CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression.

BACKGROUND: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung. METHODS: We explored the feasibility of CRISPR-mediated transcriptional upregulation of interleukin 10 (IL-10), a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting. RESULTS: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, that is, simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients. CONCLUSIONS: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants.

Utilising the dental workforce in England, United Kingdom, to optimise the response to COVID-19.

COVID-19 presents a unique and significant challenge to healthcare systems across the globe. Dental workforce redeployment, in England, during the response to Coronavirus (COVID-19), is the first reported national effort to redeploy a professional body into new clinical environments. The policy decision to facilitate redeployment of the dental workforce, in March 2020, by the Office of the Chief Dental Officer (OCDO), increased flexibility within workforce systems and allowed increasing demand on healthcare services to be managed safely and effectively. This paper outlines how this policy change was achieved via a multi-professional approach, mapping competencies of the dental workforce to high-priority areas of healthcare need. The dental workforce has a varied and often specialised skill set, offering expertise in infection prevention and control, airway management and often, behaviour management. These skills can be an important contribution to tackling a pandemic where expertise in these areas is vital. This increase in workforce supply allows healthcare systems to improve their surge response capabilities. Additionally, redeployment presents an opportunity to create greater and sustained collaboration between the medical and dental professions, leading to greater understanding of the contribution of oral health to wider medical wellbeing.

Robust segregation of donor and recipient cells from single-cell RNA-sequencing of transplant samples.

Background: Single-cell RNA-sequencing (scRNA-seq) technology has revealed novel cell populations in organs, uncovered regulatory relationships between genes, and allowed for tracking of cell lineage trajectory during development. It demonstrates promise as a method to better understand transplant biology; however, fundamental bioinformatic tools for its use in the context of transplantation have not been developed. One major need has been a robust method to identify cells as being either donor or recipient genotype origin, and ideally without the need to separately sequence the donor and recipient. Methods: We implemented a novel two-stage genotype discovery method (scTx) optimized for transplant samples by being robust to disparities in cell number and cell type. Using both in silico and real-world scRNA-seq transplant data, we benchmarked our method against existing demultiplexing methods to profile their limitations in terms of sequencing depth, donor and recipient cell imbalance, and single nucleotide variant input selection. Results: Using in silico data, scTx could more accurately separate donor from recipient cells and at much lower genotype ratios than existing methods. This was further validated using solid-organ scRNA-seq data where scTx could more reliably identify when a second genotype was present and at lower numbers of cells from a second genotype. Conclusion: scTx introduces the capability to accurately segregate donor and recipient gene expression at the single-cell level from scRNA-seq data without the need to separately genotype the donor and recipient. This will facilitate the use of scRNA-seq in the context of transplantation.

Interferon-stimulated and metallothionein-expressing macrophages are associated with acute and chronic allograft dysfunction after lung transplantation.

BACKGROUND: Lung transplant recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). ALAD episodes are a risk factor for chronic lung allograft dysfunction (CLAD), the major cause of death after lung transplantation. Our objective was to determine key cellular elements in dysfunctional lung allografts, with a focus on macrophages. METHODS: We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage cells from stable and ALAD patients and to cells from explanted CLAD lung tissue. RESULTS: We identified 2 alveolar macrophage (AM) subsets uniquely represented in ALAD. Using pathway analysis and differentially expressed genes, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) AMs. Functional analysis of an independent set of AMs in vitro revealed that ALAD AMs exhibited a higher expression of CXCL10, a marker of ISG AMs, and increased secretion of pro-inflammatory cytokines compared to AMs from stable patients. Using publicly available bronchoalveolar lavage scRNAseq datasets, we found that ISG and MT AMs are associated with more severe inflammation in COVID-19 patients. Analysis of cells from 4 explanted CLAD lungs revealed similar macrophage populations. Donor and recipient cells were identified using expressed single nucleotide variations. We demonstrated contributions of donor and recipient cells to all AM subsets early post-transplant, with loss of donor-derived cells over time. CONCLUSIONS: Our data reveal extensive heterogeneity among lung macrophages after lung transplantation and indicates that specific sub-populations may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.

A changing climate and the dental profession.

Climate change is the defining crisis of our time and experts fear it is happening faster than first predicted. In November 2021, the UK hosted COP26 where world leaders met to coordinate actions and renew commitments to tackle the problem head on. Whether COP26 galvanised the international community enough to turn a corner remains to be seen; however, as dental professionals, we face significant considerations regarding our opportunities to effect positive change. The aim of this paper is to provide a short account of the impact of dentistry on the environment, as well as to highlight some challenges and the potential for change available to the dental profession to become more sustainability-conscious. In dentistry, the main sources of carbon emissions are travel, procurement and energy use. Prevention of oral and dental disease is the single most important factor in reducing the environmental impact of dentistry long-term. It is essential that clinicians, manufacturers and relevant stakeholders are united in dealing with the environmental crisis to bring about effective change. Clinicians and patients should be encouraged to consciously think about their impact on the environment and to consider what adjustments they can make to their clinical practice and oral health habits.

