Amino acids and amines stimulate gastrin release from canine antral G-cells via different pathways.
The major determinant of meal-stimulated gastric acid secretion is the antral hormone gastrin. Decarboxylated amine derivatives of amino acids have been proposed as the final common mediators of gastrin secretion stimulated by a meal. We explored the cellular basis for this hypothesis using a recently developed isolated canine G-cell model. Both amino acids and, more potently, their corresponding amines, directly stimulated gastrin release. Amino acid-stimulated gastrin secretion was unaffected by decarboxylase inhibitors (alpha methyldopa, aminooxyacetic acid, and 4-deoxypyridoxine) but enhanced by bombesin, isobutylmethylxanthine, and dibutyryl cAMP. Somatostatin inhibited amino acid-stimulated gastrin release via a pertussis toxin-sensitive GTP-binding protein. In contrast, gastrin secretion induced by amines was unaltered by any of the various treatments. Our data indicate that amino acids and amines, either as primary constituents of an ingested meal or as metabolites of dietary proteins, act directly via separate mechanisms to stimulate gastrin secretion from G-cells.


First Confirmed Report that Puccinia horiana, Causal Agent of Chrysanthemum White Rust, Can Overwinter in Pennsylvania.
Chrysanthemum white rust (CWR) is a quarantine-significant pest in the United States (Title 7, Code of Federal Regulations, Part 319.37-2). The causal agent of CWR, Puccinia horiana Henn., is an autoecious, microcyclic rust that is pathogenic on chrysanthemum species (Chrysanthemum spp.) and close relatives within the family Asteraceae. CWR is indigenous to Japan, where it was first reported in 1895 (4). By the 1960s, CWR was found throughout Europe and later spread to Africa, Oceana, South America, and other parts of Asia. In North America, CWR was reported in Mexico and in the United States (New Jersey and Pennsylvania [1977], Oregon and Washington [1990], and California [1991]). Additional detections of CWR were later reported in 22 Pennsylvania counties (2004, 2006 to 2010) (3). These later Pennsylvania reports stated that eradication was attempted at some sites, but unconfirmed observations suggested that the rust pathogen might overwinter in volunteer plants (3). Since "CWR is known to overwinter in Europe where chrysanthemums overwinter (average minimum temperatures ranging from -10°F to 10°F)" (2), the unconfirmed Pennsylvania observations prompted us to determine if P. horiana can overwinter in Pennsylvania. During October 2010, we identified CWR on perennial mums planted at six outdoor garden locations in University Park, PA. Symptomatic plants were quarantined and eradication attempted. Eradicated sites were routinely surveyed and CWR confirmed in July 2011 on volunteer plants at two of the originally infested sites. An additional outdoor garden site with CWR was observed in State College, PA, during October 2011 and eradication attempted. The three infested sites were surveyed throughout the fall and winter of 2011 to 2012. During February 2012, two asymptomatic volunteer plants arising from root pieces were collected from each of the three sites. Each sample was washed with tap water to remove excess soil, examined morphologically, surface sterilized with 10% bleach, and divided into two subsamples. One subsample from each site was divided into crown and root portions and DNA extracted using a Qiagen DNeasy Plant Mini Kit. Molecular analysis was performed using modifications of published primers ITS 5 and Rust1 (1,4). Puccinia horiana was detected in plant roots from one site and in plant crowns from two sites. The remaining two subsamples from each site were transplanted into sterilized potting soil and placed in a clean controlled environment chamber at 18°C and 85% relative humidity (RH). After 6 weeks, six actively growing plants were transferred to a second clean controlled environment chamber at 17°C and 90 to 100% RH. On 6 April 2012, CWR symptoms and signs were confirmed morphologically on two plants that had been removed from one site. On 19 April 2012, CWR signs and symptoms were confirmed morphologically and by molecular analysis on leaves of volunteer plants at one University Park site. DNA extractions were sequenced and shared a 100% maximum identity to a known P. horiana accession (EU816920.1) in GenBank. To our knowledge, this is the first confirmed report of P. horiana overwintering in Pennsylvania. References: (1) H. Alaei et al. Mycol. Res. 113:668, 2009. (2) Anon. Chrysanthemum White Rust Bulletin, Syngenta Flowers Inc., Gilroy, CA, 2010. (3) S. Kim et al. Phytopathology 101:S91, 2011. (4) K. Pedley. Plant Dis. 93:1252, 2009.


A neurophysiological marker of impaired preparation in an 11-year follow-up study of attention-deficit/hyperactivity disorder (ADHD).
This longitudinal electrophysiological study investigated the course of multiple impaired cognitive brain functions in attention-deficit/hyperactivity disorder (ADHD) from childhood to adulthood by comparing developmental trajectories of individuals with ADHD and typically developing controls. Subjects with ADHD (N = 11) and normal controls (N = 12) diagnosed in childhood [mean age ADHD/CTRL = 10.9 years [SD 1.72]/10.0 years (SD 1.03)] were followed up after 1.1 and 2.4 years, and as young adults [ADHD/CTRL: 21.9 years (SD 1.46)/21.1 years (SD 1.29)]. At all four times, event-related potential (ERP) maps were recorded during a cued continuous performance test (CPT). We focused on residual deficits as adults, and on developmental trajectories (time and time × group effects) for CPT performance and attentional (Cue P300), preparatory (CNV: contingent negative variation) and inhibitory (NoGo P300) ERP components. All ERP components developed without significant time × group interactions. Only the CNV remained reduced in the ADHD group, although 8/11 individuals no longer met a full ADHD diagnosis as adults. Cue P300 and NoGo P300 group differences became nonsignificant in early adulthood. The CNV parameters correlated with reaction time (RT) and RT-SD. Perceptual sensitivity improved and the groups' trajectories converged with development, while RT-SD continued to be elevated in adult ADHD subjects. Attentional and preparatory deficits in ADHD continue into adulthood, and the attenuated CNV appears to reflect a particularly stable ADHD marker. Although some deficit reductions may have gone undetected due to small sample size, the findings challenge those developmental lag models postulating that most ADHD-related deficits become negligible with brain maturation.


Deep brain stimulation in the management of neuropathic pain and multiple sclerosis tremor.
Deep brain stimulation (DBS) of the central gray matter was an important component of the surgical management of chronic, drug-refractory, central neuropathic pain until only a decade ago. However, in the recent past, this technique has been increasingly neglected and has been largely replaced by motor cortex stimulation (MCS). The results of MCS, however, are far from uniform, and the best reports quote a range of 50% to 75% success in providing satisfactory pain relief. In recent years, there has been considerable success in treating various movement disorders, particularly in Parkinson's disease (PD) and dystonia, by chronic high-frequency DBS of nuclear structures in the basal ganglia. This technique has also been shown to be relatively effective in some selected cases of tremulous conditions like essential tremor and posttraumatic tremor. However, when the same techniques have been applied to patients with multiple sclerosis tremor (MST), the results have been mixed. As a result, DBS for MST has often been perceived as an unreliable and inconsistent therapeutic intervention. The authors present their experience with the application of DBS in these two relatively unpopular areas for neuromodulation in the current practice of functional stereotactic neurosurgery. The results demonstrate that with careful patient selection, DBS can offer significant functional benefit in both of these difficult clinical conditions.


Complete rectal prolapse in adults: a Tanzanian experience.
This is a retrospective report of nine patients with complete rectal prolapse managed by the authors at the Muhimbili Medical Centre, Dar es Salaam between 1990 and 1993. The average age of patients was 36 years and eight of the patients were males. Six of the patients presented as emergency admissions of whom three had irreducibility and required perineal proctosigmoidectomy. This was the procedure of choice for irreducible complete rectal prolapse.


CCL21 chemokine regulates chemokine receptor CCR7 bearing malignant melanoma cells.
The chemokine CC-ligand 21/secondary lymphoid tissue chemokine (CCL21/SLC) regulates the homing of naïve T cells and dendritic cells that express CC-chemokine receptor 7 (CCR7) from distant sites to lymphoid tissue such as lymph nodes. We hypothesized that CCL21/SLC regulates the migration of CCR7-bearing melanoma cells from a primary lesion to regional tumor-draining lymph nodes. Quantitative real-time reverse transcriptase-PCR (qRT) assay and immunohistochemistry (IHC) were used to assess the level of CCR7 expression in melanoma cell lines and in primary and metastatic melanoma tumors. Cell migration assay using melanoma cell lines was performed under the induction of CCL21/SLC. The CCL21/SLC expression level in tumor-draining sentinel lymph nodes (SLNs) was assessed by both qRT assay and IHC. Melanoma cell lines and tumors demonstrated heterogeneous expression of CCR7 mRNA by qRT assay. There was strong functional correlation between CCR7 mRNA expression and cell migration induced by CCL21/SLC. IHC evidence of CCR7 expression in primary melanomas significantly (P = 0.02) correlated with Breslow thickness. Assessment of SLN from 55 melanoma patients by qRT assay demonstrated that CCL21/SLC mRNA expression level was significantly (P = 0.008) higher in pathologically melanoma-negative SLNs than in melanoma-positive SLNs. This report demonstrates a potential mechanism for recruitment and homing of CCR7(+) metastatic melanoma cells to tumor-draining lymph nodes, which express CCL21/SLC. The study also suggests that lymph nodes bearing metastasis may suppress CCL21/SLC production.


