A pipeline for facile cloning of antibody Fv domains and their expression, purification, and characterization as recombinant His-tagged IgGs.

We report a functional pipeline for facile conversion of variable Fv domains, typically discovered in antibody discovery programs, into chimeric monoclonal antibodies (mAbs). Often, in initial screenings, a set of candidate mAbs is produced in small volumes and purified from supernatant for testing. Our pipeline also simplifies purification of mAbs by using an extended histidine tag (His-10) fused to the C-terminus of the light chain. Both the length of the His-10 and its location have been shown to affect the efficacy of mAb purification using an inexpensive nickel-based resin at neutral pH. Our antibody cloning and purification pipeline, when followed together with detection and affinity measurements, can be smoothly incorporated into an antibody discovery workflow.

How can quality be measured within a physician-led Community Emergency Medical service? A scoping review protocol.

BACKGROUND: Quality measurement as part of quality improvement in healthcare is integral for service delivery and development. This is particularly pertinent for health services that deliver care in ways that differ from traditional practice. Community Emergency Medicine (CEM) is a novel and evolving concept of care delivered by services in parts of the UK and Ireland. This scoping review aims to provide a broad overview of how quality may be measured within services delivering CEM. METHODS AND ANALYSIS: The methodology follows both the Preferred Reporting Items for Systematic Review and Meta-Analysis extension for Scoping Reviews (PRISMA-ScR). It is guided by recognised work of Arksey and O'Malley and the guidelines developed by the Joanna Briggs Institute. Several databases will be searched: MEDLINE, EMbase, EMcare, CINAHL, Scopus, the Cochrane Library and grey literature. Search terms have been developed by representatives within Community Emergency Medicine services. Two reviewers will independently screen eligible studies for final study selection. Results will be collected and analysed in descriptive and tabular form to illustrate the breadth of quality indicators that may be applicable to CEM services. This scoping review protocol has been registered with the Open Science Framework platform (osf.io/e7qxg). DISCUSSION: This is the first stage of a larger research study aimed at developing national quality indicators for CEM. The purpose of this scoping review is to provide a comprehensive review of quality indicators that could be used within CEM. The results will be mapped using a framework and identify gaps in the literature to help guide future-focused research.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Phototherapeutic Induction of Immunogenic Cell Death and CD8+ T Cell-Granzyme B Mediated Cytolysis in Human Lung Cancer Cells and Organoids.

Augmenting T cell mediated tumor killing via immunogenic cancer cell death (ICD) is the cornerstone of emerging immunotherapeutic approaches. We investigated the potential of methylene blue photodynamic therapy (MB-PDT) to induce ICD in human lung cancer. Non-Small Cell Lung Cancer (NSCLC) cell lines and primary human lung cancer organoids were evaluated in co-culture killing assays with MB-PDT and light emitting diodes (LEDs). ICD was characterised using immunoblotting, immunofluorescence, flow cytometry and confocal microscopy. Phototherapy with MB treatment and low energy LEDs decreased the proliferation of NSCLC cell lines inducing early necrosis associated with reduced expression of the anti-apoptotic protein, Bcl2 and increased expression of ICD markers, calreticulin (CRT), intercellular cell-adhesion molecule-1 (ICAM-1) and major histocompatibility complex I (MHC-I) in NSCLC cells. MB-PDT also potentiated CD8+ T cell-mediated cytolysis of lung cancer via granzyme B in lung cancer cells and primary human lung cancer organoids.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Comparative outcomes of radiofrequency ablation and cryoballoon ablation in dysplastic Barrett's esophagus: a propensity score-matched cohort study.

BACKGROUND AND AIMS: Strong evidence supports the use of radiofrequency ablation (RFA) in the management of dysplastic/neoplastic Barrett's esophagus (BE). Recently, the efficacy of the cryoballoon ablation (CBA) system was demonstrated in multicenter cohort studies. We aimed to assess the comparative effectiveness and safety of these 2 ablation modalities for endoscopic eradication therapy (EET) in a cohort study. METHODS: Data were abstracted on patients with dysplastic BE or intramucosal carcinoma undergoing EET using RFA or CBA as the primary ablation modality at 2 referral centers. The primary outcome was the rate of complete remission intestinal metaplasia (CRIM). Secondary outcomes were rates of complete remission of dysplasia (CRD) and adverse events. Cox proportional hazards models and propensity scored-matched analyses were conducted to compare outcomes. RESULTS: Three hundred eleven patients (CBA, 85 patients; RFA, 226 patients) with a median follow-up of 1.5 years (interquartile range, .8, 2.5) in the RFA group and 2.0 years (interquartile range, 1.3, 2.5) in the CBA group were studied. On multivariable analyses, the chances of reaching CRD and CRIM were not influenced by ablation modality. Propensity score-matched analysis revealed a comparable chance of achieving CRIM (CBA vs RFA: hazard ratio, 1.24; 95% confidence interval, .79-1.96; P = .35) and CRD (CBA vs RFA: hazard ratio, 1.19; 95% confidence interval, .82-1.73; P = .36). The CBA group had a higher stricture rate compared with the RFA group (10.4% vs 4.4%, P = .04). CONCLUSIONS: Histologic outcomes of EET using CBA and RFA for dysplastic BE appear to be comparable. A randomized trial is needed to definitively compare outcomes between these 2 modalities.