The Federal Retail Pharmacy Program (FRPP) Impact on COVID-19 Vaccine Distribution During Pandemic: Effectiveness, Limitations, and Implications.

The coronavirus disease 2019 (COVID-19) has altered the healthcare landscape for pharmacy practice and continues its global onslaught. As the COVID-19 vaccines began to reach the general population, many were left wondering where and when they would get the vaccine. With 90% of the American population living within 5 miles of a community pharmacy, vaccine distribution to these locations is vital for a successful vaccine campaign. The Biden Administration launched the Federal Retail Pharmacy Program (FRPP) in February 2021, a public-private partnership with 21 national pharmacy partners representing over 40,000 pharmacy locations to help expand the vaccine rollout. Community pharmacists are uniquely positioned to fulfill this mission by being a point of contact for the COVID-19 vaccination efforts. The FRPP has experienced some limitations, including the variable vaccine allocation, limited support from the healthcare system, and the lack of overwhelming public confidence in the vaccines. Improving the FRPP would require strong partnership with other healthcare professionals and the adoption of flexible vaccine dissemination. These can stem future pandemics.

Compliance problems in dental infection prevention and control in England: an analysis of Care Quality Commission inspections between 2016 and 2019.

Introduction All dental practices in England must be registered with the Care Quality Commission (CQC). The CQC inspects approximately 10% of practices each year to ensure premises are safe. Compliance with infection prevention and control is assessed during inspections.Aims The aim of this study was to identify common areas of non-compliance in infection prevention and control within general dental practice in England.Materials and methods The CQC inspection reports, for all practices found not safe following an inspection, were thematically analysed for non-compliance specifically in infection prevention and control.Results Between April 2016 and March 2019, 88 practices were deemed not safe. The most common areas of non-compliance were the management of risks associated with legionella, audit and the validation of decontamination equipment.Discussion Infection prevention and control compliance requires good management and leadership. Other factors are also thought to influence compliance including cost and morality.Conclusions Identification of recurrent infection prevention and control compliance issues highlights relevant areas to all CQC dental registrants that merit greater focus.

Can oral surgery be performed safely when COVID-19 status is unknown?

Design Cross-sectional study/special report.Study population This paper presents the early experience of the oral and maxillofacial surgery department at Hadassah University Medical Centre in Jerusalem during the first wave of the COVID-19 pandemic in 2020. The study involved both medical staff and patients.Data analysis A retrospective analysis of an eight-week period (February-April 2020) collated 1,471 patient records and examined diagnoses, procedures performed and COVID-19 status of patients and staff. Any attempts made to access routine dental care before presentation in secondary care were recorded.Results In the study period, one member of staff was confirmed as COVID-19-positive. Sixty-three patients had formal COVID-19 tests; all were negative. Forty-three patients were admitted for drainage of odontogenic fascial space infections; 53% reported delayed or failed attempts to access dental care before their infection. Additionally, the authors describe a screening process, personal protective equipment (PPE) allocation and staff/patient testing protocols employed in their surgical unit throughout this period.Conclusions The authors suggest a series of triage and screening measures to limit the risk of unknowingly exposing clinical staff to the COVID-19 virus and offer advice on safely delaying non-emergency treatment where necessary. Recommendations for use of PPE for aerosol and non-aerosol generating procedures are made, but it is important to recognise that the efficacy of these measures cannot be determined by the methodology employed. This paper demonstrates an early example of complications developing from absent or delayed routine dental services resulting from lockdowns. This 'excess morbidity' is likely to have an impact on healthcare services as the pandemic recovery unfolds and services begin to return to normal.

scSNV: accurate dscRNA-seq SNV co-expression analysis using duplicate tag collapsing.

Identifying single nucleotide variants has become common practice for droplet-based single-cell RNA-seq experiments; however, presently, a pipeline does not exist to maximize variant calling accuracy. Furthermore, molecular duplicates generated in these experiments have not been utilized to optimally detect variant co-expression. Herein, we introduce scSNV designed from the ground up to "collapse" molecular duplicates and accurately identify variants and their co-expression. We demonstrate that scSNV is fast, with a reduced false-positive variant call rate, and enables the co-detection of genetic variants and A>G RNA edits across twenty-two samples.

