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1.  Stomach infarction in an ex-premature infant 
BMJ Case Reports  2014;2014:bcr2013202814.
Gastric pneumatosis and thickened gastric wall are rare radiological findings that may be indicative of severe gastrointestinal tract ischaemia or necrosis; we report a case with a brief discussion of the literature. The premature neonate conveyed an interesting series of rare X-ray findings which were secondary to extensive gastric, duodenal and proximal jejunal infarction. She was managed palliatively and died.
doi:10.1136/bcr-2013-202814
PMCID: PMC3912369  PMID: 24488664
2.  DNA repair factor BRCA1 depletion occurs in Alzheimer brains and impairs cognitive function in mice 
Nature Communications  2015;6:8897.
Maintaining DNA integrity is vital for all cells and organisms. Defective DNA repair may contribute to neurological disorders, including Alzheimer's disease (AD). We found reduced levels of BRCA1, but not of other DNA repair factors, in the brains of AD patients and human amyloid precursor protein (hAPP) transgenic mice. Amyloid-β oligomers reduced BRCA1 levels in primary neuronal cultures. In wild-type mice, knocking down neuronal BRCA1 in the dentate gyrus caused increased DNA double-strand breaks, neuronal shrinkage, synaptic plasticity impairments, and learning and memory deficits, but not apoptosis. Low levels of hAPP/Amyloid-β overexpression exacerbated these effects. Physiological neuronal activation increased BRCA1 levels, whereas stimulating predominantly extrasynaptic N-methyl-D-aspartate receptors promoted the proteasomal degradation of BRCA1. We conclude that BRCA1 is regulated by neuronal activity, protects the neuronal genome, and critically supports neuronal integrity and cognitive functions. Pathological accumulation of Aβ depletes neuronal BRCA1, which may contribute to cognitive deficits in AD.
DNA repair deficits have been suggested to play a role in Alzheimer's pathology. Here, the authors report reduced levels of the DNA repair factor BRCA1 in patient brains, and provide evidence that loss of BRCA1 in the dentate gyrus leads to spatial learning and memory deficits in mice.
doi:10.1038/ncomms9897
PMCID: PMC4674776  PMID: 26615780
3.  Accuracy of Continuous Glucose Monitoring During Three Closed-Loop Home Studies Under Free-Living Conditions 
Diabetes Technology & Therapeutics  2015;17(11):801-807.
Abstract
Objectives: Closed-loop (CL) systems modulate insulin delivery based on glucose levels measured by a continuous glucose monitor (CGM). Accuracy of the CGM affects CL performance and safety. We evaluated the accuracy of the Freestyle Navigator® II CGM (Abbott Diabetes Care, Alameda, CA) during three unsupervised, randomized, open-label, crossover home CL studies.
Materials and Methods: Paired CGM and capillary glucose values (10,597 pairs) were collected from 57 participants with type 1 diabetes (41 adults [mean±SD age, 39±12 years; mean±SD hemoglobin A1c, 7.9±0.8%] recruited at five centers and 16 adolescents [mean±SD age, 15.6±3.6 years; mean±SD hemoglobin A1c, 8.1±0.8%] recruited at two centers). Numerical accuracy was assessed by absolute relative difference (ARD) and International Organization for Standardization (ISO) 15197:2013 15/15% limits, and clinical accuracy was assessed by Clarke error grid analysis.
Results: Total duration of sensor use was 2,002 days (48,052 h). Overall sensor accuracy for the capillary glucose range (1.1–27.8 mmol/L) showed mean±SD and median (interquartile range) ARD of 14.2±15.5% and 10.0% (4.5%, 18.4%), respectively. Lowest mean ARD was observed in the hyperglycemic range (9.8±8.8%). Over 95% of pairs were in combined Clarke error grid Zones A and B (A, 80.1%, B, 16.2%). Overall, 70.0% of the sensor readings satisfied ISO criteria. Mean ARD was consistent (12.3%; 95% of the values fall within ±3.7%) and not different between participants (P=0.06) within the euglycemic and hyperglycemic range, when CL is actively modulating insulin delivery.
Conclusions: Consistent accuracy of the CGM within the euglycemic–hyperglycemic range using the Freestyle Navigator II was observed and supports its use in home CL studies. Our results may contribute toward establishing normative CGM performance criteria for unsupervised home use of CL.
doi:10.1089/dia.2015.0062
PMCID: PMC4649721  PMID: 26241693
4.  Rapid Modulation of Axon Initial Segment Length Influences Repetitive Spike Firing 
Cell Reports  2015;13(6):1233-1245.
