Primary Nasal Epithelial Cells as a Surrogate Cell Culture Model for Type-II Alveolar Cells to Study ABCA-3 Deficiency

ATP Binding Cassette Subfamily A Member 3 (ABCA-3) is a lipid transporter protein highly expressed in type-II alveolar (AT-II) cells. Mutations in ABCA3 can result in severe respiratory disease in infants and children. To study ABCA-3 deficiency in vitro, primary AT-II cells would be the cell culture of choice although sample accessibility is limited. Our aim was to investigate the suitability of primary nasal epithelial cells, as a surrogate culture model for AT-II cells, to study ABCA-3 deficiency. Expression of ABCA3, and surfactant protein genes, SFTPB and SFTPC, was detected in primary nasal epithelial cells but at a significantly lower level than in AT-II cells. ABCA-3, SP-B, and SP-C were detected by immunofluorescence microscopy in primary nasal epithelial cells. However, SP-B and SP-C were undetectable in primary nasal epithelial cells using western blotting. Structurally imperfect lamellar bodies were observed in primary nasal epithelial cells using transmission electron microscopy. Functional assessment of the ABCA-3 protein demonstrated that higher concentrations of doxorubicin reduced cell viability in ABCA-3 deficient nasal epithelial cells compared to controls in an assay-dependent manner. Our results indicate that there may be a role for primary nasal epithelial cell cultures to model ABCA-3 deficiency in vitro, although additional cell culture models that more effectively recapitulate the AT-II phenotype may be required

Cognitive, behavioural and psychological barriers to the prevention of severe hypoglycaemia: A qualitative study of adults with type 1 diabetes

Objectives: Severe hypoglycaemia affects approximately one in three people with type 1 diabetes and is the most serious side effect of insulin therapy. Our aim was to explore individualistic drivers of severe hypoglycaemia events. Methods: In-depth semi-structured interviews were conducted with a purposive sample of 17 adults with type 1 diabetes and a history of recurrent severe hypoglycaemia, to elicit experiences of hypoglycaemia (symptoms/awareness, progression from mild to severe and strategies for prevention/treatment). Interviews were analysed using an adapted grounded theory approach. Results: Three main themes emerged: hypoglycaemia-induced cognitive impairment, behavioural factors and psychological factors. Despite experiencing early hypoglycaemic symptoms, individuals often delayed intervention due to impaired/distracted attention, inaccurate risk assessment, embarrassment, worry about rebound hyperglycaemia or unavailability of preferred glucose source. Delay coupled with use of a slow-acting glucose source compromised prevention of severe hypoglycaemia. Conclusion: Our qualitative data highlight the multifaceted, idiosyncratic nature of severe hypoglycaemia and confirm that individuals with a history of recurrent severe hypoglycaemia may have specific thought and behaviour risk profiles. Individualised prevention plans are required, emphasising both the need to attend actively to mild hypoglycaemic symptoms and to intervene promptly with an appropriate, patient-preferred glucose source to prevent progression to severe hypoglycaemia

Seismic data reveal eastern Black Sea Basin structure

Rifted continental margins are formed by progressive extension of the lithosphere. The development of these margins plays an integral role in the plate tectonic cycle, and an understanding of the extensional process underpins much hydrocarbon exploration. A key issue is whether the lithosphere extends uniformly, or whether extension varies\ud with depth. Crustal extension may be determined using seismic techniques. Lithospheric extension may be inferred from the waterloaded subsidence history, determined from\ud the pattern of sedimentation during and after rifting. Unfortunately, however, many rifted margins are sediment-starved, so the subsidence history is poorly known.\ud To test whether extension varies between the crust and the mantle, a major seismic experiment was conducted in February–March 2005 in the eastern Black Sea Basin (Figure 1), a deep basin where the subsidence history is recorded\ud by a thick, post-rift sedimentary sequence. The seismic data from the experiment indicate the presence of a thick, low-velocity zone, possibly representing overpressured sediments. They also indicate that the basement and\ud Moho in the center of the basin are both several kilometers shallower than previously inferred. These initial observations may have considerable impact on thermal models of the petroleum system in the basin. Understanding\ud the thermal history of potential source rocks is key to reducing hydrocarbon exploration risk. The experiment, which involved collaboration between university groups in the United Kingdom, Ireland, and Turkey, and BP and\ud Turkish Petroleum (TPAO), formed part of a larger project that also is using deep seismic reflection and other geophysical data held by the industry partners to determine the subsidence history and hence the strain evolution of\ud the basin

The potential of antisense oligonucleotide therapies for inherited childhood lung diseases

