Systematic review and mixed treatment comparison of intravitreal aflibercept with other therapies for diabetic macular edema (DME).
This was an indirect comparison of the effectiveness of intravitreal aflibercept (IVT-AFL) 2 mg every 8 weeks after 5 initial monthly doses (or if different periods, after an initial monthly dosing period) (2q8) and other diabetic macular edema (DME) therapies at doses licensed outside the USA. A comprehensive search was undertaken to source relevant studies. Feasibility networks were prepared to identify viable comparisons of 12-month outcomes between IVT-AFL 2q8 and therapies licensed outside the USA, which were assessed for clinical and statistical homogeneity. Pooled effect sizes (mean difference [MD] and relative risk/risk ratio [RR]) were calculated using fixed- and random-effects models. Indirect comparisons were performed using Bucher analysis. If at least one 'head-to-head' study was found then a mixed treatment comparison (MTC) was performed using Bayesian methods. Two 12-month comparisons could be undertaken based on indirect analyses: IVT-AFL 2q8 versus intravitreal ranibizumab (IVR) 0.5 mg as needed (PRN) (10 studies) and IVT-AFL 2q8 versus dexamethasone 0.7 mg implants (three studies). There was an increase in mean best-corrected visual acuity (BCVA) with IVT-AFL 2q8 over IVR 0.5 mg PRN by 4.67 letters [95% credible interval (CrI): 2.45-6.87] in the fixed-effect MTC model (10 studies) and by 4.82 letters [95% confidence interval (CI): 2.52-7.11] in the Bucher indirect analysis (four studies). IVT-AFL 2q8 doubled the proportion of patients gaining ≥ 10 Early Treatment Diabetic Retinopathy Study letters at 12 months compared with dexamethasone 0.7 mg implants (RR = 2.10 [95% CI: 1.29-3.40]) in the fixed-effect model. There were no significant differences in safety outcomes between IVT-AFL 2q8 and IVR 0.5 mg PRN or dexamethasone 0.7 mg implants. Studies of IVT-AFL 2q8 showed improved 12-month visual acuity measures compared with studies of IVR 0.5 mg PRN and dexamethasone 0.7 mg implants based on indirect comparisons. These analyses are subject to a number of limitations which are inherent in indirect data comparisons.


Toward panoramic in situ mapping of action potential propagation in transgenic hearts to investigate initiation and therapeutic control of arrhythmias.
To investigate the dynamics and propensity for arrhythmias in intact transgenic hearts comprehensively, optical strategies for panoramic fluorescence imaging of action potential (AP) propagation are essential. In particular, mechanism-oriented molecular studies usually depend on transgenic mouse hearts of only a few millimeters in size. Furthermore, the temporal scales of the mouse heart remain a challenge for panoramic fluorescence imaging with heart rates ranging from 200 min(-1) (e.g., depressed sinus node function) to over 1200 min(-1) during fast arrhythmias. To meet these challenging demands, we and others developed physiologically relevant mouse models and characterized their hearts with planar AP mapping. Here, we summarize the progress toward panoramic fluorescence imaging and its prospects for the mouse heart. In general, several high-resolution cameras are synchronized and geometrically arranged for panoramic voltage mapping and the surface and blood vessel anatomy documented through image segmentation and heart surface reconstruction. We expect that panoramic voltage imaging will lead to novel insights about molecular arrhythmia mechanisms through quantitative strategies and organ-representative analysis of intact mouse hearts.


Mutualism-parasitism paradigm synthesized from results of root-endophyte models.
Plant tissues host a variety of fungi. One important group is the dark septate endophytes (DSEs) that colonize plant roots and form characteristic intracellular structures - melanized hyphae and microsclerotia. The DSE associations are common and frequently observed in various biomes and plant taxa. Reviews suggest that the proportion of plant species colonized by DSE equal that colonized by AM and microscopic studies show that the proportion of the root system colonized by fungi DSE can equal, or even exceed, the colonization by AM fungi. Despite the high frequency and suspected ecological importance, the effects of DSE colonization on plant growth and performance have remained unclear. Here, we draw from over a decade of experimentation with the obscure DSE symbiosis and synthesize across large bodies of published and unpublished data from Arabidopsis thaliana and Allium porrum model systems as well as from experiments that use native plants to better resolve the host responses to DSE colonization. The data indicate similar distribution of host responses in model and native plant studies, validating the use of model plants for tractable dissection of DSE symbioses. The available data also permit empirical testing of the environmental modulation of host responses to DSE colonization and refining the "mutualism-parasitism-continuum" paradigm for DSE symbioses. These data highlight the context dependency of the DSE symbioses: not only plant species but also ecotypes vary in their responses to populations of conspecific DSE fungi - environmental conditions further shift the host responses similar to those predicted based on the mutualism-parasitism-continuum paradigm. The model systems provide several established avenues of inquiry that permit more detailed molecular and functional dissection of fungal endophyte symbioses, identifying thus likely mechanisms that may underlie the observed host responses to endophyte colonization.


Interactions of leukocyte integrins with intercellular adhesion molecule 1 in the production of inflammatory vascular injury in vivo. The Shwartzman reaction revisited.
We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by 99mTc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CD11b (Mac-1) showed partial inhibition of hemorrhage, while mAb against CD11a (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition.


G-protein-gated potassium channels containing Kir3.2 and Kir3.3 subunits mediate the acute inhibitory effects of opioids on locus ceruleus neurons.
Acute opioid administration causes hyperpolarization of locus ceruleus (LC) neurons. A G-protein-gated, inwardly rectifying potassium (GIRK/K(G)) conductance and a cAMP-dependent cation conductance have both been implicated in this effect; the relative contribution of each conductance remains controversial. Here, the contribution of K(G) channels to the inhibitory effects of opioids on LC neurons was examined using mice that lack the K(G) channel subunits Kir3.2 and Kir3.3. Resting membrane potentials of LC neurons in brain slices from Kir3.2 knock-out, Kir3.3 knock-out, and Kir3.2/3.3 double knock-out mice were depolarized by 15-20 mV relative to LC neurons from wild-type mice. [Met](5)enkephalin-induced hyperpolarization and whole-cell current were reduced by 40% in LC neurons from Kir3.2 knock-out mice and by 80% in neurons from Kir3.2/3.3 double knock-out mice. The small opioid-sensitive current observed in LC neurons from Kir3.2/3.3 double knock-out mice was virtually eliminated with the nonselective potassium channel blockers barium and cesium. We conclude that the acute opioid inhibition of LC neurons is mediated primarily by the activation of G-protein-gated potassium channels and that the cAMP-dependent cation conductance does not contribute significantly to this effect.


Environmental Pollution as a Risk Factor in Testicular Tumour Development: Focus on the Interaction between Bisphenol A and the Associated Immune Response.
Bisphenol A (BPA) is an endocrine disruptor to which animals and humans are highly exposed. Many reports have established a relationship between BPA exposure and breast cancer incidence, especially during critical periods of development. However, its effects on the immune response in testicular tumour growth have not yet been described. Thus, we wanted to analyse the effect of perinatal BPA exposure in pregnant female mice and the immune response modulation and tumour growth in an intratesticular cancer model in offspring male mice. Pregnant female mice were exposed to a dose of 250 mg/kg/day/body weight of BPA in their drinking water. In adulthood, male offspring underwent intrascrotal inoculation with 4T1 cancer cells. On day 21 after inoculation, mice were euthanised, and serum was obtained to measure BPA levels using HPLC coupled to mass spectrometry. The percentages of immune cell populations in peripheral lymph nodes (PLN), the spleen and tumours were evaluated by flow cytometry. In addition, the tumour expression of IL-10, TNF-α and TGF-β was analysed by RT-PCR. Of note, we found detectable circulating levels of BPA in the offspring of mothers exposed to it while pregnant. Remarkably, BPA treatment promoted tumour growth by about 75% compared to mice coming from female mice that did not receive the compound. Perinatal exposure to BPA modulated the percentages of different immune cells in the spleen and PLN. In addition, the expression of inflammatory-related cytokines (IL-10 and TNF-α) in the tumours was significantly enhanced compared to control and vehicle groups. In conclusion, the perinatal BPA administration in pregnant female mice modulated different cellular and molecular immune components that resulted in outstanding testicular tumour size in male offspring.


From structure to function: mitochondrial morphology, motion and shaping in vascular smooth muscle.
The diversity of mitochondrial arrangements, which arise from the organelle being static or moving, or fusing and dividing in a dynamically reshaping network, is only beginning to be appreciated. While significant progress has been made in understanding the proteins that reorganise mitochondria, the physiological significance of the various arrangements is poorly understood. The lack of understanding may occur partly because mitochondrial morphology is studied most often in cultured cells. The simple anatomy of cultured cells presents an attractive model for visualizing mitochondrial behaviour but contrasts with the complexity of native cells in which elaborate mitochondrial movements and morphologies may not occur. Mitochondrial changes may take place in native cells (in response to stress and proliferation), but over a slow time-course and the cellular function contributed is unclear. To determine the role mitochondrial arrangements play in cell function, a crucial first step is characterisation of the interactions among mitochondrial components. Three aspects of mitochondrial behaviour are described in this review: (1) morphology, (2) motion and (3) rapid shape changes. The proposed physiological roles to which various mitochondrial arrangements contribute and difficulties in interpreting some of the physiological conclusions are also outlined.


