Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization.
Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.


A high-capacity cell macroencapsulation system supporting the long-term survival of genetically engineered allogeneic cells.
The rapid increase in the number of approved therapeutic proteins, including recombinant antibodies, for diseases necessitating chronic treatments raises the question of the overall costs imposed on healthcare systems. It is therefore important to investigate alternative methods for recombinant protein administration. The implantation of genetically engineered cells is an attractive strategy for the chronic long-term delivery of recombinant proteins. Here, we have developed a high-capacity cell encapsulation system for the implantation of allogeneic myoblasts, which survive at high density for at least one year. This flat sheet device is based on permeable polypropylene membranes sealed to a mechanically resistant frame which confine cells seeded in a tailored biomimetic poly(ethylene glycol) (PEG)-based hydrogel matrix. In order to quantitate the number of cells surviving in the device and optimize initial conditions leading to high-density survival, we implant devices containing C2C12 mouse myoblasts expressing a luciferase reporter in the mouse subcutaneous tissue. We show that initial cell load, hydrogel stiffness and permeable membrane porosity are critical parameters to achieve long-term implant survival and efficacy. Optimization of these parameters leads to the survival of encapsulated myogenic cells at high density for several months, with minimal inflammatory response and dense neovascularization in the adjacent host tissue. Therefore, this encapsulation system is an effective platform for the implantation of genetically engineered cells in allogeneic conditions, which could be adapted to the chronic administration of recombinant proteins.


Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit.
ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the 'slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.


Multiple myeloma: disease response assessment.
Precise assessment of response to therapy is of high importance in every phase of multiple myeloma (MM). In addition to the well-established role of monoclonal protein for clinical monitoring, several methods of minimal residual disease evaluation, both inside and outside the bone marrow (BM), are to date available. Next generation flow cytometry and sequencing are probably the best approaches at the BM level, being highly sensitive and uniformly applied. FDG PET/CT is the best imaging technique for evaluating and monitoring response to therapy outside the BM. Functional whole-body MRI techniques (DCE and DWI) seem promising for response evaluation and need further studies. Standardization of most of these techniques is in progress.


Parental perceptions of information needs and service provision for children with developmental disabilities in rural Australia.
Rural Australians comprise a third of the population. However, there are relatively few research studies that have focused on issues for children with developmental disabilities in rural regions. In particular, there is very limited research that gives voice to parents regarding challenges faced by them due to their location in rural regions. This article is based on the qualitative component of a mixed-methods study undertaken in rural settings. In-depth, face-to-face interviews were conducted with 17 parents yielding 30 h of information rich taped data. Thematic analysis techniques were used to identify major issues. Three core themes emerged from analysis of the qualitative data regarding information and support needs: an ongoing lack of timely and relevant information about disabilities and support services; inadequacies in interactions with service providers particularly doctors and allied health staff; and considerable challenges and barriers to access and use of health services. Within the constraints of limited rural service provision, there are still opportunities for considerable improvements, through focussed in-service training to narrow the information gap, improve provider-client interaction around attitudinal issues and uptake of tele-health to minimise the long waiting times and the need to regularly travel long distances to access services and setting up online support groups. There is limited information on challenges faced by parents of children with developmental disabilities in rural Australia. The challenges around lack of information about support systems available add to parental stress as does limited experience, frequent staff turnover and poor attitude of many service providers. There is a need to improve rural service provision. Feasible options within resource constraints include focussed in-service training to narrow the information gap, improve provider-client interaction around attitudinal issues, and uptake of tele-health services. Setting up of parent-professional support groups as well peer support groups using digital technologies will help reduce the sense of isolation for rural carers and minimise impediments related to travelling long distances.


Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.
Lysosomes are ubiquitous acidified organelles that degrade intracellular and extracellular material trafficked via multiple pathways. Lysosomes also sense cellular nutrient levels to regulate target of rapamycin (TOR) kinase, a signaling enzyme that drives growth and suppresses activity of the MiT/TFE family of transcription factors that control biogenesis of lysosomes. In this study, we subjected worms lacking basic helix-loop-helix transcription factor 30 (hlh-30), the Caenorhabditis elegans MiT/TFE ortholog, to starvation followed by refeeding to understand how this pathway regulates survival with variable nutrient supply. Loss of HLH-30 markedly impaired survival in starved larval worms and recovery upon refeeding bacteria. Remarkably, provision of simple nutrients in a completely defined medium (C. elegans maintenance medium [CeMM]), specifically glucose and linoleic acid, restored lysosomal acidification, TOR activation, and survival with refeeding despite the absence of HLH-30. Worms deficient in lysosomal lipase 2 (lipl-2), a lysosomal enzyme that is transcriptionally up-regulated in starvation in an HLH-30-dependent manner, also demonstrated increased mortality with starvation-refeeding that was partially rescued with glucose, suggesting a critical role for LIPL-2 in lipid metabolism under starvation. CeMM induced transcription of vacuolar proton pump subunits in hlh-30 mutant worms, and knockdown of vacuolar H+-ATPase 12 (vha-12) and its upstream regulator, nuclear hormone receptor 31 (nhr-31), abolished the rescue with CeMM. Loss of Ras-related GTP binding protein C homolog 1 RAGC-1, the ortholog for mammalian RagC/D GTPases, conferred starvation-refeeding lethality, and RAGC-1 overexpression was sufficient to rescue starved hlh-30 mutant worms, demonstrating a critical need for TOR activation with refeeding. These results show that HLH-30 activation is critical for sustaining survival during starvation-refeeding stress via regulating TOR. Glucose and linoleic acid bypass the requirement for HLH-30 in coupling lysosome nutrient sensing to survival.