Mortality in association with antipsychotic medication use and clinical outcomes among geriatric psychiatry outpatients with COVID-19.

OBJECTIVES: Older adults are particularly vulnerable to the negative consequences of antipsychotic exposure and are disproportionally affected by higher mortality from coronavirus disease 2019 (COVID-19). Our goal was to determine whether concurrent antipsychotic medication use was associated with increased COVID-19 mortality in older patients with preexisting behavioral health problems. We also report on findings from post-COVID follow-ups. DESIGN: Retrospective observational study. PARTICIPANTS: Outpatients at a geriatric psychiatric clinic in New York City. MEASUREMENTS: Demographic and clinical data including medication, diagnosis and Clinical Global Impression Severity (CGI-S) scales on outpatients who had COVID-19 between February 28th and October 1st 2020 were extracted from the electronic health records (EHR) from the hospital. RESULTS: A total of 56 patients were diagnosed with COVID-19 (mean age 76 years; median age 75 years) and 13 (23.2%) died. We found an increased mortality risk for patients who were prescribed at least one antipsychotic medication at the time of COVID-19 infection (Fisher's exact test P = 0.009, OR = 11.1, 95% confidence interval: 1.4-96.0). This result remains significant after adjusting for age, gender, housing context and dementia (Logistic regression P = 0.035, Beta = 2.4). Furthermore, we found that most patients who survived COVID-19 (88.4%) recovered to pre-COVID baseline in terms of psychiatric symptoms. Comparison of pre- and post-COVID assessments of CGI-S for 33 patients who recovered from COVID-19 were not significantly different. CONCLUSION: We observed a higher COVID-19 mortality associated with concurrent antipsychotics use in older patients receiving behavioral health services. The majority of patients in our geriatric clinic who recovered from COVID-19 appeared to return to their pre-COVID psychiatric function. More precise estimates of the risk associated with antipsychotic treatment in older patients with COVID-19 and other underlying factors will come from larger datasets and meta-analyses.

Near-Infrared Fluorescent Probes for the Detection of Cancer-Associated Proteases.

Proteases are enzymes capable of catalyzing protein breakdown, which is critical across many biological processes. There are several families of proteases, each of which perform key functions through the degradation of specific proteins. As our understanding of cancer improves, it has been demonstrated that several proteases can be overactivated during the progression of cancer and contribute to malignancy. Optical imaging systems that employ near-infrared (NIR) fluorescent probes to detect protease activity offer clinical promise, both for early detection of cancer as well as for the assessment of personalized therapy. In this Review, we review the design of NIR probes and their successful application for the detection of different cancer-associated proteases.

Human kidney stones: a natural record of universal biomineralization.

GeoBioMed - a new transdisciplinary approach that integrates the fields of geology, biology and medicine - reveals that kidney stones composed of calcium-rich minerals precipitate from a continuum of repeated events of crystallization, dissolution and recrystallization that result from the same fundamental natural processes that have governed billions of years of biomineralization on Earth. This contextual change in our understanding of renal stone formation opens fundamentally new avenues of human kidney stone investigation that include analyses of crystalline structure and stratigraphy, diagenetic phase transitions, and paragenetic sequences across broad length scales from hundreds of nanometres to centimetres (five Powers of 10). This paradigm shift has also enabled the development of a new kidney stone classification scheme according to thermodynamic energetics and crystalline architecture. Evidence suggests that ≥50% of the total volume of individual stones have undergone repeated in vivo dissolution and recrystallization. Amorphous calcium phosphate and hydroxyapatite spherules coalesce to form planar concentric zoning and sector zones that indicate disequilibrium precipitation. In addition, calcium oxalate dihydrate and calcium oxalate monohydrate crystal aggregates exhibit high-frequency organic-matter-rich and mineral-rich nanolayering that is orders of magnitude higher than layering observed in analogous coral reef, Roman aqueduct, cave, deep subsurface and hot-spring deposits. This higher frequency nanolayering represents the unique microenvironment of the kidney in which potent crystallization promoters and inhibitors are working in opposition. These GeoBioMed insights identify previously unexplored strategies for development and testing of new clinical therapies for the prevention and treatment of kidney stones.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

The adaptive immune receptor repertoire community as a model for FAIR stewardship of big immunology data.