Longitudinal trajectories of illness perceptions among adolescents with type 1 diabetes.
To examine development in illness perceptions of type 1 diabetes across adolescence and relationships with intelligence, diabetes responsibility, and diabetes outcomes. Illness perceptions were measured via the Illness Perceptions Questionnaire at 3 times, every 6 months in 213 adolescents (M age = 13.00; SD = 1.54) with type 1 diabetes. Intelligence and adolescents' perceived responsibility for diabetes were examined, and adolescents' report of adherence and quality of life (QOL), and glycosylated hemoglobin (HbA1c) from medical records addressed diabetes-related outcomes. Linear growth models showed significant increases in perceptions of diabetes coherence, chronicity, consequences, personal and treatment control, and decreases in diabetes cyclicality and parental control across time. More favorable illness perceptions were generally associated with adolescent intelligence at baseline, more adolescent responsibility for management, better adherence and QOL, and lower HbA1c at each time point. Results suggest that adolescents develop complex illness perceptions, which are associated with better diabetes management.


The structural basis for 14-3-3:phosphopeptide binding specificity.
The 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Using phosphoserine-oriented peptide libraries to probe all mammalian and yeast 14-3-3s, we identified two different binding motifs, RSXpSXP and RXY/FXpSXP, present in nearly all known 14-3-3 binding proteins. The crystal structure of 14-3-3zeta complexed with the phosphoserine motif in polyoma middle-T was determined to 2.6 A resolution. The bound peptide is in an extended conformation, with a tight turn created by the pS +2 Pro in a cis conformation. Sites of peptide-protein interaction in the complex rationalize the peptide library results. Finally, we show that the 14-3-3 dimer binds tightly to single molecules containing tandem repeats of phosphoserine motifs, implicating bidentate association as a signaling mechanism with molecules such as Raf, BAD, and Cbl.


Twist 1 regulates the expression of PPARγ during hormone-induced 3T3-L1 preadipocyte differentiation: a possible role in obesity and associated diseases.
Twist 1 is highly expressed in adipose tissue and has been associated with obesity and related disorders. However, the molecular function of Twist 1 in adipose tissue is unclear. Twist 1 has been implicated in cell lineage determination and differentiation. Therefore, we investigated both the role of Twist 1 in adipocyte precursor mobilization and the relationship of Twist 1 with other molecular determinants of adipocyte differentiation. We examined Twist 1 mRNA and protein expression in subcutaneous adipose tissues from diet-induced obese C57/BL6 mice and Wistar rats and in obese patients undergoing liposuction or adipose transplant surgeries. Twist 1 expression was measured on days 0, 2, 4, 8, and 12 of 3T3-L1 differentiation in vitro. The role of Twist 1 in adipogenesis was explored using retroviral interference of Twist 1 expression. Adipokine secretion was evaluated using a RayBio® Biotin Label-based Adipokine Array. Twist 1 mRNA and protein levels were reduced in diet-induced obese mice and rats and in obese humans. Twist 1 was upregulated during 3T3-L1 preadipocyte differentiation in vitro, beginning from the fourth day of differentiation induction. Retroviral interference of Twist 1 expression did not significantly impair lipid formation; however, retroviral interference induced PPARγ mRNA and protein expression on day 4 of differentiation induction. Adipokine array analyses revealed increased secretion of CXCR4 (19.55-fold), VEGFR1 (92.13-fold), L-21 R (63.55-fold), and IL-12 R beta 1 (59.66-fold) and decreased secretion of VEGFR3 (0.01-fold), TSLP R (0.071-fold), MIP-1 gamma (0.069-fold), TNF RI/TNFRSF1A (0.09-fold), and MFG-E8 (0.06-fold). Twist 1 is a regulator of adipocyte gene expression although it is not likely to regulate differentiation. We identified PPARγ as a potential target of Twist 1 and found variation in the secretion of multiple adipokines, which might indicate a prospective mechanism linking Twist 1 expression with obesity or associated diseases.


A mathematical model of the single freeform surface design for collimated beam shaping.
Incoherent collimated beam has a wide application, and reshaping the collimated beam with freeform optics has become a popular and challenging topic of noniamging design. In this paper, we address this issue, embedded in three-dimensional space without any symmetry, with a freeform surface from a new perspective. A mathematical model is established for achieving the one-freeform surface design based on the problem of optimal mass transport. A numerical technique for solving this design model is disclosed for the first time, and boundary conditions for balancing light are presented. Besides, some key issues in achieving complex illuminations are addressed, and the influence of caustic surface on this design model is also discussed. Design examples are given to verify these theories. The results show elegance of the design model in tackling complex illumination tasks. The conclusions obtained in this paper can be generalized to achieve LED illumination and tackle multiple freeform surfaces illumination design.


Binding and cytotoxicity of conjugated and recombinant fusion proteins targeted to the gonadotropin-releasing hormone receptor.
Pokeweed antiviral protein (PAP) is a plant-derived, highly potent ribosome inactivating protein that causes inhibition of protein translation and rapid cell death. We and others have delivered this protein to various cell types, including cancer cells, using hormones to specifically target cells bearing the hormone receptor. Here, we compare binding and cytotoxicity of GnRH-PAP hormonotoxins prepared either by protein conjugation (GnRH-PAP conjugate) or through recombinant DNA technology (GnRH-PAP fusion). Although GnRH-PAP conjugate protein bound specifically to and caused cell death in cells bearing the gonadotropin-releasing hormone (GnRH) receptor, we could not detect binding or cytotoxicity using two different versions of the fusion protein in receptor-positive cells. We conclude that generation of an active GnRH-PAP fusion protein may not be feasible either because both ends of the GnRH molecule are required for receptor binding, but only the NH(2) terminus is free in the fusion protein and/or that more potent analogues of GnRH (inclusion of which is not feasible in the fusion protein) are needed for efficient targeting. In contrast, the GnRH-PAP conjugate shows promise as a novel anticancer agent, capable of targeting cancer cells expressing the GnRH receptor such as prostate, breast, ovarian, endometrial, and pancreatic cells. It may also be useful as a therapeutic agent to eliminate pituitary gonadotrophs, eliminating the need for chronic GnRH analogue administration to treat hormone-sensitive diseases.


Contractile activity of the bladder urothelium/lamina propria and its regulation by nitric oxide.
In the bladder, nitric oxide (NO) is released from neuronal and non-neuronal sources, but its actions are unclear. Strips of urothelium plus lamina propria contract in response to agonists and develop spontaneous phasic contractions, and the aim of this study was to investigate the influence of NO on this activity. Isolated strips of urothelium/lamina propria from porcine bladder developed spontaneous contractions (3.5 ± 0.3 cycles/min) and contracted in response to carbachol and electrical field stimulation (EFS). The NO synthase inhibitor N(ω)-nitro-l-arginine (L-NNA, 100 μM) had no effects on the tissues, but the NO donors diethylamine NONOate (DEANO, 100 μM) and nitroprusside (10 μM) caused relaxation, slowed the spontaneous rate of contractions and inhibited responses to carbachol. Maximum tonic contractions to carbachol were reduced by 17 ± 4% (P<0.001) and 35 ± 5% (P<0.001) by DEANO and nitroprusside respectively and the potency of carbachol was also reduced. Carbachol also increased the spontaneous frequency of contraction and these rate responses were again inhibited by DEANO and nitroprusside, but unaffected by L-NNA. Similarly, responses to EFS were significantly depressed (52-70%) by DEANO (P<0.05), but were unaffected by L-NNA. These data demonstrate spontaneous contractile activity and also nerve and agonist-induced tonic contractile activity within the urothelium and lamina propria. This activity is sensitive to depression by NO, but NO does not appear to be spontaneously released to influence this activity, nor does it appear to be released by muscarinic receptor stimulation. However the results suggest that in situations where NO production is increased, NO can influence the contractile activity of this tissue.


Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin.
FK-506 and the structurally related macrolide rapamycin (RAP) were investigated in comparison with cyclosporin A (CsA) for their immunosuppressive effects on murine T cells. All three agents suppressed the proliferation of splenic T cells triggered by lectins or antibodies to CD3 and Ly-6C. FK-506 or CsA also inhibited proliferation, IL-2 production, and IL-2R expression in splenic T cells activated with ionomycin + PMA. However, RAP minimally affected IL-2 production and IL-2R expression in these cells, although it reduced proliferation. Similarly, FK-506 and CsA, but not RAP, suppressed IL-2 production by activated DO.11.10 T hybridoma cells. In such a system, as well as in normal T cells stimulated with high ionomycin concentrations, FK-506 and CsA enhanced proliferation, indicating that they both abrogate negative signals associated with T cell activation. On the contrary, RAP diminished the autonomous proliferation of hybridoma cells, whereas FK-506 and CsA had little effect. The proliferative response induced in D10.G4 cells by IL-1 + ionomycin but not that induced by IL-1 + PMA was sensitive to inhibition by FK-506 and CsA. In contrast, RAP inhibited equally well both types of stimulation. Finally, T cell proliferation driven by IL-2 or IL-4 was found to be relatively resistant to FK-506 or CsA but sensitive to RAP. Altogether, these data demonstrate that FK-506 and CsA alter similar calcium-associated events of T cell activation and block T cell proliferation primarily by suppressing lymphokine production. RAP interferes with a different set of events and inhibits T cells by impairing their response to growth-promoting lymphokines.


