Advances in single-cell long-read sequencing technologies.

With an increase in accuracy and throughput of long-read sequencing technologies, they are rapidly being assimilated into the single-cell sequencing pipelines. For transcriptome sequencing, these techniques provide RNA isoform-level information in addition to the gene expression profiles. Long-read sequencing technologies not only help in uncovering complex patterns of cell-type specific splicing, but also offer unprecedented insights into the origin of cellular complexity and thus potentially new avenues for drug development. Additionally, single-cell long-read DNA sequencing enables high-quality assemblies, structural variant detection, haplotype phasing, resolving high-complexity regions, and characterization of epigenetic modifications. Given that significant progress has primarily occurred in single-cell RNA isoform sequencing (scRiso-seq), this review will delve into these advancements in depth and highlight the practical considerations and operational challenges, particularly pertaining to downstream analysis. We also aim to offer a concise introduction to complementary technologies for single-cell sequencing of the genome, epigenome and epitranscriptome. We conclude by identifying certain key areas of innovation that may drive these technologies further and foster more widespread application in biomedical science.

English translation and cross-cultural validation of the patient-reported outcome measurement-haemorrhoidal impact and satisfaction score (PROM-HISS).

AIM: The aim of this study was to translate the Dutch patient-reported outcome measure-haemorrhoidal impact and satisfaction score (PROM-HISS) to English and perform a cross-cultural validation. METHOD: The ISPOR good practice guidelines for the cross-cultural validation of PROMs were followed and included two steps: (1) Two forward and two backward translations. The forward translation concerned the translation from the source language (Dutch) to the target language (English), performed by two independent English speakers, one medical doctor and one nonmedical. Subsequently, a discussion about discrepancies in the reconciled version was performed by a stakeholder group. (2) Cognitive interviews were held with patients with haemorrhoidal disease (HD), probing the comprehensibility and comprehensiveness of the PROM-HISS. RESULTS: Discrepancies in the reconciled forward translation concerned the terminology of HD symptoms. Furthermore, special attention was paid to the response options, ranging from "not at all", indicating minor symptoms, to "a lot", implying many symptoms. Consensus among the stakeholder group about the final version of the translated PROM-HISS was reached. Interviews were conducted with 10 native English-speaking HD patients (30% female), with a mean age of 44 years (24-83) and primarily diagnosed with grade II HD (80%). The mean time to complete the PROM-HISS was 1 min 43 s. Patients showed a good understanding of the questions and response options, found all items relevant and did not miss important symptoms or topics. CONCLUSION: The translated English language PROM-HISS is a valid tool to assess symptoms of HD, its impact on daily activities and patient satisfaction with HD treatment.

Improving Access and Timeliness of Early Palliative Care Specialist Assessment for Patients With Advanced Lung Cancer in a Rapid Assessment Clinic.

Background: Integrating palliative care in the management of patients with lung cancer improves quality of life, patient satisfaction, and overall survival. However, few patients receive timely palliative care consultation. The Lung Diagnostic Assessment Program (LDAP) in Southeastern Ontario is a multidisciplinary rapid assessment clinic that expedites the diagnosis and management of patients with suspected lung cancer. Objectives: We sought to increase the percentage of LDAP patients with stage IV lung cancer receiving palliative care consultation within three months of diagnosis. Design: We integrated a palliative care specialist in LDAP to facilitate in-person, same-visit consultation for patients with a new lung cancer diagnosis. Setting/Subjects: Five hundred fifty patients in a Canadian academic center (154 initial baseline, 104 COVID baseline, 292 post-palliative care integration). Measurements: Baseline data were established using retrospective chart review (February-June 2020 and December 2020-March 2021 due to COVID-19 pandemic). Data were collected prospectively to assess improvement (March-August 2021). Statistical Process Control charts assessed for special cause variation; chi-square tests assessed for differences between groups. Results: The percentage of patients with stage IV lung cancer seen by palliative care within three months increased from 21.8% (12/55) during early-COVID baseline to 49.2% (32/65) after palliative care integration (p < 0.006). Palliative care integration in LDAP reduced mean time from referral to consultation from 24.8 to 12.3 days, including same-day consultation for 15/32 (46.8%) patients with stage IV disease. Conclusions: Integrating palliative care specialists into LDAP improved the timeliness of palliative care assessment for patients with stage IV lung cancer.

Predicting necrotising soft tissue infections in people who inject drugs: poor performance of the Laboratory Risk Indicator for Necrotising Fasciitis score and development of a novel clinical predictive nomogram in a retrospective cohort with internal validation.

