scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.

BACKGROUND: The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. RESULTS: This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific. CONCLUSIONS: In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

A technical note on variable inter-frame interval as a cause of non-physiological experimental artefacts in ultrasound.

Ultrasound (US) imaging is a well-recognized technique for the study of static tissues but its suitability for studying tissue dynamics depends upon accurate frame time information, which may not always be available to users. Here we present methods to quantify the inter-frame interval (IFI) variability, and evaluate different procedures for collecting temporal information from two US-imaging devices. The devices tested exhibited variable IFIs that could only be confirmed by direct measures of timing signals, available by means of electrical signals (triggers) and/or temporal information contained in the software used for the US data collection. Interpolating frame-by-frame measures of dynamic changes within image sequences using individual IFI values provided improved synchronization between measures of skeletal muscle movement and activation; validating US as a valuable technique for the study of musculoskeletal tissue dynamics, when correctly implemented.

Prevalence and incidence of systemic lupus erythematosus in the adult population of Estonia.

Studies have demonstrated considerable variability in systemic lupus erythematosus (SLE) incidence and prevalence estimates. Lack of reliable epidemiological data may hinder evidence-based health care planning. The aim of the present study was to estimate the prevalence and incidence of SLE in the Estonian adult population. The SLE billing cases were extracted from the Estonian Health Insurance Fund database 2006-2010 and verified using health care providers' databases. The patients' life status data for January 1, 2011, were retrieved from the Estonian Population Register. The calculations for the estimates' lower limits were based on verified cases only; the upper limits calculations also accounted for the billing cases for which clinical data were unavailable. The period prevalence of SLE was between 39 and 48 per 100,000 and incidence rate between 1.5 and 1.8 per 100,000 person-years. The point prevalence on January 1, 2011, was between 37 and 40 per 100,000. The estimates are comparable with internationally published figures and can be used to enhance evidence-based health care planning. The high percentage of billing cases that could not be verified using clinical data supports the argument that epidemiological studies based solely on administrative databases are usually of low reliability.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and proteolytic processing by a disintegrin and metalloproteinases (ADAM): a regulator of several pathways.

HB-EGF is a member of the EGF family of ligands that is initially synthesized as a membrane-bound growth factor termed, proHB-EGF. The membrane bound proHB-EGF undergoes extensive proteolytic processing by several metalloproteinases capable of stimulating cellular proliferation. Soluble, mature HB-EGF binds to and activates EGF receptors. HB-EGF is a critical molecular component to a number of normal physiological processes including but not limited to tissue injury and wound healing, reproduction, angiogenesis and recently, adipogenesis. Misexpression of HB-EGF is linked to tumor formation and cancer including hepatocellular, pancreatic, gastric, breast, colon and melanoma, gliomas and glioblastomas. HB-EGF is a likely tool for therapeutic approaches to enhance treatment of injuries as well as a target for prevention of several cancers and obesity.

Alcohol Limits and Public Safety.

On May 14, 2013, the National Transportation Safety Board (NTSB) recommended lowering the legal blood-alcohol limit to 0.05 g/dL for motor vehicle operators in the United States, in an effort to reduce the risk of injuries and deaths caused by a driver's alcohol impairment (NTSB/SR-13/01). This recommendation has prompted other organizations and agencies, including the National Safety Council, to evaluate and consider supporting this action. In order to determine the scientific and legal feasibility and advisability of lowering or establishing 0.05 per se laws, we examined 554 alcohol-related publications. Risk factors, instrument reliability, law enforcement, and adjudication issues were considered in this overview of the literature. The extensive scientific literature reviewed provides ample support for lowering the operation of motor vehicle alcohol limits to 0.05, and for supporting the NTSB recommendations. Research clearly demonstrates that impairment begins at very low concentrations, well below the recommended NTSB limit, and increases with concentration. Lowering the limit to 0.05 will save many lives and prevent injuries. Breath, blood, and saliva samples have proved to be accurate and reliable specimens for legal acceptability in a court of law.

Cellular reprogramming into a brown adipose tissue-like phenotype by co-expression of HB-EGF and ADAM 12S.

