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1.  HPV E6/E7 RNA In Situ Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade 
PLoS ONE  2014;9(3):e91142.
Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.
PMCID: PMC3953338  PMID: 24625757
2.  Evaluating the Accuracy and Large Inaccuracy of Two Continuous Glucose Monitoring Systems 
This study evaluated the accuracy and large inaccuracy of the Freestyle® Navigator (FSN) (Abbott Diabetes Care, Alameda, CA) and Dexcom® SEVEN® PLUS (DSP) (Dexcom, Inc., San Diego, CA) continuous glucose monitoring (CGM) systems during closed-loop studies.
Research Design and Methods
Paired CGM and plasma glucose values (7,182 data pairs) were collected, every 15–60 min, from 32 adults (36.2±9.3 years) and 20 adolescents (15.3±1.5 years) with type 1 diabetes who participated in closed-loop studies. Levels 1, 2, and 3 of large sensor error with increasing severity were defined according to absolute relative deviation greater than or equal to±40%,±50%, and±60% at a reference glucose level of ≥6 mmol/L or absolute deviation greater than or equal to±2.4 mmol/L,±3.0 mmol/L, and±3.6 mmol/L at a reference glucose level of <6 mmol/L.
Median absolute relative deviation was 9.9% for FSN and 12.6% for DSP. Proportions of data points in Zones A and B of Clarke error grid analysis were similar (96.4% for FSN vs. 97.8% for DSP). Large sensor over-reading, which increases risk of insulin over-delivery and hypoglycemia, occurred two- to threefold more frequently with DSP than FSN (once every 2.5, 4.6, and 10.7 days of FSN use vs. 1.2, 2.0, and 3.7 days of DSP use for Level 1–3 errors, respectively). At Levels 2 and 3, large sensor errors lasting 1 h or longer were absent with FSN but persisted with DSP.
FSN and DSP differ substantially in the frequency and duration of large inaccuracy despite only modest differences in conventional measures of numerical and clinical accuracy. Further evaluations are required to confirm that FSN is more suitable for integration into closed-loop delivery systems.
PMCID: PMC3558677  PMID: 23256605
3.  Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids 
Free radical research  2012;46(4):10.3109/10715762.2012.659248.
A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.
PMCID: PMC3864884  PMID: 22263561
reactive oxygen species; reactive nitrogen species; biomarkers; DNA oxidation; free radicals; hydroxyl radical
4.  Calcineurin signalling mediates activity-dependent relocation of the axon initial segment 
The axon initial segment (AIS) is a specialised neuronal sub-compartment located at the beginning of the axon that is crucially involved in both the generation of action potentials and in the regulation of neuronal polarity. We recently showed that prolonged neuronal depolarisation produces a distal shift of the entire AIS structure away from the cell body, a change associated with a decrease in neuronal excitability. Here, we utilised dissociated rat hippocampal cultures, with a major focus on the dentate granule cell (DGC) population, to explore the signalling pathways underlying activity-dependent relocation of the AIS. Firstly, a pharmacological screen of voltage-gated calcium channels (VGCCs) showed that AIS relocation is triggered by activation of L-type Cav1 VGCCs with negligible contribution from any other VGCC subtypes. Further pharmacological analysis revealed that downstream signalling events are mediated by the calcium-sensitive phosphatase calcineurin; inhibition of calcineurin with either FK506 or cyclosporin A totally abolished both depolarisation- and optogenetically-induced activity-dependent AIS relocation. Furthermore, calcineurin activation is sufficient for AIS plasticity, as expression of a constitutively active form of the phosphatase resulted in relocation of the AIS of DGCs without a depolarising stimulus. Finally, we assessed the role of calcineurin in other forms of depolarisation-induced plasticity. Neither membrane resistance changes nor spine density changes were affected by FK506 treatment, suggesting that calcineurin acts via a separate pathway to modulate AIS plasticity. Taken together, these results emphasise calcineurin as a vital player in the regulation of intrinsic plasticity as governed by the AIS.
PMCID: PMC3743026  PMID: 23595753
5.  Thymosin β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing 
Nature communications  2013;4:2081.
The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide (H2O2) released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that Thymosin β4-Sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, lies downstream of H2O2 in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T cell/CD14+ monocyte co-cultures, Tβ4-SO inhibits IFN-γ and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, Tβ4-SO is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.
