Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles

Preparation of multi-omics grade extracellular vesicles by density-based fractionation of urine

The multidimensional cargo of extracellular vesicles (EV) released in urine is a reflection of the pathophysiological processes occurring within their cells and tissues of origin in the urogenital system. Here, we describe a step-by-step protocol for density-based separation of urinary EV with high specificity and repeatability. The implementation of integrative omics allows the study of the molecular complexity of highly purified urinary EV, supporting the identification of EV-specific functions and biomarkers

Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated T-s (S-phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity-based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object-based colocalization approach was devised to score the relative number of double-positive cells. Using this approach, T-s (S-phase duration) in the main population of neoblasts was similar to 13 h. During early regeneration, no significant change in T-s was observed

Detection of bladder cancer with aberrantly fucosylated ITGA3

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay

Detection of bladder cancer with aberrantly fucosylated ITGA3

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-dopednanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCaderived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay.</p

Impact of the COVID-19 pandemic on emergency adult surgical patients and surgical services: an international multi-center cohort study and department survey.

OBJECTIVES: The PREDICT study aimed to determine how the COVID-19 pandemic affected surgical services and surgical patients and to identify predictors of outcomes in this cohort. BACKGROUND: High mortality rates were reported for surgical patients with COVID-19 in the early stages of the pandemic. However, the indirect impact of the pandemic on this cohort is not understood, and risk predictors are yet to be identified. METHODS: PREDICT is an international longitudinal cohort study comprising surgical patients presenting to hospital between March and August 2020, conducted alongside a survey of staff redeployment and departmental restructuring. A subgroup analysis of 3176 adult emergency patients, recruited by 55 teams across 18 countries is presented. RESULTS: Among adult emergency surgical patients, all-cause in-hospital mortality (IHM) was 3 6%, compared to 15 5% for those with COVID-19. However, only 14 1% received a COVID-19 test on admission in March, increasing to 76 5% by July.Higher Clinical Frailty Scale scores (CFS >7 aOR 18 87), ASA grade above 2 (aOR 4 29), and COVID-19 infection (aOR 5 12) were independently associated with significantly increased IHM.The peak months of the first wave were independently associated with significantly higher IHM (March aOR 4 34; April aOR 4 25; May aOR 3 97), compared to non-peak months.During the study, UK operating theatre capacity decreased by a mean of 63 6% with a concomitant 27 3% reduction in surgical staffing. CONCLUSION: The first wave of the COVID-19 pandemic significantly impacted surgical patients, both directly through co-morbid infection and indirectly as shown by increasing mortality in peak months, irrespective of COVID-19 status.Higher CFS scores and ASA grades strongly predict outcomes in surgical patients and are an important risk assessment tool during the pandemic

Tetraspanin immunoassay for the detection of extracellular vesicles and renal cell carcinoma

Half of patients with renal cell carcinoma (RCC) develop metastases. New and noninvasive biomarkers are needed for the diagnosis of RCC. The study aims to develop an EV-based assay for the detection of RCC using a highly sensitive nanoparticle-aided time-resolved fluorescence immunoassay (NP-TRFIA). To confirm the presence of tetraspanins on EVs, size exclusion chromatography is used to separate EV- and PE-fractions from RCC4, 786-O, and HEK293 cell lines. EV- and PE-fractions are quantified using NP-TRFIA assays established for CD9, CD63, CD81, and CD151. Tetraspanins are measured from RCC CCM and serum samples of RCC (n = 14), benign (n = 17), and healthy (n = 9) individuals. Among the tetraspanins, CD63 exhibits 3-5-fold higher expression on RCC4 and 786-O CCM compared to HEK293. A sandwich CD63-CD63 assay demonstrates significant discrimination of RCC patients from benign (p = 0.0003), and healthy (p = 0.005) individuals, respectively. Similarly, the CD81-CD81 assay also enables significant separation of RCC patients compared to benign (p = 0.014), and healthy (p = 0.003) controls, respectively. This suggests that RCC cell lines and serum of RCC patients show higher amounts of CD63- and CD81-EVs compared to controls. Detection of these EVs using NP-TRFIA approach may play a vital role in the detection of RCC.</p