Abnormal adipogenesis leads to excessive fat accumulation and several health disorders. Mouse fibroblasts (MLC) transfected with ADAM 12S and HB-EGF promoted lipid accumulation. Addition of KBR-7785, an ADAM 12S inhibitor, to HB-EGF/ADAM 12S expressing cells suppressed adipogenesis. BrdU incorporation was attenuated and enhanced mitotracker staining was observed in HB-EGF/ADAM 12S cells. Quantitative real time RT-PCR resulted in elevated levels of expression of three brown adipose tissue (BAT) genes (PRDM16, PGC-1α, and UCP-1), while expression levels of the three white adipose tissue (WAT) genes (PPARγ, C/EBPα, and AKT-1) were unaltered in HB-EGF/ADAM 12S cells. Amino- or carboxy-terminal deletions of HB-EGF (HB-EGFΔN and HB-EGFΔC) co-expressed with ADAM 12S stimulated lipid accumulation. Human epidermoid carcinoma cells (A431) also exhibited lipid accumulation by HB-EGF/ADAM 12S co-expression. These studies suggest ADAM 12S and HB-EGF are involved in cellular plasticity resulting in the production of BAT-like cells and offers insight into novel therapeutic approaches for fighting obesity.

Comparison of two approaches to performing an inferior alveolar nerve block in the horse.

OBJECTIVES: To compare two approaches to performing the inferior alveolar nerve block in the horse and to evaluate the consistency of described topographical landmarks. DESIGN: Experimental cadaver model. METHODS: Eleven cadaver heads were positioned to mimic a standing sedated horse and the position of the mandibular foramen approximated. The vertical approach to the approximate location of the mandibular foramen was undertaken and red dye was deposited. The angled approach was then undertaken and blue ink was used to identify it. The heads were then dissected to determine the location of the dye. Placement was categorised as a hit or a miss for each technique for each side of the head. The distance of the dye from the nerve was recorded. Straight lateral radiographs of the sectioned heads were taken to evaluate the topographical landmarks for performing this nerve block. RESULTS: Each method was performed 22 times. A hit was achieved 16 times (73%) for the angled approach and 13 times (59%) for the vertical approach. There was no significant difference between the two approaches (P = 0.34). Radiographs revealed that the topographical landmarks used to approximate the mandibular foramen were relatively accurate. CONCLUSION: Both methods were found to be equivalently accurate. The previously reported topographic landmarks for locating the approximate position of the mandibular foramen on the medial aspect of the mandible were found to be accurate, but currently recommended doses of local anaesthetic may be excessive.

Photosensitive epilepsy and image safety.

Photosensitive epilepsy came to prominence in the 1950s with the advent of television. Photosensitive epilepsy occurs in 1 in 4000 of the population. The incidence is 1.1 per 100,000 per annum, however amongst 7-19 year-olds the incidence is more than five times as common. Photosensitive epilepsy is twice as common in females as in males. The onset is around puberty, but less than 25 per cent of patients lose their photosensitivity in their twenties. Patients are investigated in the EEG laboratory using intermittent photic stimulation. Peak sensitivity is between 16 and 20 flashes/s but 49 per cent of patients are sensitive to 50 flashes/s, explaining the sensitivity to PAL television systems. From 1993 the development of broadcast guidelines was developed restricting both flash rates and the areas of screen involved, as well as the use of long-wavelength red. Automatic analysis systems can now test material for compliance with guidelines in real time.

Polymorphism at the avpr1a locus in male prairie voles correlated with genetic but not social monogamy in field populations.

