Systematic comparison of adeno‐associated virus and biotinylated dextran amine reveals equivalent sensitivity between tracers and novel projection targets in the mouse brain

As an anterograde neuronal tracer, recombinant adeno‐associated virus (AAV) has distinct advantages over the widely used biotinylated dextran amine (BDA). However, the sensitivity and selectivity of AAV remain uncharacterized for many brain regions and species. To validate this tracing method further, AAV (serotype 1) was systematically compared with BDA as an anterograde tracer by injecting both tracers into three cortical and 15 subcortical regions in C57BL/6J mice. Identical parameters were used for our sequential iontophoretic injections, producing injections of AAV that were more robust in size and in density of neurons infected compared with those of BDA. However, these differences did not preclude further comparison between the tracers, because the pairs of injections were suitably colocalized and contained some percentage of double‐labeled neurons. A qualitative analysis of projection patterns showed that the two tracers behave very similarly when injection sites are well matched. Additionally, a quantitative analysis of relative projection intensity for cases targeting primary motor cortex (MOp), primary somatosensory cortex (SSp), and caudoputamen (CP) showed strong agreement in the ranked order of projection intensities between the two tracers. A detailed analysis of the projections of two brain regions (SSp and MOp) revealed many targets that have not previously been described in the mouse or rat. Minor retrograde labeling of neurons was observed in all cases examined, for both AAV and BDA. Our results show that AAV has actions equivalent to those of BDA as an anterograde tracer and is suitable for analysis of neural circuitry throughout the mouse brain. J. Comp. Neurol. 522:1989–2012, 2014. © 2014 Wiley Periodicals, Inc.     

Double deletion of orexigenic neuropeptide Y and dynorphin results in paradoxical obesity in mice

Objective

Orexigenic neuropeptide Y (NPY) and dynorphin (DYN) regulate energy homeostasis. Single NPY or dynorphin deletion reduces food intake or increases fat loss. Future developments of obesity therapeutics involve targeting multiple pathways. We hypothesised that NPY and dynorphin regulate energy homeostasis independently, thus double NPY and dynorphin ablation would result in greater weight and/or fat loss than the absence of NPY or dynorphin alone.

Design and methods

We generated single and double NPY and dynorphin knockout mice (NPYΔ, DYNΔ, NPYDYNΔ) and compared body weight, adiposity, feeding behaviour, glucose homeostasis and brown adipose tissue uncoupling protein-1 (UCP-1) expression to wildtype counterparts.

Results

Body weight and adiposity were significantly increased in NPYDYNΔ, but not in NPYΔ or DYNΔ. This was not due to increased food intake or altered UCP-1 expression, which were not significantly altered in double knockouts. NPYDYNΔ mice demonstrated increased body weight loss after a 24-h fast, with no effect on serum glucose levels after glucose injection.

Conclusions

Contrary to the predicted phenotype delineated from single knockouts, double NPY and dynorphin deletion resulted in heavier mice, with increased adiposity, despite no significant changes in food intake or UCP-1 activity. This indicates that combining long-term opioid antagonism with blockade of NPY-ergic systems may not produce anti-obesity effects.     

Does anxiety predict the use of urgent care by people with long term conditions? A systematic review with meta-analysis

Objective

The role of anxiety in the use of urgent care in people with long term conditions is not fully understood. A systematic review was conducted with meta-analysis to examine the relationship between anxiety and future use of urgent healthcare among individuals with one of four long term conditions: diabetes; coronary heart disease, chronic obstructive pulmonary disease and asthma.

Methods

Electronic searches of MEDLINE, EMBASE, PSYCINFO, CINAHL, the British Nursing Library and the Cochrane Library were conducted These searches were supplemented by hand-searching bibliographies, citation tracing eligible studies and asking experts within the field about relevant studies. Studies were eligible for inclusion if they: a) used a standardised measure of anxiety, b) used prospective cohort design, c) included adult patients diagnosed with coronary heart disease (CHD), asthma, diabetes or chronic obstructive pulmonary disease (COPD), d) assessed urgent healthcare use prospectively. Data regarding participants, methodology, and association between anxiety and urgent care use was extracted from studies eligible for inclusion. Odds ratios were calculated for each study and pooled using random effects models.

Results

8 independent studies were identified for inclusion in the meta-analysis, with a total of 28,823 individual patients. Pooled effects indicate that anxiety is not associated with an increase in the use of urgent care (OR = 1.078, p = 0.476), regardless of the type of service, or type of medical condition.