INTRODUCTION: Necrotising soft tissue infections (NSTI) can threaten life and limb. Early identification and urgent surgical debridement are key for improved outcomes. NSTI can be insidious. Scoring systems, like the Laboratory Risk Indicator for Necrotising Fasciitis (LRINEC), exist to aid diagnosis. People who inject drugs (PWID) are high risk for NSTI. This study aimed to assess the utility of the LRINEC in PWID with lower limb infections and develop a predictive nomogram. METHODS: A retrospective database of all hospital admissions due to limb-related complications secondary to injecting drug use between December 2011 and December 2020 was compiled through discharge codes and a prospectively maintained Vascular Surgery database. All lower limb infections were extracted from this database, dichotomised by NSTI and non-NSTI with the LRINEC applied. Specialty management times were evaluated. Statistical analyses involved: chi-square; Analysis of "variance"; Kaplan-Meier, and receiver operating characteristic curves. Nomograms were developed to facilitate diagnosis and predict survival. RESULTS: There were 557 admissions for 378 patients, with 124 (22.3%; 111 patients) NSTI. Time from admission to: theatre and computed tomography imaging respectively varied significantly between specialties ( P =0.001). Surgical specialties were faster than medical ( P =0.001). Vascular surgery received the most admissions and had the quickest time to theatre. During follow-up there were 79 (20.9%) deaths: 27 (24.3%) NSTI and 52 (19.5%) non-NSTI. LRINEC ≥6 had a positive predictive value of 33.3% and sensitivity of 74% for NSTI. LRINEC <6 had a negative predictive value of 90.7% and specificity of 63.2% for non-NSTI. Area under the curve was 0.697 (95% CI: 0.615-0.778). Nomogram models found age, C-reactive protein, and non-linear albumin to be significant predictors of NSTI, with age, white cell count, sodium, creatinine, C-reactive protein, and albumin being significant in predicting survival on discharge. CONCLUSION: There was reduced performance of the LRINEC in this PWID cohort. Diagnosis may be enhanced through use of this predictive nomogram.

Single-Cell DNA Methylation Analysis in Cancer.

Morphological, transcriptomic, and genomic defects are well-explored parameters of cancer biology. In more recent years, the impact of epigenetic influences, such as DNA methylation, is becoming more appreciated. Aberrant DNA methylation has been implicated in many types of cancers, influencing cell type, state, transcriptional regulation, and genomic stability to name a few. Traditionally, large populations of cells from the tissue of interest are coalesced for analysis, producing averaged methylome data. Considering the inherent heterogeneity of cancer, analysing populations of cells as a whole denies the ability to discover novel aberrant methylation patterns, identify subpopulations, and trace cell lineages. Due to recent advancements in technology, it is now possible to obtain methylome data from single cells. This has both research and clinical implications, ranging from the identification of biomarkers to improved diagnostic tools. As with all emerging technologies, distinct experimental, bioinformatic, and practical challenges present themselves. This review begins with exploring the potential impact of single-cell sequencing on understanding cancer biology and how it could eventually benefit a clinical setting. Following this, the techniques and experimental approaches which made this technology possible are explored. Finally, the present challenges currently associated with single-cell DNA methylation sequencing are described.

Efficacy of low-dose and/or adjuvant methadone in palliative medicine.

OBJECTIVES: To summarise the current body of published evidence on the use of low-dose and/or adjuvant methadone in the palliative care setting. METHODS: The authors searched multiple databases (PubMED, SCORPUS, EMBASE and the Cochrane library) for relevant articles using the terms 'methadone', 'palliative', 'low dose' and 'adjuvant'. The review was restricted to articles published between 2003 and 2018. Paediatric and single-case studies were also excluded. Evidence quality was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) method. RESULTS: Our search yielded 171 results, of which seven met the inclusion criteria. Four were retrospective chart reviews, one was a retrospective cohort study, one was a case series and one was a double-blind randomised control trial. The overall quality was found to be very low. Of the seven articles, all seven reported some improvement in pain with the addition of low-dose or adjuvant methadone. This improvement was statistically significant in four out of seven articles; statistical significance was not commented on in the remaining three articles. CONCLUSION: While case series and chart reviews offer promising results about the utility of adjuvant and/or low-dose methadone in the management of complex pain, the very low evidence quality, relative dearth of studies and near absence of randomised controlled trials make it impossible to draw firm conclusions. Thus, while very preliminary evidence suggests methadone is a potentially effective and valuable agent, further research must be performed before such findings can be implemented into clinical practice.

Structure of phospholipase Cε reveals an integrated RA1 domain and previously unidentified regulatory elements.

