Probing Apoptotic Caspase Allostery and Exosite Interactions for Alternative Regulation

Programmed cell death, or apoptosis is a critical homeostatic pathway that monitors the balance of cell life and death. Apoptosis is regulated by a class of enzymes known as the cysteine aspartic proteases, or the caspases. The 12 human caspases that play important roles in the progression and regulation of apoptosis and inflammation. Caspases are tightly regulated by numerous factors including enzymatic activation, post-translational modifications, metal ligand binding, and protein modulation. Aberrant caspase activation and regulation has been implicated in the progression of numerous diseases such as proliferative diseases and neurodegeneration. The deeply entwined nature of caspases and apoptosis makes them interesting targets for therapeutic intervention of apoptotic diseases. However, the highly conserved fold and overlapping active site specificities makes the development of specific caspase modulators difficult. Therefore, it is critical to understand the mechanistic details of each caspases function, dynamics and specificity to further differentiate these homologous enzymes and uncover new scaffolds for select targeting of these enzymes. In this work, we aim to identify distal residues that comprise protein exosites which mediate the recruitment of proteins for enzymatic hydrolysis. First we developed an evolved specificity caspase that may be utilized for the identification of specific substrate that utilize exosite for caspase recognition and recruitment. Next we probed the details of caspase regulation by zinc validating the emerging signaling atom as an essential regulator of the caspases. Next we identified a putative exosite of caspase-6 located within the N-terminal domain that is essential for protein substrate recruitment and also modulates the dynamics of unique 130’s helix to strand interconversion of the enzyme. Lastly, we identified an active site adjacent inhibitor of caspase-6 that takes advantage of a unique evolutionarily conserved cysteine of caspase-6. Modulation of this distal cysteine serves as an anchor for the most selective, potent and cell permeable inhibitor of caspase-6 to date. The identification of exosites provides new scaffolds for development of specific inhibitors that take advantage of the unique characteristics of this family of enzymes. Interestingly, uncovering the details of protein exosites also provides the unique opportunity block substrate hydrolysis by targeting either the enzyme exosite or the substrate exosite, thereby expanding the chemical space for effective modulation and therapeutic intervention

A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for thera- peutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus–host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivi- rus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B–NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluo- rescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus inter- nal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbec- co’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus–host interactions and facilitate future applications in antiviral drug research to manage flavivi- rus infections.International Centre for Genetic Engineering and Biotechnology Grant CRP/CRI18-02.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Accuracy and consequences of using trial-of-antibiotics for TB diagnosis (ACT-TB study) : protocol for a randomised controlled clinical trial

The clinical trial is funded by the Commonwealth Scholarship Commission and the Helse Nord RHF grant awarded to THD. This work is part of THD’s PhD work at London School of Hygiene & Tropical Medicine (LSHTM). LSHTM is the sponsor of this clinical trial (sponsor address: Keppel Street, Bloomsbury, London WC1E 7HT). ELC is funded by a Wellcome Trust Senior Research Fellowship in Clinical Science: WT200901.Introduction Over 40% of global tuberculosis case notifications are diagnosed clinically without mycobacteriological confirmation. Standard diagnostic algorithms include ‘trial-of-antibiotics’—empirical antibiotic treatment given to mycobacteriology-negative individuals to treat infectious causes of symptoms other than tuberculosis, as a ‘rule-out’ diagnostic test for tuberculosis. Potentially 26.5 million such antibiotic courses/year are prescribed globally for the 5.3 million/year mycobacteriology-negative patients, making trial-of-antibiotics the most common tuberculosis diagnostic, and a global-scale risk for antimicrobial resistance (AMR). Our systematic review found no randomised controlled trial (RCT) to support use of trial-of-antibiotic. The RCT aims to determine the diagnostic and clinical value and AMR consequences of trial-of-antibiotics. Methods and analysis A three-arm, open-label, RCT randomising (1:1:1) Malawian adults (≥18 years) seeking primary care for cough into: (a) azithromycin 500 mg one time per day for 3 days or (b) amoxicillin 1 g three times per day for 5 days or (c) standard-of-care (no immediate antibiotic). We will perform mycobacteriology tests (microscopy, Xpert MTB/RIF (Mycobacterium tuberculosis/rifampicin) and Mycobacterium tuberculosis culture) at baseline. We will use audiocomputer-assisted self-interview to assess clinical improvement at day 8. First primary outcome will be proportion of patients reporting day 8 improvement out of those with negative mycobacteriology (specificity). Second primary outcome will be day 29 incidence of a composite endpoint of either death or hospitalisation or missed tuberculosis diagnosis. To determine AMR impact we compare proportion of resistant nasopharyngeal Streptococcus pneumoniae isolates on day 29. 400 mycobacteriology-negative participants/arm will be required to detect a ≥10% absolute difference in diagnostic specificity with 80% power. We will estimate measures of effect by comparing outcomes in antibiotic arms (combined and individually) to standard-of-care. Ethics and dissemination The study has been reviewed and approved by Malawi College of Medicine Research and Ethics Committee, London School of Hygiene & Tropical Medicine (LSHTM) Research Ethics Committee and Regional Committee for Health and Research Ethics – Norway, and Malawi Pharmacy, Medicines and Poisons Board. We will present abstracts at relevant conferences, and prepare a manuscript for publication in a peer-reviewed journal.Publisher PDFPeer reviewe