Non-invasive imaging of beta cell mass: a quantitative analysis.
Currently there are major efforts to develop strategies for the in vivo imaging of pancreatic beta cell mass as a clinical and investigational tool for detecting and tracking the loss of beta cells that underlies the progression of Type I diabetes. However, beta cells constitute only about 1% of pancreatic mass and are distributed throughout the pancreas within tiny islets of Langerhans that are each less than the spatial resolution of non-invasive imaging technologies. To estimate the requisite binding characteristics of a candidate beta cell imaging agent, calculations of the beta cell contribution to a positron emission tomography signal were made using simple equations. These were based on the relative population of beta cells and non-beta cells within the pancreas and surrounding tissue and an equation describing equilibrium ligand binding. The calculations show that two criteria must be met: (1) The low-volume fraction of beta cells within the exocrine pancreas (about 1:100) requires that beta cells retain labeled imaging agents at least 1,000-fold more strongly than exocrine cells. (2) Agents that label cell surface receptors, even if beta cell-specific, must do so at a high enough level so that the imaging signal arising from unbound label retained in extracellular spaces must not overwhelm signals from labeled beta cells. The limits developed here can serve as criteria for identifying candidate imaging agents for the in vivo imaging of beta cell mass, thereby avoiding expensive preclinical development using compounds that have no chance of success.


Decision analysis for secondline maintenance treatment of platinum sensitive recurrent ovarian cancer: a review.
Most women with ovarian cancer experience disease relapse, presenting numerous treatment challenges for clinicians. Maintenance therapy in the relapsed setting aims to extend the time taken for a cancer to progress, thus delaying the need for additional treatments. Four therapies are currently approved in the USA for secondline maintenance treatment of platinum sensitive, recurrent ovarian cancer: one antivascular endothelial growth factor agent (bevacizumab) and three poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors (olaparib, niraparib, and rucaparib). In addition to efficacy, maintenance therapies must have a good tolerability profile and no significant detrimental impact on quality of life, as patients who receive maintenance are generally free from cancer related symptoms. Data from key bevacizumab trials (OCEANS, NCT00434642; GOG-0213, NCT00565851; MITO16B, NCT01802749) and PARP inhibitor trials (Study 19, NCT00753545; SOLO2, NCT01874353; NOVA, NCT01847274; ARIEL3, NCT01968213) indicate that bevacizumab and the PARP inhibitors are effective in patients with platinum sensitive, recurrent ovarian cancer but differ in their tolerability profiles. In addition, the efficacy of PARP inhibitors is dependent on the presence of homologous recombination repair deficiency, with patients with the deficiency experiencing greater responses from treatment compared with those who are homologous recombination repair proficient. Allowing for caveats of cross trial comparisons, we advise that clinicians account for the following points when choosing whether and when to administer a secondline maintenance treatment for a specific patient: presence of a homologous recombination repair deficient tumor; the patient's baseline characteristics, such as platelet count and blood pressure; mode of administration of therapy; and consideration of future treatment options for thirdline and later therapy.


Bacterial succession in Antarctic soils of two glacier forefields on Larsemann Hills, East Antarctica.
Antarctic glacier forefields are extreme environments and pioneer sites for ecological succession. Increasing temperatures due to global warming lead to enhanced deglaciation processes in cold-affected habitats, and new terrain is becoming exposed to soil formation and microbial colonization. However, only little is known about the impact of environmental changes on microbial communities and how they develop in connection to shifting habitat characteristics. In this study, using a combination of molecular and geochemical analysis, we determine the structure and development of bacterial communities depending on soil parameters in two different glacier forefields on Larsemann Hills, East Antarctica. Our results demonstrate that deglaciation-dependent habitat formation, resulting in a gradient in soil moisture, pH and conductivity, leads to an orderly bacterial succession for some groups, for example Cyanobacteria, Bacteroidetes and Deltaproteobacteria in a transect representing 'classical' glacier forefields. A variable bacterial distribution and different composed communities were revealed according to soil heterogeneity in a slightly 'matured' glacier forefield transect, where Gemmatimonadetes, Flavobacteria, Gamma- and Deltaproteobacteria occur depending on water availability and soil depth. Actinobacteria are dominant in both sites with dominance connected to certain trace elements in the glacier forefields.


Review and Meta-analysis of Emerging Risk Factors for Agricultural Injury.
Agricultural injury is a significant public health problem globally. Extensive research has addressed this problem, and a growing number of risk factors have been reported. The authors evaluated the evidence for frequently reported risk factors earlier. The objective in the current study was to identify emerging risk factors for agricultural injury and calculate pooled estimates for factors that were assessed in two or more studies. A total of 441 (PubMed) and 285 (Google Scholar) studies were identified focusing on occupational injuries in agriculture. From these, 39 studies reported point estimates of risk factors for injury; 38 of them passed the Newcastle-Ottawa criteria for quality and were selected for the systematic review and meta-analysis. Several risk factors were significantly associated with injury in the meta-analysis. These included older age (vs. younger), education up to high school or higher (vs. lower), non-Caucasian race (vs. Caucasian), Finnish language (vs. Swedish), residence on-farm (vs. off-farm), sleeping less than 7-7.5 hours (vs. more), high perceived injury risk (vs. low), challenging social conditions (vs. normal), greater farm sales, size, income, and number of employees on the farm (vs. smaller), animal production (vs. other production), unsafe practices conducted (vs. not), computer use (vs. not), dermal exposure to pesticides and/or chemicals (vs. not), high cooperation between farms (vs. not), and machinery condition fair/poor (vs. excellent/good). Eighteen of the 25 risk factors were significant in the meta-analysis. The identified risk factors should be considered when designing interventions and selecting populations at high risk of injury.


No evidence for increased self-reported cognitive failure in Type 1 and Type 2 diabetes: a cross-sectional study.
Mild cognitive deficits have been determined in both types of diabetes using neurocognitive tests. Little is known about the degree to which patients complain about their cognitive functioning. This study set out to investigate the magnitude and correlates of self-reported cognitive failure in adult out-patients with Type 1 and Type 2 diabetes. Subjective cognitive functioning was measured in 187 diabetic patients using the Cognitive Failures Questionnaire (CFQ). Demographic and clinical characteristics were retrieved from the medical records. The Patient Health Questionnaire 9 items (PHQ-9) was self-administered along with the CFQ to correct for the confounding effect of depression. Analyses were based on 55 patients with Type 1 diabetes and 100 patients with Type 2 diabetes. No difference in mean CFQ score was observed between Type 1 and Type 2 diabetic patients or between Type 1 diabetic patients and healthy control subjects. Female patients with Type 2 diabetes reported significantly fewer cognitive complaints compared with female healthy control subjects. None of the demographic variables and diabetes-related complications was associated with subjective cognitive complaints. A strong positive association was found between depression symptomatology and frequency of self-reported cognitive failure. Our study could not confirm elevated subjective cognitive complaints in a group of Type 1 and Type 2 diabetes patients, as might be expected given the observed elevated rates of mild cognitive dysfunction in patients with diabetes. Self-reported cognitive failure appears largely determined by depressive symptomatology. Therefore, affective status should be included in any cognitive assessment procedure.


The Role of Gender, Child Maltreatment, Alcohol Expectancies, and Personality Pathology on Relationship Violence Among Undergraduates.
Male (n = 1,104) and female (n = 1,337) college students' self-report surveys on childhood maltreatment, alcohol expectancies, and narcissistic personality traits are examined to determine their associations with relationship violence. Intimate partner violence was measured using the violence subscales of the revised Conflict Tactics. Because we were interested in the effects of gender and because z tests of the correlations and t tests of means indicated men and women differed significantly on several variables, ordinary least squares regression models were run separately for men and women. Results suggest there are both gender similarities and differences. Relationship violence was associated with child sexual abuse for both men and women, whereas high scores on negative alcohol expectancies and vulnerable narcissism, and low scores on grandiose narcissism, were significantly associated with violence for men only. In addition, z tests revealed the regression coefficients for child sexual abuse and negative alcohol expectancies were significantly different for men and women. Child sexual abuse and alcohol expectancies had stronger associations with men's than women's violent behavior. In addition, the models explained a greater amount of variance in men's than in women's behavior. Policy implications of the research will be discussed.


How should a nurse approach truth-telling? A virtue ethics perspective.
Truth-telling is a key issue within the nurse-patient relationship. Nurses make decisions on a daily basis regarding what information to tell patients. This paper analyses truth-telling within an end of life scenario. Virtue ethics provides a useful philosophical approach for exploring decisions on information disclosure in more detail. Virtue ethics allows appropriate examination of the moral character of the nurse involved, their intention, ability to use wisdom and judgement when making decisions and the virtue of truth-telling. It is appropriate to discuss nursing as a 'practice' in relation to virtue ethics. This is achieved through consideration of the implications of arguments made by Alasdair MacIntyre who believes that qualities such as honesty, courage and justice are virtues because they enable us to achieve the internal goods of practices.


Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction.
Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.


Belimumab: a guide to its use in systemic lupus erythematosus.
Belimumab (Benlysta®), a fully human recombinant IgG1λ monoclonal antibody that inhibits the binding of soluble B lymphocyte stimulator to B cells, is indicated for the treatment of adult patients with active, autoantibody-positive systemic lupus erythematosus (SLE) with a high degree of disease activity despite receiving standard therapy. In multinational trials, significantly more belimumab recipients than placebo recipients achieved an SLE Responder Index response at 52 weeks.


A 27 kDa heat shock protein that has anomalous prognostic powers in early and advanced breast cancer.
This paper describes a prospective immunohistochemical analysis of 27 kDa heat shock protein (HSP27) in 361 patients with primary breast cancer in relation to disease-free survival (DFS) and survival from first relapse (SR). Oestradiol (ER) and progesterone (PR) receptors were also quantitated and related to the HSP27 data. While ER positively predicted a good outcome for both DFS and SR, HSP27 positivity predicted a prolonged SR but short DFS. The association between HSP27 and DFS only attained statistical significance in node-negative patients. Subgroup analysis reinforced the complementary relationship of HSP27 and ER for SR and opposing influences for DFS. In both node-negative and node-positive women, ER+ HSP27- patients had a longer DFS than ER- HSP27+ counterparts. There was no relationship between HSP27 and overall survival. HSP27 staining was highly correlated with ER but not PR, patient age, tumour size or menstrual status. There was a marginal correlation (P = 0.04) with histological grade with well-differentiated tumours having the highest HSP27. Cox multivariate regression analysis of the contribution of HSP27 in the presence of data on ER, PR, stage, nodal status and histological grade indicated that HSP27 was not of independent prognostic importance for DFS or overall survival and was only of borderline significance for OS (P < 0.07). However, in the absence of ER and PR data, HSP27 staining is an effective way of getting the same prognostic information. HSP27 staining appears to correlate with different biological features in early and advanced breast, high HSP27 being linked with short DFS in node-negative patients but with prolonged survival from first recurrence.


Disruption of long-chain base hydroxylation alters growth and impacts sphingolipid synthesis in Physcomitrella patens.
Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella patens, 4-hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long-chain base (LCB). To assess the functional significance of t18:0, CRISPR-Cas9 mutagenesis was used to generate mutant lines lacking the sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content in sbh protonemata was 2.4-fold higher than in wild-type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl-chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl-chain pairing in glucosylceramides, in which 4,8-sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. The sbh lines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content of sbh lines decreased to wild-type levels, and the mutants exhibited phenotypes more similar to wild-type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyte P. patens and angiosperm A. thaliana.


Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit.
Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic. Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM. After i.v. injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo. Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT. In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity. The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.


Risk adjustment alternatives in paying for behavioral health care under Medicaid.
To compare the performance of various risk adjustment models in behavioral health applications such as setting mental health and substance abuse (MH/SA) capitation payments or overall capitation payments for populations including MH/SA users. The 1991-93 administrative data from the Michigan Medicaid program were used. We compared mean absolute prediction error for several risk adjustment models and simulated the profits and losses that behavioral health care carve outs and integrated health plans would experience under risk adjustment if they enrolled beneficiaries with a history of MH/SA problems. Models included basic demographic adjustment, Adjusted Diagnostic Groups, Hierarchical Condition Categories, and specifications designed for behavioral health. Differences in predictive ability among risk adjustment models were small and generally insignificant. Specifications based on relatively few MH/SA diagnostic categories did as well as or better than models controlling for additional variables such as medical diagnoses at predicting MH/SA expenditures among adults. Simulation analyses revealed that among both adults and minors considerable scope remained for behavioral health care carve outs to make profits or losses after risk adjustment based on differential enrollment of severely ill patients. Similarly, integrated health plans have strong financial incentives to avoid MH/SA users even after adjustment. Current risk adjustment methodologies do not eliminate the financial incentives for integrated health plans and behavioral health care carve-out plans to avoid high-utilizing patients with psychiatric disorders.


Study of thyroid hormones free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) in subjects with dental fluorosis.
Apart from its well-known deleterious dental and skeletal effects, fluoride excess can have toxic effects on many other tissues. Fluoride, when in excess, is known to interfere with thyroid gland function. Fluoride-induced thyroid disturbances similar to those observed in iodine deficiency state in spite of adequate iodine intake have been documented. Similar thyroid disturbances in individuals with dental fluorosis have not been well studied in populations with endemic fluorosis. This work was undertaken to study the effects of fluoride-induced thyroid disturbances in individuals with dental fluorosis. The study group included 65 subjects with dental fluorosis from endemic fluorosis populations. An additional control group was comprised of 10 subjects without dental fluorosis. The drinking water fluoride levels of the study populations were analyzed. Serum free FT3, FT4, and TSH levels of both groups were assessed. All subjects with dental fluorosis had serum levels of thyroid hormones (FT3, FT4, and TSH) within the normal range, with the exception of 1 individual, who had elevated levels of TSH. Statistical significance was found when FT3 and TSH values were compared with different Dean's index groups by a 1-way ANOVA test: FT3 (F = 3.4572; P=.0377) and TSH (F = 3.2649 and P=.0449). Findings of this study did not show any significant alterations in the levels of the thyroid hormones FT3, FT4, and TSH in subjects with dental fluorosis. Our observations suggest that thyroid hormone levels were not altered in subjects with dental fluorosis. Hence, future studies of this kind, along with more detailed investigations are needed.


Campylobacter jejuni colonization in wild birds: results from an infection experiment.
Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.


Sulfation of estrone and 17 beta-estradiol in human liver. Catalysis by thermostable phenol sulfotransferase and by dehydroepiandrosterone sulfotransferase.
Sulfation is a major pathway in humans for the biotransformation of estrogens. However, the nature of the enzymes that catalyze the sulfation of estrone (E1) and 17 beta-estradiol (E2) in human liver is unclear. Human liver contains at least three well-characterized cytoplasmic sulfotransferases, the thermostable (TS) and thermolabile (TL) forms of phenol sulfotransferase (PST) and dehydroepiandrosterone sulfotransferase (DHEA ST). Therefore, we determined optimal conditions for the assay of E1 and E2 ST activities in human hepatic cytosol to compare their properties and regulation with those of the three well-characterized human liver ST activities. Thermal inactivation studies showed that human liver E2 ST and TS PST had very similar thermal stabilities. The thermal inactivation profile of E1 ST suggested that this activity might be related to both DHEA ST and TS PST. Inhibition studies performed with 2,6-dichloro-4-nitrophenol (DCNP) also showed similar inhibition profiles for E2 ST and TS PST. Neither thermal inactivation nor DCNP inhibition studies indicated a possible relationship between TL PST activity and E1 or E2 sulfation. Experiments performed with 20 individual human liver samples showed highly significant correlations between activity levels of E2 ST and TS PST (rs = 0.944, p less than 0.0001), E1 ST and DHEA ST (rs = 0.845, p less than 0.0001), and, to a lesser degree, E1 ST and TS PST (rs = 0.608, p less than 0.01). Ion exchange chromatography of a human liver preparation, followed by assay of all five ST activities, confirmed the important roles played by TS PST and DHEA ST in the sulfation of E2 and E1. Similar results were found by study of the elution patterns of ST activities after ion exchange chromatography of human jejunal mucosal preparations. Partially purified TL PST, however, was unable to catalyze the sulfate conjugation of either E1 or E2. All of these results were compatible with the conclusion that, in human liver, TS PST is the enzyme predominantly responsible for the sulfate conjugation of E2, DHEA ST is the major enzyme responsible for the sulfation of E1, and TL PST does not appear to catalyze the sulfation of either E1 or E2.


Side view thrombosis microfluidic device with controllable wall shear rate and transthrombus pressure gradient.
Hemodynamic conditions vary throughout the vasculature, creating diverse environments in which platelets must respond. To stop bleeding, a growing platelet deposit must be assembled in the presence of fluid wall shear stress (τw) and a transthrombus pressure gradient (ΔP) that drives bleeding. We designed a microfluidic device capable of pulsing a fluorescent solute through a developing thrombus forming on collagen ± tissue factor (TF), while independently controlling ΔP and τw. Computer control allowed step changes in ΔP with a rapid response time of 0.26 mm Hg s(-1) at either venous (5.2 dynes cm(-2)) or arterial (33.9 dynes cm(-2)) wall shear stresses. Side view visualization of thrombosis with transthrombus permeation allowed for quantification of clot structure, height, and composition at various ΔP. Clot height was reduced 20% on collagen/TF and 28% on collagen alone when ΔP was increased from 20.8 to 23.4 mm Hg at constant arterial shear stress. When visualized with a platelet-targeting thrombin sensor, intrathrombus thrombin levels decreased by 62% as ΔP was increased from 0 to 23.4 mm Hg across the thrombus-collagen/TF barrier, consistent with convective removal of thrombogenic solutes due to pressure-driven permeation. Independent of ΔP, the platelet deposit on collagen had a permeability of 5.45 × 10(-14) cm(2), while the platelet/fibrin thrombus on collagen/TF had a permeability of 2.71 × 10(-14) cm(2) (comparable to that of an intact endothelium). This microfluidic design allows investigation of the coupled processes of platelet deposition and thrombin/fibrin generation in the presence of controlled transthrombus permeation and wall shear stress.