PMCID: PMC3797509  PMID: 23820300
7.  5-HT2C Receptor Agonist Anorectic Efficacy Potentiated by 5-HT1B Receptor Agonist Coapplication: An Effect Mediated via Increased Proportion of Pro-Opiomelanocortin Neurons Activated 
An essential component of the neural network regulating ingestive behavior is the brain 5-hydroxytryptamine2C receptor (5-HT2CR), agonists of which suppress food intake and were recently approved for obesity treatment by the US Food and Drug Administration. 5-HT2CR-regulated appetite is mediated primarily through activation of hypothalamic arcuate nucleus (ARC) pro-opiomelanocortin (POMC) neurons, which are also disinhibited through a 5-HT1BR-mediated suppression of local inhibitory inputs. Here we investigated whether 5-HT2CR agonist anorectic potency could be significantly enhanced by coadministration of a 5-HT1BR agonist and whether this was associated with augmented POMC neuron activation on the population and/or single-cell level. The combined administration of subanorectic concentrations of 5-HT2CR and 5-HT1BR agonists produced a 45% reduction in food intake and significantly greater in vivo ARC neuron activation in mice. The chemical phenotype of activated ARC neurons was assessed by monitoring agonist-induced cellular activity via calcium imaging in mouse POMC-EGFP brain slices, which revealed that combined agonists activated significantly more POMC neurons (46%) compared with either drug alone (~25% each). Single-cell electrophysiological analysis demonstrated that 5-HT2CR/5-HT1BR agonist coadministration did not significantly potentiate the firing frequency of individual ARC POMC-EGFP cells compared with agonists alone. These data indicate a functional heterogeneity of ARC POMC neurons by revealing distinct subpopulations of POMC cells activated by 5-HT2CRs and disinhibited by 5-HT1BRs. Therefore, coadministration of a 5-HT1BR agonist potentiates the anorectic efficacy of 5-HT2CR compounds by increasing the number, but not the magnitude, of activated ARC POMC neurons and is of therapeutic relevance to obesity treatment.
PMCID: PMC3717514  PMID: 23739976
8.  Recommendations for standardised description of, and nomenclature concerning, oxidatively damaged nucleobases in DNA 
Chemical research in toxicology  2010;23(4):705-707.
The field of oxidative stress, and the study of oxidatively damaged DNA, in particular, is a subject of intense, and growing interest. This has, in part, benefited from the availability of kits from commercial suppliers which are advertised as reporting on markers of oxidative stress. Such widespread use has inevitably led to an increase in the number of concerns, amongst experts in the field, editors and referees, over appropriateness of terminology and methodology. Thus, the widely used term “oxidative DNA damage” is misleading as it implies that the damage, i.e. the lesion per se, is oxidative and thus capable of oxidising other substrates. We would encourage the use of such terms as ‘oxidatively damaged DNA’, ‘oxidatively generated DNA damage’, ‘oxidatively-derived damage to DNA’ or ‘oxidation-induced DNA damage’ to describe the consequence of the interaction of reactive oxygen species with DNA. One of the most studied nucleic acid-derived biomarkers of oxidative stress is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Yet, in the literature, this compound has been referred to using a number of different terms, sometimes leading to confusion over the designation of the modified nucleobase or (2′-deoxy)ribonucleoside. Standardisation of nomenclature would not only simplify literature searches, but also clarify the lesion in question. Herein, we provide justification for our preferred nomenclature, and suggest a number of steps by which we may work towards standardisation of calibration, and with it improved inter-laboratory agreement, for assays of 8-oxodG, in order to achieve accurate measurements.
PMCID: PMC3713252  PMID: 20235554
9.  Human and Methodological Sources of Variability in the Measurement of Urinary 8-Oxo-7,8-dihydro-2′-deoxyguanosine 
Antioxidants & Redox Signaling  2013;18(18):2377-2391.
Aims: Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed ‘spot’ samples showed strong correlations with 24 h excretion (the ‘gold’ standard) of urinary 8-oxodG (rp 0.67–0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended. Antioxid. Redox Signal. 18, 2377–2391.
PMCID: PMC3671631  PMID: 23198723
10.  A Giant Pliosaurid Skull from the Late Jurassic of England 
PLoS ONE  2013;8(5):e65989.
