Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Use of a rapid electronic survey methodology to estimate blood donors' potential exposure to emerging infectious diseases: Application of a statistically representative sampling methodology to assess risk in US blood centers.

Risk assessments of transfusion-transmitted emerging infectious diseases (EIDs) are complicated by the fact that blood donors' demographics and behaviors can be different from the general population. Therefore, when assessing potential blood donor exposure to EIDs, the use of general population characteristics, such as U.S. travel statistics, may invoke uncertainties that result in inaccurate estimates of blood donor exposure. This may, in turn, lead to the creation of donor deferral policies that do not match actual risk. STUDY DESIGN AND METHODS: This article reports on the development of a system to rapidly assess EID risks for a nationally representative portion of the U.S. blood donor population. To assess the effectiveness of this system, a test survey was developed and deployed to a statistically representative sample frame of blood donors from five blood collecting organizations. Donors were directed to an online survey to ascertain their recent travel and potential exposure to Middle East respiratory syndrome coronavirus (MERS-CoV). RESULTS: A total of 7128 responses were received from 54 256 invitations. The age-adjusted estimated total number of blood donors potentially exposed to MERS-CoV was approximately 15 640 blood donors compared to a lower U.S. general population-based estimate of 9610 blood donors. CONCLUSION: The structured donor demographic sample-based data provided an assessment of blood donors' potential exposure to an emerging pathogen that was 63% larger than the U.S. population-based estimate. This illustrates the need for tailored blood donor-based EID risk assessments that provide more specific demographic risk intelligence and can inform appropriate regulatory decision making.

Transcriptomic characterisation of the optimised rat model of Walker 256 breast cancer cell-induced bone pain.

In patients with breast cancer, metastases of cancer cells to the axial skeleton may cause excruciating pain, particularly in the advanced stages. The current drug treatments available to alleviate this debilitating pain condition often lack efficacy and/or produce undesirable side effects. Preclinical animal models of cancer-induced bone pain are key to studying the mechanisms that cause this pain and for the success of drug discovery programs. In a previous study conducted in our laboratory, we validated and characterised the rat model of Walker 256 cell-induced bone pain, which displayed several key resemblances to the human pain condition. However, gene level changes that occur in the pathophysiology of cancer-induced bone pain in this preclinical model are unknown. Hence, in this study, we performed the transcriptomic characterisation of the Walker 256 cell line cultured in vitro to predict the molecular genetic profile of this cell line. We also performed transcriptomic characterisation of the Walker 256 cell-induced bone pain model in rats using the lumbar spinal cord and lumbar dorsal root ganglia tissues. Here we show that the Walker 256 cell line resembles the basal-B molecular subtype of human breast cancer cell lines. We also identify several genes that may underpin the progression of pain hypersensitivities in this condition, however, this needs further confirmatory studies. These transcriptomic insights have the potential to direct future studies aimed at identifying various mechanisms underpinning pain hypersensitivities in this model that may also assist in discovery of novel pain therapeutics for breast cancer-induced bone pain.

Correlation of West Nile Virus Incidence in Donated Blood with West Nile Neuroinvasive Disease Rates, United States, 2010-2012.

Over the past decade, West Nile virus (WNV) has spread across the United States. We aggregated blood donor data from 2010-2012 and then calculated the incidence of WNV RNA-positive donations and compared the incidence with neuroinvasive disease (NID) case data from the ArboNET surveillance system. Of 10,107,853 donations, 640 were confirmed positive. The seasonal WNV incidence rate per 100,000 persons was 33.4 (95% CI 22-45) in 2010, 25.7 (95% CI 15-34) in 2011, and 119.9 (95% CI 98-141) in 2012. NID to blood donor ratios were 1 in 164 (95% CI 152-178) in 2010, 1 in 158 (95% CI 145-174) in 2011, and 1 in 131 (95% CI 127-136) in 2012. We updated estimates of the ratio of NID to WNV infection rates, demonstrating stable disease penetrance over the study period. Blood donor WNV RNA screening is a valuable public health tool for WNV surveillance.

Recent viral infection in US blood donors and health-related quality of life (HRQOL).

