Evaluating an mRNA based body fluid identification test using SYBR green fluorescent dye and real-time PCR

The requirement to have more definitive and wider ranging body fluid identification (BFID) tests has resulted in a range of mRNA based real-time PCR BFI assays utilising Taqman fluorescent dye. An attempt to make a reliable and cost effective BFI test utilising the alternative SYBR Green fluorescent dye was carried out. RNA was extracted from blood and saliva stains from both male and female donors, this was then reverse transcribed using M-MLV and random hexamers. Using real-time PCR, relative quantitation of blood and saliva specific markers was carried out on the cDNA from the blood and saliva samples using the SYBR® Green fluorescent dye. Melting curve analysis was also performed immediately following PCR amplification. The relative quantitation values were calculated using the formula 2-ΔΔCT and all samples were normalised to reference gene 18s rRNA. The results revealed good specificity for a number of markers using this chemistry, however some markers were undetected. Blood markers NCF2, SPTB, PBDG and saliva specific markers HTN3, SPRR1A, KRT4 and KRT13 were investigated. In the SYBR green studies, the most specific markers were NCF2, KRT4, KRT13 and SPRR1A, showing reproducible results in a number of studies. Blood marker SPTB also appeared to be specific to blood however the melt curve data for this marker in each study was questionable given the low melting temperature for the amplified products. Blood specific marker PBGD, and saliva specific marker HTN3 were not detected using SYBR Green and saliva marker STATH was detected however in each case appeared to be non-specific in nature when them melt curves were analysed. Analysis of the 18s rRNA Ct values showed a higher expression in saliva than in blood in almost all instances, this may be due to collection of a higher number of cells when using a buccal swab, coupled with the inability to accurately quantify the RNA extracts before reverse transcription. Taqman assays were run on all markers as an additional test, to compare with the SYBR green data. All markers except SPTB showed very good specificity for their respective body fluids. SPTB, like in the SYBR green studies was detected in blood more than saliva, however detection was never consistent in each sample. It can therefore be said that real-time PCR using SYBR Green dye was capable of identifying specific mRNA markers blood and saliva however, the lack of specificity for this type of assay makes its use as a routine identification of body fluids in forensic casework not suitable. The main aim of this study was to develop a more cost effective BFID and as such involved the use of SYBR Green as a cheaper alternative to TaqMan. However, throughout these studies, it appeared to be quite costly in terms of validating a SYBR Green experiment, as more reagents were required in the long run due to vast amount of no template controls required per experiment. It therefore would appear that while SYBR Green is cheaper to buy, the cost to validate these type of experiments can be quite high, due to the non-specific nature of the dye itself. The SYBR Green studies were also much more time consuming with regards to data interpretation as post analysis of the amplification plot and melt curves is a necessity with this detection chemistry to ensure successful interpretation of the data

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Evaluating an MRNA based body fluid identification test using sybr green fluorescent dye and real-time PCR

The requirement to have more definitive and wider ranging body fluid identification (BFI) tests has resulted in a range of mRNA based real-time PCR BFI assays utilising Taqman fluorescent dye. An attempt to make a reliable BFI test utilising the alternative SYBR Green fluorescent dye was carried out. Samples were extracted from blood and saliva stains and then reverse transcribed using M-MLV and random hexamers. Using real-time PCR, relative quantitation of SPTB (blood), NCF2 (blood), KRT4 (saliva) and SPRR1A (saliva) was carried out on cDNA from the blood and saliva samples using the SYBR Green reagents. Melting curve analysis was also conducted following the amplification step. The RQ values were calculated using the formula 2-ΔΔCT. In all cases, the blood markers (SPTB and NCF2) were under expressed in saliva and over expressed in blood. The saliva markers (KRT4 and SPRR1A) were over expressed in saliva and under expressed in blood. Verification of the amplicons was carried out using melting curve analysis. Relatively high levels of fluorescence were also detected in the reverse transcription blanks and negative controls; despite stringent anti-contamination procedures. This along with the melting curve analysis results suggest that the amplification may be a by-product of the test itself rather than contamination; due to the lack of specificity of the SYBR Green fluorescent dye. This was verified by contamination monitoring using negative controls along with SYBR Green and Taqman fluorescent dyes. This shows that a SYBR Green based BFI test could be developed and it may be more appropriate to use than a Taqman based BFI test in a commercial forensic science environment. However, the amplification detected in the reverse transcription blanks and negative controls may render the results of a SYBR Green based BFI test unreliable and insufficiently robust for use in a court of law