Pliosaurids were a long-lived and cosmopolitan group of marine predators that spanned 110 million years and occupied the upper tiers of marine ecosystems from the Middle Jurassic until the early Late Cretaceous. A well-preserved giant pliosaurid skull from the Late Jurassic Kimmeridge Clay Formation of Dorset, United Kingdom, represents a new species, Pliosaurus kevani. This specimen is described in detail, and the taxonomy and systematics of Late Jurassic pliosaurids is revised. We name two additional new species, Pliosaurus carpenteri and Pliosaurus westburyensis, based on previously described relatively complete, well-preserved remains. Most or all Late Jurassic pliosaurids represent a globally distributed monophyletic group (the genus Pliosaurus, excluding ‘Pliosaurus’ andrewsi). Despite its high species diversity, and geographically widespread, temporally extensive occurrence, Pliosaurus shows relatively less morphological and ecological variation than is seen in earlier, multi-genus pliosaurid assemblages such as that of the Middle Jurassic Oxford Clay Formation. It also shows less ecological variation than the pliosaurid-like Cretaceous clade Polycotylidae. Species of Pliosaurus had robust skulls, large body sizes (with skull lengths of 1.7–2.1 metres), and trihedral or subtrihedral teeth suggesting macropredaceous habits. Our data support a trend of decreasing length of the mandibular symphysis through Late Jurassic time, as previously suggested. This may be correlated with increasing adaptation to feeding on large prey. Maximum body size of pliosaurids increased from their first appearance in the Early Jurassic until the Early Cretaceous (skull lengths up to 2360 mm). However, some reduction occurred before their final extinction in the early Late Cretaceous (skull lengths up to 1750 mm).
PMCID: PMC3669260  PMID: 23741520
11.  Brain Glucose Sensors Play a Significant Role in the Regulation of Pancreatic Glucose-Stimulated Insulin Secretion 
Diabetes  2012;61(2):321-328.
As patients decline from health to type 2 diabetes, glucose-stimulated insulin secretion (GSIS) typically becomes impaired. Although GSIS is driven predominantly by direct sensing of a rise in blood glucose by pancreatic β-cells, there is growing evidence that hypothalamic neurons control other aspects of peripheral glucose metabolism. Here we investigated the role of the brain in the modulation of GSIS. To examine the effects of increasing or decreasing hypothalamic glucose sensing on glucose tolerance and insulin secretion, glucose or inhibitors of glucokinase, respectively, were infused into the third ventricle during intravenous glucose tolerance tests (IVGTTs). Glucose-infused rats displayed improved glucose handling, particularly within the first few minutes of the IVGTT, with a significantly lower area under the excursion curve within the first 10 min (AUC0-10). This was explained by increased insulin secretion. In contrast, infusion of the glucokinase inhibitors glucosamine or mannoheptulose worsened glucose tolerance and decreased GSIS in the first few minutes of IVGTT. Our data suggest a role for brain glucose sensors in the regulation of GSIS, particularly during the early phase. We propose that pharmacological agents targeting hypothalamic glucose-sensing pathways may represent novel therapeutic strategies for enhancing early phase insulin secretion in type 2 diabetes.
PMCID: PMC3266403  PMID: 22210318
12.  Comparison of Optimised MDI versus Pumps with or without Sensors in Severe Hypoglycaemia (the Hypo COMPaSS trial) 
Severe hypoglycaemia (SH) is one of the most feared complications of type 1 diabetes (T1DM) with a reported prevalence of nearly 40%. In randomized trials of Multiple Daily Injections (MDI) and Continuous Subcutaneous Insulin Infusion (CSII) therapy there is a possible benefit of CSII in reducing SH. However few trials have used basal insulin analogues as the basal insulin in the MDI group and individuals with established SH have often been excluded from prospective studies. In published studies investigating the effect of Real Time Continuous Glucose Monitoring (RT-CGM) benefit in terms of reduced SH has not yet been demonstrated. The primary objective of this study is to elucidate whether in people with T1DM complicated by impaired awareness of hypoglycaemia (IAH), rigorous prevention of biochemical hypoglycaemia using optimized existing self-management technology and educational support will restore awareness and reduce risk of recurrent SH.