Flourishing workplaces: a multisensory approach to design and POE

How can we design workplaces which occupants thrive in, which are functional but are also expressive? Drawing on research about the senses and office related studies this paper demonstrates how buildings can be designed to allow for positive multi-sensory experiences. In order to design a creative and productive workplace, it is essential to consider how the environment is making us feel, behave and act within it. As the workplace continues to evolve, the case is made for a sensory palette framework to drive a systems approach to building environmental design enabling the integration of the multi-modal sensory relationship of people’s reactions within various environmental settings. Technological advances, in the form of wearables that monitor our physiological and stress responses offer the opportunity to capture empirical data, further enabling the investigation to see how a diverse range of environmental settings affect our physical, mental and social wellbeing. The paper goes on to develop the established conceptual theories of ‘Flourish’ proposing a move beyond comfort when designing the interiors and the mechanics of facility controls towards a sensory impacts framework that considers a whole life costing approach using the Flourish Model sets the basis for a design and post-occupancy evaluation toolkit

Emergent global patterns of ecosystem structure and function from a mechanistic general ecosystem model

Anthropogenic activities are causing widespread degradation of ecosystems worldwide, threatening the ecosystem services upon which all human life depends. Improved understanding of this degradation is urgently needed to improve avoidance and mitigation measures. One tool to assist these efforts is predictive models of ecosystem structure and function that are mechanistic: based on fundamental ecological principles. Here we present the first mechanistic General Ecosystem Model (GEM) of ecosystem structure and function that is both global and applies in all terrestrial and marine environments. Functional forms and parameter values were derived from the theoretical and empirical literature where possible. Simulations of the fate of all organisms with body masses between 10 µg and 150,000 kg (a range of 14 orders of magnitude) across the globe led to emergent properties at individual (e.g., growth rate), community (e.g., biomass turnover rates), ecosystem (e.g., trophic pyramids), and macroecological scales (e.g., global patterns of trophic structure) that are in general agreement with current data and theory. These properties emerged from our encoding of the biology of, and interactions among, individual organisms without any direct constraints on the properties themselves. Our results indicate that ecologists have gathered sufficient information to begin to build realistic, global, and mechanistic models of ecosystems, capable of predicting a diverse range of ecosystem properties and their response to human pressures

Trial-of-antibiotics to assist tuberculosis diagnosis in symptomatic adults in Malawi (ACT-TB study): a randomised controlled trial.

BACKGROUND: Clinical practice and diagnostic algorithms often assume that tuberculosis can be ruled out in mycobacteriology-negative individuals whose symptoms improve with a trial-of-antibiotics. We aimed to investigate diagnostic performance, clinical benefit, and antimicrobial resistance using a randomised controlled trial. METHODS: In this three-arm, individually randomised, open-label, controlled trial, we enrolled Malawian adults (aged ≥18 years) attending primary care who reported being unwell for at least 14 days (including cough) with no immediate indication for hospitalisation at Limbe and Ndirande Health Centres in Blantyre. Participants were randomly allocated (1:1:1) to azithromycin (500 mg taken once per day for 3 days), amoxicillin (1 g taken three times per day for 5 days), or standard of care with no immediate antibiotics, stratified by study site. Sputum at enrolment and day 8 was tested for tuberculosis (microscopy, Xpert MTB/RIF, and culture). The primary efficacy outcome was day 8 specificity (percentage with symptom improvement among mycobacteriology-negative participants), and day 29 clinical outcome (death, hospitalisation, or missed tuberculosis diagnosis) among all randomised participants. This study is registered with ClinicalTrials.gov, NCT03545373. FINDINGS: Between Feb 25, 2019, and March 14, 2020, 5825 adults were screened and 1583 (mean age 36 years; 236 [14·9%] HIV positive) were randomly assigned to standard of care (530 participants), azithromycin (527 participants), or amoxicillin (526 participants) groups. Overall, 6·3% (100 of 1583 participants) had positive baseline sputum mycobacteriology. 310 (79·1%) of 392 patients receiving standard of care reported symptom improvement at day 8, compared with 340 (88·7%) of 383 patients receiving azithromycin (adjusted difference 8·6%, 95% CI 3·9-13·3%; p<0·0004) and 346 (89·4%) of 387 receiving amoxicillin (adjusted difference 8·8%, 4·0-13·6%; p=0·0003). The proportion of participants with day 29 composite clinical outcomes was similar between groups (standard of care 1% [7 of 530 participants], azithromycin 1% [6 of 527 participants], amoxicillin 2% [12 of 526 participants]). INTERPRETATION: Routine outpatient trial-of-antibiotics during tuberculosis investigations modestly improved diagnostic specificity for mycobacteriologically confirmed tuberculosis but had no appreciable effect on death, hospitalisation, and missed tuberculosis diagnosis. These results confirm the limited benefit of trial-of-antibiotics, presenting an opportunity for discontinuation of trial-of-antibiotics and improved antimicrobial stewardship during tuberculosis screening, without affecting clinical outcomes. FUNDING: Northern Norway Regional Health Authority (Helse Nord RHF), Commonwealth Scholarship Commission in the UK, Wellcome Trust, UK Medical Research Council, and the UK Department for International Development

Trial-of-antibiotics to assist tuberculosis diagnosis in symptomatic adults in Malawi (ACT-TB study): a randomised controlled trial.