Role of resting metabolic rate and energy expenditure in hunger and appetite control: a new formulation.
A long-running issue in appetite research concerns the influence of energy expenditure on energy intake. More than 50 years ago, Otto G. Edholm proposed that "the differences between the intakes of food [of individuals] must originate in differences in the expenditure of energy". However, a relationship between energy expenditure and energy intake within any one day could not be found, although there was a correlation over 2 weeks. This issue was never resolved before interest in integrative biology was replaced by molecular biochemistry. Using a psychobiological approach, we have studied appetite control in an energy balance framework using a multi-level experimental system on a single cohort of overweight and obese human subjects. This has disclosed relationships between variables in the domains of body composition [fat-free mass (FFM), fat mass (FM)], metabolism, gastrointestinal hormones, hunger and energy intake. In this Commentary, we review our own and other data, and discuss a new formulation whereby appetite control and energy intake are regulated by energy expenditure. Specifically, we propose that FFM (the largest contributor to resting metabolic rate), but not body mass index or FM, is closely associated with self-determined meal size and daily energy intake. This formulation has implications for understanding weight regulation and the management of obesity.


Mutations in fast skeletal troponin I, troponin T, and beta-tropomyosin that cause distal arthrogryposis all increase contractile function.
Distal arthrogryposes (DAs) are a group of disorders characterized by congenital contractures of distal limbs without overt neurological or muscle disease. Unexpectedly, mutations in genes encoding the fast skeletal muscle regulatory proteins troponin T (TnT), troponin I (TnI), and beta-tropomyosin (beta-TM) have been shown to cause autosomal dominant DA. We tested how these mutations affect contractile function by comparing wild-type (WT) and mutant proteins in actomyosin ATPase assays and in troponin-replaced rabbit psoas fibers. We have analyzed all four reported mutants: Arg63His TnT, Arg91Gly beta-TM, Arg174Gln TnI, and a TnI truncation mutant (Arg156ter). Thin filaments, reconstituted using actin and WT troponin and beta-TM, activated myosin subfragment-1 ATPase in a calcium-dependent, cooperative manner. Thin filaments containing either a troponin or beta-TM DA mutant produced significantly enhanced ATPase rates at all calcium concentrations without alternating calcium-sensitivity or cooperativity. In troponin-exchanged skinned fibers, each mutant caused a significant increase in Ca2+ sensitivity, and Arg156ter TnI generated significantly higher maximum force. Arg91Gly beta-TM was found to have a lower actin affinity than WT and form a less stable coiled coil. We propose the mutations cause increased contractility of developing fast-twitch skeletal muscles, thus causing muscle contractures and the development of the observed limb deformities.


A novel role for an APC2-Diaphanous complex in regulating actin organization in Drosophila.
The rearrangement of cytoskeletal elements is essential for many cellular processes. The tumor suppressor Adenomatous polyposis coli (APC) affects the function of microtubules and actin, but the mechanisms by which it does so are not well understood. Here we report that Drosophila syncytial embryos null for Apc2 display defects in the formation and extension of pseudocleavage furrows, which are cortical actin structures important for mitotic fidelity in early embryos. Furthermore, we show that the formin Diaphanous (DIA) functions with APC2 in this process. Colocalization of APC2 and DIA peaks during furrow extension, and localization of APC2 to furrows is DIA-dependent. Furthermore, APC2 binds DIA directly through a region of APC2 not previously shown to interact with DIA-related formins. Consistent with these results, reduction of dia enhances actin defects in Apc2 mutant embryos. Thus, an APC2-DIA complex appears crucial for actin furrow extension in the syncytial embryo. Interestingly, EB1, a microtubule +TIP and reported partner of vertebrate APC and DIA1, may not function with APC2 and DIA in furrow extension. Finally, whereas DIA-related formins are activated by Rho family GTPases, our data suggest that the APC2-DIA complex might be independent of RHOGEF2 and RHO1. Furthermore, although microtubules play a role in furrow extension, our analysis suggests that APC2 and DIA function in a novel complex that affects actin directly, rather than through an effect on microtubules.


Structural and mechanistic consequences of polypeptide binding by GroEL.
The remarkable ability of the chaperonin GroEL to recognise a diverse range of non-native states of proteins constitutes one of the most fascinating molecular recognition events in protein chemistry. Recent structural studies have revealed a possible model for substrate binding by GroEL and a high-resolution image of the GroEL-GroES folding machinery has provided important new insights into our understanding of the mechanism of action of this chaperonin. Studies with a variety of model substrates reveal that the binding of substrate proteins to GroEL is not just a passive event, but can result in significant changes in the structure and stability of the bound polypeptide. The potential impact of this on the mechanism of chaperonin-assisted folding is not fully understood, but provides exciting scope for further experiment.


Motion-based video monitoring for early detection of livestock diseases: The case of African swine fever.
Early detection of infectious diseases can substantially reduce the health and economic impacts on livestock production. Here we describe a system for monitoring animal activity based on video and data processing techniques, in order to detect slowdown and weakening due to infection with African swine fever (ASF), one of the most significant threats to the pig industry. The system classifies and quantifies motion-based animal behaviour and daily activity in video sequences, allowing automated and non-intrusive surveillance in real-time. The aim of this system is to evaluate significant changes in animals' motion after being experimentally infected with ASF virus. Indeed, pig mobility declined progressively and fell significantly below pre-infection levels starting at four days after infection at a confidence level of 95%. Furthermore, daily motion decreased in infected animals by approximately 10% before the detection of the disease by clinical signs. These results show the promise of video processing techniques for real-time early detection of livestock infectious diseases.


An Enhanced Map-Matching Algorithm for Real-Time Position Accuracy Improvement with a Low-Cost GPS Receiver.
This paper proposes a real-time position accuracy improvement method for a low-cost global positioning system (GPS), which uses geographic data for forming a digital road database in the digital map information. We link the vehicle's location to the position on the digital map using the map-matching algorithm to improve the position accuracy. In the proposed method, we can distinguish the vehicle direction on the road and enhance the horizontal accuracy using the geographic data composed of the vector point set of the digital map. We use the iterative closest point (ICP) algorithm that calculates the rotation matrix and the translation vector to compensate for the disparity between the GPS and the digital map information. We also use the least squares method to correct the error caused by the rotation of the ICP algorithm and link on the digital map to eliminate the residual disparity. Finally, we implement the proposed method in real time with a low-cost embedded system and demonstrate the effectiveness of the proposed method through various experiments.


Risk of Unnatural Mortality in People With Epilepsy.
People with epilepsy are at increased risk of mortality, but, to date, the cause-specific risks of all unnatural causes have not been reported. To estimate cause-specific unnatural mortality risks in people with epilepsy and to identify the medication types involved in poisoning deaths. This population-based cohort study used 2 electronic primary care data sets linked to hospitalization and mortality records, the Clinical Practice Research Datalink (CPRD) in England (from January 1, 1998, to March 31, 2014) and the Secure Anonymised Information Linkage (SAIL) Databank in Wales (from January 1, 2001, to December 31, 2014). Each person with epilepsy was matched on age (within 2 years), sex, and general practice with up to 20 individuals without epilepsy. Unnatural mortality was determined using International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes V01 through Y98 in the Office for National Statistics mortality records. Hazard ratios (HRs) were estimated in each data set using a stratified Cox proportional hazards model, and meta-analyses were conducted using DerSimonian and Laird random-effects models. The analysis was performed from January 5, 2016, to November 16, 2017. People with epilepsy were identified using primary care epilepsy diagnoses and associated antiepileptic drug prescriptions. Hazard ratios (HRs) for unnatural mortality and the frequency of each involved medication type estimated as a percentage of all medication poisoning deaths. In total, 44 678 individuals in the CPRD and 14 051 individuals in the SAIL Databank were identified in the prevalent epilepsy cohorts, and 891 429 (CPRD) and 279 365 (SAIL) individuals were identified in the comparison cohorts. In both data sets, 51% of the epilepsy and comparison cohorts were male, and the median age at entry was 40 years (interquartile range, 25-60 years) in the CPRD cohorts and 43 years (interquartile range, 24-64 years) in the SAIL cohorts. People with epilepsy were significantly more likely to die of any unnatural cause (HR, 2.77; 95% CI, 2.43-3.16), unintentional injury or poisoning (HR, 2.97; 95% CI, 2.54-3.48) or suicide (HR, 2.15; 95% CI, 1.51-3.07) than people in the comparison cohort. Particularly large risk increases were observed in the epilepsy cohorts for unintentional medication poisoning (HR, 4.99; 95% CI, 3.22-7.74) and intentional self-poisoning with medication (HR, 3.55; 95% CI, 1.01-12.53). Opioids (56.5% [95% CI, 43.3%-69.0%]) and psychotropic medication (32.3% [95% CI, 20.9%-45.3%)] were more commonly involved than antiepileptic drugs (9.7% [95% CI, 3.6%-19.9%]) in poisoning deaths in people with epilepsy. Compared with people without epilepsy, people with epilepsy are at increased risk of unnatural death and thus should be adequately advised about unintentional injury prevention and monitored for suicidal ideation, thoughts, and behaviors. The suitability and toxicity of concomitant medication should be considered when prescribing for comorbid conditions.


Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System.
Successful developments of new therapeutic strategies often rely on the ability to deliver exogenous molecules into cytosol. We have developed a versatile on-chip vortex-assisted electroporation system, engineered to conduct sequential intracellular delivery of multiple molecules into various cell types at low voltage in a dosage-controlled manner. Micro-patterned planar electrodes permit substantial reduction in operational voltages and seamless integration with an existing microfluidic technology. Equipped with real-time process visualization functionality, the system enables on-chip optimization of electroporation parameters for cells with varying properties. Moreover, the system's dosage control and multi-molecular delivery capabilities facilitate intracellular delivery of various molecules as a single agent or in combination and its utility in biological research has been demonstrated by conducting RNA interference assays. We envision the system to be a powerful tool, aiding a wide range of applications, requiring single-cell level co-administrations of multiple molecules with controlled dosages.


The role of tryptophans in the UV-B absorption of a UVR8 photoreceptor--a computational study.
Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) has been identified as a photoreceptor for ultraviolet-B (UV-B). Tryptophan (Trp) residues have been shown to play a critical role in the response to UV-B irradiation in UVR8. In this work, we explore the spectroscopic behaviors of Trps in different protein environments of the UVR8 structure using the time-dependent density functional tight-binding (TD-DFTB) scheme. We show that W233 exhibits the longest absorption wavelength, highlighting its potential as a terminal Trp chromophore in the UV-B harvesting antenna. Our electronic and optical property analyses using various amino acid models support the important roles of W285 and W233 in sensing UV-B light at longer absorption wavelengths (∼290 nm). We also provide evidence for the specific function of W94 in absorption at the longest wavelengths (305.8 nm in cluster II and 304.5 nm in cluster III). To these findings, we also add information about the influence of the arginine and aspartic acid residues surrounding the Trp pyramid on the particular absorption bands (280-300 nm) that are characteristic of the UV-B photoreceptor.


Diagnostic Accuracy of Chest Computed Tomography Scans for Suspected Patients With COVID-19: Receiver Operating Characteristic Curve Analysis.
Computed tomography (CT) scans are increasingly available in clinical care globally. They enable a rapid and detailed assessment of tissue and organ involvement in disease processes that are relevant to diagnosis and management, particularly in the context of the COVID-19 pandemic. The aim of this paper is to identify differences in the CT scan findings of patients who were COVID-19 positive (confirmed via nucleic acid testing) to patients who were confirmed COVID-19 negative. A retrospective cohort study was proposed to compare patient clinical characteristics and CT scan findings in suspected COVID-19 cases. A multivariable logistic model with LASSO (least absolute shrinkage and selection operator) selection for variables was used to identify the good predictors from all available predictors. The area under the curve (AUC) with 95% CI was calculated for each of the selected predictors and the combined selected key predictors based on receiver operating characteristic curve analysis. A total of 94 (56%) patients were confirmed positive for COVID-19 from the suspected 167 patients. We found that elderly people were more likely to be infected with COVID-19. Among the 94 confirmed positive patients, 2 (2%) patients were admitted to an intensive care unit. No patients died during the study period. We found that the presence, distribution, and location of CT lesions were associated with the presence of COVID-19. White blood cell count, cough, and a travel history to Wuhan were also the top predictors for COVID-19. The overall AUC of these selected predictors is 0.97 (95% CI 0.93-1.00). Taken together with nucleic acid testing, we found that CT scans can allow for the rapid diagnosis of COVID-19. This study suggests that chest CT scans should be more broadly adopted along with nucleic acid testing in the initial assessment of suspected COVID-19 cases, especially for patients with nonspecific symptoms.


Nd:YAG laser machining with multilevel resist kinoforms.
We demonstrate that resist kinoforms can be used for laser micromachining. A 10-level resist kinoform, manufactured by electron-beam lithography, was shown to have a diffraction efficiency of 68%. Nine diffraction-limited holes were simultaneously drilled in 0.10-mm-thick stainless steel. Marking in a silicon wafer is also demonstrated.


Comparison of methods for constructing confidence intervals of standardized indirect effects.
Mediation models are often used as a means to explain the psychological mechanisms between an independent and a dependent variable in the behavioral and social sciences. A major limitation of the unstandardized indirect effect calculated from raw scores is that it cannot be interpreted as an effect-size measure. In contrast, the standardized indirect effect calculated from standardized scores can be a good candidate as a measure of effect size because it is scale invariant. In the present article, 11 methods for constructing the confidence intervals (CIs) of the standardized indirect effects were evaluated via a computer simulation. These included six Wald CIs, three bootstrap CIs, one likelihood-based CI, and the PRODCLIN CI. The results consistently showed that the percentile bootstrap, the bias-corrected bootstrap, and the likelihood-based approaches had the best coverage probability. Mplus, LISREL, and Mx syntax were included to facilitate the use of these preferred methods in applied settings. Future issues on the use of the standardized indirect effects are discussed.


Risk of suboptimal iron and zinc nutriture among adolescent girls in Australia and New Zealand: causes, consequences, and solutions.
Surveys in Australia, New Zealand and other industrialised countries report that many adolescent girls have dietary intakes of iron and zinc that fail to meet their high physiological requirements for growing body tissues, expanding red cell mass, and onset of menarche. Such dietary inadequacies can be attributed to poor food selection patterns, and low energy intakes. Additional exacerbating non-dietary factors may include high menstrual losses, strenuous exercise, pregnancy, low socioeconomic status and ethnicity. These findings are cause for concern because iron and zinc play essential roles in numerous metabolic functions and are required for optimal growth, immune and cognitive function, work capacity, sexual maturation, and bone mineralization. Moreover, if adolescents enter pregnancy with a compromised iron and zinc status, and continue to receive intakes of iron and zinc that do not meet their increased needs, their poor iron and zinc status could adversely affect the pregnancy outcome. Clearly, intervention strategies may be needed to improve the iron and zinc status of high risk adolescent subgroups in Australia and New Zealand. The recommended treatment for iron deficiency anaemia and moderate zinc deficiency is supplementation. Although dietary intervention is often recommended for treating non-anaemic iron deficiency and mild zinc deficiency, it is probably more effective and appropriate for prevention than for the treatment of suboptimal iron and zinc status. Many of the strategies for enhancing the content and bioavailability of dietary iron are also appropriate for zinc.


An Empirical Study of Applying Statistical Disclosure Control Methods to Public Health Research.
Patient data or information collected from public health and health care surveys are of great research value. Usually, the data contain sensitive personal information. Doctors, nurses, or researchers in the public health and health care sector do not analyze the available datasets or survey data on their own, and may outsource the tasks to third parties. Even though all identifiers such as names and ID card numbers are removed, there may still be some occasions in which an individual can be re-identified via the demographic or particular information provided in the datasets. Such data privacy issues can become an obstacle in health-related research. Statistical disclosure control (SDC) is a useful technique used to resolve this problem by masking and designing released data based on the original data. Whilst ensuring the released data can satisfy the needs of researchers for data analysis, there is high protection of the original data from disclosure. In this research, we discuss the statistical properties of two SDC methods: the General Additive Data Perturbation (GADP) method and the Gaussian Copula General Additive Data Perturbation (CGADP) method. An empirical study is provided to demonstrate how we can apply these two SDC methods in public health research.


Food culture in the home environment: family meal practices and values can support healthy eating and self-regulation in young people in four European countries.
Overweight epidemics, including among children and adolescents, are fuelled by contemporary obesogenic environments. Recent research and theory highlight the importance of socio-cultural factors in mitigating adverse impacts of the abundance of food in high-income countries. The current study examines whether family meal culture shapes young people's eating behaviors and self-regulation. Young people aged 10-17 years were recruited through schools in four European countries: the Netherlands, Poland, Portugal and the United Kingdom. A total of 2,764 participants (mean age 13.2 years; 49.1% girls) completed a self-report questionnaire in class, providing information on healthy and unhealthy eating, joint family meals and communal meal values and use of eating-related self-regulation strategies. Path analysis found that family meal culture variables were significantly associated with young people's eating behaviors, as was self-regulation. Significant indirect effects of family meal culture were also found, through self-regulation. Results confirm that family meal culture, encompassing values as well as practices, shapes young people's eating behaviors. Findings extend and link previously separate lines of enquiry by showing how food cultures can play out in the home environment. Importantly, the study contributes novel evidence suggesting that self-regulation is shaped by the home environment and mediates its influence.