This is a multicentre prospective RCT comparing hypoglycaemia avoidance with optimized MDI and CSII with or without RT-CGM in a 2×2 factorial design in people with type 1 diabetes who have IAH. The primary outcome measure for this study is the difference in IAH (Gold score) at 24 weeks. Secondary outcomes include biomedical measures such as HbA1c, SH incidence, blinded CGM analysis, self monitored blood glucose (SMBG) and response to hypoglycaemia in gold standard clamp studies. Psychosocial measures including well-being and quality of life will also be assessed using several validated and novel measures. Analysis will be on an intention-to-treat basis.
Most existing RCTs using this study’s interventions have been powered for change in HbA1c rather than IAH or SH. This trial will demonstrate whether IAH can be reversed and SH prevented in people with T1DM in even those at highest risk by using optimized conventional management and existing technology.
Trial Registration
ISRCTN52164803 Eudract No: 2009-015396-27
PMCID: PMC3556156  PMID: 23237320
13.  Neurochemical Characterization of Body Weight-Regulating Leptin Receptor Neurons in the Nucleus of the Solitary Tract 
Endocrinology  2012;153(10):4600-4607.
The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.
PMCID: PMC3507354  PMID: 22869346
14.  In Situ Hybridization Signal Patterns in Recurrent Laryngeal Squamous Papillomas Indicate that HPV Integration Occurs at an Early Stage 
Head and Neck Pathology  2011;6(1):32-37.
Laryngeal papillomas are benign tumors that frequently recur and can compromise airways. We investigated HPV genotype, physical status, and protein expression in juveniles versus adults. Thirty-five laryngeal papilloma specimens were obtained from ten juveniles (1–16 years) and eleven adults (24–67 years). In cases of recurrent papillomatosis (7 juveniles, 7 adults), the first and last papillomas were assayed. HPV type was determined by GP5+/6+ PCR and dot blot hybridization. In situ hybridization (ISH) was performed on 34 specimens; the data were recorded in terms of diffuse (episomal HPV) and punctate (integrated HPV) signal patterns. Immunohistochemistry for the HPV L1 capsid protein, a marker of HPV productive status, was performed on 32 samples. All samples tested HPV positive: HPV 11 in 2/10 (20.0%) juveniles and 5/11 (45.5%) adults; HPV 6 in 7/10 (70%) juveniles and 5/11 (45.5%) adults; and HPV 6/11 double infection was noted in one juvenile and one adult. ISH signals (punctate ± diffuse) were detected among 7/10 (70.0%) juveniles and 7/11 (63.6%) adults. L1 staining was detected in 1/9 (11.1%) juveniles and 6/10 (60.0%) adults (P = 0.06). These data support the idea that integration of low-risk HPV types into the cell genome is an early and common event in the etiology of juvenile and adult recurrent laryngeal papillomas. Productive HPV infections may be more common in adults; accordingly, constant laryngeal re-infection by HPV shed from a productive lesion may contribute to adult recurrent lesions, whereas the mechanism of papilloma recurrence in juveniles may be more attributable to HPV integration.
PMCID: PMC3311939  PMID: 22052184
Recurrent respiratory papilloma; Laryngeal papilloma; Human papillomavirus; HPV; Larynx
15.  Meta-Analysis of Overnight Closed-Loop Randomized Studies in Children and Adults with Type 1 Diabetes: The Cambridge Cohort 
We reviewed the safety and efficacy of overnight closed-loop insulin delivery compared with conventional continuous subcutaneous insulin infusion (CSII) in two distinct age groups with type 1 diabetes mellitus (T1DM), young people aged 5 to 18 years and adults, combining data of previously published randomized studies.
We evaluated four randomized crossover studies in 17 children and adolescents [13.4 ± 3.6 years; mean ± standard deviation (SD)] and 24 adults (37.5 ± 9.1 years) on 45 closed-loop (intervention) and 45 CSII (control) visits. Each subject attended for two overnight study visits, using either closed-loop or conventional pump therapy, in random order. In each age group, studies were designed to mimic realistic likely scenarios. In the children and adolescent studies, closed loop was used following a standard evening meal and following 40 min of moderate-intensity exercise. In the adult studies, closed loop was commenced following a 60 g carbohydrate meal or a 100 g carbohydrate meal accompanied by alcohol. The primary outcome measure was time for which plasma glucose was within target range (3.91–8.0 mmol/liter).