Conclusions

Anxiety is not associated with increased use of urgent care. This finding is in contrast to similar studies which have investigated the role of depression as a risk factor for use of urgent care.     

Management of Positive Sub‐areolar/Nipple Duct Margins in Nipple‐Sparing Mastectomies

We evaluated management of positive sub‐areolar/nipple duct margins in nipple‐sparing mastectomies (NSM) at our institution. Retrospective chart review of all NSM from January 2007 to April 2012 was performed and patient, tumor, and treatment information was collected. Sub‐areolar/nipple duct margins included ductal tissue from within the nipple. Of 438 NSM, 22 (5%) had positive sub‐areolar/nipple duct margins; 21 of 220 cancer‐bearing breasts (10%) and 1 of 218 prophylactic mastectomies (0.5%). Positive margins included four with invasive lobular carcinoma and 18 with ductal carcinoma in situ (DCIS). Management included removal of eight nipples and nine nipple areola complexes (NAC). Four of 17 nipple/NAC specimens had evidence of residual DCIS and none had residual invasive cancer. The majority of nipple/NAC specimens excised for a positive margin had no residual malignancy. Future studies are needed to determine the extent of NAC tissue removal required for positive margins.     

Genetics of New-Onset Diabetes after Transplantation

New-onset diabetes after transplantation is a common complication that reduces recipient survival. Research in renal transplant recipients has suggested that pancreatic β-cell dysfunction, as opposed to insulin resistance, may be the key pathologic process. In this study, clinical and genetic factors associated with new-onset diabetes after transplantation were identified in a white population. A joint analysis approach, with an initial genome-wide association study in a subset of cases followed by de novo genotyping in the complete case cohort, was implemented to identify single-nucleotide polymorphisms (SNPs) associated with the development of new-onset diabetes after transplantation. Clinical variables associated with the development of diabetes after renal transplantation included older recipient age, female sex, and percentage weight gain within 12 months of transplantation. The genome-wide association study identified 26 SNPs associated with new-onset diabetes after transplantation; this association was validated for eight SNPs (rs10484821, rs7533125, rs2861484, rs11580170, rs2020902, rs1836882, rs198372, and rs4394754) by de novo genotyping. These associations remained significant after multivariate adjustment for clinical variables. Seven of these SNPs are associated with genes implicated in β-cell apoptosis. These results corroborate recent clinical evidence implicating β-cell dysfunction in the pathophysiology of new-onset diabetes after transplantation and support the pursuit of therapeutic strategies to protect β cells in the post-transplant period.One-year graft survival after renal transplantation is now excellent, exceeding 93% for organs donated after brain death and 96% for those from living donors.^1–3 Technical advancements in surgery, improved understanding of immunology, and innovative developments in pharmacology have altered the landscape of renal transplantation. The goal of preventing early graft loss has largely been achieved and arguably the greatest challenge now is the avoidance of late graft failure. Although there has been a considerable improvement in 1-year renal transplant survival, the rate of graft attrition after the first year remains frustratingly constant.^2,4New-onset diabetes after transplantation (NODAT) is a common and serious disorder that curtails recipient survival.^5–7 NODAT is associated with cardiovascular complications^8–11 and develops in 2%–50%¹² of renal transplant recipients. Approximately 50% of recipients with NODAT require insulin therapy.^{6–8,13–15} A number of clinical variables have been associated with NODAT, including black ethnicity, older recipient age, female sex, obesity, immunosuppression, and viral infections.^{5,6,8,13,16,17}Until recently, the pathophysiology of NODAT was considered to be analogous to type 2 diabetes mellitus. Renal transplant recipients have increased insulin resistance compared with transplant-naïve persons with normal renal function.¹⁸ In a nondiabetic renal transplant population, the main determinants of insulin resistance are obesity and corticosteroid therapy.¹⁹ Insulin resistance improves in renal transplant recipients after successful transplantation^20,21 and recipients have enhanced insulin sensitivity compared with dialysis patients.²² At 1 year, there is no significant difference in insulin resistance between renal transplant recipients with NODAT and those with normal glucose tolerance.^18,23 Furthermore, insulin resistance indices before transplantation and in the early post-transplant period do not predict NODAT development.¹¹Pancreatic β-cell dysfunction may prove to be the main pathologic contributor to NODAT. In glucose clamp studies, a deficit in insulin secretion was common to renal transplant recipients with NODAT.^18,20,21,24 There are a number of possible mechanisms for β-cell toxicity in renal transplantation, including hyperglycemia,²⁵ elevated free fatty acids,²⁶ and the effect of immunosuppressive medication.²⁷ A recent proof-of-concept clinical trial demonstrated that aggressive management of post-transplant hyperglycemia with insulin significantly reduced the 1-year incidence of NODAT.²⁸ This provides further evidence that post-transplant hyperglycemia plays a key role in NODAT development.This study investigates clinical and genetic factors associated with NODAT in a relatively large, white renal transplant population. Clinical variables were identified in a carefully phenotyped, ethnically homogeneous cohort. Initial exploratory analysis was conducted via a genome-wide association study (GWAS) in a subgroup of NODAT cases patients and controls to identify genetic variants associated with NODAT. De novo genotyping was then performed in a larger cohort of NODAT patients and controls to validate the findings.     