Characterising degradation profiles of RNA molecules within blood stains: Pilot studies

Introduction One of the capability gaps in a forensic investigation is the estimation or determination of the age of the stain. For example, was the blood stain deposited today or two months ago; was the semen stain deposited yesterday or five days ago, and was the balaclava worn by the suspect at the time of the robbery? A number of strategies have been proposed, one of which is the characterisation of RNA molecules as they degrade over time. Preliminary work targeting housekeeping genes have demonstrated a proof of principle [1]. Other strategies have included chemical options, for example Raman spectroscopy [2, 3]; however, to maximise the usefulness of the data, a genetic strategy would be preferred – in order that closer links with DNA profiles can be made. Consequently, characterising the pattern of RNA expression over time since deposition should produce some insights in the degradation profile. Such insights may lead to being able to offer an opinion as to the age of the stain in criminal case work. Different time scales were explored, including a 2 year study (collection at 2 year and 10 months), a 22 days study (with a collection everyday) and a 12 hour study (with a collection every hour). The data was brought together from three different studies

The Use of Forensic Messenger RNA (mRNA) Analysis to Determine Stain Age

After attending this presentation, attendees will understand the principles of forensic mRNA analysis and learn how it may be used as a method to determine the age of biological stains found at crime scenes. This presentation will impact the forensic science community by proposing a novel method for determining the age of biological stains. The proposed method could prove crucial for forensic casework and could run parallel to the predominant DNA analysis method. Where DNA analysis is used to identify a suspect, mRNA analysis could be used to determine the age of biological stains, that in turn could place the suspect at the scene of a crime at a specific time. RNA has been presumed to be very unstable, as one of its features is that it degrades rapidly as it is broken down by ribonucleases. In previous literature however; it has been shown that RNA may be extracted from biological stains up to 16-years-old. For stain age determination, it has been suggested that using a decay rate ratio, derived from two endogenous controls differentially expressed within the body fluid stain, should eliminate the effect of any external decomposition factors. This should then be expressed as a linear change in mRNA expression over time. The majority of previous research concentrates on determining the age of bloodstains. In this study, blood and saliva stains were used. The goal of the study was to try to determine the age of these biological stains by isolating mRNA and quantifying the expression of housekeeping genes 18s, GAPDH, and ACTB also known as β-Actin. This study aims to demonstrate proof of principle and explore any limitations to a decay rate ratio, to see if there is any correlation between the age of a stain and the amount of genetic material present. Blood andsaliva samples havebeencollected ona regular basis overthe past two years. Bloodstains were prepared using the finger prick method and depositing blood on to a sterile filter paper. Saliva stains were prepared by swabbing the inside of the cheek with a buccal swab. All samples were taken from healthy individuals. The samples were stored at room temperature and protected from sunlight. Both blood and saliva samples were extracted using Qiagen RNeasy Mini Kit with the appropriate modifications for each body fluid. The extracted samples then underwent RNA quantitation, DNase digestion, and reverse transcription using M-MLV reverse transcriptase and random hexamers. The resulting complimentary DNA (cDNA) was then quantified using absolute quantitation on a 7500 Fast Real-Time PCR System, using pre-designed Taqman Gene Expression Assays for human housekeeping genes 18s, GAPDH, and ACTB (β-Actin). Only two housekeeping genes were run together at one time, this was to compare the differences between all three, for example the decay rate ratio between 18s and GAPDH as well as 18s with ACTB and then GAPDH with ACTB. The two values from quantifying the two housekeeping genes were then expressed as a ratio. The proof of principle would be demonstrated by a linear downward expression of the two genes over time. The results from initial experiments show that mRNA can be extracted from stains up to two years old. Further experiments are being carried out to obtain enough data to determine the proof of principle. Future work will involve optimising the protocols and identifying the shortest stain age per body fluid as well as exploring the use of more housekeeping genes. With bloodstains being the current focus in most of the literature, experiments will be carried out to determine the expression levels of human housekeeping genes in semen to see if it is possible to identify the time of deposition. Identifying the time a semen stain was deposited in a sexual offence case could prove to be vital when trying to convict or exonerate an individual