Theoretical Study of Triboelectric-Potential Gated/Driven Metal-Oxide-Semiconductor Field-Effect Transistor.
Triboelectric nanogenerator has drawn considerable attentions as a potential candidate for harvesting mechanical energies in our daily life. By utilizing the triboelectric potential generated through the coupling of contact electrification and electrostatic induction, the "tribotronics" has been introduced to tune/control the charge carrier transport behavior of silicon-based metal-oxide-semiconductor field-effect transistor (MOSFET). Here, we perform a theoretical study of the performances of tribotronic MOSFET gated by triboelectric potential in two working modes through finite element analysis. The drain-source current dependence on contact-electrification generated triboelectric charges, gap separation distance, and externally applied bias are investigated. The in-depth physical mechanism of the tribotronic MOSFET operations is thoroughly illustrated by calculating and analyzing the charge transfer process, voltage relationship to gap separation distance, and electric potential distribution. Moreover, a tribotronic MOSFET working concept is proposed, simulated and studied for performing self-powered FET and logic operations. This work provides a deep understanding of working mechanisms and design guidance of tribotronic MOSFET for potential applications in micro/nanoelectromechanical systems (MEMS/NEMS), human-machine interface, flexible electronics, and self-powered active sensors.


Intersexual trophic niche partitioning in an ant-eating spider (Araneae: Zodariidae).
Divergence in trophic niche between the sexes may function to reduce competition between the sexes ("intersexual niche partitioning hypothesis"), or may be result from differential selection among the sexes on maximizing reproductive output ("sexual selection hypothesis"). The latter may lead to higher energy demands in females driven by fecundity selection, while males invest in mate searching. We tested predictions of the two hypotheses underlying intersexual trophic niche partitioning in a natural population of spiders. Zodarion jozefienae spiders specialize on Messor barbarus ants that are polymorphic in body size and hence comprise potential trophic niches for the spider, making this system well-suited to study intersexual trophic niche partitioning. Comparative analysis of trophic morphology (the chelicerae) and body size of males, females and juveniles demonstrated highly female biased SSD (Sexual Size Dimorphism) in body size, body weight, and in the size of chelicerae, the latter arising from sex-specific growth patterns in trophic morphology. In the field, female spiders actively selected ant sub-castes that were larger than the average prey size, and larger than ants captured by juveniles and males. Female fecundity was highly positively correlated with female body mass, which reflects foraging success during the adult stage. Females in laboratory experiments preferred the large ant sub-castes and displayed higher capture efficiency. In contrast, males occupied a different trophic niche and showed reduced foraging effort and reduced prey capture and feeding efficiency compared with females and juveniles. Our data indicate that female-biased dimorphism in trophic morphology and body size correlate with sex-specific reproductive strategies. We propose that intersexual trophic niche partitioning is shaped primarily by fecundity selection in females, and results from sex-differences in the route to successful reproduction where females are selected to maximize energy intake and fecundity, while males switch from foraging to invest in mating effort.


Variations in glycoprotein B contribute to immunogenic difference between PRV variant JS-2012 and Bartha-K61.
A newly emerged pseudorabies virus (PRV) variant has been identified in many Bartha-K61-vaccinated pig farms. This variant has caused great economic losses to the swine industry in China since 2011. Sequence analysis demonstrated that the gB gene of the emerging PRV variant JS-2012 had multiple variations compared with the vaccine strain Bartha-K61. In the study, a specific CRISPR/Cas9 system combined with homologous recombination was used to construct two recombinant viruses, BJB (Bartha-K61+JS-2012gB) and JBJ (JS-2012-ΔgE/gI+Bartha-K61gB), by interchanging the full-length gB genes between Bartha-K61 and JS-2012-ΔgE/gI. The two recombinant viruses showed similar characteristics in growth kinetics in vitro and similar pathogenicity in mice, as compared to their parental strains. Immunization of mice with inactivated BJB or JBJ followed by challenge of JS-2012 showed that BJB could increase protective efficacy to 80%, compared to only 40% protection by the parental Bartha-K61 strain. JBJ had a decreased protective efficacy of 65%, as compared to 90% protection by its parental JS-2012-ΔgE/gI strain. Exchange of the gB gene markedly altered the immunogenicity of the recombinant PRV. These data suggest that variations in gB might play an important role in the virulence of the reemergent PRV variant in China. Our results demonstrate the importance of gB in protective immunity and suggest that the recombinant virus BJB could be a promising vaccine candidate for eradication of the PRV variant.


The new 8th TNM staging system of lung cancer and its potential imaging interpretation pitfalls and limitations with CT image demonstrations.
The tumor, node, metastasis (TNM) staging system approved by International Association for the Study of Lung Cancer (IASLC) and the American Joint Committee on Cancer (AJCC) to stage lung cancer was recently revised. The latest revision is the 8th edition published in January, 2017. This new edition made some important changes to the previous edition, including modification of the T classification based on 1 cm increment, downstage of T descriptor including endobronchial tumor disregarding its distance from carina (T2), merging total and partial atelectasis/pneumonitis into the same T category (T2), upstage diaphragmatic invasion to T4, new classification concept of adenocarcinoma in situ and minimally invasive adenocarcinoma for pure and part-solid ground-glass nodules, and further division of extrathoracic metastasis into M1b and M1c based on the number and sites of extrathoracic metastases. Consensus is reached for debating situations not covered in the previous edition of staging system, such as the classification of pancoast tumor based on its invasion depth and staging tumors that extend directly across the fissure as T2a. Classification of multiple sites of pulmonary involvement, including multiple primary lung cancer, separate lung cancer nodules, multiple ground-glass or lepidic lesions, and consolidation, is also discussed. Even though the 8th edition of the TNM lung staging system provides us with more precise classification based on prognostic analysis of each TNM descriptors, there are still some potential limitations and clinical situations that have not yet been clarified in terms of clinical staging by imaging. It is important for radiologists to understand the major changes introduced in the 8th edition of TNM staging and to recognize the potential pitfalls and limitations of imaging interpretation to precisely classify the clinical stage of lung cancer.


A National Wastewater Monitoring Program for a better understanding of public health: A case study using the Australian Census.
Wastewater contains a large range of biological and chemical markers of human activity and exposures. Through systematic collection and analysis of these markers within wastewater samples it is possible to measure the public health of whole populations. The analysis of effluent and biosolids can also be used to understand the release of chemicals from wastewater treatment plants into the environment. Wastewater analysis and comparison with catchment specific data (e.g. demographics) however remains largely unexplored. This manuscript describes a national wastewater monitoring study that combines influent, effluent and biosolids sampling with the Australian Census. An archiving program allows estimation of per capita exposure to and consumption of chemicals, public health information, as well as per capita release of chemicals into the environment. The paper discusses the study concept, critical steps in setting up a coordinated national approach and key logistical and other considerations with a focus on lessons learnt and future applications. The unique combination of archived samples, analytical data and associated census-derived population data will provide a baseline dataset that has wide and potentially increasing applications across many disciplines that include public health, epidemiology, criminology, toxicology and sociology.


A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP).
The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).


Design and challenges of a large HIV prevention clinical study on mother-to-child transmission: ANRS 12397 PROMISE-EPI study in Zambia and Burkina Faso.
Post-natal HIV infection through breastfeeding remains a challenge in many low and middle-income countries, particularly due to non-availability of alternative infant feeding options and the suboptimal Prevention of Mother to Child Transmission of HIV-1 (PMTCT) cascade implementation and monitoring. The PROMISE-EPI study aims to address the latter by identifying HIV infected mothers during an almost never-missed visit for their infant, the second extended program on immunization visit at 6-8 weeks of age (EPI-2). The study is divided into 3 components inclusive of an open-label randomized controlled trial aiming to assess the efficacy of a responsive preventive intervention compared to routine intervention based on the national PMTCT guidelines for HIV-1 uninfected exposed breastfeeding infants. The preventive intervention includes: a) Point of care testing for early infant HIV diagnosis and maternal viral load; b) infant, single-drug Pre-Exposure Prophylaxis (PrEP) (lamivudine) if mothers are virally unsuppressed. The primary outcome is HIV-transmission rate from EPI-2 to 12 months. The study targets to screen 37,000 mother/infant pairs in Zambia and Burkina Faso to identify 2000 mother/infant pairs for the clinical trial. The study design and challenges faced during study implementation are described, including the COVID-19 pandemic and the amended HIV guidelines in Zambia in 2020 (triple-drug PrEP in HIV exposed infants guided by quarterly maternal viral load). The changes in the Zambian guidelines raised several questions including the equipoise of PrEP options, the standard of care-triple-drug (control arm in Zambia) versus the study-single-drug (intervention arm). Trial registration number (www.clinicaltrials.gov): NCT03869944. Submission category: Study Design, Statistical Design, Study Protocols.