Computational inference of scenarios for alpha-proteobacterial genome evolution.
The alpha-proteobacteria, from which mitochondria are thought to have originated, display a 10-fold genome size variation and provide an excellent model system for studies of genome size evolution in bacteria. Here, we use computational approaches to infer ancestral gene sets and to quantify the flux of genes along the branches of the alpha-proteobacterial species tree. Our study reveals massive gene expansions at branches diversifying plant-associated bacteria and extreme losses at branches separating intracellular bacteria of animals and humans. Alterations in gene numbers have mostly affected functional categories associated with regulation, transport, and small-molecule metabolism, many of which are encoded by paralogous gene families located on auxiliary chromosomes. The results suggest that the alpha-proteobacterial ancestor contained 3,000-5,000 genes and was a free-living, aerobic, and motile bacterium with pili and surface proteins for host cell and environmental interactions. Approximately one third of the ancestral gene set has no homologs among the eukaryotes. More than 40% of the genes without eukaryotic counterparts encode proteins that are conserved among the alpha-proteobacteria but for which no function has yet been identified. These genes that never made it into the eukaryotes but are widely distributed in bacteria may represent bacterial drug targets and should be prime candidates for future functional characterization.


Distributed neural system for emotional intelligence revealed by lesion mapping.
Cognitive neuroscience has made considerable progress in understanding the neural architecture of human intelligence, identifying a broadly distributed network of frontal and parietal regions that support goal-directed, intelligent behavior. However, the contributions of this network to social and emotional aspects of intellectual function remain to be well characterized. Here we investigated the neural basis of emotional intelligence in 152 patients with focal brain injuries using voxel-based lesion-symptom mapping. Latent variable modeling was applied to obtain measures of emotional intelligence, general intelligence and personality from the Mayer, Salovey, Caruso Emotional Intelligence Test (MSCEIT), the Wechsler Adult Intelligence Scale and the Neuroticism-Extroversion-Openness Inventory, respectively. Regression analyses revealed that latent scores for measures of general intelligence and personality reliably predicted latent scores for emotional intelligence. Lesion mapping results further indicated that these convergent processes depend on a shared network of frontal, temporal and parietal brain regions. The results support an integrative framework for understanding the architecture of executive, social and emotional processes and make specific recommendations for the interpretation and application of the MSCEIT to the study of emotional intelligence in health and disease.


Rural independent and corporate Supplemental Nutrition Assistance Program (SNAP)-authorized store owners' and managers' perceived feasibility to implement marketing-mix and choice-architecture strategies to encourage healthy consumer purchases.
Supplemental Nutrition Assistance Program (SNAP) participants could benefit from exposure to marketing-mix and choice-architecture (MMCA) strategies that encourage healthy consumer purchases. However, the perceived feasibility of independent and corporate SNAP-authorized store owners and managers (e.g., retailers) to implement healthy MMCA strategies is understudied. The purpose of this study was to inform a healthy food retail program that meets both public health and business expectations by prioritizing retailer perspectives. A mixed methods approach was used. Retailers completed a card sorting exercise to determine perceived feasibility to implement MMCA strategies place, profile, portion, pricing, promotion, priming, prompting, and proximity. This process was audio-recorded. Chi-square was used to identify potential differences in perceived feasibility to implement healthy MMCA strategies between independent and corporate SNAP-authorized retailers. Qualitative data were coded among a panel to construct themes. Themes were organized by barriers and facilitators and coded for strategy acceptability, appropriateness, and feasibility. SNAP-authorized retailers' (n = 29) considered prompting (e.g., labeling; 83%) and proximity (e.g., location; 90%) strategies to encourage healthy consumer purchases highly feasible. Few differences were detected between independent and corporate retailers' perceived feasibility to implement healthy MMCA strategies. The largest barriers to implementing healthy MMCA strategies were related to strategy appropriateness. Priorities for healthy food retail initiatives included prompting and proximity changes that highlight products aligned with the DGA, without altering products available to consumers that are misaligned with the DGA. Future work is required to understand how other healthy MMCA strategies may be adapted to enhance their appropriateness for these settings.


Pharmacological profile of UP 5145-52, an original antiulcer and antisecretory agent.
The gastric and antiulcer effects of UP 5145-52, a new naphthyridinone derivative (1-phenyl-1,2-dihydro-2-oxo-1,8-naphthyridin-3-yl)-4-acetoxy butane, were studied in rats. UP 5145-52 dose dependently prevented the formation of gastric lesions induced by a necrotizing agent such as absolute ethanol, indomethacin or stress (ED50 values were 0.065, 1.26 and 0.72 mg/kg p.o., respectively). A beneficial action was also observed against indomethacin-induced intestinal lesions. UP 5145-52 inhibited total acid output in the pylorus-ligated rat (ED50 on total acid output was 0.16 mg/kg p.o.) and in the rat chronic gastric fistula model. The mechanism of antisecretory activity currently remains unknown as UP 5145-52 fails to bind to receptors involved in gastric secretion (H2 or muscarinic) and to inhibit H+/K+ adenosine triphosphatase. The ability of UP 5145-52 (1 mg/kg p.o.) to restore synthesis of gastric acid glycoproteins depressed by previous administration of absolute ethanol suggests that cytoprotection could play a role in the antiulcer efficacy of the drug.


Improving the sterile sperm identification method for its implementation in the Area-wide Sterile Insect Technique Program against Ceratitis capitata (Diptera: Tephritidae) in Spain.
The success of sterile males in area-wide sterile insect technique (aw-SIT) programs against Ceratitis capitata (Wiedemann) is currently measured by using indirect methods as the wild:sterile male ratio captured in monitoring traps. In the past decade, molecular techniques have been used to improve these methods. The development of a polymerase chain reaction-restriction fragment-length polymorphism-based method to identify the transfer of sterile sperm to wild females, the target of SIT, was considered a significant step in this direction. This method relies on identification of sperm by detecting the presence of Y chromosomes in spermathecae DNA extract complemented by the identification of the genetic origin of this sperm: Vienna-8 males or wild haplotype. However, the application of this protocol to aw-SIT programs is limited by handling time and personnel cost. The objective of this work was to obtain a high-throughput protocol to facilitate the routine measurement in a pest population of sterile sperm presence in wild females. The polymerase chain reaction-restriction fragment-length polymorphism markers previously developed were validated in Mediterranean fruit fly samples collected from various locations worldwide. A laboratory protocol previously published was modified to allow for the analysis of more samples at the same time. Preservation methods and preservation times commonly used for Mediterranean fruit fly female samples were assessed for their influence on the correct molecular detection of sterile sperm. This high-throughput methodology, as well as the results of sample management presented here, provide a robust, efficient, fast, and economical sterile sperm identification method ready to be used in all Mediterranean fruit fly SIT programs.


Sex difference in strength and size ratios between reciprocal muscle groups in the lower leg.
This study compared strength and size of reciprocal muscle groups in the lower leg between sexes. 20 young men and 14 young women volunteered as subjects. Joint torques developed during isometric maximal voluntary plantar flexion (TQPF) and dorsiflexion (TQDF) were measured using a dynamometer. Muscle volumes of plantar flexors (MVPF) and dorsiflexors (MVDF) were determined by magnetic resonance imaging. In each of the muscle groups, joint torque was significantly correlated with muscle volume in young men and women (r=0.610-0.848) and the y-intercept of the regression line between them was not significantly different from zero. Based on these observations, the dependencies of muscle strength ratio on muscle size ratio between the plantar flexors and dorsiflexors were investigated using joint torque and muscle volume. The correlations between the MVPF per MVDF and the TQPF per TQDF were significant both in young men (r=0.608) and women (r=0.773), suggesting that strength ratio is strongly affected by size ratio between the plantar flexors and dorsiflexors in young men and women.


Y-chromosome lineages trace diffusion of people and languages in southwestern Asia.
The origins and dispersal of farming and pastoral nomadism in southwestern Asia are complex, and there is controversy about whether they were associated with cultural transmission or demic diffusion. In addition, the spread of these technological innovations has been associated with the dispersal of Dravidian and Indo-Iranian languages in southwestern Asia. Here we present genetic evidence for the occurrence of two major population movements, supporting a model of demic diffusion of early farmers from southwestern Iran-and of pastoral nomads from western and central Asia-into India, associated with Dravidian and Indo-European-language dispersals, respectively.


Serologic prevalence of coxsackievirus group B in Greece.
Coxsackieviruses are human enteroviruses, which have been associated with myocarditis/pericarditis and sudden death. In one investigation (Spanakis N, Manolis EN, Tsakris A, Tsiodras S, Panagiotopoulos T, Saroglou G, and Legakis NJ: J Clin Pathol 2005;58:357-360), a cluster of cases of fatal myocarditis in Greece was linked to coxsackievirus B3. The information from this investigation prompted us to study serologically the prevalence of coxsackieviruses B throughout Greece. Sera were obtained from 506 healthy blood donors from various transfusion centers, covering the entire country. All sera were tested for the presence of IgG and IgM antibodies, using ELISAs with various antigenic specificities: (1) heat-denatured coxsackievirus type B1 and B5 virions, (2) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B3, and (3) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B4. Sera positive for IgG antibodies against coxsackieviruses B1/B5, B3, and B4 were detected in 6.7 to 21.6% of the individuals tested in the various regions of Greece. Statistical analysis revealed that the highest prevalence of IgG antibodies against coxsackieviruses B1/B5 was found in blood donors from Crete (p = 0.025), whereas the highest prevalence against coxsackievirus B4 was detected in blood donors from Athens (p = 0.01). IgM antibodies against coxsackievirus B were detected at low percentage, less than 5%, with no significant viral preference for particular geographic regions. The preference of anti-coxsackievirus IgG antibodies for particular geographic regions could be potentially related to the previously reported clustering of cases of insulin-dependent diabetes mellitus and myocarditis in Athens and Crete, respectively.


Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader-Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons.
Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.


Inhibitory autocrine factors produced by the mesenchyme-derived hair follicle dermal papilla may be a key to male pattern baldness.
Androgenetic alopecia, or male pattern baldness, is a common, progressive disorder where large, terminal scalp hairs are gradually replaced by smaller hairs in precise patterns until only tiny vellus hairs remain. This balding can cause a marked reduction in the quality of life. Although these changes are driven by androgens, most molecular mechanisms are unknown, limiting available treatments. The mesenchyme-derived dermal papilla at the base of the mainly epithelial hair follicle controls the type of hair produced and is probably the site through which androgens act on follicle cells by altering the regulatory paracrine factors produced by dermal papilla cells. During changes in hair size the relationship between the hair and dermal papilla size remains constant, with alterations in both dermal papilla volume and cell number. This suggests that alterations within the dermal papilla itself play a key role in altering hair size in response to androgens. Cultured dermal papilla cells offer a useful model system to investigate this as they promote new hair growth in vivo, retain characteristics in vitro which reflect their parent follicle's response to androgens in vivo and secrete mitogenic factors for dermal papilla cells and keratinocytes. To investigate whether cultured dermal papilla cells from balding follicles secrete altered amounts/types of mitogenic factors for dermal papilla cells than those from larger, normal follicles. We also aimed to determine whether rodent cells would recognize mitogenic signals from human cells in vitro and whether factors produced by balding dermal papilla cells could alter the start of a new mouse hair cycle in vivo. Dermal papilla cells were cultured from normal, balding and almost clinically normal areas of balding scalps and their ability to produce mitogenic factors compared using both human and rat whisker dermal papilla cells as in vitro targets and mouse hair growth in vivo. Normal scalp cells produced soluble factors which stimulated the growth of both human scalp and rat whisker dermal papilla cells in vitro, demonstrating dose-responsive mitogenic capability across species. Although balding cells stimulated some growth, this was much reduced and they also secreted inhibitory factor(s). Balding cell media also delayed new hair growth when injected into mice. Human balding dermal papilla cells secrete inhibitory factors which affect the growth of both human and rodent dermal papilla cells and factors which delay the onset of anagen in mice in vivo. These inhibitory factor(s) probably cause the formation of smaller dermal papillae and smaller hairs in male pattern baldness. Identification of such factor(s) could lead to novel therapeutic approaches.


Characterizing microcrack orientation distribution functions in osteonal bone samples.
Prefailure microdamage in bone tissue is considered to be the most detrimental factor in defining its strength and toughness with respect to age and disease. To understand the influence of microcracks on bone mechanics it is necessary to assess their morphology and three-dimensional distribution. This requirement reaches beyond classic histology and stereology, and methods to obtain such information are currently missing. Therefore, the aim of the study was to develop a methodology that allows to characterize three-dimensional microcrack distributions in bulk bone samples. Four dumbbell-shaped specimens of human cortical bone of a 77-year-old female donor were loaded beyond yield in either tension, compression or torsion (one control). Subsequently, synchrotron radiation micro-computed tomography (SRμCT) was used to obtain phase-contrast images of the damaged samples. A microcrack segmentation algorithm was developed and used to segment microcrack families for which microcrack orientation distribution functions were determined. Distinct microcrack families were observed for each load case that resulted in distinct orientation distribution functions. Microcracks had median areas of approximately 4.7 μm2 , 33.3 μm2 and 64.0 μm2 for tension, compression and torsion. Verifying the segmentation algorithm against a manually segmented ground truth showed good results when comparing the microcrack orientation distribution functions. A size dependence was noted when investigating the orientation distribution functions with respect to the size of the volume of interest used for their determination. Furthermore, a scale separation between tensile, compressive and torsional microcracks was noticeable. Visual comparison to classic histology indicated that microcrack families were successfully distinguished. We propose a methodology to analyse three-dimensional microcrack distributions in overloaded cortical bone. Such information could improve our understanding of bone microdamage and its impact on bone failure in relation to tissue age and disease.


Increased cardiac awareness in panic disorder.
We investigated cardiac perception in panic disorder with both self-report and objective measures. In Study 1, 120 patients with panic disorder, 86 infrequent panickers, and 38 patients with other anxiety disorders reported greater cardiac and gastrointestinal awareness than 62 normal control subjects. Subjects with panic attacks reported greater cardiac awareness, but not gastrointestinal awareness, than those with other anxiety disorders. Studies 2 and 3 included a test of heart rate perception in which subjects silently counted their heart-beats without taking their pulse. In Study 2, 65 panic disorder patients showed better performance than 50 infrequent panickers, 27 patients with simple phobias, and 46 normal control subjects. No group differences were found in ability to estimate time intervals. In Study 3, 13 patients with panic disorder and 15 with generalized anxiety disorder showed better heart rate perception than 16 depressed patients.


A real-time PCR assay for the monitoring of influenza A virus in wild birds.
A screening system including a new real-time PCR assay for the monitoring of influenza A virus in wild birds was developed. The real-time PCR assay uses SYBR green chemistry and the primers are targeting the matrix gene of influenza A virus. The performance of the assay was compared with two other assays, one assay also using SYBR green chemistry and one assay using TaqMan chemistry, i.e. a specific probe. A total of 45 fecal bird samples were analysed for influenza A virus in three different PCR reactions. Overall, 26 samples were positive in at least one of the three real-time PCR assays. Of the 26 samples, 18 were positive by all three reactions. Eight samples were found positive exclusively by the two SYBR green reactions, six of which were detected by both SYBR green reactions. Of the 26 positive samples, 15 samples were verified as positive either by virus isolation or influenza A M2-gene PCR. The results showed that the two SYBR green systems had a higher performance regarding the detection of influenza A as compared to the PCR reaction using a specific probe.


Self-Poling of BiFeO3 Thick Films.
Bismuth ferrite (BiFeO3) is difficult to pole because of the combination of its high coercive field and high electrical conductivity. This problem is particularly pronounced in thick films. The poling, however, must be performed to achieve a large macroscopic piezoelectric response. This study presents evidence of a prominent and reproducible self-poling effect in few-tens-of-micrometer-thick BiFeO3 films. Direct and converse piezoelectric measurements confirmed that the as-sintered BiFeO3 thick films yield d33 values of up to ∼20 pC/N. It was observed that a significant self-poling effect only appears in cases when the films are heated and cooled through the ferroelectric-paraelectric phase transition (Curie temperature TC ∼ 820 °C). These self-poled films exhibit a microstructure with randomly oriented columnar grains. The presence of a compressive strain gradient across the film thickness cooled from above the TC was experimentally confirmed and is suggested to be responsible for the self-poling effect. Finally, the macroscopic d33 response of the self-poled BiFeO3 film was characterized as a function of the driving-field frequency and amplitude.


Pneumococcal polysaccharide immunization in patients with active nephrotic syndrome.
We have studied the IgG and IgM antibody responses to pneumococcal polysaccharides types 3 and 19 in eleven patients with active nephrotic syndrome and fifteen normal adult controls. Immunization in both patients with nephrotic syndrome and normal subjects resulted in significant increases in serum IgM antibody to types 3 and 19 pneumococcal polysaccharides. While controls also had a significant rise in IgG antibody to both types 3 and 19, patients with nephrotic syndrome had an increase in IgG antibody only to type 3. Also, after immunization, geometric mean titers of IgG antibodies to both types 3 and 19 were lower in patients with active nephrotic syndrome than in normal subjects (p less than 0.001). In patients with nephrotic syndrome, a significant correlation was found between serum albumin level and type 3 (p less than 0.01) and type 19 (p less than 0.05) antipneumococcal IgG antibody concentration. Furthermore, antipneumococcal IgG antibody was found in the urine of nephrotic patients, while no IgM antibody was excreted in the urine of nephrotic and adult controls. Our data demonstrate that patients with active nephrotic syndrome are able to mount a normal IgM immune response to pneumococcal polysaccharide antigens. The low IgG antibody levels are likely due to increased urinary losses and/or to a partial inability of these patients to produce IgG antibodies. Moreover, since protection may depend on antipneumococcal IgG antibody, these data raise questions as to the benefits of pneumococcal vaccination in patients with active nephrotic syndrome.