Integrative studies of genetics, neurobiology and behaviour indicate that polymorphism in specific genes contributes to variation observed in some complex social behaviours. The neuropeptide arginine vasopressin plays an important role in the regulation of a variety of social behaviours, including social attachment of males to females, through its action on the vasopressin 1a receptor (V1aR). In socially monogamous prairie voles (Microtus ochrogaster), polymorphism in the length of microsatellite DNA within the regulatory region of the gene (avpr1a) encoding the V1aR predicts differences among males in neural expression of V1aRs and partner preference under laboratory conditions. However, understanding the extent to which V1aR mediates variation in prairie vole social and reproductive behaviour observed in nature requires investigating the consequences of avpr1a polymorphism and environmental influences under ecologically relevant conditions. We examined the relationship between avpr1a length polymorphism and monogamy among male prairie voles living in 0.1 ha enclosures during a time similar to their natural lifespan. We found no evidence that avpr1a genotype of males predicts variation in social monogamy measured in the field but some indices of social monogamy were affected by population density. Parentage data indicated that a male's avpr1a genotype significantly influenced the number of females with which he sired offspring and the total number of offspring sired. Total brain concentrations of V1aR mRNA were not associated with either male behaviour or avpr1a genotype. These data show that melding ecological field studies with neurogenetics can substantially augment our understanding of the effects of genes and environment on social behaviours.

The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach.

A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser(108/121), HB-EGF-Cys/Ser(116/132), and HB-EGF-Cys/Ser(134/143)) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M(r) of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser(108/121) and HB-EGF-Cys/Ser(116/132) stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF(134/143) proteins competed with 125I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser(108/121) and HB-EGF-Cys/Ser(116/132) failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser(134/143) antagonizes EGFRs.

Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study.

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.

Amino-terminal deletion of heparin-binding epidermal growth factor-like growth factor4-127 stimulates cell proliferation but lacks insulin-like activity.

UNLABELLED: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) Northern analysis demonstrated a novel 0.8-kb liver-specific HB-EGF transcript in addition to the endogenous 2.5-kb HB-EGF full-length transcript present in kidney, lung and liver tissues. Reverse transcriptase-polymerase chain reaction screening of liver RNA suggests that the 0.8-kb HB-EGF transcript lacks at least a portion of the amino-terminal EGF-like domain. In light of these data, we have constructed a human HB-EGF cDNA (HB-EGF(DeltaN)) which lacks 373 bp, encoding the majority of the extracellular EGF-like domain, while maintaining the ectodomain 'shedding' site, transmembrane and cytoplasmic domains. OBJECTIVE: The goal of this study is to characterize the ability of HB-EGF(DeltaN) to (i) stimulate cell proliferation and (ii) determine whether down-regulation of insulin-like growth factor-binding protein (IGFBP)-3 and -4 mRNA is regulated by soluble, mature HB-EGF or HB-EGF C. MATERIALS AND METHODS: HB-EGF(DeltaN) encodes nucleotides +1-10 of exon 1 linked to nucleotides 383-627 of the carboxy-terminal portion of exon 3 through exon 5. RESULTS: Expression of HB-EGF(DeltaN) in mouse fibroblasts (MLC) resulted in 6.5- and 8-kDa HB-EGF immunoreactive proteins, stimulated tyrosine phosphorylation of p42 kDa and cell proliferation in MLC, but lacked the ability to bind EGF receptors. Finally, HB-EGF(DeltaN) failed to down-regulate IGFBP-3 and -4 mRNA when expressed in normal rat kidney cells. CONCLUSIONS: These findings demonstrate that amino-terminally truncated, membrane-bound form of HB-EGF stimulates cell proliferation but lacks insulin-like signalling, suggesting that insulin-like signalling is mediated by soluble, mature HB-EGF binding to EGF receptors.

The influence of antisense oligonucleotide length on dystrophin exon skipping.

Antisense oligonucleotides (AOs) can be used to redirect dystrophin pre-messenger RNA (mRNA) processing, to remove selected exons from the mature dystrophin mRNA, to overcome nonsense mutations, and/or restore the reading frame. Redundancy within the dystrophin protein allows some domains to be removed without seriously compromising function. One of the challenges for splicing blockade is to design AOs that efficiently remove targeted exons across the dystrophin pre-mRNA. AOs are initially designed to anneal to the more obvious motifs implicated in the splicing process, such as acceptor or donor splice sites and in silico predicted exonic splicing enhancers. The AOs are evaluated for their ability to induce targeted exon skipping after transfection into cultured myoblasts. Although no single motif has been implicated in the consistent induction of exon skipping, the length of the AO has emerged as an important parameter in designing compounds that redirect dystrophin pre-mRNA processing. We present data from in vitro studies in murine and human cells showing that appropriately designed AOs of 25-31 nucleotides are generally more effective at inducing exon skipping than shorter counterparts. However, there appears to be an upper limit in optimal length, which may have to be established on a case-by-case basis.