Overnight closed loop increased the time in target plasma glucose in both young (from 40% to 60%, p = .002) and adults (from 50% to 76%, p < .001) compared with conventional CSII. Combined analysis showed an increase from 43% to 71% with closed loop (p < .001). Additionally, closed loop reduced the time spent below 3.91 mmol/liter and above 8.0 mmol/liter, from 4.1% to 2.1% (p = .01) and 33% to 20% (p = .03), respectively. Glycemic variability, as measured by the SD of plasma glucose, was lower during closed loop compared with CSII (1.5 versus 2.1 mmol/liter, p = .007).
Overnight closed loop may improve glycemic control and reduce nocturnal hypoglycemia in both young people and adults with T1DM.
PMCID: PMC3262701  PMID: 22226252
adults; alcohol; closed loop; exercise; large meal; young
16.  Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem 
BMC Infectious Diseases  2012;12:212.
Viral myocarditis is a major cause of sudden unexpected death in children and young adults. Until recently, coxsackievirus B3 (CVB3) has been the most commonly implicated virus in myocarditis. At present, no standard diagnosis is generally accepted due to the insensitivity of traditional diagnostic tests. This has prompted health professionals to seek new diagnostic approaches, which resulted in the emergence of new molecular pathological tests and a more detailed immunohistochemical and histopathological analysis. When supplemented with immunohistochemistry and molecular pathology, conventional histopathology may provide important clues regarding myocarditis underlying etiology.
This study is based on post-mortem samples from sudden unexpected death victims and controls who were investigated prospectively. Immunohistochemical investigations for the detection of the enteroviral capsid protein VP1 and the characterization and quantification of myocardial inflammatory reactions as well as molecular pathological methods for enteroviral genome detection were performed.
Overall, 48 sudden unexpected death victims were enrolled. As for controls, 37 cases of unnatural traffic accident victims were studied. Enterovirus was detected in 6 sudden unexpected death cases (12.5 %). The control samples were completely enterovirus negative. Furthermore, the enteroviral capsid protein VP1 in the myocardium was detected in enterovirus-positive cases revealed by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike control samples, immunohistochemical investigations showed a significant presence of T and B lymphocytes in sudden unexpected death victims.
Our findings demonstrate clearly a higher prevalence of viral myocarditis in cases of sudden unexpected death compared to control subjects, suggesting that coxsackie B enterovirus may contribute to myocarditis pathogenesis significantly.
PMCID: PMC3462138  PMID: 22966951
17.  Insulin Pump Therapy With Automated Insulin Suspension in Response to Hypoglycemia 
Diabetes Care  2011;34(9):2023-2025.
To evaluate a sensor-augmented insulin pump with a low glucose suspend (LGS) feature that automatically suspends basal insulin delivery for up to 2 h in response to sensor-detected hypoglycemia.
The LGS feature of the Paradigm Veo insulin pump (Medtronic, Inc., Northridge, CA) was tested for 3 weeks in 31 adults with type 1 diabetes.
There were 166 episodes of LGS: 66% of daytime LGS episodes were terminated within 10 min, and 20 episodes lasted the maximum 2 h. LGS use was associated with reduced nocturnal duration ≤2.2 mmol/L in those in the highest quartile of nocturnal hypoglycemia at baseline (median 46.2 vs. 1.8 min/day, P = 0.02 [LGS-OFF vs. LGS-ON]). Median sensor glucose was 3.9 mmol/L after 2-h LGS and 8.2 mmol/L at 2 h after basal restart.
Use of an insulin pump with LGS was associated with reduced nocturnal hypoglycemia in those at greatest risk and was well accepted by patients.
PMCID: PMC3161284  PMID: 21868778
18.  Stool submission by general practitioners in SW England - when, why and how? A qualitative study 
BMC Family Practice  2012;13:77.
We know little about when and why general practitioners (GPs) submit stool specimens in patients with diarrhoea. The recent UK-wide intestinal infectious disease (IID2) study found ten GP consultations for every case reported to national surveillance. We aimed to explore what factors influence GP’s decisions to send stool specimens for laboratory investigation, and what guidance, if any, informs them.
