A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

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.

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.

TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA.

Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

RNASequel: accurate and repeat tolerant realignment of RNA-seq reads.

RNA-seq is a key technology for understanding the biology of the cell because of its ability to profile transcriptional and post-transcriptional regulation at single nucleotide resolutions. Compared to DNA sequencing alignment algorithms, RNA-seq alignment algorithms have a diminished ability to accurately detect and map base pair substitutions, gaps, discordant pairs and repetitive regions. These shortcomings adversely affect experiments that require a high degree of accuracy, notably the ability to detect RNA editing. We have developed RNASequel, a software package that runs as a post-processing step in conjunction with an RNA-seq aligner and systematically corrects common alignment artifacts. Its key innovations are a two-pass splice junction alignment system that includes de novo splice junctions and the use of an empirically determined estimate of the fragment size distribution when resolving read pairs. We demonstrate that RNASequel produces improved alignments when used in conjunction with STAR or Tophat2 using two simulated datasets. We then show that RNASequel improves the identification of adenosine to inosine RNA editing sites on biological datasets. This software will be useful in applications requiring the accurate identification of variants in RNA sequencing data, the discovery of RNA editing sites and the analysis of alternative splicing.

Mouthwash use and associated head and neck cancer risk.

DATA SOURCES: All studies with questionnaire items on mouthwash use in the International Head and Neck Cancer Epidemiology Consortium (INHANCE). DATA EXTRACTION AND SYNTHESIS: Pooled analysis data from case controlled studies using Individual Patient Data (IPD) meta-analysis methods. Logistic regression was used to assess the association of mouthwash use with cancers of the oral cavity, oropharynx, hypopharynx and larynx, adjusting for study, age, sex, pack-years of tobacco smoking, number of alcoholic drinks/day and education. RESULTS: Eight thousand, nine hundred and eighty-one cases of head and neck cancer and 10,090 controls from 12 case-control studies with comparable information on mouthwash use were included in the analysis. Compared with never users of mouthwash, the odds ratio (OR) of all head and neck cancers was 1.01 [95% confidence interval (CI): 0.94-1.08] for ever users, based on 12 studies. The corresponding ORs of cancer of the oral cavity and oropharynx were 1.11 (95% CI: 1.00-1.23) and 1.28 (95% CI: 1.06-1.56), respectively. CONCLUSIONS: Although limited by the retrospective nature of the study and the limited ability to assess risks of mouthwash use in nonusers of tobacco and alcohol, this large investigation shows potential risks for head and neck cancer subsites and in long-term and frequent users of mouthwash.

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.

Population-based comparison of two feeding tube approaches for head and neck cancer patients receiving concurrent systemic-radiation therapy: is a prophylactic feeding tube approach harmful or helpful?

PURPOSE: The purpose of this study is to compare patient outcomes between a therapeutic versus a prophylactic gastrostomy tube (GT) placement approach in patients treated with concurrent systemic and radiation (SRT) therapy for head and neck cancer (HNC). METHODS: Outcomes were compared between all HNC patients treated with concurrent SRT from January 2001 to June 2009 from a center that only places GTs therapeutically when clinically necessary (center A) versus a center that generally places them prophylactically (center B). RESULTS: A total of 445 patients with HNC were identified, with 63 % from center A. As anticipated, GTs were placed less commonly in center A compared to B (31 versus 88 %; p < 0.001). Center B had a significantly higher number of GT complications (p < 0.001), including infection (16 versus 5 %), leakage (10 versus 2 %), and blockage (3 versus 1 %). Conversely, center A had a higher admission rate (27 versus 13 %, p = 0.001), most prominent for GT-related issues (15 versus 6 %). Center B had higher GT dependence at 90 days post-radiation therapy (34 versus 12 %; p < 0.001), but not at 1 year (11 versus 10 %; p = 0.74). There was no significant difference in the proportion of head and neck patients who had a 10 % weight loss at 1 year (compared to baseline) between centers A and B (42 versus 53 %, p = 0.07). There was no significant difference in the overall survival (A versus B, HR = 0.99; p = 0.96). CONCLUSION: A prophylactic GT approach results in exposing higher number of patients to GT complications. The higher rate of hospitalizations using a therapeutic approach suggests that patients are sicker when GTs are required. Given the similar weight loss and survival, a therapeutic approach at an earlier stage of need may be a preferable approach, when access to prompt GT placement is available.

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.

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.

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.

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.

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.

Bacillus Calmette-Guérin vaccination reduces the severity and progression of tuberculosis in badgers.

Control of bovine tuberculosis (TB) in cattle has proven particularly challenging where reservoirs of infection exist in wildlife populations. In Britain and Ireland, control is hampered by a reservoir of infection in Eurasian badgers (Meles meles). Badger culling has positive and negative effects on bovine TB in cattle and is difficult, costly and controversial. Here we show that Bacillus Calmette-Guérin (BCG) vaccination of captive badgers reduced the progression, severity and excretion of Mycobacterium bovis infection after experimental challenge. In a clinical field study, BCG vaccination of free-living badgers reduced the incidence of positive serological test results by 73.8 per cent. In common with other species, BCG did not appear to prevent infection of badgers subjected to experimental challenge, but did significantly reduce the overall disease burden. BCG vaccination of badgers could comprise an important component of a comprehensive programme of measures to control bovine TB in cattle.

Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII.

Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.

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.

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.

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.