Transforming growth factor beta regulates cyclooxygenase-2 in glomerular mesangial cells.

This study examines the hypothesis that transforming growth factor beta (TGFbeta) regulates cyclooxygenase-2 (COX-2) and induces prostaglandin E synthase (mPGES-1) in rat mesangial cells. COX-2 expression was determined by Northern blot analysis after treatment with either TGFbeta1 or the selective COX-2 inhibitor, NS398. mPGES-1 expression was determined by real-time polymerase chain reaction. The effect of TGFbeta1 on COX-2 gene transcription was assessed using a luciferase reporter assay, and mRNA stability was also determined. To determine whether TGFbeta1 activates elements of the COX-2 promoter, we performed gel shift analyses to examine activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB). Prostaglandin E(2) (PGE(2)) and thromboxane B2 (TxB2) production was assayed by enzyme immunoassay. Finally, the pathophysiological relevance of COX-2 inhibition on the downstream effects of TGFbeta was assessed by examining collagen type I mRNA and net collagen production. COX-2 mRNA and mPGES-1 were induced after treatment with TGFbeta1 for 4 h, and this rise was accompanied by a three-fold increase in PGE(2) production that could be antagonized by selective inhibition of COX-2 with NS398. TGFbeta1 increased transcription by approximately 50% and activated both AP-1 and NF-kappaB. These effects were antagonized by co-treatment with NS398. Treatment with TGFbeta1 also doubled the half-life of COX-2 mRNA. Neither collagen type I mRNA nor net collagen production were altered by co-treatment with NS398. In conclusion, these results indicate that TGFbeta stimulates COX-2 and mPGES-1, with additional effects on transcription and stability of COX-2 mRNA.

Distribution of Ixodes ricinus in the British Isles: investigation of historical records.

Ixodes ricinus Linnaeus (Acari: Ixodidae) is the most abundant and widely distributed tick in the British Isles, and is a vector for a number of bacterial, viral and protozoal pathogens of both medical and veterinary importance. This report provides an update to the historical distribution data of I. ricinus, published by the Biological Records Centre (BRC), Monks Wood in The Provisional Atlas of the Ticks (Ixodidae) of the British Isles by K. P. Martyn (1988), and is supplemented with additional BRC records since 1988, additional data from published scientific literature and unpublished field studies, and enhanced with spatial and temporal information on tick stages collected and their host associations. Records have been mapped at 10 km resolution and enhanced to 5 km, 1 km and 0.1 km. Differentiation between records representing one-off collections from those representing populations of I. ricinus has been achieved through the classification of the records into either reported or established populations. Detailed seasonality and host associations of records are investigated, highlighting the value in obtaining additional detailed contemporary data to aid risk assessments and research within this field.

A comparison of polyacrylamide gels and radiochromic film for source measurements in intravascular brachytherapy.

For intravascular brachytherapy with catheter-based systems, AAPM Task Group 60 has recommended measurements that should be made to characterize the sources. Beta emitters, including (90)Sr/(90)Y are ideal for intravascular brachytherapy, but problems arise in measuring dose distributions in the high dose gradient region at short distances from the source. In this paper, measurements of radial and orthogonal dose distributions and dose profiles for a (90)Sr/(90)Y source train using polyacrylamide gel (PAG) dosimetry and a high-field 4.7 Tesla MRI scanner are presented and compared with measurements made with two types of radiochromic film, MD-55 and HD-810. For the PAG system, the dose distributions were determined with in-plane resolutions of 0.4 mm and 0.2 mm. The measurements of absorbed dose distributions both orthogonal and parallel to the source axis show good agreement between the PAG and radiochromic film. The absolute dose at a radial distance of 2 mm in the central 32 mm of a line parallel to the axis was measured. For the PAG the measured absorbed dose was 1.25% lower, for MD-55 4% higher and for the HD-810 1.6% higher when compared with the value given by the source calibration. These results confirm that both absorbed dose and dose distributions for high gradient vascular brachytherapy sources can be measured using PAG but the disadvantages of gel manufacture and the need for access to a high resolution scanner suggests that the use of radiochromic film is the method of choice.