Summary
Neurons implement a variety of plasticity mechanisms to alter their function over timescales ranging from seconds to days. One powerful means of controlling excitability is to directly modulate the site of spike initiation, the axon initial segment (AIS). However, all plastic structural AIS changes reported thus far have been slow, involving days of neuronal activity perturbation. Here, we show that AIS plasticity can be induced much more rapidly. Just 3 hr of elevated activity significantly shortened the AIS of dentate granule cells in a calcineurin-dependent manner. The functional effects of rapid AIS shortening were offset by dephosphorylation of voltage-gated sodium channels, another calcineurin-dependent mechanism. However, pharmacological separation of these phenomena revealed a significant relationship between AIS length and repetitive firing. The AIS can therefore undergo a rapid form of structural change over timescales that enable interactions with other forms of activity-dependent plasticity in the dynamic control of neuronal excitability.
Graphical Abstract
Highlights
•Structural plasticity at the axon initial segment can occur within hours•Ankyrin-G and sodium channel distributions shorten after 3 hr of elevated activity•Rapid plasticity depends on calcineurin signaling opposed by CDK5•All else being equal, AIS shortening correlates with lowered neuronal excitability
Evans et al. show that activity-dependent structural plasticity at the axon initial segment (AIS) can be surprisingly rapid. Elevated activity shortens the AIS in dentate granule cells after just 3 hr, an effect associated with lower excitability and reduced repetitive spiking.
doi:10.1016/j.celrep.2015.09.066
PMCID: PMC4646840  PMID: 26526995
5.  Home use of closed loop insulin delivery improves overnight glucose control in adults with type 1 diabetes: A four-week multicentre randomised crossover study 
Summary
Background
We assessed whether overnight home use of automated closed loop insulin delivery (artificial pancreas) improves glucose control.
Methods
We studied 24 adults with type 1 diabetes in a multicentre crossover study design comparing four weeks of overnight closed loop using a model predictive control algorithm to direct insulin delivery, with four weeks of insulin pump therapy in which participants used real-time display of continuous glucose monitoring independent of their pumps as control. Primary outcome was time when glucose was in the target range of 3·9 and 8·0mmol/l between midnight to 07:00. Analyses were by intention to treat. Trial registration ClinicalTrials.gov NCT01440140.
Findings
Closed loop was utilised over median 8·3 (interquartile range 6·0, 9·6)hours on 555nights (86%). Proportion of time when overnight glucose was in target range was significantly higher during closed loop compared to control by 13·5% (95% CI, 7·3–19·7; p<0·001). Mean overnight glucose (8·2±0·9 vs. 9·0±1·3mmol/l; p=0·005) and time spent above target (44·3%±11·9 vs. 57·1%±15·6; p=0·001) were significantly lower during closed loop. Time spent below target was low and comparable [1·8%(0·6, 3·6) vs. 2·1%(0·7, 3·9); p=0·28]. Lower mean overnight glucose was brought about by increased overnight insulin delivery [6·4 (4·5, 8·1) vs. 4·9 (3·7, 6·3)units; p<0·001) without changing the total daily insulin amount [34·5 (29·3, 48·4) vs. 35·4 (29·7, 45·2)units; p=0·32]. No severe hypoglycaemia episodes occurred during control period and two during closed loop not related to algorithm instructions.
Interpretation
Unsupervised overnight closed loop at home is feasible and may improve glucose control in adults with type 1 diabetes.
doi:10.1016/S2213-8587(14)70114-7
PMCID: PMC4165604  PMID: 24943065
6.  Epidermal growth factor receptor pathway mutation and expression profiles in cervical squamous cell carcinoma: therapeutic implications 
Background
Cervical squamous cell carcinoma (CSCC) is a major cause of female mortality worldwide. This study has examined epidermal growth factor receptor (EGFR) pathway markers that represent actionable pharmacological targets.
Methods
HPV16 positive CSCCs (n = 105 patients) from Madhya Pradesh, India were screened for KRAS and PIK3CA mutations by PNA-clamp real-time PCR. Immunohistochemistry (IHC) was performed for EGFR, PIK3CA, PTEN, phospho-AKT, phospho-mTOR and phospho-44/42 MAPK (ERK1/2).
Results
KRAS mutations were detected in 0/91 (0%) and PIK3CA mutations in 19/95 (20.0%) informative specimens: exon 9, E542 (n = 3) and E545 (n = 15); exon 20, H1047R (n = 1). PIK3CA mutation detection was associated with older mean patient age [48.2 vs. 56.6 years (P = 0.007)] and with post-menopausal age: 5/45 (11.1%) patients <50 years vs. 14/50 (28.0%) patients ≥50 years (P = 0.045; OR = 3.11). EGFR expression was present in 60/101 (59.4%) CSCCs and was associated with PIK3CA mutation detection (P < 0.05) but not age (P > 0.05). EGFR and phospho-AKT staining showed associations with tumor grade and/or lymph node status (P < 0.05). Significant associations were not found for the other study markers (P > 0.05).