PURPOSE: Blood donors are considered to be one of the healthiest populations, but relatively little is known about their perceived quality of life. The objective was to examine HRQOL in donors infected with HIV, HBV, HCV or HTLV and a comparison group. METHODS: Donors with confirmed viral infection (cases) and donors who tested false-positive (controls) participated in a multicenter study of US blood donors (2010-2013), funded by the National Heart, Lung and Blood Institute (NHLBI). HRQOL was measured by the EuroQol Five Dimension (EQ-5D) instrument and EQ-5D visual analogue scale (VAS). The lower 25th ‰ of EQ-5D index or VAS score of controls was defined as a "lower HRQOL." RESULTS: A total of 1574 controls completed the HRQOL assessment with a mean EQ-5D index of 0.94 (SD = 0.10) and EQ-VAS of 87.6 (SD = 10.6). Mean EQ-5D index for 192 HIV-, 315 HCV- and 195 HTLV-positive donors were significantly lower than the controls (0.86, 0.83 and 0.87; SD = 0.18, 0.20 and 0.16, respectively, p < 0.001). HBV-positive donors (n = 290) had a similar mean EQ-5D index (0.93, SD = 0.14, p = 0.05) to controls. Anxiety/depression was reported by 34 % of cases, compared with 13 % of controls. In multivariable modeling, the odds of lower HRQOL in HIV, HBV, HCV and HTLV cases were 2.1, 1.6, 2.6 and 2.3 times that of controls, respectively (p < 0.05). CONCLUSIONS: HRQOL reported by blood donors with recent viral infections was relatively high but lower than controls. On average, HRQOL among HCV-positive donors was the lowest and HBV-positive donors reported scores similar to donors without infection.

Motivations for donating and attitudes toward screening policies in US blood donors with viral infection.

BACKGROUND: Differences in motivating factors that contribute to the decision to donate blood between infected and uninfected donors may help to identify areas for improving donor education. STUDY DESIGN AND METHODS: As part of a risk factor study, confirmed-positive donors (cases) based on serology-only (human T-lymphotropic virus [HTLV]) or serology and nucleic acid testing (NAT) or NAT-only (human immunodeficiency virus [HIV], hepatitis B virus [HBV], hepatitis C virus [HCV]), and serology-unconfirmed, NAT-negative false-positive donors (controls) were asked about motivations and opinions toward blood donation. "Test seeking" was inferred if a donor answered "yes" to "I wanted to get my test results" and one of the following: "blood center testing is confidential," "free," "more accurate than other test centers," or "tests will identify problems with my blood." Cases were compared to controls using descriptive and multivariable analyses. RESULTS: Whether a case or control, the most common donation reason was "to help someone in need" (>90% in each group). After adjusting for demographic characteristics, test seeking was not significantly associated with infection status. Test seeking was more common in first-time, younger males and nonwhite, non-Hispanic donors. Of donors with HIV, 13% considered selection policies to be unfair, compared with 1, 2, 0.5, and 6% of donors with HBV, HCV, and HTLV and controls, respectively (adjusted odds ratio for HIV cases vs. controls, 3.9; 95% confidence interval, 2.3-6.7). CONCLUSIONS: Most donors give to help those in need, including HIV-positive donors. Our results establish a baseline from which additional studies can be compared focused on alternate ways to reduce noncompliance and improved messaging to ensure that high-risk potential donors understand the reasons for blood donor screening policies.

Risk factors for retrovirus and hepatitis virus infections in accepted blood donors.

BACKGROUND: Risk factor surveillance among infected blood donors provides information on the effectiveness of eligibility assessment and is critical for reducing risk of transfusion-transmitted infection. STUDY DESIGN AND METHODS: American Red Cross, Blood Systems, Inc., New York Blood Center, and OneBlood participated in a case-control study from 2010 to 2013. Donors with serologic and nucleic acid testing (NAT) or NAT-only confirmed human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), or serology-confirmed human T-lymphotropic virus (HTLV) infections (cases) and donors with false-positive results (controls) were interviewed for putative behavioral and demographic risks. Frequencies and adjusted odds ratios (AORs) from multivariable logistic regression analyses for each exposure in cases compared to controls are reported. RESULTS: In the study, 196 HIV, 292 HBV, 316 HCV, and 198 HTLV cases, and 1587 controls were interviewed. For HIV, sex with an HIV+ person (AOR, 132; 95% confidence interval [CI], 27-650) and male-male sex (AOR, 62; 95% CI, 27-140) were primary risk factors. For HBV, first-time donor status (AOR, 16; 95% CI, 10-27), sex with an injection drug user (IDU; AOR, 11; 95% CI, 5-28), and black race (AOR, 11; 95% CI, 6-19) were primary. For HCV, IDU (AOR, 42; 95% CI, 13-136), first time (AOR, 18; 95% CI, 10-30), and a family member with hepatitis (AOR, 15; 95% CI, 6-40) were primary. For HTLV, sex with an IDU (AOR, 22; 95% CI, 10-48), 55 years old or more (AOR, 21; 95% CI, 8-52], and first time (AOR, 15; 95% CI, 9-24) were primary. CONCLUSIONS: Despite education efforts and risk screening, individuals with deferrable risks still donate; they may fail to understand or ignore or do not believe they have risk. Recipients have potential transfusion-transmitted infection risk because of nondisclosure by donors.