Xenopus SMOC-1 Inhibits bone morphogenetic protein signaling downstream of receptor binding and is essential for postgastrulation development in Xenopus.
The bone morphogenetic protein (BMP) family of signaling molecules and their antagonists are involved in patterning of the body axis and numerous aspects of organogenesis. Classical biochemical purification and protein sequencing of highly purified fractions containing potent bone forming activity from bovine cartilage identified several BMPs together with a number of other proteins. One such protein was SMOC-2 (secreted modular calcium-binding protein-2), classified as belonging to the BM-40 family of modular extracellular proteins. Data regarding the biological function of SMOC-2 and closely related SMOC-1 remain limited, and their expression or function during embryological development is unknown. We therefore isolated the Xenopus ortholog of human SMOC-1 (XSMOC-1) and explored its function in Xenopus embryos. In gain-of-function assays, XSMOC-1 acted similarly to a BMP antagonist. However, in contrast to known extracellular ligand-binding BMP antagonists, such as noggin, SMOC antagonizes BMP activity in the presence of a constitutively active BMP receptor, indicating a mechanism of action downstream of the receptor. We provide several lines of evidence to suggest that SMOC acts downstream of the BMP receptor via MAPK-mediated phosphorylation of the Smad linker region. Loss-of-function studies, using antisense morpholino oligonucleotides, revealed XSMOC-1 to be essential for postgastrulation development. The catastrophic developmental failure observed following XSMOC knockdown resembles that observed following simultaneous depletion of three ligand-binding BMP antagonists described in prior studies. These findings provide a direct link between the extracellular matrix-associated protein SMOC and a signaling pathway of general importance in anatomic patterning and cell or tissue fate specification.


Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells.
Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT), to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells. Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined. Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1). In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation. Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways. In addition, genistein cooperates with vorinostat to induce cell death in prostate cancer cell lines with a greater effect on early stage prostate cancer.


The Changing Epidemiology of Autism Spectrum Disorders.
Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with lifelong impacts. Genetic and environmental factors contribute to ASD etiology, which remains incompletely understood. Research on ASD epidemiology has made significant advances in the past decade. Current prevalence is estimated to be at least 1.5% in developed countries, with recent increases primarily among those without comorbid intellectual disability. Genetic studies have identified a number of rare de novo mutations and gained footing in the areas of polygenic risk, epigenetics, and gene-by-environment interaction. Epidemiologic investigations focused on nongenetic factors have established advanced parental age and preterm birth as ASD risk factors, indicated that prenatal exposure to air pollution and short interpregnancy interval are potential risk factors, and suggested the need for further exploration of certain prenatal nutrients, metabolic conditions, and exposure to endocrine-disrupting chemicals. We discuss future challenges and goals for ASD epidemiology as well as public health implications.


Phosphatidylethanolamine-esterified eicosanoids in the mouse: tissue localization and inflammation-dependent formation in Th-2 disease.
In this study, murine peritoneal macrophages from naïve lavage were found to generate four phospholipids that contain 12-hydroxyeicosatetraenoic acid (12-HETE). They comprise three plasmalogen and one diacyl phosphatidylethanolamines (PEs) (16:0p, 18:1p, 18:0p, and 18:0a at sn-1) and are absent in macrophages from 12/15-lipoxygenase (12/15-LOX)-deficient mice. They are generated acutely in response to calcium mobilization, are primarily cell-associated, and are detected on the outside of the plasma membrane. Levels of 12-HETE-PEs in naïve lavage are in a similar range to those of free 12-HETE (5.5 +/- 0.2 ng or 18.5 +/- 1.03 ng/lavage for esterified versus free, respectively). In healthy mice, 12/15-LOX-derived 12-HETE-PEs are found in the peritoneal cavity, peritoneal membrane, lymph node, and intestine, with a similar distribution to 12/15-LOX-derived 12-HETE. In vivo generation of 12-HETE-PEs occurs in a Th2-dependent model of murine lung inflammation associated with interleukin-4/interleukin-13 expression. In contrast, in Toll receptor-dependent peritonitis mediated either by live bacteria or bacterial products, 12-HETE-PEs are rapidly cleared during the acute phase then reappear during resolution. The human homolog, 18:0a/15-HETE-PE inhibited human monocyte generation of cytokines in response to lipopolysaccharide. In summary, a new family of lipid mediators generated by murine macrophages during Th2 inflammation are identified and structurally characterized. The studies suggest a new paradigm for lipids generated by 12/15-LOX in inflammation involving formation of esterified eicosanoids.


Sexual Dimorphism of Body Size Is Controlled by Dosage of the X-Chromosomal Gene Myc and by the Sex-Determining Gene tra in Drosophila.
Drosophila females are larger than males. In this article, we describe how X-chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced expression of a key X-linked growth-regulating gene, and second, through female-specific activation of the sex-determination pathway. X-chromosome dosage determines phenotypic sex by regulating the genes of the sex-determining pathway. In the presence of two sets of X-chromosome signal elements (XSEs), Sex-lethal (Sxl) is activated in female (XX) but not male (XY) animals. Sxl activates transformer (tra), a gene that encodes a splicing factor essential for female-specific development. It has previously been shown that null mutations in the tra gene result in only a partial reduction of body size of XX animals, which shows that other factors must contribute to size determination. We tested whether X dosage directly affects animal size by analyzing males with duplications of X-chromosomal segments. Upon tiling across the X chromosome, we found four duplications that increase male size by >9%. Within these, we identified several genes that promote growth as a result of duplication. Only one of these, Myc, was found not to be dosage compensated. Together, our results indicate that both Myc dosage and tra expression play crucial roles in determining sex-specific size in Drosophila larvae and adult tissue. Since Myc also acts as an XSE that contributes to tra activation in early development, a double dose of Myc in females serves at least twice in development to promote sexual size dimorphism.


Gravitational wave content and stability of uniformly, rotating, triaxial neutron stars in general relativity.
Targets for ground-based gravitational wave interferometers include continuous, quasiperiodic sources of gravitational radiation, such as isolated, spinning neutron stars. In this work, we perform evolution simulations of uniformly rotating, triaxially deformed stars, the compressible analogs in general relativity of incompressible, Newtonian Jacobi ellipsoids. We investigate their stability and gravitational wave emission. We employ five models, both normal and supramassive, and track their evolution with different grid setups and resolutions, as well as with two different evolution codes. We find that all models are dynamically stable and produce a strain that is approximately one-tenth the average value of a merging binary system. We track their secular evolution and find that all our stars evolve toward axisymmetry, maintaining their uniform rotation, rotational kinetic energy, and angular momentum profiles while losing their triaxiality.


Continuous-time adaptive critics.
A continuous-time formulation of an adaptive critic design (ACD) is investigated. Connections to the discrete case are made, where backpropagation through time (BPTT) and real-time recurrent learning (RTRL) are prevalent. Practical benefits are that this framework fits in well with plant descriptions given by differential equations and that any standard integration routine with adaptive step-size does an adaptive sampling for free. A second-order actor adaptation using Newton's method is established for fast actor convergence for a general plant and critic. Also, a fast critic update for concurrent actor-critic training is introduced to immediately apply necessary adjustments of critic parameters induced by actor updates to keep the Bellman optimality correct to first-order approximation after actor changes. Thus, critic and actor updates may be performed at the same time until some substantial error build up in the Bellman optimality or temporal difference equation, when a traditional critic training needs to be performed and then another interval of concurrent actor-critic training may resume.


Symptoms of gastroesophageal reflux disease improve after parathyroidectomy.
Primary hyperparathyroidism can be associated with symptoms related to GERD, but it is unclear which symptoms of GERD improve after parathyroidectomy. Our goal was to assess prospectively for changes in specific GERD symptoms after parathyroidectomy using a validated questionnaire. Using the GERD health-related quality of life (GERD-HRQL) questionnaire, symptoms of heartburn were prospectively assessed before and 6 months after treatment of hyperparathyroidism with parathyroidectomy. This validated questionnaire includes 10 items, with a Likert scale of 0-5. Scores range from 0 to 45, a lesser score indicates fewer/less severe symptoms. Pre- and postoperative surveys were available for 51 patients. Parathyroidectomy improved the overall questionnaire score (12.5 ± 1.3 vs 4.5 ± 0.9, P < .0001). Overall scores for each question improved after parathyroidectomy, including symptoms of dysphagia (P = .001) and overall satisfaction with symptoms (P < .0001). However, the number of patients taking antireflux medication before and after parathyroidectomy was not substantially different (34 vs 28 patients, P = .17). All symptoms of GERD improved after parathyroidectomy for hyperparathyroidism. Despite the decrease in symptoms, there was not a change in the number of patients who remained on anti-reflux therapy. For patients with symptoms of GERD, a trial off antireflux medications after parathyroidectomy should be considered.


Performance of the NINDS-CSN 5-minute protocol in a national population-based sample.
In 2006, the National Institute of Neurological Disorders and Stroke-Canadian Stroke Network (NINDS-CSN) Vascular Cognitive Impairment Harmonization Standards recommended a 5-Minute Protocol as a brief screening instrument for vascular cognitive impairment (VCI). We report demographically adjusted norms for the 5-Minute Protocol and its relation to other measures of cognitive function and cerebrovascular risk factors. We performed a cross-sectional analysis of 7199 stroke-free adults in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study on the NINDS-CSN 5-Minute Protocol score. Total scores on the 5-Minute Protocol were inversely correlated with age and positively correlated with years of education, and performance on the Six-Item Screener, Word List Learning, and Animal Fluency (all p-values <.001). Higher cerebrovascular risk on the Framingham Stroke Risk Profile (FSRP) was associated with lower total 5-Minute Protocol scores (p <.001). The 5-Minute Protocol also differentiated between participants with and without confirmed stroke and with and without stroke symptom histories (p <.001). The NINDS-CSN 5-Minute Protocol is a brief, easily administered screening measure that is sensitive to cerebrovascular risk and offers a valid method of screening for cognitive impairment in populations at risk for VCI.