The impact of COVID-19 on oral surgery training.

Aim: Coronavirus disease, caused by severe acute respiratory syndrome coronavirus 2, originated in December 2019 from Wuhan, China. This virus has spread worldwide, with over 16 million cases and over 650,000 deaths. The novel coronavirus disease (COVID-19) has resulted in significant impact on the livelihoods of the British public and has had implications for healthcare delivery. The cancellation of elective procedures is likely to affect oral surgery (OS) specialty training. This paper aims to ascertain the extent of any impact of COVID-19 on OS specialty training. Material and Methods: A survey was created for OS specialty trainees in the United Kingdom. A variety of questions were used, including multiple choice, yes/no, Likert scales and free text answers. All questions were related to the impact of COVID-19 on training. Results: A total of 34 full responses were recorded. Results showed that COVID-19 has had an immense impact on OS training, with most trainees recording high anxiety levels regarding the future of their training. The overall experience of most trainees involved a reduction in logbook procedures, cancelled study days, courses or conferences. Conclusion: Although OS training has been deficient during this period, some trainees reported positive experiences while redeployed in other fields or specialties. In addition, we highlight the significant effect on trainees' mental health. Most trainees suggested a training period extension to remedy deficiencies. From this paper, we identify the wide-ranging effects of the pandemic, and OS trainees now await decisions on the future of specialty training.

Estimating wildlife vaccination coverage using genetic methods.

Vaccination is a useful approach for the control of disease in wildlife populations. However, its effectiveness is dependent in part on delivery to a sufficient proportion of the target population. Measuring the proportions of wild animal populations that have been vaccinated is challenging and so there is a need to develop robust approaches that can contribute to our understanding of the likely efficacy of wildlife vaccination campaigns. We used a modified capture mark recapture technique to estimate vaccine coverage in a wild population of European badgers (Meles meles) vaccinated by live-trapping and injecting with Bacillus Calmette-Guérin as part of a bovine tuberculosis control initiative in Wales, United Kingdom. Our approach used genetic matching of vaccinated animals to a sample of the wider population to estimate the percentage of badgers that had been vaccinated. Individual-specific genetic profiles were obtained using microsatellite genotyping of hair samples, which were collected directly from trapped and vaccinated badgers and non-invasively from the wider population using hair traps deployed at badger burrows (setts). With two nights of trapping at each sett in an annual campaign, an estimated 50 % (95 % confidence interval 40-60 %) of the badger population received at least one dose of vaccine in a single year. Using a simple population model this suggested that the proportion of the population that would have received at least one dose of vaccine over the course of the four year vaccination campaign was between 67 % and 83 %. This is the first attempt, outside of field trials, to quantify the level of vaccine coverage achieved by trapping and injecting badgers, which is currently the only option for delivering BCG vaccine to this species. The results therefore have specific application to bTB control policy and the novel approach may have wider value in wildlife management and research.

Towards personalized induction therapy for esophageal adenocarcinoma: organoids derived from endoscopic biopsy recapitulate the pre-treatment tumor.

Esophageal adenocarcinoma has few known recurrent mutations and therefore robust, reliable and reproducible patient-specific models are needed for personalized treatment. Patient-derived organoid culture is a strategy that may allow for the personalized study of esophageal adenocarcinoma and the development of personalized induction therapy. We therefore developed a protocol to establish EAC organoids from endoscopic biopsies of naïve esophageal adenocarcinomas. Histologic characterization and molecular characterization of organoids by whole exome sequencing demonstrated recapitulation of the tumors' histology and genomic (~ 60% SNV overlap) characteristics. Drug testing using clinically appropriate chemotherapeutics and targeted therapeutics showed an overlap between the patient's tumor response and the corresponding organoids' response. Furthermore, we identified Barrett's esophagus epithelium as a potential source of organoid culture contamination. In conclusion, organoids can be robustly cultured from endoscopic biopsies of esophageal adenocarcinoma and recapitulate the originating tumor. This model demonstrates promise as a tool to better personalize therapy for esophageal adenocarcinoma patients.

A comprehensive hospital agile preparedness (CHAPs) tool for pandemic preparedness, based on the COVID-19 experience.

COVID-19 presents an unprecedented challenge to hospitals and the systems in which they operate. The primary exponential surge of COVID-19 cases is arguably the most devastating event a hospital will face. In some countries, these surges during the initial outbreak of the disease have resulted in hospitals suffering from significant resource strain, leading to excess patient mortality and negatively impacting staff wellbeing. As experience builds in managing these surges, it has become evident that agile, tailored planning tools are required. The comprehensive hospital agile preparedness (CHAPs) tool provides clinical planners with six key domains to consider that frequently create resource strain during COVID-19; it also allows local planners to identify issues unique to their hospital, system or region. Although this tool has been developed from COVID-19 experiences, it has potential to be modified for a variety of pandemic scenarios according to transmission modes, rates and critical care resource requirements.