We used qualitative methods that enabled us to explore opinions and ask open questions through 20 telephone interviews with GPs with a range of stool submission rates in England, and a discussion group with 24 GPs. Interviews were transcribed and subjected to content analysis.
Interviews: GPs only sent stool specimens to microbiology if diarrhoea persisted for over one week, after recent travel, or the patient was very unwell. Very few had a systematic approach to determine the clinical or public health need for a stool specimen. Only two GPs specifically asked patients about blood in their stool; only half asked about recent antibiotics, or potential food poisoning, and few asked about patients’ occupations. Few GPs gave patients advice on how to collect specimens.
Results from interviews and discussion group in relation to guidance: All reported that the HPA stool guidance and patient collection instructions would be useful in their clinical work, but only one GP (an interviewee) had previously accessed them. The majority of GPs would value links to guidance on electronic requests. Most GPs were surprised that a negative stool report did not exclude all the common causes of IID.
GPs value stool culture and laboratories should continue to provide it. Patient instructions on how to collect stool specimens should be within stool collection kits. Through readily accessible guidance and education, GPs need to be encouraged to develop a more systematic approach to eliciting and recording details in the patient’s history that indicate greater risk of severe infection or public health consequences. Mild or short duration IID (under one week) due to any cause is less likely to be picked up in national surveillance as GPs do not routinely submit specimens in these cases.
PMCID: PMC3481435  PMID: 22870944
Stool specimens; Microbiology; Laboratory submission; Diarrhoea; Primary care; Qualitative; National guidance
19.  Neurochemical Characterization of Body Weight-Regulating Leptin Receptor Neurons in the Nucleus of the Solitary Tract 
Endocrinology  2012;153(10):4600-4607.
The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.
PMCID: PMC3507354  PMID: 22869346
20.  Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry 
Head and Neck Pathology  2011;5(4):344-348.
Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16INK4a immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16INK4a staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16INK4a negative and two HPV negative samples were p16INK4a positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16INK4a positive, PCR and CISH HPV positive/p16 INK4a negative, or PCR HPV positive/p16 INK4a and CISH negative, also requires further investigation.
PMCID: PMC3210222  PMID: 21786153
Tonsillar carcinoma; Human papillomavirus (HPV); p16INK4a
21.  Rapid measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in human biological matrices using ultra-high-performance liquid chromatography–tandem mass spectrometry 
Free Radical Biology & Medicine  2012;52(10):2057-2063.
Interaction of reactive oxygen species with DNA results in a variety of modifications, including 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), which has been extensively studied as a biomarker of oxidative stress. Oxidative stress is implicated in a number of pathophysiological processes relevant to obstetrics and gynecology; however, there is a lack of understanding as to the precise role of oxidative stress in these processes. We aimed to develop a rapid, validated assay for the accurate quantification of 8-oxodG in human urine using solid-phase extraction and ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and then investigate the levels of 8-oxodG in several fluids of interest to obstetrics and gynecology. Using UHPLC–MS/MS, 8-oxodG eluted after 3.94 min with an RSD for 15 injections of 0.07%. The method was linear between 0.95 and 95 nmol/L with LOD and LOQ of 5 and 25 fmol on-column, respectively. Accuracy and precision were 98.7–101.0 and <10%, respectively, over three concentrations of 8-oxodG. Recovery from urine was 88% with intra- and interday variations of 4.0 and 10.2%, respectively. LOQ from urine was 0.9 pmol/ml. Rank order from the greatest to lowest 8-oxodG concentration was urine>seminal plasma>amniotic fluid>plasma>serum>peritoneal fluid, and it was not detected in saliva. Urine concentrations normalized to creatinine (n=15) ranged between 0.55 and 1.95 pmol/μmol creatinine. We describe, for the first time, 8-oxodG concentrations in human seminal plasma, peritoneal fluid, amniotic fluid, and breast milk, as well as in urine, plasma, and serum, using a rapid UHPLC–MS/MS method that will further facilitate biomonitoring of oxidative stress.
► We report a novel, rapid solid-phase extraction UHPLC–MS/MS method for urinary 8-oxodG. ► We examined 8-oxodG in biological matrices of interest to reproductive medicine. ► We quantified 8-oxodG in plasma, serum, and seminal, amniotic, and peritoneal fluids. ► Oxidative stress can be accurately and rapidly quantified in clinical samples.
