. tuberculosis is often found before treatment, albeit at lower proportionate levels than in earlier reports. Strikingly, the prevalence and proportionate representation of DD M. tuberculosis increased during standard treatment. Sublethal exposure to certain antibiotics may help generate DD M. tuberculosis cells or enrich their representation among the surviving bacteria, and this may contribute to the need for prolonged treatment with those agents in order to achieve durable cures., (Copyright © 2018 McAulay et al.)

A cobalt rhenium catalyst active for ammonia synthesis at 400 °C and ambient pressure was studied using in situ XAS to elucidate the reducibility and local environment of the two metals during reaction conditions. The ammonia reactivity is greatly affected by the gas mixture used in the pre-treatment step. Following H 2 /Ar pre-treatment, a subsequent 20 min induction period is also observed before ammonia production occurs whereas ammonia production commences immediately following comparable H 2 /N 2 pre-treatment. In situ XAS at the Co K-edge and Re L III -edge show that cobalt initiates reduction, undergoing reduction between 225 and 300 °C, whereas reduction of rhenium starts at 300 °C. The reduction of rhenium is near complete below 400 °C, as also confirmed by H 2 -TPR measurements. A synergistic co-metal effect is observed for the cobalt rhenium system, as complete reduction of both cobalt and rhenium independently requires higher temperatures. The phases present in the cobalt rhenium catalyst during ammonia production following both pre-treatments are largely bimetallic Co-Re phases, and also monometallic Co and Re phases. The presence of nitrogen during the reduction step strongly promotes mixing of the two metals, and the bimetallic Co-Re phase is believed to be a pre-requisite for activity.

Convergent total syntheses of the furanocembranoids 7-epi-pukalide and 7-acetylsinumaximol B have been achieved using a one-pot Knoevenagel condensation and thioether-mediated furan-forming reaction. Furan formation proceeds via a sulfur ylide and results in rapid introduction of structural complexity during the coupling of two highly functionalised fragments. The targets have been prepared in 16 steps from (R)-perillyl alcohol., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections., Competing Interests: The authors have declared that no competing interests exist.