Chaotic activity in a mathematical model of the vagally driven sinoatrial node.
Phase-locking behavior and irregular dynamics were studied in a mathematical model of the sinus node driven with repetitive vagal input. The central region of the sinus node was simulated as a 15 x 15 array of resistively coupled pacemakers with each cell randomly assigned one of 10 intrinsic cycle lengths (range 290-390 msec). Coupling of the pacemakers resulted in their mutual entrainment to a common frequency and the emergence of a dominant pacemaker region. Repetitive acetylcholine (ACh; vagal) pulses were applied to a randomly selected 60% of the cells. Over a wide range of stimulus intensities and basic cycle lengths, such perturbations resulted in a large variety of stimulus/response patterns, including phase locking (1:1, 3:2, 2:1, etc.) and irregular (i.e., chaotic) dynamics. At a low ACh concentration (1 microM), the patterns followed the typical Farey sequence of phase-locked behavior. At a higher concentration (5 microM), period doubling and aperiodic patterns were found. When a single pacemaker cell was perturbed with repetitive ACh pulses, qualitatively similar results were obtained. In both types of simulation, chaotic behavior was investigated using phase-plane (orbital) plots, Poincaré mapping, and return mapping. Period-doubling bifurcations (2:2, 4:4, and 8:8) were found temporally and spatially within the array. Under certain conditions of stimulation, the attractor in the return map during chaotic activity of the single cell resembled the Lorenz tent map. However, when electrical coupling between cells was allowed, the interactions with neighboring cells exhibiting chaotic dynamics resulted in characteristic alterations of the attractor geometry. Our results suggest that irregular dynamics obeying the rules derived from other chaotic systems are present during vagal stimulation of the sinus node. In addition, application of the same analytical tools to the analysis of simulation of reflex vagal control of sinus rate suggests that chaotic dynamics can be obtained in the physiologically relevant case of the baroreceptor reflex loop. These results may provide insight into the mechanisms of dynamic vagal control of heart rate and may help to provide insights into clinically relevant disturbances of cardiac rate and rhythm.


Parietal lobe contribution to mental rotation demonstrated with rTMS.
A large number of imaging studies have identified a role for the posterior parietal lobe, in particular Brodmann's area 7 and the intraparietal sulcus (IPS), in mental rotation. Here we investigated whether neural activity in the posterior parietal lobe is essential for successful mental rotation performance by observing the effects of interrupting this activity during the execution of a mental rotation task. Repetitive transcranial magnetic stimulation (rTMS) was applied to posterior parietal locations estimated to overlie Brodmann's area 7 in the right and the left hemisphere, or to a posterior midline location (sham condition). In three separate experiments, rTMS (four pulses, 20 Hz) was delivered at these locations either 200-400, 400-600, or 600-800 msec after the onset of a mental rotation trial. Disrupting neural activity in the right parietal lobe interfered with task performance, but only when rTMS was delivered 400 to 600 msec after stimulus onset. Stimulation of the left parietal lobe did not reliably affect mental rotation performance at any of the time points investigated. The time-limited effect of rTMS was replicated in a fourth experiment that directly compared the effects of rTMS applied to the right parietal lobe either 200-400 or 400-600 msec into the mental rotation trial. The results indicate that the right superior posterior parietal lobe plays an essential role in mental rotation, consistent with its involvement in a variety of visuospatial and visuomotor transformations.


Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease.
A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application.


Androgen receptor phosphorylation status at serine 578 predicts poor outcome in prostate cancer patients.
Prostate cancer growth is dependent upon androgen receptor (AR) activation, regulated via phosphorylation. Protein kinase C (PKC) is one kinase that can mediate AR phosphorylation. This study aimed to establish if AR phosphorylation by PKC is of prognostic significance. Immunohistochemistry for AR, AR phosphorylated at Ser-81 (pARS81), AR phosphorylated at Ser-578 (pARS578), PKC and phosphorylated PKC (pPKC) was performed on 90 hormone-naïve prostate cancer specimens. Protein expression was quantified using the weighted histoscore method and examined with regard to clinico-pathological factors and outcome measures; time to biochemical relapse, survival from biochemical relapse and disease-specific survival. Nuclear PKC expression strongly correlated with nuclear pARS578 (c.c. 0.469, p=0.001) and cytoplasmic pARS578 (c.c. 0.426 p=0.002). High cytoplasmic and nuclear pARS578 were associated with disease-specific survival (p<0.001 and p=0.036 respectively). High nuclear PKC was associated with lower disease-specific survival when combined with high pARS578 in the cytoplasm (p=0.001) and nucleus (p=0.038). Combined high total pARS81 and total pARS578 was associated with decreased disease-specific survival (p=0.005). pARS578 expression is associated with poor outcome and is a potential independent prognostic marker in hormone-naïve prostate cancer. Furthermore, PKC driven AR phosphorylation may promote prostate cancer progression and provide a novel therapeutic target.


Effects on patients of their healthcare practitioner's or institution's participation in clinical trials: a systematic review.
Systematic reviews have shown uncertainty about the size or direction of any 'trial effect' for patients in trials compared to those treated outside trials. We are not aware of any systematic review of whether there is a 'trial effect' related to being treated by healthcare practitioners or institutions that take part in research. We searched the Cochrane Methodology Register and MEDLINE (most recently in January 2009) for studies in which patients were allocated to treatment in one or other setting, and cohort studies reporting the outcomes of patients from different settings. We independently assessed study quality, including the control of bias in the generation of the comparison groups, and extracted data. We retrieved and checked more than 15,000 records. Thirteen articles were eligible: five practitioner studies and eight institution studies. Meta-analyses were not possible because of heterogeneity. Two practitioner studies were judged to be 'controlled' or better. A Canadian study among nurses found that use of research evidence was higher for those who took part in research working groups and a Danish study on general practitioners found that trial doctors were more likely to prescribe in accordance with research evidence and guidelines. Five institution studies were 'controlled' but provided mixed results. A study of North American patients at hospitals that had taken part in trials for myocardial infarction found no statistically significant difference in treatment for patients in trial and non-trial hospitals. A Canadian study of myocardial infarction patients found that trial participants had better survival than patients in the same hospitals who were not in trials or those in non-trial hospitals. A study of general practices in Denmark did not detect differences in guideline adherence between trial and non-trial practices but found that trial practices were more likely to prescribe the trial sponsor's drugs. The other two 'controlled' studies of institutions found lower mortality in trial than non-trial hospitals. The available findings from existing research suggest that there might be a 'trial effect' of better outcomes, greater adherence to guidelines and more use of evidence by practitioners and institutions that take part in trials. However, the consequences for patient health are uncertain and the most robust conclusion may be that there is no apparent evidence that patients treated by practitioners or in institutions that take part in trials do worse than those treated elsewhere.


Interpenetrating polymer networks of collagen, hyaluronic acid, and chondroitin sulfate as scaffolds for brain tissue engineering.
Stem cells can provide neuro-protection and potentially neuro-replacement to patients suffering from traumatic brain injuries (TBI), with a practical option being delivery via engineered scaffolds. Collagen (Coll) and glycosaminoglycan (GAG) have been used as scaffolds for brain tissue engineering yet they often do not support cell differentiation and survival. In this study, we developed interpenetrating polymer network scaffolds comprising Coll, and incorporating two commonly found GAGs in the brain, chondroitin sulfate (CS) and/or hyaluronic acid (HA). We seeded these scaffolds with mouse neural stem cells from the subventricular zone (SVZ) niche. Compared to Coll-alone, all other substrates decreased the percent of nestin+ stem cells. Coll-CS-HA was more efficient at suppressing nestin expression than the other scaffolds; all SVZ cells lost nestin expression within 7 days of culture. In contrast to nestin, the percentage of microtubule associated protein 2 (MAP2+) neurons was greater in scaffolds containing, CS, HA or CS-HA, compared to Coll alone. Finally, Coll-CS increased the percentage of glial fibrillary acidic protein (GFAP+) astrocytes compared to Coll scaffolds. Overall, this work shows that Coll-HA and Coll-CS-HA scaffolds selectively enhance neurogenesis and may be advantageous in tissue engineering therapy for TBI. STATEMENT OF SIGNIFICANCE: Brain injury is devastating yet with few options for repair. Stem cells that reside in the subventricular zone (SVZ) only repair damage inefficiently due to poor control of their cellular progeny and unsuitable extracellular matrix substrates. To solve these problems, we have systematically generated collagen (Coll) scaffolds with interpenetrating polymer networks (IPN) of hyaluronic acid (HA) or chondroitin sulfate proteoglycans (CS) or both. The scaffolds had defined pore sizes, similar mechanical properties and all three stimulated neurogenesis, whereas only CS stimulated astrocyte genesis. Overall, this work suggests that Coll-HA and Coll-CS-HA scaffolds selectively enhance neurogenesis and may be advantageous in tissue engineering therapy for brain repair.


Adolescent psychosocial development: A review of longitudinal models and research.
This review used 4 types of longitudinal models (descriptive models, prediction models, developmental sequence models and longitudinal mediation models) to identify regular patterns of psychosocial development in adolescence. Eight patterns of adolescent development were observed across countries: (1) adolescent maturation in multiple developmental domains; (2) heterogeneous continuity of personal relationships; (3) good goes together with good, and bad with bad, across time in adolescence; (4) parents transmit values and behaviors to their adolescent children over time; (5) adolescent psychopathology leads to erosion of personal relationships with parents and peers; (6) adolescent psychopathology prevents adolescent independence from parents; (7) parental interference in personal issues of adolescents has counterproductive effects over time; (8) mood variability and (social and personal) uncertainty are mechanisms that maintain psychopathology in adolescence. Principles of life span developmental psychology are used to discuss adolescent maturation, and a developmental contextual perspective is used to discuss links between the various developmental patterns. Strengths and limitations of the various longitudinal models, and links between longitudinal and experimental research are discussed. (PsycINFO Database Record


Polygenic risk scores predict diabetes complications and their response to intensive blood pressure and glucose control.
Type 2 diabetes increases the risk of cardiovascular and renal complications, but early risk prediction could lead to timely intervention and better outcomes. Genetic information can be used to enable early detection of risk. We developed a multi-polygenic risk score (multiPRS) that combines ten weighted PRSs (10 wPRS) composed of 598 SNPs associated with main risk factors and outcomes of type 2 diabetes, derived from summary statistics data of genome-wide association studies. The 10 wPRS, first principal component of ethnicity, sex, age at onset and diabetes duration were included into one logistic regression model to predict micro- and macrovascular outcomes in 4098 participants in the ADVANCE study and 17,604 individuals with type 2 diabetes in the UK Biobank study. The model showed a similar predictive performance for cardiovascular and renal complications in different cohorts. It identified the top 30% of ADVANCE participants with a mean of 3.1-fold increased risk of major micro- and macrovascular events (p = 6.3 × 10-21 and p = 9.6 × 10-31, respectively) and a 4.4-fold (p = 6.8 × 10-33) higher risk of cardiovascular death. While in ADVANCE overall, combined intensive blood pressure and glucose control decreased cardiovascular death by 24%, the model identified a high-risk group in whom it decreased the mortality rate by 47%, and a low-risk group in whom it had no discernible effect. High-risk individuals had the greatest absolute risk reduction with a number needed to treat of 12 to prevent one cardiovascular death over 5 years. This novel multiPRS model stratified individuals with type 2 diabetes according to risk of complications and helped to target earlier those who would receive greater benefit from intensive therapy.