Conclusion
These data show that PIK3CA mutation acquisition is related to patient age and EGFR expression. The absence of KRAS mutations supports the potential of anti-EGFR therapies for CSCC treatment. The relatively high PIK3CA mutation rates indicate that PI3K may be a therapeutic target for a significant subset of CSCC patients. Qualitatively distinct IHC staining profiles for the marker panel were noted patient to patient; however, across patients, consistent linear relationships between up- and downstream pathway markers were not observed. Evaluation of the expression status of potential cancer pathway targets may be of value in addition to molecular profiling for choosing among therapeutic options.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0611-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-015-0611-0
PMCID: PMC4513684  PMID: 26209091
Cervical cancer; HPV16; EGFR; PIK3CA; KRAS; PTEN; AKT; mTOR; MAPK; ERK1/2
7.  A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation 
Neuroepigenetics  2015;3:1-6.
Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.
doi:10.1016/j.nepig.2015.06.001
PMCID: PMC4659419  PMID: 26702400
DNA methylation; Neuronal activity; Calcium channels; Reduced representation bisulfite sequencing; Hippocampal; Pharmacological manipulation
8.  Up-Regulation of miR-21 Is Associated with Cervicitis and Human Papillomavirus Infection in Cervical Tissues 
PLoS ONE  2015;10(5):e0127109.
MicroRNA-21 (miR-21) is recognized as an oncomir and shows up-regulation in many types of human malignancy. The aim of this study was to investigate the association of miR-21 expression associated with HPV infection in normal and abnormal cervical tissues. Cervical tissue samples with different cytological or histopathological grades were investigated for HPV by PCR and for miR-21 and programmed cell death, protein 4 (PDCD4) expression using quantitative real-time PCR (qRT-PCR). Laser capture microdissection (LCM) of stromal and epithelial tissues and in situ hybridization (ISH) using locked nucleic acid (LNA) probes were performed on a subset of fixed specimens. Cell line experiments were conducted on fibroblasts stimulated in culture media from HeLa cells, which were then assessed for miR-21, PDCD4, IL-6 and α-SMA expression by qRT-PCR. Twenty normal cervical cell, 12 cervicitis, 14 cervical intraepithelial neoplastic I (CIN I), 22 CIN II-III and 43 cervical squamous cell carcinoma (SCC) specimens were investigated. miR-21 levels were significantly lower in normal than in abnormal tissues. The expression of miR-21 in HPV negative normal cytology was significantly lower than in HPV positive samples in abnormal tissue and SCC. The miR-21 expression was significantly higher in HPV negative cervicitis than HPV negative normal cells. LCM and ISH data showed that miR-21 is primarily expressed in the tumor-associated stromal cell microenvironment. Fibroblasts treated with HeLa cell culture media showed up-regulated expression of miR-21, which correlated with increased expression of α-SMA and IL-6 and with down-regulation of PDCD4. These results demonstrate that miR-21 is associated with HPV infection and involved in cervical lesions as well as cervicitis and its up-regulation in tumor-stroma might be involved in the inflammation process and cervical cancer progression.
doi:10.1371/journal.pone.0127109
PMCID: PMC4444121  PMID: 26010154
9.  Evaluating the Performance of a Novel Embedded Closed-loop System 
The objective was to assess the reliability of a novel automated closed-loop glucose control system developed within the AP@home consortium in adults with type 1 diabetes. Eight adults with type 1 diabetes on insulin pump therapy (3 men; ages 40.5 ± 14.3 years; HbA1c 8.2 ± 0.8%) participated in an open-label, single-center, single-arm, 12-hour overnight study performed at the clinical research facility. A standardized evening meal (80 g CHO) accompanied by prandial insulin boluses were given at 19:00 followed by an optional snack of 15 g at 22:00 without insulin bolus. Automated closed-loop glucose control was started at 19:00 and continued until 07:00 the next day. Basal insulin delivery (Accu-Chek Spirit, Roche) was automatically adjusted by Cambridge model predictive control algorithm, running on a purpose-built embedded device, based on real-time continuous glucose monitor readings (Dexcom G4 Platinum). Closed-loop system was operational as intended over 99% of the time. Overnight plasma glucose levels (22:00 to 07:00) were within the target range (3.9 to 8.0 mmol/l) for 75.4% (37.5, 92.9) of the time without any time spent in hypoglycemia (<3.9 mmol/l). Mean overnight glucose was 7.8 ± 1.3 mmol/l. For the entire 12-hour closed-loop period (19:00 until 07:00) plasma glucose levels were within the target range (3.9 to 10.0 mmol/l) for 84.4% (63.3, 100) of time. There were no adverse events noted during the trial. We observed a high degree of reliability of the automated closed-loop system. The time spent in target glucose level overnight was comparable to results of previously published studies. Further developments to miniaturize the system for home studies are warranted.
doi:10.1177/1932296813519882
PMCID: PMC4455420  PMID: 24876577
closed-loop; continuous glucose monitoring; Dexcom G4 Platinum; MPC algorithm; AP@home consortium
10.  HIV Type 1 Polymerase Gene Polymorphisms Are Associated With Phenotypic Differences in Replication Capacity and Disease Progression 
Background. Determinants of intersubtype differences in human immunodeficiency virus type 1 (HIV-1) clinical disease progression remain unknown.