Antisense oligonucleotides are an emerging therapeutic option to treat diseases with known genetic origin. In the age of personalised medicines, antisense oligonucleotides can sometimes be designed to target and bypass or overcome a patient’s genetic mutation, in particular those lesions that compromise normal pre-mRNA processing. Antisense oligonucleotides can alter gene expression through a variety of mechanisms as determined by the chemistry and antisense oligomer design. Through targeting the pre-mRNA, antisense oligonucleotides can alter splicing and induce a specific spliceoform or disrupt the reading frame, target an RNA transcript for degradation through RNaseH activation, block ribosome initiation of protein translation or disrupt miRNA function. The recent accelerated approval of eteplirsen (renamed Exondys 51™) by the Food and Drug Administration, for the treatment of Duchenne muscular dystrophy, and nusinersen, for the treatment of spinal muscular atrophy, herald a new and exciting era in splice-switching antisense oligonucleotide applications to treat inherited diseases. This review considers the potential of antisense oligonucleotides to treat inherited lung diseases of childhood with a focus on cystic fibrosis and disorders of surfactant protein metabolism

Deficits in mitochondrial spare respiratory capacity contribute to the neuropsychological changes of alzheimer’s disease

Alzheimer’s disease (AD) is diagnosed using neuropsychological testing, supported by amyloid and tau biomarkers and neuroimaging abnormalities. The cause of neuropsychological changes is not clear since they do not correlate with biomarkers. This study investigated if changes in cellular metabolism in AD correlate with neuropsychological changes. Fibroblasts were taken from 10 AD patients and 10 controls. Metabolic assessment included measuring total cellular ATP, extracellular lactate, mitochondrial membrane potential (MMP), mitochondrial respiration and glycolytic function. All participants were assessed with neuropsychological testing and brain structural MRI. AD patients had significantly lower scores in delayed and immediate recall, semantic memory, phonemic fluency and Mini Mental State Examination (MMSE). AD patients also had significantly smaller left hippocampal, left parietal, right parietal and anterior medial prefrontal cortical grey matter volumes. Fibroblast MMP, mitochondrial spare respiratory capacity (MSRC), glycolytic reserve, and extracellular lactate were found to be lower in AD patients. MSRC/MMP correlated significantly with semantic memory, immediate and delayed episodic recall. Correlations between MSRC and delayed episodic recall remained significant after controlling for age, education and brain reserve. Grey matter volumes did not correlate with MRSC/MMP. AD fibroblast metabolic assessment may represent an emergent disease biomarker of AD

The Dynamic Insulin Sensitivity and Secretion Test (DISST) - a novel measure of insulin sensitivity

Objective: To validate the methodology for the Dynamic Insulin Sensitivity and Secretion Test (DISST) and to demonstrate its potential in clinical and research settings. Methods: 123 men and women had routine clinical and biochemical measurements, an oral glucose tolerance test and a DISST. For the DISST, participants were cannulated for blood sampling and bolus administration. Blood samples were drawn at t=0, 10, 15, 25 and 35 minutes for measurement of glucose, insulin and C-peptide. A 10g bolus of intravenous glucose at t=5 minutes and 1U of intravenous insulin immediately after the t=15 minute sample were given. Fifty participants also had a hyperinsulinaemic euglycaemic clamp. Relationships between DISST insulin sensitivity (SI) and the clamp, and both DISST SI and secretion and other metabolic variables were measured. Results: A Bland-Altman plot showed little bias in the comparison of DISST with the clamp; with DISST underestimating the glucose clamp by 0.1·10-2·mg·l·kg-1·min-1·pmol-1 (90%CI -0.2 to 0). The correlation between SI as measured by DISST and the clamp was 0.82, the c unit for the ROC analysis for the two tests was 0.96. Metabolic variables showed significant correlations with DISST IS, and the second phase of insulin release. DISST also appears able to distinguish different insulin secretion patterns in individuals with identical SI values. Conclusions: DISST is a simple, dynamic test that compares favourably with the clamp in assessing SI and allows simultaneous assessment of insulin secretion. DISST has the potential to provide even more information about the pathophysiology of diabetes than more complicated tests

Intra-sexual competition alters the relationship between testosterone and ornament expression in a wild territorial bird

Open Access funded by Natural Environment Research Council Under a Creative Commons licensePeer reviewedPublisher PD

Phosphate steering by Flap Endonuclease 1 promotes 5´-flap specificity and incision to prevent genome instability

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 50polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 50-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 50-flap specificity and catalysis, preventing genomic instability

Phosphate steering by Flap Endonuclease 1 promotes 5´-flap specificity and incision to prevent genome instability

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 50polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 50-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 50-flap specificity and catalysis, preventing genomic instability