PMCID: PMC3404459  PMID: 22542794
ROS, reactive oxygen species; 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; HPLC, high-performance liquid chromatography; RSD, relative standard deviation; SPE, solid-phase extraction; UHPLC, ultra-high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; 8-Oxo-7,8-dihydro-2′-deoxyguanosine; Mass spectrometry; Human; Biomarkers; Oxidative stress; DNA repair; Oxidatively damaged DNA; Urine; Free radicals
22.  Leptin does not directly affect CNS serotonin neurons to influence appetite 
Cell metabolism  2011;13(5):584-591.
Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sertcre) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sertcre promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sertcre-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.
PMCID: PMC3087147  PMID: 21531340
serotonin; leptin; food intake; PCPA; Sert-Cre; electrophysiology; raphe
24.  Neuropeptide Y Cells Represent a Distinct Glucose-Sensing Population in the Lateral Hypothalamus 
Endocrinology  2011;152(11):4046-4052.
The maintenance of appropriate glucose levels is necessary for survival. Within the brain, specialized neurons detect glucose fluctuations and alter their electrical activity. These glucose-sensing cells include hypothalamic arcuate nucleus neurons expressing neuropeptide Y (NPY) and lateral hypothalamic area (LHA) neurons expressing orexin/hypocretins (ORX) or melanin-concentrating hormone (MCH). Within the LHA, a population of NPY-expressing cells exists; however, their ability to monitor energy status is unknown. We investigated whether NPY neurons located in the LHA, a classic hunger center, detect and respond to fluctuations in glucose availability and compared these responses with those of known LHA glucose sensors expressing ORX or MCH. Using mice expressing green fluorescent protein under the control of NPY regulatory elements, we identified LHA NPY cells and explored their anatomical distribution, neurochemical and electrical properties, in vivo responses to fasting and insulin-induced hypoglycemia, and in situ electrical responses to extracellular glucose. We report that NPY, ORX, and MCH are expressed in nonoverlapping populations within the LHA. Subpopulations of LHA NPY neurons were activated in vivo by both a 6-h fast and insulin-induced hypoglycemia. Likewise, increased extracellular glucose suppressed the electrical activity of approximately 70% of LHA NPY neurons in situ, eliciting hyperpolarization and activating background K+ currents. Furthermore, we report that the glucose sensitivity of LHA NPY neurons is significantly different from neighboring ORX and MCH neurons. These data suggest that NPY-expressing cells in the LHA are a novel population of glucose-sensing neurons that represent a new player in the brain circuitry integrating information about glucose homeostasis.
PMCID: PMC3328128  PMID: 21914773
25.  Recurrent Hypoglycemia Is Associated with Loss of Activation in Rat Brain Cingulate Cortex 
Endocrinology  2012;153(4):1908-1914.
A subset of people with diabetes fail to mount defensive counterregulatory responses (CRR) to hypoglycemia. Although the mechanisms by which this occurs remain unclear, recurrent exposure to hypoglycemia may be an important etiological factor. We hypothesized that loss of CRR to recurrent exposure to hypoglycemia represents a type of stress desensitization, in which limbic brain circuitry involved in modulating stress responses might be implicated. Here, we compared activation of limbic brain regions associated with stress desensitization during acute hypoglycemia (AH) and recurrent hypoglycemia (RH). Healthy Sprague Dawley rats were exposed to either acute or recurrent 3-d hypoglycemia. We also examined whether changes in neuronal activation were caused directly by the CRR itself by infusing epinephrine, glucagon, and corticosterone without hypoglycemia. AH increased neuronal activity as quantified by c-fos immunoreactivity (FOS-IR) in the cingulate cortex and associated ectorhinal and perirhinal cortices but not in an adjacent control area (primary somatosensory cortex). FOS-IR was not observed after hormone infusion, suggesting that AH-associated activation was caused by hypoglycemia rather than by CRR. Importantly, AH FOS-IR activation was significantly blunted in rats exposed to RH. In conclusion, analogous with other models of stress habituation, activation in the cingulate cortex and associated brain areas is lost with exposure to RH. Our data support the hypothesis that limbic brain areas may be associated with the loss of CRR to RH in diabetes.
PMCID: PMC3328129  PMID: 22396449

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