Methods. HIV-1 subtype was independently determined for 5 separate genomic regions in 396 HIV-1 seroconverters from Rakai, Uganda, using a multiregion hybridization assay. Replication capacities (RC) in samples from a subset of 145 of these subjects were determined. HIV-1 genomic regions and pol RC were examined for association with disease progression. Amino acid polymorphisms were examined for association with pol RC.
Results. In multivariate analyses, the hazard for progression to the composite end point (defined as a CD4+ T-cell count <250 cells/mm3, antiretroviral therapy initiation, or death) among patients with subtype D pol infection was 2.4 times the hazard for those infected with subtype A pol infection (P = .001). Compared with subtype A pol (the reference group), the hazard for progression to the composite end point for subtype D pol infection with a pol RC >67% (ie, the median pol RC) was significantly greater (HR, 4.6; 95% confidence interval [CI], 1.9–11.0; P = .001), whereas the hazard for progression to the composite end point for subtype D pol infection with a pol RC ≤67% was not significantly different (HR, 2.2; 95% CI, 1.0–4.9; P = .051). Amino acid substitutions at protease positions 62 and 64 and at reverse transcriptase position 272 were associated with significant differences in pol RC.
Conclusions. HIV-1 pol gene intersubtype and RC differences are associated with disease progression and may be influenced by amino acid polymorphisms.
doi:10.1093/infdis/jit425
PMCID: PMC3864385  PMID: 23922373
HIV-1 Subtype; subtype A; subtype D; disease progression; polymerase; replication capacity; amino acid polymorphisms
11.  Management of suspected infectious diarrhoea by English GPs: are they right? 
Background
The criteria used when GPs submit stool specimens for microbiological investigation are unknown.
Aim
To determine what criteria GPs use to send stool specimens, and if they are consistent with national guidance, and whether GPs would prescribe an antibiotic before they receive a result.
Design and setting
Questionnaire survey of 974 GPs in 172 surgeries in England.
Method
GPs were sent a questionnaire (23 questions) based on national guidance.
Results
Questionnaires were returned by 90% (154/172) of surgeries and 49% (477/968) of GPs. GPs reported sending stool specimens in about 50% of cases of suspected infectious diarrhoea, most commonly because of individual symptoms, rather than public health implications. Fewer considered sampling with antibiotic-associated diarrhoea post hospitalisation, or children with acute, painful, bloody diarrhoea; only 14% mentioned outbreaks as a reason. Nearly one-half of GPs reported they would consider antibiotics in suspected cases of Escherichia coli O157, which is contraindicated. Only 23% of GPs would send the recommended three specimens for ova, cysts, and parasites (OCP) examination. Although 89% of GPs gave some verbal advice on how to collect stool specimens, only 2% of GPs gave patients any written instructions.
Conclusion
GPs need more education to address gaps in knowledge about the risks and diagnosis of different infections in suspected infectious diarrhoea, especially Clostridium difficile post-antibiotics, E. coli O157, and requesting OCPs. Advice on reports, tick boxes, or links to guidance on electronic request forms may facilitate this.
doi:10.3399/bjgp14X676429
PMCID: PMC3876145  PMID: 24567579
antibiotics; diarrhoea; E. coli O157; general practice; guidance; investigation
12.  Randomized Trial of Infusion Set Function: Steel Versus Teflon 
Abstract
Background: This study compared infusion set function for up to 1 week using either a Teflon® (Dupont™, Wilmington, DE) catheter or a steel catheter for insulin pump therapy in type 1 diabetes mellitus.
Subjects and Methods: Twenty subjects participating in a randomized, open-labeled, crossover study were asked to wear two Quick-Set® and two Sure-T® infusion sets (both from Medtronic Minimed, Northridge, CA) until the infusion set failed or was worn for 1 week. All subjects wore a MiniMed continuous glucose monitoring system for the duration of the study.