Systems biology involves network-oriented, computational approaches to modeling biological systems through analysis of big biological data. To contribute maximally to scientific progress, big biological data should be FAIR: findable, accessible, interoperable, and reusable. Here, we describe high-throughput sequencing data that characterize the vast diversity of B- and T-cell clones comprising the adaptive immune receptor repertoire (AIRR-seq data) and its contribution to our understanding of COVID-19 (coronavirus disease 19). We describe the accomplishments of the AIRR community, a grass-roots network of interdisciplinary laboratory scientists, bioinformaticians, and policy wonks, in creating and publishing standards, software and repositories for AIRR-seq data based on the FAIR principles.

Re-contact rates with a UK ambulance service following paramedic referral to a falls prevention service for those aged ≥ 65 years: a retrospective cohort study.

BACKGROUND: Falls in older populations constitute a large proportion of the workload for UK ambulance services, and cost the NHS over £2.3 billion per year. A large proportion of older fallers are not conveyed to an emergency department (ED), representing a vulnerable group of patients. New pathways have been developed for paramedics to refer this group directly to falls prevention services. OBJECTIVES: This study aimed to investigate the re-contact rates and characteristics of service users aged ≥ 65 years who fell and were referred to a falls prevention service by paramedics, and to describe the characteristics of those who re-contacted the ambulance service after referral. METHODS: A retrospective cross-sectional cohort study was carried out in the geographical area covered by the South Eastern division of the Northern Ireland Ambulance Service (NIAS) between 1 July and 30 September 2017. The primary outcome was the rate of subsequent contacts with the ambulance service following referral. RESULTS: There were 1079 incidents of falls in service users aged ≥ 65 years. A referral rate of 7% (n = 75) was reported, constituting the study cohort. Re-contact rates were 37.3% (n = 28) within 1 month and 70.7% (n = 53) within 6 months. Women and those exposed to a 'long lie' were more likely to re-contact, while those with cognitive impairment appeared particularly vulnerable to falls and repeat falls. Repeat falls were common. Documentation by attending clinicians was generally poor. CONCLUSION: Future research should investigate the efficacy of paramedic referral to falls prevention services. Interventions targeted at reducing long lies and investigating optimal interventions for those with cognitive impairments should also be explored. Improving clinical documentation will facilitate future research.

BODIPY-Labeled Cyclobutanes by Secondary C(sp3 )-H Arylations for Live-Cell Imaging.

Arylated cyclobutanes were accessed by a versatile palladium-catalyzed secondary C(sp3 )-H activation, exploiting chelation assistance by modular triazoles. The C-H arylation led to cyclobutane natural product derivatives in a highly regioselective fashion, setting the stage for the easy access to novel fluorogenic boron-dipyrrin (BODIPY)-labeled probes for live-cell imaging.

Natural product-inspired profluorophores for imaging NQO1 activity in tumour tissues.

Herein we designed a collection of trimethyl-lock quinone profluorophores as activity-based probes for imaging NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells and tumour tissues. Profluorophores were prepared via synthetic routes from naturally-occurring quinones and characterised in vitro using recombinant enzymes, to be further validated in cells and fresh frozen canine tumour tissues as potential new tools for cancer detection and imaging.

Inferred Allelic Variants of Immunoglobulin Receptor Genes: A System for Their Evaluation, Documentation, and Naming.

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC-focusing, to begin with, on human IGHV genes-with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.

Tricarbocyanine N-triazoles: the scaffold-of-choice for long-term near-infrared imaging of immune cells in vivo.

Herein tricarbocyanine N-triazoles are first described as a rationally-designed near-infrared (NIR) structure overcoming the brightness and photostability limitations of tricarbocyanines for long-term in vivo imaging. The straightforward synthetic approach and the wide availability of alkynes makes this strategy a versatile methodology for the preparation of highly stable N-substituted tricarbocyanines. Furthermore, we validated CIR38M as a non-transferable marker to monitor the fate of therapeutic T cells non-invasively in vivo, showing enhanced performance over conventional NIR fluorophores (i.e. DiR, IR800CW and indocyanine green) as well as compatibility with human cells for translational studies. CIR38M is able to track over time smaller numbers of T cells than current NIR agents, and to visualise antigen-driven accumulation of immune cells at specific sites in vivo. This chemical technology will improve longitudinal imaging studies to assess the efficacy of cell-based immunotherapies in preclinical models and in human samples.