BODIPY Fluorophores for Membrane Potential Imaging.
Fluorophores based on the BODIPY scaffold are prized for their tunable excitation and emission profiles, mild syntheses, and biological compatibility. Improving the water-solubility of BODIPY dyes remains an outstanding challenge. The development of water-soluble BODIPY dyes usually involves direct modification of the BODIPY fluorophore core with ionizable groups or substitution at the boron center. While these strategies are effective for the generation of water-soluble fluorophores, they are challenging to implement when developing BODIPY-based indicators: direct modification of BODIPY core can disrupt the electronics of the dye, complicating the design of functional indicators; and substitution at the boron center often renders the resultant BODIPY incompatible with the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To highlight the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based, voltage-sensitive fluorophores (VF). The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in the context of a water-soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY dyes for functional indicators.


Building Force Fields: An Automatic, Systematic, and Reproducible Approach.
The development of accurate molecular mechanics force fields is a significant challenge that must be addressed for the continued success of molecular simulation. We developed the ForceBalance method to automatically derive accurate force field parameters using flexible combinations of experimental and theoretical reference data. The method is demonstrated in the parametrization of two rigid water models, yielding new parameter sets (TIP3P-FB and TIP4P-FB) that accurately describe many physical properties of water.


The mesoscopic dynamics of thermodynamic systems.
Concepts of everyday use such as energy, heat, and temperature have acquired a precise meaning after the development of thermodynamics. Thermodynamics provides the basis for understanding how heat and work are related and the general rules that the macroscopic properties of systems at equilibrium follow. Outside equilibrium and away from macroscopic regimes, most of those rules cannot be applied directly. Here we present recent developments that extend the applicability of thermodynamic concepts deep into mesoscopic and irreversible regimes. We show how the probabilistic interpretation of thermodynamics together with probability conservation laws can be used to obtain Fokker-Planck equations for the relevant degrees of freedom. This approach provides a systematic method to obtain the stochastic dynamics of a system directly from its equilibrium properties. A wide variety of situations can be studied in this way, including many that were thought to be out of reach of thermodynamic theories, such as nonlinear transport in the presence of potential barriers, activated processes, slow relaxation phenomena, and basic processes in biomolecules, such as translocation and stretching.


Engineering de novo reciprocal chromosomal translocations associated with Mll to replicate primary events of human cancer.
The etiology of human tumors often involves chromosomal translocations. Models that emulate translocations are essential to understanding the determinants of frank malignancy, those dictating the restriction of translocations to specific lineages, and as a basis for development of rational therapeutic methods. We demonstrate that developmentally regulated Cre-loxP-mediated interchromosomal recombination between the Mll gene, whose human counterpart is involved in a spectrum of leukemias, and the Enl gene creates reciprocal chromosomal translocations that cause myeloid tumors. There is a rapid onset and high penetrance of leukemogenesis in these translocator mice, and high proportions of cells carrying chromosomal translocations can be found in bone marrow as early as 12 days after birth. This de novo strategy is a direct recapitulation of naturally occurring human cancer-associated translocations.


Independent practice associations and physician-hospital organizations can improve care management for smaller practices.
Pay-for-performance, public reporting, and accountable care organization programs place pressures on physicians to use health information technology and organized care management processes to improve the care they provide. But physician practices that are not large may lack the resources and size to implement such processes. We used data from a unique national survey of 1,164 practices with fewer than twenty physicians to provide the first information available on the extent to which independent practice associations (IPAs) and physician-hospital organizations (PHOs) might make it possible for these smaller practices to share resources to improve care. Nearly a quarter of the practices participated in an IPA or a PHO that accounted for a significant proportion of their patients. On average, practices participating in these organizations provided nearly three times as many care management processes for patients with chronic conditions as nonparticipating practices did (10.4 versus 3.8). Half of these processes were provided only by IPAs or PHOs. These organizations may provide a way for small and medium-size practices to systematically improve care and participate in accountable care organizations.


Individual and Societal Wisdom: Explaining the Paradox of Human Aging and High Well-Being.
Objective: Although human aging is characterized by loss of fertility and progressive decline in physical abilities, later life is associated with better psychological health and well-being. Furthermore, there has been an unprecedented increase in average lifespan over the past century without corresponding extensions of fertile and healthy age spans. We propose a possible explanation for these paradoxical phenomena. Method: We reviewed the relevant literature on aging, well-being, and wisdom. Results: An increase in specific components of individual wisdom in later life may make up for the loss of fertility as well as declining physical health. However, current data on the relationship between aging and individual wisdom are not consistent and do not explain increased longevity in the general population during the past century. We propose that greater societal wisdom (including compassion) may account for the notable increase in average lifespan over the last century. Data in older adults with serious mental illnesses are limited, but suggest that many of them too experience improved psychosocial functioning, although their longevity has not yet increased, suggesting persistent stigma against mental illness and inadequate societal compassion. Conclusions: The proposed construct of societal wisdom needs more investigation. Research should also focus on the reasons for discrepant findings related to age-associated changes in different components of individual wisdom. Studies of wisdom and well-being are warranted in older people with serious mental illnesses, along with campaigns to enhance societal compassion for these disenfranchised individuals. Finally, effective interventions to enhance wisdom need to be developed and tested.


Modulation of fibroblast inflammatory response by surface modification of a perfluorinated ionomer.
An ideal surface for implantable glucose sensors would be able to evade the events leading to chronic inflammation and fibrosis, thereby extending its utility in an in vivo environment. Nafion™, a perfluorinated ionomer, is the membrane material preferred for in situ glucose sensors. Unfortunately, the surface properties of Nafion™ promote random protein adsorption and eventual foreign body encapsulation, thus leading to loss of glucose signal over time. Details of the techniques to render Nafion™ nonprotein fouling are given in a previous article [T. I. Valdes et al., Biomaterials 29, 1356 (2008)]. Once random protein adsorption is prevented, a biologically active peptide can be covalently bonded to the treated Nafion™ to induce cellular adhesion. Cellular responses to these novel decorated Nafion™ surfaces are detailed here, including cell viability, cell spreading, and type I collagen synthesis. Normal human dermal fibroblasts (NHDFs) were cultured on control and modified Nafion™ surfaces. Findings indicate that Nafion™ modified with 10% 2-hydroxyethyl methacrylate and 90% tetraglyme created a nonfouling surface that was subsequently decorated with the YRGDS peptide. NHDFs were shown to have exhibited decreased type I collagen production in comparison to NHDF cells on unmodified Nafion™ surfaces. Here, the authors report evidence that proves that optimizing conditions to prevent protein adsorption and enhance cellular adhesion may eliminate fibrous encapsulation of an implant.


VEGF stimulates RCAN1.4 expression in endothelial cells via a pathway requiring Ca2+/calcineurin and protein kinase C-delta.
Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system. We show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes. Our data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca(2+) and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.


Intraarticular hip injection and early revision surgery following total hip arthroplasty: a retrospective cohort study.
Therapeutic intraarticular injections are used in the management of hip osteoarthritis (OA). Some studies suggest that their use increases the risk of infection and subsequent revision surgery after primary total hip arthroplasty (THA), while others do not. We undertook this study to clarify the relationship between prior intraarticular injection and the risk of complication in a subsequent primary THA. In a cohort of patients with hip OA who underwent a primary elective THA between 2002 and 2009, we identified those who received ≥1 intraarticular injection performed by a radiologist in the 5 years preceding their THA. Multivariable Cox proportional hazards models were used to determine the relationship between receipt of a presurgical injection (no injection, 1-5 years prior to THA, or <1 year prior to THA) and the occurrence of postsurgical joint infection and revision THA in the following 2 years, while controlling for confounders. Of 37,881 eligible THA recipients, 2,468 (6.5%) received an intraarticular injection performed by a radiologist within 5 years of their THA (1,691 at <1 year, 777 at 1-5 years). Controlling for age, sex, comorbidity, frailty, income, and provider volume, those who had an injection in the year preceding surgery were at increased risk of infection (adjusted hazard ratio [HR] 1.37, P = 0.03) and revision THA (adjusted HR 1.53, P = 0.03) within 2 years of the primary THA, relative to patients who did not. The association between prior injection and revision arthroplasty was attenuated and became nonsignificant (adjusted HR 1.41, P = 0.13) after occurrence of postoperative infection was controlled for in the regression model. No effect was found for injection 1-5 years prior to surgery. Intraarticular injection in the year preceding THA independently predicted increased risk of infection leading to early revision surgery. Further studies are warranted to elucidate explanations for these findings.