Results: One subject withdrew from the study. There were 38 weeks of Sure-T wear and 39 weeks of Quick-Set wear with no difference in the survival curves of the infusion sets. There was, however, a 15% initial failure rate with the Teflon infusion set. After 7 days, both types of infusion sets had a 64% failure rate. Overall, 30% failed because of hyperglycemia and a failed correction dose, 13% were removed for pain, 10% were pulled out by accident, 10% had erythema and/or induration of>10 mm, 5% fell out because of loss of adhesion, and 4% were removed for infection. The main predictor of length of wear was the individual subject. There was no increase in hyperglycemia or daily insulin requirements when an infusion set was successfully used for 7 days (n=25 of 77 weeks).
Conclusions: We found no difference between steel and Teflon infusion sets in their function over 7 days, although 15% of Teflon sets failed because of kinking on insertion. The strongest predictor of prolonged 7-day infusion set function was the individual subject, not the type of infusion set.
doi:10.1089/dia.2013.0119
PMCID: PMC3887416  PMID: 24090124
13.  Lysosomal Two-pore Channel Subtype 2 (TPC2) Regulates Skeletal Muscle Autophagic Signaling* 
The Journal of Biological Chemistry  2014;290(6):3377-3389.
Background: The endolysosomal TPC2 ion channel interacts with mTOR to regulate cellular energy utilization.
Results: Mice lacking TPC2 display muscle atrophy phenotype with reduced muscle endurance, altered autophagy, and lysosomal enzymatic activities.
Conclusion: TPC2 regulates autophagic signaling in skeletal muscle.
Significance: TPC2 impacts protein turnover via regulating autophagy signaling in the process of tissue homeostasis and aging.
Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2−/− mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.
doi:10.1074/jbc.M114.608471
PMCID: PMC4319008  PMID: 25480788
Autophagy; Calcium Channel; Lysosome; Muscle Atrophy; Protein Turnover
14.  A Parabrachial-Hypothalamic Cholecystokinin Neurocircuit Controls Counterregulatory Responses to Hypoglycemia 
Cell Metabolism  2014;20(6):1030-1037.
Summary
Hypoglycemia engenders an autonomically mediated counterregulatory (CR)-response that stimulates endogenous glucose production to maintain concentrations within an appropriate physiological range. Although the involvement of the brain in preserving normoglycemia has been established, the neurocircuitry underlying centrally mediated CR-responses remains unclear. Here we demonstrate that lateral parabrachial nucleus cholecystokinin (CCKLPBN) neurons are a population of glucose-sensing cells (glucose inhibited) with counterregulatory capacity. Furthermore, we reveal that steroidogenic-factor 1 (SF1)-expressing neurons of the ventromedial nucleus of the hypothalamus (SF1VMH) are the specific target of CCKLPBN glucoregulatory neurons. This discrete CCKLPBN→SF1VMH neurocircuit is both necessary and sufficient for the induction of CR-responses. Together, these data identify CCKLPBN neurons, and specifically CCK neuropeptide, as glucoregulatory and provide significant insight into the homeostatic mechanisms controlling CR-responses to hypoglycemia.
Graphical Abstract
Highlights
•CCKLPBN neurons are glucose inhibited and activated by hypoglycemia•CCKLPBN neurons are necessary and sufficient for counterregulatory (CR)-responses•CCK neuropeptide is the key mediator of CCKLPBN neuron-mediated CR-responses•CCKLPBN neuron-induced CR-responses require downstream SF1VMH neurons
The counterregulatory response (CRR) to hypoglycemia is critical for the maintenance of normoglycemia and governed by the brain. Garfield et al. identify a population of brainstem CCK neurons that directly sense extracellular glucose concentrations and, via their connection to SF1 hypothalamic neurons, promote CRR.
doi:10.1016/j.cmet.2014.11.006
PMCID: PMC4261079  PMID: 25470549
15.  Restoration of Self-Awareness of Hypoglycemia in Adults With Long-Standing Type 1 Diabetes 
Diabetes Care  2013;36(12):4063-4070.
OBJECTIVE
Impaired awareness of hypoglycemia (IAH) and defective counterregulation significantly increase severe hypoglycemia risk in type 1 diabetes (T1D). We evaluated restoration of IAH/defective counterregulation by a treatment strategy targeted at hypoglycemia avoidance in adults with T1D with IAH (Gold score ≥4) participating in the U.K.-based multicenter HypoCOMPaSS randomized controlled trial.
RESEARCH DESIGN AND METHODS
Eighteen subjects with T1D and IAH (mean ± SD age 50 ± 9 years, T1D duration 35 ± 10 years, HbA1c 8.1 ± 1.0% [65 ± 10.9 mmol/mol]) underwent stepped hyperinsulinemic-hypoglycemic clamp studies before and after a 6-month intervention. The intervention comprised the HypoCOMPaSS education tool in all and randomized allocation, in a 2 × 2 factorial study design, to multiple daily insulin analog injections or continuous subcutaneous insulin infusion therapy and conventional glucose monitoring or real-time continuous glucose monitoring. Symptoms, cognitive function, and counterregulatory hormones were measured at each glucose plateau (5.0, 3.8, 3.4, 2.8, and 2.4 mmol/L), with each step lasting 40 min with subjects kept blinded to their actual glucose value throughout clamp studies.
