Boosting of post-exposure human T-cell and B-cell recall responses in vivo by Burkholderia pseudomallei-related proteins.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T-cell and B-cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon-γ-secreting cells, and induced antibody affinity maturation. Moreover, antigen-specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N-PilO2 can stimulate human memory T and B cells and highlight the potential of the hu-PBL-SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063

The 1.8\u2005\uc5 resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using \u3b2-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a “lid” domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a “lid” domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG

Durvalumab (MEDI 4736) in combination with extended neoadjuvant regimens in rectal cancer : a study protocol of a randomised phase II trial (PRIME-RT)

Acknowledgements We are grateful to Mr George Davidson and Ms Monica Jeffers for their input with writing the PRIME-RT protocol and patient information sheet. This study is co-sponsored by the University of Glasgow and NHS Greater Glasgow and Clyde. Funding PRIME-RT is funded by Astrazeneca and receives core funding from CRUK Clinical Trials Unit Glasgow for the purposes of trial set-up and data collection. The trial is co-sponsored by the University Of Glasgow and NHS Greater Glasgow and Clyde.Peer reviewedPublisher PD

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Physical, cognitive, and mental health impacts of COVID-19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research

An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form

The identification and 3D structural characterization of a homolog of the (R)-selective transaminase (RTA) from Aspergillus terreus (AtRTA), from the thermotolerant fungus Thermomyces stellatus (TsRTA) is here reported. The thermostability of TsRTA (40% retained activity after 7 days at 40°C) was initially attributed to its tetrameric form in solution, however subsequent studies of AtRTA revealed it also exists predominantly as a tetramer yet, at 40°C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with TsRTA_G205C retaining almost full activity after incubation at 50°C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. TsRTA accepts the common amine donors (R)-methylbenzylamine, isopropylamine, and D-alanine as well as aromatic and aliphatic ketones and aldehydes