Novel soluble HLA-A2/MELAN-A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma.
Multimeric MHC I-peptide complexes containing phycoerythrin-streptavidin are widely used to detect and investigate antigen-specific CD8+ (and CD4+) T cells. Because such reagents are heterogeneous, we compared their binding characteristics with those of monodisperse dimeric, tetrameric and octameric complexes containing linkers of variable length and flexibility on Melan-A-specific CD8+ T cell clones and peripheral blood mononuclear cells (PBMC) from HLA-A*0201(+) melanoma patients. Striking binding differences were observed for different defined A2/Melan-A(26-35) complexes on T cells depending on their differentiation stage. In particular, short dimeric but not octameric A2/Melan-A(26-35) complexes selectively and avidly stained incompletely differentiated effector-memory T cells clones and populations expressing CD27 and CD28 and low levels of cytolytic mediators (granzymes and perforin). This subpopulation was found in PBMC from all six melanoma patients analyzed and proliferated on peptide stimulation with only modest phenotypic changes. By contrast influenza matrix(58-66) -specific CD8+ PBMC from nine HLA-A*0201(+) healthy donors were efficiently stained by A2/Flu matrix(58-61) multimers, but not dimer and upon peptide stimulation proliferated and differentiated from memory into effector T cells. Thus PBMC from melanoma patients contain a differentiation defective sub-population of Melan-A-specific CD8+ T cells that can be selectively and efficiently stained by short dimeric A2/Melan- A(26-35) complexes, which makes them directly accessible for longitudinal monitoring and further investigation.


Detection of basal cell carcinomas in Mohs excisions with fluorescence confocal mosaicing microscopy.
High-resolution real-time imaging of human skin is possible with a confocal microscope either in vivo or in freshly excised tissue ex vivo. Nuclear and cellular morphology is observed in thin optical sections, similar to that in conventional histology. Contrast agents such as acridine orange in fluorescence and acetic acid in reflectance have been used in ex vivo imaging to enhance nuclear contrast. To evaluate the sensitivity and specificity of ex vivo real-time imaging with fluorescence confocal mosaicing microscopy, using acridine orange, for the detection of residual basal cell carcinoma (BCC) in Mohs fresh tissue excisions. Forty-eight discarded skin excisions were collected following completion of Mohs surgery, consisting of excisions with and without residual BCC of all major subtypes. The tissue was stained with acridine orange and imaged with a fluorescent confocal mosaicing microscope. Confocal mosaics were matched to the corresponding haematoxylin and eosin-stained Mohs frozen sections. Each mosaic was divided into subsections, resulting in 149 submosaics for study. Two Mohs surgeons, who were blinded to the cases, independently assessed confocal submosaics and recorded the presence or absence of BCC, location, and histological subtype(s). Assessment of confocal mosaics was by comparison with corresponding Mohs surgery maps. The overall sensitivity and specificity of detecting residual BCC was 96.6% and 89.2%, respectively. The positive predictive value was 92.3% and the negative predictive value 94.7%. Very good correlation was observed between confocal mosaics and matched Mohs frozen sections for benign and malignant skin structures, overall tumour burden and location, and identification of all major histological subtypes of BCC. Fluorescent confocal mosaicing microscopy using acridine orange enables detection of residual BCC of all subtypes in Mohs fresh tissue excisions with high accuracy. This observation is an important step towards the long-term clinical goal of using a noninvasive imaging modality for potential real-time surgical pathology-at-the-bedside for skin and other tissues.


Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin.
The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or the presence of thrombomodulin between the inactivation rates of high molecular weight scu-PA, and a low molecular weight scu-PA which lacked the growth factor and kringle domains. Enzyme kinetic experiments with varying concentrations of scu-PA showed that thrombomodulin decreased the Km of thrombin for scu-PA from 7.8 to 0.43 microM and increased the kcat from 0.30 to 1.2 s-1, corresponding to a 70-fold increase in the second-order rate constant kcat/Km. SDS-polyacrylamide gel electrophoresis showed that scu-PA was cleaved into two chains upon inactivation by thrombin, and confirmed the acceleration effect of thrombomodulin on inactivation of scu-PA. Thrombomodulin thus not only has anticoagulant properties but is also antifibrinolytic. The acceleration may imply a new mechanism for the regulation of local plasminogen activator activity on the cell surface.


Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia.
Proinflammatory signaling mechanisms are implicated in the induction of retinal neovascularization (NV) during ischemic retinopathies. This study examined transcription factor (TF) AP-1, HIF-1, and NF-kappaB DNA-binding in relation to cyclooxygenase (COX)-2 and VEGF RNA and protein levels in hypoxia-triggered monkey choroidal retinal (RF/6A) endothelial cells. Effects of the carboxamide CGP43182 were tested on COX-2 and VEGF activation and prostaglandin (PG)E(2) release. RF/6A cells were subjected to hypoxia for 1 and 3 hours, at which times RNA and proteins were isolated. Potential AP-1, hypoxia-inducible factor (HIF)-1 and NF-kappaB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE(2) levels were quantified by RT-PCR, Western analysis, and enzyme immunoassay, respectively. Tubular morphogenesis was analyzed with phase-contrast imaging microscopy. Nuclear AP-1, HIF-1 and NF-kappaB promoter DNA binding increased 1.5-, 4-, and 3-fold, respectively, after 1 hour of hypoxia. COX-2 RNA was elevated five- and fourfold after 1 and 3 hours of hypoxia, respectively. VEGF RNA and protein abundance lagged behind COX-2 induction but were each increased two- to threefold 3 hours after hypoxia. CGP43182 was found to inhibit NF-kappaB DNA binding, COX-2 and VEGF gene expression, PGE(2) release, and hypoxia-induced tubular morphogenesis. Maximum HIF-1 and NF-kappaB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1beta emulated AP-1, HIF-1, and NF-kappaB DNA binding during hypoxia and may be a novel cytokine trigger for NV. CGP43182 appears to be an effective inhibitor of NV. VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 directly and indirectly through NF-kappaB-mediated COX-2 expression and PGE(2) production.


Angiotensin II type-2 receptor-specific effects on the cardiovascular system.
The renin-angiotensin system (RAS) is intricately involved in cardiovascular homeostasis. It is well known that angiotensin II, the key effector in RAS, contributes to a range of cardiovascular pathologies and diseases via angiotensin II type-1 receptor (AT1R) activation. However, the role of angiotensin II type-2 receptor (AT2R) regulation is less well understood. Recent studies describe the role of the AT2R on cardiovascular function in normal and pathologic conditions. The data describe an important role of AT2R in blood pressure regulation, cardiac hypertrophy and fibrosis, myocardial infarction and vascular homeostasis.


Toxicity of fruit fly baits to beneficial insects in citrus.
Two fruit fly baits, Nu-Lure/malathion and GF-120 (Spinosad) were evaluated in the laboratory for non-target impacts on beneficial insects. Nu-Lure/malathion proved attractive and toxic to adults and larvae of the coccinellid species, Curinus coeruleus Mulsant, Cycloneda sanguinea L. and Harmonia axyridis Pallas, a lacewing species, Chrysoperla rufilabris Burmeister. The coccinellids Olla v-nigrum Mulsant, Scymnus sp. and nymphs of the insidious flower bug, Orius insidiosus (Say) did not succumb to Nu-Lure baits, even in no-choice situations. Nu-Lure was also attractive and lethal to adults of two aphidophagous flies; Leucopis sp. and the syrphid fly Pseudodorus clavatus (F.). Both Nu-Lure and GF-120 caused significant mortality to the parasitoid wasps, Aphytis melinus De Bach and Lysiphlebus testaceipes Cresson, within 24 h of exposure. However, GF-120 caused no significant mortality to any coccinellid in either choice or no-choice situations, despite considerable consumption of baits. Adults of P. clavatus tended to avoid GF-120, although mortality was significant in no-choice tests. Although larvae and adults of the lacewing C. rufilabris consumed GF-120, mortality was delayed; adults died 48 -96 h post-exposure and those exposed as larvae died two weeks later in the pupal stage. The Nu-Lure bait did not appear palatable to any of the insects, but the high concentration of malathion (195,000 ppm) caused rapid mortality to susceptible insects. Nu-Lure bait without malathion also caused significant mortality to flies and lacewings in cage trials. Although GF-120 bait appeared more benign overall, further research efforts are warranted to increase its selectivity for target fly species and reduce its attractiveness to parasitoids and lacewings. I conclude that the Florida "fly free zone" protocol in its current form is not compatible with an IPM approach to commercial citrus production.