RESULTS
After intervention, glucose concentrations at which subjects first felt hypoglycemic increased (mean ± SE from 2.6 ± 0.1 to 3.1 ± 0.2 mmol/L, P = 0.02), and symptom and plasma metanephrine responses to hypoglycemia were higher (median area under curve for symptoms, 580 [interquartile range {IQR} 420–780] vs. 710 [460–1,260], P = 0.02; metanephrine, 2,412 [−3,026 to 7,279] vs. 5,180 [−771 to 11,513], P = 0.01). Glycemic threshold for deterioration of cognitive function measured by four-choice reaction time was unchanged, while the color-word Stroop test showed a degree of adaptation.
CONCLUSIONS
Even in long-standing T1D, IAH and defective counterregulation may be improved by a clinical strategy aimed at hypoglycemia avoidance.
doi:10.2337/dc13-1004
PMCID: PMC3836150  PMID: 24130355
16.  Day and Night Closed-Loop Control in Adults With Type 1 Diabetes 
Diabetes Care  2013;36(12):3882-3887.
OBJECTIVE
To compare two validated closed-loop (CL) algorithms versus patient self-control with CSII in terms of glycemic control.
RESEARCH DESIGN AND METHODS
This study was a multicenter, randomized, three-way crossover, open-label trial in 48 patients with type 1 diabetes mellitus for at least 6 months, treated with continuous subcutaneous insulin infusion. Blood glucose was controlled for 23 h by the algorithm of the Universities of Pavia and Padova with a Safety Supervision Module developed at the Universities of Virginia and California at Santa Barbara (international artificial pancreas [iAP]), by the algorithm of University of Cambridge (CAM), or by patients themselves in open loop (OL) during three hospital admissions including meals and exercise. The main analysis was on an intention-to-treat basis. Main outcome measures included time spent in target (glucose levels between 3.9 and 8.0 mmol/L or between 3.9 and 10.0 mmol/L after meals).
RESULTS
Time spent in the target range was similar in CL and OL: 62.6% for OL, 59.2% for iAP, and 58.3% for CAM. While mean glucose level was significantly lower in OL (7.19, 8.15, and 8.26 mmol/L, respectively) (overall P = 0.001), percentage of time spent in hypoglycemia (<3.9 mmol/L) was almost threefold reduced during CL (6.4%, 2.1%, and 2.0%) (overall P = 0.001) with less time ≤2.8 mmol/L (overall P = 0.038). There were no significant differences in outcomes between algorithms.
CONCLUSIONS
Both CAM and iAP algorithms provide safe glycemic control.
doi:10.2337/dc12-1956
PMCID: PMC3836152  PMID: 24170747
18.  Assessing the effectiveness of 3 months day and night home closed-loop insulin delivery in adults with suboptimally controlled type 1 diabetes: a randomised crossover study protocol 
BMJ Open  2014;4(9):e006075.
Introduction
Despite therapeutic advances, many people with type 1 diabetes are still unable to achieve optimal glycaemic control, limited by the occurrence of hypoglycaemia. The objective of the present study is to determine the effectiveness of day and night home closed-loop over the medium term compared with sensor-augmented pump therapy in adults with type 1 diabetes and suboptimal glycaemic control.
Methods and analysis
The study will adopt an open label, three-centre, multinational, randomised, two-period crossover study design comparing automated closed-loop glucose control with sensor augmented insulin pump therapy. The study will aim for 30 completed participants. Eligible participants will be adults (≥18 years) with type 1 diabetes treated with insulin pump therapy and suboptimal glycaemic control (glycated haemoglobin (HbA1c) ≥7.5% (58 mmol/mmol) and ≤10% (86 mmol/mmol)). Following a 4-week optimisation period, participants will undergo a 3-month use of automated closed-loop insulin delivery and sensor-augmented pump therapy, with a 4–6 week washout period in between. The order of the interventions will be random. All analysis will be conducted on an intention to treat basis. The primary outcome is the time spent in the target glucose range from 3.9 to 10.0 mmol/L based on continuous glucose monitoring levels during the 3 months free living phase. Secondary outcomes include HbA1c changes; mean glucose and time spent above and below target glucose levels. Further, participants will be invited at baseline, midpoint and study end to participate in semistructured interviews and complete questionnaires to explore usability and acceptance of the technology, impact on quality of life and fear of hypoglycaemia.
Ethics and dissemination
Ethical approval has been obtained at all sites. Before screening, all participants will be provided with oral and written information about the trial. The study will be disseminated by peer-review publications and conference presentations.