Expression of Phosphofructokinase in Skeletal Muscle Is Influenced by Genetic Variation and Associated With Insulin Sensitivity

Using an integrative approach in which genetic variation, gene expression, and clinical phenotypes are assessed in relevant tissues may help functionally characterize the contribution of genetics to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (expression quantitative trait loci [eQTLs]) as well as expression associated with measures of insulin sensitivity. We investigated associations of 3,799,401 genetic variants in expression of >7,000 genes from three cohorts (n = 104). We identified 287 genes with cis-acting eQTLs (false discovery rate [FDR] <5%; P < 1.96 × 10⁻⁵) and 49 expression–insulin sensitivity phenotype associations (i.e., fasting insulin, homeostasis model assessment–insulin resistance, and BMI) (FDR <5%; P = 1.34 × 10⁻⁴). One of these associations, fasting insulin/phosphofructokinase (PFKM), overlaps with an eQTL. Furthermore, the expression of PFKM, a rate-limiting enzyme in glycolysis, was nominally associated with glucose uptake in skeletal muscle (P = 0.026; n = 42) and overexpressed (Bonferroni-corrected P = 0.03) in skeletal muscle of patients with T2D (n = 102) compared with normoglycemic controls (n = 87). The PFKM eQTL (rs4547172; P = 7.69 × 10⁻⁶) was nominally associated with glucose uptake, glucose oxidation rate, intramuscular triglyceride content, and metabolic flexibility (P = 0.016–0.048; n = 178). We explored eQTL results using published data from genome-wide association studies (DIAGRAM and MAGIC), and a proxy for the PFKM eQTL (rs11168327; r² = 0.75) was nominally associated with T2D (DIAGRAM P = 2.7 × 10⁻³). Taken together, our analysis highlights PFKM as a potential regulator of skeletal muscle insulin sensitivity.     

The effect of docosahexaenoic acid on bone microstructure in young mice and bone fracture in neonates

Background

As low bone mineral density is a risk factor for fracture in childhood, optimizing age appropriate bone mass is recommended and might lower the impact of bone loss related to age. Consumption of omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic and docosahexaenoic (DHA) acids have been shown to beneficially modulate bone metabolism. The objective of this study was to determine the incidence of fracture in neonates receiving a fish compared with soybean oil–based intravenous lipid emulsion and evaluate the effect of varying dietary omega-3 PUFA consumption on growing bone in young mice.

Materials and methods

Eligibility criteria for the clinical study included gestational age ≤37 wk and parenteral nutrition–dependence for ≥4 wk. Radiographs were reviewed after lipid initiation to identify radiologic bone fracture. The animal study evaluated female C57/Bl6 mice randomized into one of five groups from age 3–12 wk, at which time femurs were harvested for micro–computed tomography and light microscopy analysis.

Results

A lower incidence of bone fracture was found in neonates maintained on fish compared with soybean oil. In the animal study, findings suggest the DHA diet provides the best protection against trabecular bone loss as evidenced by increased bone volume fraction, increased trabecular number, and decreased trabecular separation on micro–computed tomography. These protective effects appeared to affect the bone microstructure alone.

Conclusions

The lower fracture risk observed in fish oil fed neonates in combination with the protective effects of DHA observed in the femurs of young C57/BL6 mice suggest an important role for omega-3 PUFAs on bone growth.