The importance of pacing in basketball players with intellectual impairment: Input for evidence-based classification.
Pacing places a high demand on intellectual functioning and has been found useful for classification of athletes with intellectual impairments (II). This may also be true in open-loop sports like basketball. The current study aimed to investigate the pacing behaviour of basketball players with and without II. Using time-motion analysis, the activity of elite basketball players with II (n = 37) and amateur players without II (n = 34) was coded into four movement categories over eight periods of each game: standing, walking, running and jumping. Following two-way ANOVA, an effect of group showed differences between groups in duration and frequency of the movement categories within each period of the games. Additionally, an effect of time suggested that players in both groups paced their performances. However, no interaction was found, indicating that pacing may not be different between groups. In conclusion, the results suggest that due to the dynamic nature of basketball, the included players paced more intuitively by responding to environmental cues and using less deliberate planning. The players with II demonstrated slower games, which may be due to an impaired ability to make quick moment-to-moment deliberate decisions. These skills should be further studied in the context of evidence-based classification.


Comparison of the toxicity using body residues of DDE and select PCB congeners to the midge, Chironomus riparius, in partial-life cycle tests.
Due to the long time course required to achieve steady state with highly lipophilic contaminants such as PCBs (polychlorinated biphenyls), data derived from short-term toxicity tests may lead to an erroneous interpretation of hazard. In addition, PCBs bioaccumulated over time can cause sublethal impairments in organisms at concentrations much lower than required for mortality. Here, the body residues of 1,1-dichloro-2,2-bis-p-chlorophenyl ethane (DDE) and select PCB congeners associated with a spectrum of chronic effects in the midge, Chironomus riparius, were evaluated. The route of exposure was ingestion of the PCB-contaminated alga, Chlorella vulgarus, and trout chow loaded with the selected test compound. Two separate exposures of midges were performed. In the first experiment, midges were exposed from the second instar to the pupal stage. In the second exposure, midges were exposed from the second instar to the adult stage. A variety of sublethal endpoints was monitored, including developmental time within a stadium, body weight, and fecundity for the female adult. The dose was assessed as the whole body residue concentration of the contaminant. Overall, the midge concentration increased with increasing exposure concentration in algae and trout chow. Body weight at the end of each stadium was the assessment parameter that was least significantly affected among the test endpoints monitored. In contrast, a significant increase in development time was the endpoint that was most frequently observed in response to contaminant exposure. Reduction in fecundity was found only for DDE-exposed midges. These data, in which chronic endpoints are related to body residues, suggest that body residues will be useful in defining sublethal hazards of DDE and some PCB congeners.


Genetically defined elevated homocysteine levels do not result in widespread changes of DNA methylation in leukocytes.
DNA methylation is affected by the activities of the key enzymes and intermediate metabolites of the one-carbon pathway, one of which involves homocysteine. We investigated the effect of the well-known genetic variant associated with mildly elevated homocysteine: MTHFR 677C>T independently and in combination with other homocysteine-associated variants, on genome-wide leukocyte DNA-methylation. Methylation levels were assessed using Illumina 450k arrays on 9,894 individuals of European ancestry from 12 cohort studies. Linear-mixed-models were used to study the association of additive MTHFR 677C>T and genetic-risk score (GRS) based on 18 homocysteine-associated SNPs, with genome-wide methylation. Meta-analysis revealed that the MTHFR 677C>T variant was associated with 35 CpG sites in cis, and the GRS showed association with 113 CpG sites near the homocysteine-associated variants. Genome-wide analysis revealed that the MTHFR 677C>T variant was associated with 1 trans-CpG (nearest gene ZNF184), while the GRS model showed association with 5 significant trans-CpGs annotated to nearest genes PTF1A, MRPL55, CTDSP2, CRYM and FKBP5. Our results do not show widespread changes in DNA-methylation across the genome, and therefore do not support the hypothesis that mildly elevated homocysteine is associated with widespread methylation changes in leukocytes.


Dimethyl fumarate (Tecfidera): a new oral agent for multiple sclerosis.
To describe the clinical evidence supporting the safety, efficacy, and clinical utility of oral dimethyl fumarate for the treatment of multiple sclerosis (MS). A comprehensive PubMed search was conducted in July 2013 using the search terms dimethyl fumarate and Tecfidera. Reference lists of abstracted publications were reviewed to identify relevant works that were not retrieved via the electronic search. Additional information was obtained from the FDA Web site, manufacturer prescribing information, and Clinicaltrials.gov. Clinical trials and review articles that included the use of dimethyl fumarate in the treatment of MS and were available in English were abstracted for review. The safety and efficacy of dimethyl fumarate for the treatment of relapsing remitting MS was confirmed in 2 phase III trials, DEFINE and CONFIRM. Relative to placebo, twice-daily dimethyl fumarate was found to reduce the proportion of patients with relapses at 2 years by 34% to 49% and the annualized relapse rate by 44% to 53%. Although the incidence of serious adverse effects did not differ from that of placebo, intolerable flushing and gastrointestinal adverse effects prompted discontinuation in 3% and 4% of patients, respectively. In March 2013, dimethyl fumarate was approved as a third oral option for patients with relapsing forms of MS. Although no head-to-head trials have been conducted, a comparison of data from phase III trials suggests that the efficacy of dimethyl fumarate is comparable to that of existing oral agents and may offer a preferable safety profile.


Understanding antibiotic prophylaxis prescribing in pediatric surgical specialties.
Overuse of antibiotics has caused secondary poor outcomes and has led to a current rate of antibiotic resistant infections that constitutes a public health crisis. In pediatric surgical specialties, children continue to receive unnecessary antibiotics. To understand the factors that contribute to pediatric surgeons' decisions regarding the use of perioperative antibiotic prophylaxis. Focus groups included pediatric proceduralists/surgeons from the following specialties: interventional cardiology, otolaryngology, orthopedic surgery, cardiothoracic surgery, and general surgery. A total of 23 surgeons with a median of 9 years of experience (range, 0.5-29 years) participated in the focus groups that lasted 30-90 minutes each. Five themes emerged influencing beliefs about antibiotic prescribing practices: (1) reliance on previous experience and early education, (2) balancing antibiotic use with risk of infection, (3) uncertainty about the state of the scientific evidence, (4) understanding importance of communication and team collaboration, and (5) a prevalence of hospital-level concerns. Surgeons describe a complex set of factors that impact their antibiotic prescribing in pediatric surgical cases. They reported initial, but not ongoing, training and a use of individual weight of risk and benefit as a major dictator of prescribing practices. Antimicrobial stewardship programs should work with surgeons to develop acceptable implementation strategies to optimize antibiotic prescribing.


Expression and regulation of the zinc finger transcription factor Churchill during zebrafish development.
During gastrulation dynamic cell movements establish the germ layers and shape the body axis of the vertebrate embryo. The zinc finger protein Churchill (chch) has been proposed to be a key regulator of these movements. We examined the expression pattern of chch in zebrafish and studied the regulation of chch by FGF signaling. We observed zygotic expression of chch during early cleavage stages. Two lines of evidence demonstrate that chch is zygotically expressed prior to the mid-blastula transition. First, blocking transcription during early cleavage stages represses chch expression. Second, endogenous levels of chch transcripts increase between 1-cell and 16-cell embryos. chch remains widely expressed during blastula and gastrula stages but scattered cells express higher levels of chch. By somitogenesis, chch is expressed in the ventral-most cells of the embryo adjacent to the yolk. In addition, transcripts are also observed in superficial cells on the surface of the yolk, in presumptive mucous cells and keratinocytes. By 30 hpf transcripts are observed in anterior neural tissue and ventral cells adjacent to the yolk. Over the next three days chch expression is indistinct until 4 dpf when we observe expression in the pharynx and gut. We show that activation of FGF signaling during gastrulation is sufficient to induce chch expression. In addition, we demonstrate that blocking FGF signaling between the 4-cell and shield stages represses chch expression.


Oxidative stress in hemodialysis patients receiving intravenous iron therapy and the role of N-acetylcysteine in preventing oxidative stress.
To determine the contribution of injectable iron administered to hemodialysis (HD) patients in causing oxidative stress and the beneficial effect of N-acetylcysteine (NAC) in reducing it, we studied in a prospective, double blinded, randomized controlled, cross over trial 14 adult HD patients who were randomized into two groups; one group received NAC in a dose of 600 mgs twice daily for 10 days prior to intravenous iron therapy and the other group received placebo. Both the groups were subjected to intravenous iron therapy, 100 mg of iron sucrose in 100 mL of normal saline given over a period of one hour. Blood samples for the markers of oxidative stress were taken before and after iron therapy. After the allowance of a week of wash out period for the effect of N-acetylcysteine we crossed over the patients to the opposite regimen. We measured the lipid peroxidation marker, malondiaaldehyde (MDA), to evaluate the oxidative stress and total anti-oxidant capacity (TAC) for the antioxidant level in addition to the highly sensitive C-reactive protein (HsCRP). Non-invasive assessment of endothelial dysfunction was measured by digital plethysmography before and after intravenous iron therapy. There was an increase of MDA (21.97 + 3.65% vs 7.06 + 3.65%) and highly sensitive C-reactive protein (HsCRP) (11.19 + 24.63% vs 13.19 + 7.7%) after iron administration both in the placebo and the NAC groups. NAC reduced the baseline acute systemic generation of oxidative stress when compared to placebo, which was statistically significant with MDA (12.76 + 4.4% vs 9.37 + 4.40%: P = 0.032) but not with HsCRP though there was a declining trend (2.85 + 22.75 % vs 8.93 + 5.19%: P = 0.112). Pre-treatment with NAC reduced the endothelial dysfunction when compared to placebo, but it was not statistically significant, except for reflection index (RI). We conclude that in our HD patients NAC reduced the oxidative stress before and after the administration of intravenous iron therapy in addition to the endothelial dysfunction induced by this treatment.