Trial registration number
NCT01961622 (ClinicalTrials.gov).
doi:10.1136/bmjopen-2014-006075
PMCID: PMC4158197  PMID: 25186158
Closed-loop; Type 1 diabetes; Continuous glucose monitoring; Artificial pancreas; Model predictive control
19.  5-HT Obesity Medication Efficacy via POMC Activation is Maintained During Aging 
Endocrinology  2014;155(10):3732-3738.
The phenomenon commonly described as the middle-age spread is the result of elevated adiposity accumulation throughout adulthood until late middle-age. It is a clinical imperative to gain a greater understanding of the underpinnings of age-dependent obesity and, in turn, how these mechanisms may impact the efficacy of obesity treatments. In particular, both obesity and aging are associated with rewiring of a principal brain pathway modulating energy homeostasis, promoting reduced activity of satiety pro-opiomelanocortin (POMC) neurons within the arcuate nucleus of the hypothalamus (ARC). Using a selective ARC-deficient POMC mouse line, here we report that former obesity medications augmenting endogenous 5-hydroxytryptamine (5-HT) activity d-fenfluramine and sibutramine require ARC POMC neurons to elicit therapeutic appetite-suppressive effects. We next investigated whether age-related diminished ARC POMC activity therefore impacts the potency of 5-HT obesity pharmacotherapies, lorcaserin, d-fenfluramine, and sibutramine and report that all compounds reduced food intake to a comparable extent in both chow-fed young lean (3–5 months old) and middle-aged obese (12–14 months old) male and female mice. We provide a mechanism through which 5-HT anorectic potency is maintained with age, via preserved 5-HT–POMC appetitive anatomical machinery. Specifically, the abundance and signaling of the primary 5-HT receptor influencing appetite via POMC activation, the 5-HT2CR, is not perturbed with age. These data reveal that although 5-HT obesity medications require ARC POMC neurons to achieve appetitive effects, the anorectic efficacy is maintained with aging, findings of clinical significance to the global aging obese population.
doi:10.1210/en.2014-1223
PMCID: PMC4164923  PMID: 25051442
20.  Evidence That Alpha-9 Human Papillomavirus Infections are a Major Etiologic Factor for Oropharyngeal Carcinoma in Black South Africans 
Head and Neck Pathology  2013;7(4):361-372.
Human papillomavirus (HPV) infection, most commonly genotype 16 of the alpha-9 family, is implicated in the etiology of a subset of oropharyngeal squamous cell carcinomas (OPSC) worldwide. Data are scarce regarding OPSC in South Africans, and three prior studies suggest no significant etiologic role for HPV. We aimed to investigate for evidence of HPV etiology in OPSCs from black South Africans by polymerase chain reaction (PCR) methodologies with determination of HPV subtype by sequencing, in situ hybridization (ISH), and p16INK4a immunohistochemistry (IHC), as a surrogate marker for an HPV-driven tumor. It was hypothesized that HPV-driven tumors would be positive by PCR plus IHC and/or ISH whereas OPSCs with HPV background infections (HPV-passenger) would be positive by PCR alone. Formalin-fixed, paraffin-embedded tissues from 51 OPSCs collected between 2005 and 2010 from 41 patients were analyzed for HPV by GP5+6+ PCR (targeting the HPV L1 region), pU-1M/pU-2R PCR (targeting the HPV E6/E7 region) and HPV-31 specific PCR (targeting the E5 region), chromogenic ISH, and p16INK4a IHC. All cases positive by PCR were subject to sequencing to determine HPV genotype. The patient mean age was 58.0 years and 88 % were male. Of the 51 evaluable tumors, 48 (94.1 %) were positive for HPV DNA by PCR: 25 (49.1 %) met criteria for an HPV-driven tumor, 23 (45.1 %) for HPV-passenger, and 3 (5.9 %) were HPV-unrelated. Sequencing of the PCR-positive cases revealed the following genotypes: combined HPV-16 and 31 (41.7 %), HPV-31 (25.0 %), HPV-16 (22.9 %), combined HPV-16 and 18 (6.3 %), and a single case each of HPV 18 and HPV 33. Studies via ISH were negative in all cases. In accordance with worldwide trends but contrary to prior South African data, HPV likely plays an etiologic role in a significant subset (at least 49.1 %) of OPSC in black South Africans. We found that the alpha-9 HPV family, particularly HPV-16 and 31 either in combination or separately, to predominate in our sample tumors. The use of multiple PCR primers increased sensitivity of viral detection, and a HPV-31 specific primer confirmed the presence of this genotype in many samples. Further studies including HPV E6/E7 mRNA assays are needed to better elucidate the pathogenic role of HPV in black South African OPSCs.
doi:10.1007/s12105-013-0453-0
PMCID: PMC3824804  PMID: 23797844
Human papillomavirus; HPV-16; HPV-31; South Africa; Oropharyngeal squamous cell carcinoma
21.  Distribution of cells responsive to 5-HT6 receptor antagonist-induced hypophagia 
Behavioural Brain Research  2014;266(100):201-206.