Safety of the blood supply.

BACKGROUND: The transfusion of blood components plays a significant role as supportive therapy in the treatment of patients with cancer. Although blood transfusions help manage complications arising from either the patient's primary condition or associated with therapeutic intervention, their use introduces a new set of risks; therefore, health care professionals must be aware of the potential morbidity introduced by using blood components and endeavor to optimize outcomes by ordering transfusions only when the benefits outweigh the inherent risks. METHODS: This article sought to review the published literature, including the epidemiology of diseases transmissible via transfusion, performance characteristics for assays used for blood donor screening, surveillance activities to detect newly emergent pathogens, and biovigilance activities reported by public health authorities. RESULTS: Effective measures have been implemented to significantly decrease the risk of transmissible diseases associated with transfusion. Reports of viral disease transmitted via transfusion have been nearly eliminated, particularly since the introduction of molecular-based detection technology. The transmission of bacteria and parasites still represents a threat to the use of cellular blood components. Transfusion-associated human prion disease has not been reported in the United States. Immune-mediated reactions due to donor-recipient incompatibility remain a challenge. CONCLUSIONS: Transmissible agents most commonly associated with risks due to transfusion are no longer a major threat; however, a significant challenge remains with regard to addressing the need for quick response mechanisms to manage emerging pathogens with the potential for rapid spread, either unintentionally (eg, globalization) or intentionally (eg, bioterrorism). The use of technology to reduce pathogens holds promise for further increasing the safety profile of blood transfusion.

Spesolimab Reduces Inflammation in Generalized Pustular Psoriasis: Molecular Characterization of Flare Treatment in EFFISAYIL 1.

EFFISAYIL 1 was a randomized, placebo-controlled study of spesolimab, an anti-IL-36 receptor antibody, in patients presenting with a generalized pustular psoriasis flare. Treatment with spesolimab led to more rapid pustular and skin clearance versus treatment with placebo in approximately half of the patients. In this study, we present histologic, transcriptomic, and proteomic analyses of lesional and nonlesional skin and whole-blood samples collected from EFFISAYIL 1. Treatment with spesolimab led to a transition toward a nonlesional profile, with a downregulation of gene expressions in the skin of IL-36 transcripts (IL36α, IL36ß, IL36γ) and those associated with neutrophil recruitment (CXCL1, CXCL6, CXCL8), proinflammatory cytokines (IL6, IL19, IL20), and skin inflammation (DEFB4A, S100A7, S100A8). Changes were manifest at week 1 and sustained to week 8. At the systemic level, reductions in serum biomarkers of inflammation (IL-17, IL-8, IL-6) were sustained until 12 weeks after spesolimab treatment. Considerable overlap was observed in the spesolimab-induced changes in gene and protein expressions from skin and blood samples, demonstrating the molecular basis of the effects of spesolimab on controlling local and systemic inflammation. Data are consistent with the mode of action of spesolimab, whereby inhibition of the IL-36 pathway leads to subsequent reductions in the key local and systemic pathologic events associated with generalized pustular psoriasis flares.

Monocyte-derived alveolar macrophages are key drivers of smoke-induced lung inflammation and tissue remodeling.