A randomised trial of the effect of appearance versus health-framed messages on engagement with an online brief screening and alcohol reduction intervention.
Objectives: To compare the impact of appearance versus health-framed messages on engagement in a brief web-based risk screening and alcohol reduction intervention. Design: Randomised trial delivered via Drinkaware's website. Visitors were exposed to appearance (n = 51,588) or health-framed messages (n = 52,639) directing them towards an AUDIT-C risk screening questionnaire. Users completing this questionnaire were given feedback on their risk level and extended frame-congruent information. Outcomes: The primary outcome is completion of the AUDIT-C questionnaire. The secondary outcome is whether the participant accessed any of four further resources. Results: The appearance-framed message led to a small but significant increase in the number of users completing the AUDIT-C compared to the health-framed message (n = 3,537, 6.86% versus n = 3,355, 6.37%, p < 0.01). Conversely, following subsequent risk feedback, users exposed to extended health-framed information were more likely to access further resources (n = 1,146, 2.17% versus n = 942, 1.83%, p < 0.01). Conclusions: Physical appearance-framed messages increased the likelihood of engagement with an online alcohol screening and brief intervention tool, whereas health-framed messages increased the likelihood of accessing further resources. This highlights the potential for the use of multi-level approaches in alcohol reduction interventions.


Imaging reflectometry in situ.
An innovative method of in situ real-time optical monitoring of thin film deposition and etching is presented. In this technique, intensity maps of a thin film corresponding to a series of wavelengths selected by a monochromator (300-800 nm) are recorded by a CCD camera. From the maps the reflectance spectra at individual points of the sample surface can be extracted. By fitting the reflectance spectra to the theoretical ones, the maps of a thin film morphology (including optical parameters) and their temporal development during technological processes can be obtained. The method was tested by in situ observation of the growth of silicon nitride and silicon oxide thin films prepared by ion beam sputtering and by the monitoring of etching of thermally grown SiO(2) thin films.


Combination therapy as initial treatment for newly diagnosed hypertension.
The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure recommends that clinicians consider the use of multidrug therapy to increase likelihood of achieving blood pressure goal. Little is known about recent patterns of combination antihypertensive therapy use in patients being initiated on hypertension treatment. We investigated combination antihypertensive therapy use in newly diagnosed hypertensive patients from the Cardiovascular Research Network Hypertension Registry. Multivariable logistic regression was used to assess the relationship between combination antihypertensive therapy and 12-month blood pressure control. Between 2002 and 2007, a total of 161,585 patients met criteria for incident hypertension and were initiated on treatment. During the study period, an increasing proportion of patients were treated initially with combination rather than with single-agent therapy (20.7% in 2002 compared with 35.8% in 2007, P < .001). This increase in combination therapy use was more pronounced in patients with stage 2 hypertension, whose combination therapy use increased from 21.6% in 2002 to 44.5% in 2007. Nearly 90% of initial combination therapy was accounted for by 2 combinations, a thiazide and a potassium-sparing diuretic (47.6%) and a thiazide and an angiotensin-converting enzyme inhibitor (41.4%). After controlling for relevant clinical factors, including subsequent intensification of treatment and medication adherence, combination therapy was associated with increased odds of blood pressure control at 12 months (odds ratio compared with single-drug initial therapy 1.20; 95% CI 1.15-1.24, P < .001). Initial treatment of hypertension with combination therapy is increasingly common and is associated with better long-term blood pressure control.


Immunogold localization of vitellogenin in the ovaries, hypopharyngeal glands and head fat bodies of honeybee workers, Apis mellifera.
Vitellogenin is a yolk precursor protein in most oviparous females. In the advanced eusocial honeybee, Apis mellifera (Hymenoptera: Apidae), vitellogenin has recently attracted much interest as this protein, in addition to a classical function in oocyte development in the reproductive queen caste, has evolved functions in the facultatively sterile female worker caste not documented in other species. However, research on the spatial dynamics of vitellogenin in various tissues is not easily performed with available tools. Here we present an immunogold staining procedure that visualizes honeybee vitellogenin in resin embedded tissue. To establish the protocol, we used ovaries of worker bees from colonies with and without a queen. Under the first condition, vitellogenin is assumed not to be present in the workers' ovaries. Under the second condition, the ovaries of worker bees become vitellogenic, with abundant opportunities for detection of complex patterns of vitellogenin uptake and storage. By use of this experimental setup, the staining method is shown to be both sensitive and specific. To demonstrate the functional significance of the protocol, it was subsequently used to identify vitellogenin protein in the hypopharyngeal glands (brood food producing head glands) of nursing worker bees and in adjacent head fat body cells for the first time. Localization of vitellogenin in these tissues supports previously hypothesized roles of vitellogenin in social behavior. This protocol thus provides deeper insights into the functions of vitellogenin in the honeybee.


Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma.
High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression. We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data. We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested. We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.


Periodontitis in RA-the citrullinated enolase connection.
Autoimmunity in rheumatoid arthritis (RA) is characterized by an antibody response to citrullinated proteins. Two of the risk factors for RA-HLA-DRB1 shared epitope alleles and smoking-are also associated with periodontitis, which is largely, but not exclusively, caused by Porphyromonas gingivalis infection. Furthermore, RA and periodontitis have a similar pathophysiology, characterized by destructive inflammation. The citrullination of proteins by P. gingivalis and the subsequent generation of autoantigens that drive autoimmunity in RA represents a possible causative link between these two diseases. Antibodies directed towards the immunodominant epitope of human citrullinated α-enolase cross-react with a conserved sequence on citrullinated P. gingivalis enolase. On the basis of this cross-reactivity, in this Perspectives article we explore the hypothesis of molecular mimicry in the etiology of RA, with citrullinated enolase as the specific antigen involved.


Metallothionein expression and nuclear size in benign, borderline, and malignant serous ovarian tumours.
Metallothioneins (MTs) are low-molecular-weight proteins involved in metalloregulatory functions such as cell proliferation, growth, and differentiation. In recent years, MT expression has been linked with carcinogenesis, resistance to cancer therapy, and tumour progression. However, the significance of MT expression in ovarian cancers is at present inadequately documented. In this study, MT immunohistochemistry was performed in 12 benign, 14 borderline, and eight malignant serous tumours of the ovary. The intensity of the immunostaining was evaluated by image analysis. There was a significantly higher number of MT-immunopositive cells in the multilayered epithelial cells of borderline serous tumours (atypical proliferative serous tumours) than in the single layered epithelial cells within the same tumour, and in the single cell layer of benign serous tumours. There was no difference in the expression of MTs in the single layered tumour cells of benign and borderline serous tumours. Significantly higher numbers of MT-immunopositive cells were observed in both the single and the multilayered epithelial cells of serous carcinomas, the highest number being observed in the multiple layers of serous carcinomas. The positively stained malignant tumour cells in both single and multiple layers were larger than the negatively stained cells in benign, borderline, and malignant serous ovarian tumours. There was moderate to intense staining. These findings indicate that there is increased expression of MTs in the progression of malignancy, which could be used as a marker in grading the three groups of ovarian serous tumours and for determining prognosis.


Integrated Digital Patient Education at the Bedside for Patients with Chronic Conditions: Observational Study.
Patient education delivered by a health care provider increases patients' understanding and adherence to medical instructions, which helps to improve patient health. Multiple challenges exist to delivering effective patient education to patients with multiple chronic conditions, including giving the necessary time, range, and types of learning materials, and assessing the level of understanding. To help overcome these challenges, it is important to study new electronic means to assist in patient education, such as the use of mobile devices, interactive media, 3-dimensional images, and multimedia educational content at the bedside. The goal of this study was to address the need for blended learning strategies combining technical and workflow integration of digital patient education systems for patients with chronic conditions within and across the regular process of care. Studies are needed to evaluate the utility and benefits of these technologies for providers and patients alike. A mixed-methods approach was employed including survey administration to 178 patients after they received digital patient education in person with a health care provider, and qualitative interviews with 16 nurse educators who used the mobile digital health education technology to deliver instruction to patients. Patient survey data were analyzed using chi-square statistical tests. Qualitative interviews were analyzed for user acceptance and perceived value themes. Patients who were counseled using a blended digital health education approach reported improved understanding of educational content (P=.034) and chronic health conditions (P<.001), were more motivated to care for themselves at home (P<.001), were more likely to say that they felt capable of making health care decisions with their doctors (P<.001) and on their own (P=.001), and were more likely to report their intention to follow their doctor's instructions (P<.001) than were patients whose education was not computer-based. Nurse educators felt that the digital education system and content enhanced their education efforts and could be easily integrated into the outpatient clinical workflow. Patient education for individuals with chronic conditions may be more effective than traditional formats when provided in blended digital formats supervised by a health care provider.