The central 5-hydroxytryptamine (5-HT; serotonin) system is well established as an important regulator of appetite and continues to remain a focus of obesity research. While much emphasis has focussed on the 5-HT2C receptor (5-HT2CR) in 5-HT's anorectic effect, pharmacological manipulation of the 5-HT6 receptor (5-HT6R) also reduces appetite and body weight and may be amenable to obesity treatment. However, the neurological circuits that underlie 5-HT6R-induced hypophagia remain to be identified. Using c-fos immunoreactivity (FOS-IR) as a marker of neuronal activation, here we mapped the neuroanatomical targets activated by an anorectic dose of the 5-HT6R antagonist SB-399885 throughout the brain. Furthermore, we quantified SB-399855 activated cells within brain appetitive nuclei, the hypothalamus, dorsal raphe nucleus (DRN) and nucleus of the solitary tract (NTS). Our results reveal that 5-HT6R antagonist-induced hypophagia is associated with significantly increased neuronal activation in two nuclei with an established role in the central control of appetite, the paraventricular nucleus of the hypothalamus (PVH) and the NTS. In contrast, no changes in FOS-IR were observed between treatment groups within other hypothalamic nuclei or DRN. The data presented here provide a first insight into the neural circuitry underlying 5-HT6R antagonist-induced appetite suppression and highlight the PVH and NTS in the coordination of 5-HT6R hypophagia.
doi:10.1016/j.bbr.2014.02.018
PMCID: PMC4003350  PMID: 24566060
htr6; 5-HT6 receptor; SB-399885; Food intake; Hypothalamus; Nucleus of the solitary tract
22.  Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling 
F1000Research  2014;3:93.
Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca 2+ waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca 2+ via L-SR junctions, in a manner that requires initial Ca 2+ release from lysosomes and subsequent Ca 2+-induced Ca 2+ release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca 2+ signal information as input data. Simulations of NAADP-dependent junctional Ca 2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca 2+ signals and simulated Ca 2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca 2+] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca 2+signal coupling and may contribute to cellular function in health and disease.
doi:10.12688/f1000research.3720.1
PMCID: PMC4126599  PMID: 25126414
23.  Predicting HIV-1 broadly neutralizing antibody epitope networks using neutralization titers and a novel computational method 
BMC Bioinformatics  2014;15:77.
Background
Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay.
282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody.
Results
We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity.
Conclusions
Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies.
doi:10.1186/1471-2105-15-77
PMCID: PMC3999910  PMID: 24646213
HIV-1 antibody; Thick patch analysis; Bioinformatics algorithms; Boosting algorithm; Machine learning; Neutralization; in-silico epitope mapping; Epitope networks; Structural mapping; Sequence and structure analysis
24.  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.
doi:10.1371/journal.pone.0091142
PMCID: PMC3953338  PMID: 24625757
25.  Absence of Anaplastic Lymphoma Kinase Translocations in Signet Ring Cell Carcinomas of the Upper Gastrointestinal Tract 
ABSTRACT
BACKGROUND:
Anaplastic lymphoma kinase (ALK) fusion oncogenes are present in multiple cancer types. The inversion of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes on chromosome 2 is present in a subset of patients with non-small-cell lung cancer (NSCLC). ALK-rearranged lung cancers demonstrate a significantly higher incidence of signet ring cell histology than do ALK-negative tumors. Based on the histologic similarities of ALK-rearranged NSCLC and signet ring cell carcinomas (SRCCs) of the gastrointestinal tract, we hypothesized that SRCC of the upper gastrointestinal (GI) tract may also harbor ALK translocations.
METHODS:
Thirty-five formalin-fixed, paraffin-embedded (FFPE) diagnostic tissue specimens of SRCC or poorly differentiated adenocarcinoma with greater than 10% signet ring cell features originating from the upper GI tract were obtained and confirmed by a board-certified, GI pathologist. SRCC specimens were analyzed by fluorescence in situ hybridization (FISH) analysis, with an ALK (2p23) break-apart probe.
RESULTS:
The FISH analysis revealed no evidence of ALK translocation. All 35 (100%) SRCC specimens showed intact ALK FISH signals.
CONCLUSIONS:
These data indicate that, despite histologic similarities between SRCC of the upper GI tract and ALK-positive NSCLC, ALK translocations are unlikely to be a significant contributor to the molecular etiology of SRCC. Further genomic investigations are ongoing.
PMCID: PMC4007674  PMID: 24799969

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