Genetic evidence further elucidates the history and extent of badger introductions from Great Britain into Ireland.

The colonization of Ireland by mammals has been the subject of extensive study using genetic methods and forms a central problem in understanding the phylogeography of European mammals after the Last Glacial Maximum. Ireland exhibits a depauperate mammal fauna relative to Great Britain and continental Europe, and a range of natural and anthropogenic processes have given rise to its modern fauna. Previous Europe-wide surveys of the European badger (Meles meles) have found conflicting microsatellite and mitochondrial DNA evidence in Irish populations, suggesting Irish badgers have arisen from admixture between human imported British and Scandinavian animals. The extent and history of contact between British and Irish badger populations remains unclear. We use comprehensive genetic data from Great Britain and Ireland to demonstrate that badgers in Ireland's northeastern and southeastern counties are genetically similar to contemporary British populations. Simulation analyses suggest this admixed population arose in Ireland 600-700 (CI 100-2600) years before present most likely through introduction of British badgers by people. These findings add to our knowledge of the complex colonization history of Ireland by mammals and the central role of humans in facilitating it.

Author Correction: Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.

Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary and metastatic tumors. Transcriptome analysis demonstrated that molecular subtypes are a product of a gene expression continuum driven by a mixture of intratumoral subpopulations, which was confirmed by single-cell analysis. Integrated whole-genome analysis uncovered that molecular subtypes are linked to specific copy number aberrations in genes such as mutant KRAS and GATA6. By mapping tumor genetic histories, tetraploidization emerged as a key mutational process behind these events. Taken together, these data support the premise that the constellation of genomic aberrations in the tumor gives rise to the molecular subtype, and that disease heterogeneity is due to ongoing genomic instability during progression.

Morphological classification of pancreatic ductal adenocarcinoma that predicts molecular subtypes and correlates with clinical outcome.

INTRODUCTION: Transcriptional analyses have identified several distinct molecular subtypes in pancreatic ductal adenocarcinoma (PDAC) that have prognostic and potential therapeutic significance. However, to date, an indepth, clinicomorphological correlation of these molecular subtypes has not been performed. We sought to identify specific morphological patterns to compare with known molecular subtypes, interrogate their biological significance, and furthermore reappraise the current grading system in PDAC. DESIGN: We first assessed 86 primary, chemotherapy-naive PDAC resection specimens with matched RNA-Seq data for specific, reproducible morphological patterns. Differential expression was applied to the gene expression data using the morphological features. We next compared the differentially expressed gene signatures with previously published molecular subtypes. Overall survival (OS) was correlated with the morphological and molecular subtypes. RESULTS: We identified four morphological patterns that segregated into two components ('gland forming' and 'non-gland forming') based on the presence/absence of well-formed glands. A morphological cut-off (≥40% 'non-gland forming') was established using RNA-Seq data, which identified two groups (A and B) with gene signatures that correlated with known molecular subtypes. There was a significant difference in OS between the groups. The morphological groups remained significantly prognostic within cancers that were moderately differentiated and classified as 'classical' using RNA-Seq. CONCLUSION: Our study has demonstrated that PDACs can be morphologically classified into distinct and biologically relevant categories which predict known molecular subtypes. These results provide the basis for an improved taxonomy of PDAC, which may lend itself to future treatment strategies and the development of deep learning models.

Integration of Genomic and Transcriptional Features in Pancreatic Cancer Reveals Increased Cell Cycle Progression in Metastases.

We integrated clinical, genomic, and transcriptomic data from 224 primaries and 95 metastases from 289 patients to characterize progression of pancreatic ductal adenocarcinoma (PDAC). Driver gene alterations and mutational and expression-based signatures were preserved, with truncations, inversions, and translocations most conserved. Cell cycle progression (CCP) increased with sequential inactivation of tumor suppressors, yet remained higher in metastases, perhaps driven by cell cycle regulatory gene variants. Half of the cases were hypoxic by expression markers, overlapping with molecular subtypes. Paired tumor heterogeneity showed cancer cell migration by Halstedian progression. Multiple PDACs arising synchronously and metachronously in the same pancreas were actually intra-parenchymal metastases, not independent primary tumors. Established clinical co-variates dominated survival analyses, although CCP and hypoxia may inform clinical practice.