Smoking is a leading risk factor of chronic obstructive pulmonary disease (COPD), that is characterized by chronic lung inflammation, tissue remodeling and emphysema. Although inflammation is critical to COPD pathogenesis, the cellular and molecular basis underlying smoking-induced lung inflammation and pathology remains unclear. Using murine smoke models and single-cell RNA-sequencing, we show that smoking establishes a self-amplifying inflammatory loop characterized by an influx of molecularly heterogeneous neutrophil subsets and excessive recruitment of monocyte-derived alveolar macrophages (MoAM). In contrast to tissue-resident AM, MoAM are absent in homeostasis and characterized by a pro-inflammatory gene signature. Moreover, MoAM represent 46% of AM in emphysematous mice and express markers causally linked to emphysema. We also demonstrate the presence of pro-inflammatory and tissue remodeling associated MoAM orthologs in humans that are significantly increased in emphysematous COPD patients. Inhibition of the IRAK4 kinase depletes a rare inflammatory neutrophil subset, diminishes MoAM recruitment, and alleviates inflammation in the lung of cigarette smoke-exposed mice. This study extends our understanding of the molecular signaling circuits and cellular dynamics in smoking-induced lung inflammation and pathology, highlights the functional consequence of monocyte and neutrophil recruitment, identifies MoAM as key drivers of the inflammatory process, and supports their contribution to pathological tissue remodeling.

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier.

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles.

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.

Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling.

MOTIVATION: Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large-scale RNA-Seq datasets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. RESULTS: We report on a comprehensive study of target identification and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive recall of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, <30% of all transcripts could be quantified reliably with a relative error<20%. Based on established tools, we then introduce a new approach for mapping and analysing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision. CONTACT: rnaseq10@boku.ac.at

Time and phenotype-dependent transcriptome analysis in AAV-TGFß1 and Bleomycin-induced lung fibrosis models.

We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFß1. Here, we provide an in-depth characterization of phenotypic and transcriptomic changes (mRNA and miRNA) in a head-to-head comparison with Bleomycin-induced lung injury over a 4-week disease course. The analyses delineate the temporal state of model-specific and commonly altered pathways, thereby providing detailed insights into the processes underlying disease development. They further guide appropriate model selection as well as interventional study design. Overall, Bleomycin-induced fibrosis resembles a biphasic process of acute inflammation and subsequent transition into fibrosis (with partial resolution), whereas the TGFß1-driven model is characterized by pronounced and persistent fibrosis with concomitant inflammation and an equally complex disease phenotype as observed upon Bleomycin instillation. Finally, based on an integrative approach combining lung function data, mRNA/miRNA profiles, their correlation and miRNA target predictions, we identify putative drug targets and miRNAs to be explored as therapeutic candidates for fibrotic diseases. Taken together, we provide a comprehensive analysis and rich data resource based on RNA-sequencing, along with a strategy for transcriptome-phenotype coupling. The results will be of value for TGFß research, drug discovery and biomarker identification in progressive fibrosing interstitial lung diseases.

Model-based probe set optimization for high-performance microarrays.

A major challenge in microarray design is the selection of highly specific oligonucleotide probes for all targeted genes of interest, while maintaining thermodynamic uniformity at the hybridization temperature. We introduce a novel microarray design framework (Thermodynamic Model-based Oligo Design Optimizer, TherMODO) that for the first time incorporates a number of advanced modelling features: (i) A model of position-dependent labelling effects that is quantitatively derived from experiment. (ii) Multi-state thermodynamic hybridization models of probe binding behaviour, including potential cross-hybridization reactions. (iii) A fast calibrated sequence-similarity-based heuristic for cross-hybridization prediction supporting large-scale designs. (iv) A novel compound score formulation for the integrated assessment of multiple probe design objectives. In contrast to a greedy search for probes meeting parameter thresholds, this approach permits an optimization at the probe set level and facilitates the selection of highly specific probe candidates while maintaining probe set uniformity. (v) Lastly, a flexible target grouping structure allows easy adaptation of the pipeline to a variety of microarray application scenarios. The algorithm and features are discussed and demonstrated on actual design runs. Source code is available on request.

In-depth transcriptomic analysis of human retina reveals molecular mechanisms underlying diabetic retinopathy.