Androgen deprivation in men with prostate cancer is associated with an increased rate of bone loss.

The objective of this work was to determine the effect of androgen deprivation therapy (ADT) on rates of bone mineral density (BMD) loss in men with prostate cancer. It was a prospective study comparing men receiving ADT to age matched controls for 2 y. Subjects received a history, physical exam, bone mineral density measurement, and laboratory evaluation every 6 months. Thirty-nine subjects receiving continuous ADT for prostate cancer (subjects) were compared to 39 age-matched controls not receiving ADT (controls). Twenty-three subjects and 30 controls completed the study through 24 months. Men in the ADT group demonstrated greater rates of bone mineral density loss than men in the control group at every site except the lumbar spine. Twenty-four month per cent of bone mineral density loss is presented as mean+/-standard error (s.e.). At the distal forearm, the ADT group value was -9.4%+/-1.0% and -4.4%+/-0.3% for controls (P<0.0005). The ADT group femoral neck values were -1.9%+/-0.7% and 0.6%+/-0.5% in the control group (P=0.0016). The ADT group total hip value was -1.5%+/-1.0% and 0.8%+/-0.5% in the control group (P=0.0018). The ADT group trochanter value was -2.0%+/-1.3% and -0.1%+/-0.5% in the control group (P=0.0019). The ADT group lumbar spine value was -0.2%+/-0.8 % and 1.1%+/-0.6% in the control group (P=0.079). Our data demonstrate greater rates of bone mineral density loss in men receiving androgen deprivation therapy for prostate cancer.

Rapid responses of British butterflies to opposing forces of climate and habitat change.

Habitat degradation and climate change are thought to be altering the distributions and abundances of animals and plants throughout the world, but their combined impacts have not been assessed for any species assemblage. Here we evaluated changes in the distribution sizes and abundances of 46 species of butterflies that approach their northern climatic range margins in Britain-where changes in climate and habitat are opposing forces. These insects might be expected to have responded positively to climate warming over the past 30 years, yet three-quarters of them declined: negative responses to habitat loss have outweighed positive responses to climate warming. Half of the species that were mobile and habitat generalists increased their distribution sites over this period (consistent with a climate explanation), whereas the other generalists and 89% of the habitat specialists declined in distribution size (consistent with habitat limitation). Changes in population abundances closely matched changes in distributions. The dual forces of habitat modification and climate change are likely to cause specialists to decline, leaving biological communities with reduced numbers of species and dominated by mobile and widespread habitat generalists.

Hysteroscopic endomyometrial resection of three uterine sarcomas.

STUDY OBJECTIVE: To describe our experience with three uterine sarcomas associated with hysteroscopic endometrial ablation. DESIGN: Cohort study (Canadian Task Force classification II-2). SETTING: University-affiliated teaching hospitals. PATIENTS: Three of 2402 women undergoing hysteroscopic endometrial ablation who had uterine sarcomas. INTERVENTION: Hysteroscopic endomyometrial resection. MEASUREMENTS AND MAIN RESULTS: One low-grade endometrial stromal sarcoma and two carcinosarcomas were resected. After hysterectomy in two patients, no residual cancer was identified in one of them. The third patient was an 82-year-old woman with moderate menorrhagia who refused hysterectomy. After endomyometrial resection she remained amenorrheic for the last 14 months of her life. CONCLUSION: From our experience the incidence of uterine sarcomas is approximately 1/800 women undergoing hysteroscopic ablation for abnormal uterine bleeding. Complete endomyometrial resection is feasible and may be offered as diagnostic and palliative therapy in women at high risk for hysterectomy.