Background: Clinical practice and diagnostic algorithms often assume that tuberculosis can be ruled out in mycobacteriology-negative individuals whose symptoms improve with a trial-of-antibiotics. We aimed to investigate diagnostic performance, clinical benefit, and antimicrobial resistance using a randomised controlled trial. Methods: In this three-arm, individually randomised, open-label, controlled trial, we enrolled Malawian adults (aged ≥18 years) attending primary care who reported being unwell for at least 14 days (including cough) with no immediate indication for hospitalisation at Limbe and Ndirande Health Centres in Blantyre. Participants were randomly allocated (1:1:1) to azithromycin (500 mg taken once per day for 3 days), amoxicillin (1 g taken three times per day for 5 days), or standard of care with no immediate antibiotics, stratified by study site. Sputum at enrolment and day 8 was tested for tuberculosis (microscopy, Xpert MTB/RIF, and culture). The primary efficacy outcome was day 8 specificity (percentage with symptom improvement among mycobacteriology-negative participants), and day 29 clinical outcome (death, hospitalisation, or missed tuberculosis diagnosis) among all randomised participants. This study is registered with ClinicalTrials.gov, NCT03545373. Findings: Between Feb 25, 2019, and March 14, 2020, 5825 adults were screened and 1583 (mean age 36 years; 236 [14·9%] HIV positive) were randomly assigned to standard of care (530 participants), azithromycin (527 participants), or amoxicillin (526 participants) groups. Overall, 6·3% (100 of 1583 participants) had positive baseline sputum mycobacteriology. 310 (79·1%) of 392 patients receiving standard of care reported symptom improvement at day 8, compared with 340 (88·7%) of 383 patients receiving azithromycin (adjusted difference 8·6%, 95% CI 3·9-13·3%; p<0·0004) and 346 (89·4%) of 387 receiving amoxicillin (adjusted difference 8·8%, 4·0-13·6%; p=0·0003). The proportion of participants with day 29 composite clinical outcomes was similar between groups (standard of care 1% [7 of 530 participants], azithromycin 1% [6 of 527 participants], amoxicillin 2% [12 of 526 participants]). Interpretation: Routine outpatient trial-of-antibiotics during tuberculosis investigations modestly improved diagnostic specificity for mycobacteriologically confirmed tuberculosis but had no appreciable effect on death, hospitalisation, and missed tuberculosis diagnosis. These results confirm the limited benefit of trial-of-antibiotics, presenting an opportunity for discontinuation of trial-of-antibiotics and improved antimicrobial stewardship during tuberculosis screening, without affecting clinical outcomes. Funding: Northern Norway Regional Health Authority (Helse Nord RHF), Commonwealth Scholarship Commission in the UK, Wellcome Trust, UK Medical Research Council, and the UK Department for International Development

Linkage to chromosome 2q32.2-q33.3 in familial serrated neoplasia (Jass syndrome)

Causative genetic variants have to date been identified for only a small proportion of familial colorectal cancer (CRC). While conditions such as Familial Adenomatous Polyposis and Lynch syndrome have well defined genetic causes, the search for variants underlying the remainder of familial CRC is plagued by genetic heterogeneity. The recent identification of families with a heritable predisposition to malignancies arising through the serrated pathway (familial serrated neoplasia or Jass syndrome) provides an opportunity to study a subset of familial CRC in which heterogeneity may be greatly reduced. A genome-wide linkage screen was performed on a large family displaying a dominantly-inherited predisposition to serrated neoplasia genotyped using the Affymetrix GeneChip Human Mapping 10 K SNP Array. Parametric and nonparametric analyses were performed and resulting regions of interest, as well as previously reported CRC susceptibility loci at 3q22, 7q31 and 9q22, were followed up by finemapping in 10 serrated neoplasia families. Genome-wide linkage analysis revealed regions of interest at 2p25.2-p25.1, 2q24.3-q37.1 and 8p21.2-q12.1. Finemapping linkage and haplotype analyses identified 2q32.2-q33.3 as the region most likely to harbour linkage, with heterogeneity logarithm of the odds (HLOD) 2.09 and nonparametric linkage (NPL) score 2.36 (P = 0.004). Five primary candidate genes (CFLAR, CASP10, CASP8, FZD7 and BMPR2) were sequenced and no segregating variants identified. There was no evidence of linkage to previously reported loci on chromosomes 3, 7 and 9

Observing the temperature dependent transition of the GP2 peptide using terahertz spectroscopy

The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue), a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (TD). In particular, we show that below TD the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above TD, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.Yiwen Sun, Zexuan Zhu, Siping Chen, Jega Balakrishnan, Derek Abbott, Anil T. Ahuja and Emma Pickwell-MacPherso