Diabetic Retinopathy (DR) is among the major global causes for vision loss. With the rise in diabetes prevalence, an increase in DR incidence is expected. Current understanding of both the molecular etiology and pathways involved in the initiation and progression of DR is limited. Via RNA-Sequencing, we analyzed mRNA and miRNA expression profiles of 80 human post-mortem retinal samples from 43 patients diagnosed with various stages of DR. We found differentially expressed transcripts to be predominantly associated with late stage DR and pathways such as hippo and gap junction signaling. A multivariate regression model identified transcripts with progressive changes throughout disease stages, which in turn displayed significant overlap with sphingolipid and cGMP-PKG signaling. Combined analysis of miRNA and mRNA expression further uncovered disease-relevant miRNA/mRNA associations as potential mechanisms of post-transcriptional regulation. Finally, integrating human retinal single cell RNA-Sequencing data revealed a continuous loss of retinal ganglion cells, and Müller cell mediated changes in histidine and ß-alanine signaling. While previously considered primarily a vascular disease, attention in DR has shifted to additional mechanisms and cell-types. Our findings offer an unprecedented and unbiased insight into molecular pathways and cell-specific changes in the development of DR, and provide potential avenues for future therapeutic intervention.

Hybridization thermodynamics of NimbleGen microarrays.

BACKGROUND: While microarrays are the predominant method for gene expression profiling, probe signal variation is still an area of active research. Probe signal is sequence dependent and affected by probe-target binding strength and the competing formation of probe-probe dimers and secondary structures in probes and targets. RESULTS: We demonstrate the benefits of an improved model for microarray hybridization and assess the relative contributions of the probe-target binding strength and the different competing structures. Remarkably, specific and unspecific hybridization were apparently driven by different energetic contributions: For unspecific hybridization, the melting temperature Tm was the best predictor of signal variation. For specific hybridization, however, the effective interaction energy that fully considered competing structures was twice as powerful a predictor of probe signal variation. We show that this was largely due to the effects of secondary structures in the probe and target molecules. The predictive power of the strength of these intramolecular structures was already comparable to that of the melting temperature or the free energy of the probe-target duplex. CONCLUSIONS: This analysis illustrates the importance of considering both the effects of probe-target binding strength and the different competing structures. For specific hybridization, the secondary structures of probe and target molecules turn out to be at least as important as the probe-target binding strength for an understanding of the observed microarray signal intensities. Besides their relevance for the design of new arrays, our results demonstrate the value of improving thermodynamic models for the read-out and interpretation of microarray signals.

Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease.

Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit- based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA-rich uEV preparation components. Our data shows that HFD,UC and the kit-based method are suitable methods to isolate uEV for miRNA-seq. However, only HFD and UC were suitable for mRNA-seq in our settings.

New method for the detection of micro-organisms in blood: application of quantitative interpretation model to aerobic blood cultures.

The physical and chemical changes occurring in blood that has been inoculated into a blood culture bottle can be used as means to detect the presence of microorganisms in blood cultures. These changes include primarily the conversion of oxy- to deoxyhemoglobin within the red blood cells (RBCs) and changes in the cell number densities. These changes in the physical and chemical properties of blood can be readily detected using spectrophometric methods thus enabling the continuous monitoring of blood culture vials to provide quantitative information on the growth behavior of the microorganisms present. This paper reports on the application of spectrophotometric information obtained from diffuse reflectance measurements of aerobic blood cultures to detect microbial growth and compares the results to those obtained using the standard blood culture system.

A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples.

One important goal of genomics is to explore the extent of alternative splicing in the transcriptome and generate a comprehensive catalog of splice forms. New computational and experimental approaches have led to an increase in the number of predicted alternatively spliced transcripts; however, validation of these predictions has not kept pace. In this work, we systematically explore different methods for the validation of cassette exons predicted by computational methods or tiling microarrays. Our goal was to find a procedure that is cost effective, sensitive and specific. We examined three ways of priming the reverse transcription (RT) reaction-poly-dT priming, random priming and pooled exon-specific priming. We also examined two strategies for PCR amplification-flanking PCR, which uses primers that hybridize to the constitutive exons flanking the predicted exon, and a semi-nested PCR with a primer that targets the predicted exon. We found that the combination of RT using a pool of gene-specific primers followed by semi-nested PCR resulted in a significant increase in sensitivity over the most commonly used methodology (97% of the test set was detected versus 14%). Our method was also highly specific-no false positives were detected using a test set of true negatives. Finally, we demonstrate that this method is able to detect alternative exons with a high sensitivity from whole-organism RNA, allowing all tissues to be sampled in a single experiment. The protocol developed here is an accurate and cost-effective way to validate predictions of alternative splicing.