Phospholipase Cε (PLCε) generates lipid-derived second messengers at the plasma and perinuclear membranes in the cardiovascular system. It is activated in response to a wide variety of signals, such as those conveyed by Rap1A and Ras, through a mechanism that involves its C-terminal Ras association (RA) domains (RA1 and RA2). However, the complexity and size of PLCε has hindered its structural and functional analysis. Herein, we report the 2.7 Å crystal structure of the minimal fragment of PLCε that retains basal activity. This structure includes the RA1 domain, which forms extensive interactions with other core domains. A conserved amphipathic helix in the autoregulatory X-Y linker of PLCε is also revealed, which we show modulates activity in vitro and in cells. The studies provide the structural framework for the core of this critical cardiovascular enzyme that will allow for a better understanding of its regulation and roles in disease.

Profiling lung adenocarcinoma by liquid biopsy: can one size fit all?

BACKGROUND: Cancer is first and foremost a disease of the genome. Specific genetic signatures within a tumour are prognostic of disease outcome, reflect subclonal architecture and intratumour heterogeneity, inform treatment choices and predict the emergence of resistance to targeted therapies. Minimally invasive liquid biopsies can give temporal resolution to a tumour's genetic profile and allow the monitoring of treatment response through levels of circulating tumour DNA (ctDNA). However, the detection of ctDNA in repeated liquid biopsies is currently limited by economic and time constraints associated with targeted sequencing. METHODS: Here we bioinformatically profile the mutational and copy number spectrum of The Cancer Genome Network's lung adenocarcinoma dataset to uncover recurrently mutated genomic loci. RESULTS: We build a panel of 400 hotspot mutations and show that the coverage extends to more than 80% of the dataset at a median depth of 8 mutations per patient. Additionally, we uncover several novel single-nucleotide variants present in more than 5% of patients, often in genes not commonly associated with lung adenocarcinoma. CONCLUSION: With further optimisation, this hotspot panel could allow molecular diagnostics laboratories to build curated primer banks for 'off-the-shelf' monitoring of ctDNA by droplet-based digital PCR or similar techniques, in a time- and cost-effective manner.

BMP2/BMPR1A is linked to tumour progression in dedifferentiated liposarcomas.

Bone Morphogenic Protein 2 (BMP2) is a multipurpose cytokine, important in the development of bone and cartilage, and with a role in tumour initiation and progression. BMP2 signal transduction is dependent on two distinct classes of serine/threonine kinase known as the type I and type II receptors. Although the type I receptors (BMPR1A and BMPR1B) are largely thought to have overlapping functions, we find tissue and cellular compartment specific patterns of expression, suggesting potential for distinct BMP2 signalling outcomes dependent on tissue type. Herein, we utilise large publicly available datasets from The Cancer Genome Atlas (TCGA) and Protein Atlas to define a novel role for BMP2 in the progression of dedifferentiated liposarcomas. Using disease free survival as our primary endpoint, we find that BMP2 confers poor prognosis only within the context of high BMPR1A expression. Through further annotation of the TCGA sarcoma dataset, we localise this effect to dedifferentiated liposarcomas but find overall BMP2/BMP receptor expression is equal across subsets. Finally, through gene set enrichment analysis we link the BMP2/BMPR1A axis to increased transcriptional activity of the matrisome and general extracellular matrix remodelling. Our study highlights the importance of continued research into the tumorigenic properties of BMP2 and the potential disadvantages of recombinant human BMP2 (rhBMP2) use in orthopaedic surgery. For the first time, we identify high BMP2 expression within the context of high BMPR1A expression as a biomarker of disease relapse in dedifferentiated liposarcomas.

BDNF Val66Met polymorphism and lithium response: a meta-analysis.

AIM: To characterize the impact of the Val66Met polymorphism on lithium response in patients with bipolar disorder. METHODS: A systematic literature search of MEDLINE, Web of Science, PsychINFO and Cochrane CENTRAL was conducted from the earliest possible date through to 1 July 2012. The search was performed using the following medical subject headings: bipolar disorder, lithium, lithium carbonate, pharmacogenomics, pharmacogenetics, polymorphism and brain-derived neurotrophic factor. Five of 71 identified studies met the inclusion criteria. Data were abstracted using a standardized data abstraction tool. For categorical end points, the pooled odds ratio with 95% CI was calculated. Random effects models were used for analysis. RESULTS: The Val66Met polymorphism did not predict response to prophylactic lithium in this combined population (odds ratio: 2.67; p = 0.078). CONCLUSION: This analysis suggests that the Val66Met polymorphism does not predict response to lithium treatment in bipolar disorder in this combined population. Prospective studies are needed to clearly define the role of Val66Met polymorphism of BDNF in lithium response.