Deterioration of kidney function by the (pro)renin receptor blocker handle region peptide in aliskiren-treated diabetic transgenic (mRen2)27 rats.
Dual renin-angiotensin system (RAS) blockade in diabetic nephropathy is no longer feasible because of the profit/side effect imbalance. (Pro)renin receptor [(P)RR] blockade with handle region peptide (HRP) has been reported to exert beneficial effects in various diabetic models in a RAS-independent manner. To what degree (P)RR blockade adds benefits on top of RAS blockade is still unknown. In the present study, we treated diabetic TGR(mREN2)27 rats, a well-established nephropathy model with high prorenin levels [allowing continuous (P)RR stimulation in vivo], with HRP on top of renin inhibition with aliskiren. Aliskiren alone lowered blood pressure and exerted renoprotective effects, as evidenced by reduced glomerulosclerosis, diuresis, proteinuria, albuminuria, and urinary aldosterone levels as well as diminished renal (P)RR and ANG II type 1 receptor expression. It also suppressed plasma and tissue RAS activity and suppressed cardiac atrial natriuretic peptide and brain natriuretic peptide expression. HRP, when given on top of aliskiren, did not alter the effects of renin inhibition on blood pressure, RAS activity, or aldosterone. However, it counteracted the beneficial effects of aliskiren in the kidney, induced hyperkalemia, and increased plasma plasminogen activator-inhibitor 1, renal cyclooxygenase-2, and cardiac collagen content. All these effects have been linked to (P)RR stimulation, suggesting that HRP might, in fact, act as a partial agonist. Therefore, the use of HRP on top of RAS blockade in diabetic nephropathy is not advisable.


Sequential Sampling for Estimation and Classification of the Incidence of Hop Powdery Mildew I: Leaf Sampling.
Hop powdery mildew (caused by Podosphaera macularis) is an important disease of hops (Humulus lupulus) in the Pacific Northwest. Sequential sampling models for estimation and classification of the incidence of powdery mildew on leaves of hop were developed based on the beta-binomial distribution, using parameter estimates of the binary power law determined in previous studies. Stop lines, models that indicate that enough information has been collected to estimate disease incidence and cease sampling, for sequential estimation were validated by bootstrap simulations of a select group of 18 data sets (out of a total of 198 data sets) from the model-construction data, and through simulated sampling of 104 data sets collected independently (i.e., validation data sets). The achieved coefficient of variation (C) approached prespecified C values as the achieved disease incidence ( ) increased. Achieving a C of 0.1 was not possible for data sets in which < 0.10. The 95% confidence interval of the median difference between the true p and included zero for 16 of 18 data sets evaluated at C = 0.2 and all data sets when C = 0.1. For sequential classification, Monte-Carlo simulations were used to determine the probability of classifying mean disease incidence as less than a threshold incidence, pt (operating characteristic [OC]), and average sample number (ASN) curves for 16 combinations of candidate stop lines and error levels (α and β). Four pairs of stop lines were selected for further evaluation based on the results of the Monte-Carlo simulations. Bootstrap simulations of the 18 selected data sets indicated that the OC and ASN curves of the sequential sampling plans for each of the four sets of stop lines were similar to OC and ASN values determined by Monte Carlo simulation. Correct classification of disease incidence as being above or below preselected thresholds was 2.0 to 7.7% higher when stop lines were determined by the beta-binomial approximation than when stop lines were calculated using the binomial distribution. Correct decision rates differed depending on the location where sampling was initiated in the hop yard; however, in all instances were greater than 86% when stop lines were determined using the beta-binomial approximation. The sequential sampling plans evaluated in this study should allow for rapid and accurate estimation and classification of the incidence of hop leaves with powdery mildew, and aid in sampling for pest management decision making.


Lithium for acute mania.
Bipolar disorder is a common condition associated with high morbidity; developing efficacious, safe treatments is therefore essential. Lithium is an effective maintenance treatment for bipolar disorder. It acts as mood stabiliser and reduces the risk of suicide. However, evidence assessing the efficacy of lithium in the treatment of acute mania is less robust. Current evidence-based guidelines cite multiple anti-dopaminergic and mood-stabilising agents as initial treatments: more definite evidence is needed to decide if lithium should be the first-line therapy. 1. To assess the effects of lithium in comparison with placebo or other active treatment in alleviating the acute symptoms of a manic or mixed episode in people with bipolar disorder.2. To review the acceptability and tolerability of treatment with lithium in comparison with placebo or other active treatments in alleviating the acute symptoms of a manic or mixed episode in people with bipolar disorder. We searched the Cochrane Common Mental Disorders Controlled Trials Register, CENTRAL, MEDLINE, Embase, and PsycINFO. We also searched the World Health Organization trials portal (ICTRP) and ClinicalTrials.gov. We checked the reference lists of all included studies and relevant systematic reviews. We have incorporated studies from searches to 18 May 2018 into the current analyses. Prospective randomised controlled studies comparing lithium with placebo or alternative drug treatment in treatment of acute mania. We included anyone with bipolar disorder, male and female, of any age. At least two review authors independently extracted data and assessed methodological quality. We used odds ratios (ORs) to analyse binary efficacy outcomes, and mean differences (MDs) or standardised mean differences (SMDs) for continuously distributed outcomes. We used a fixed-effect model unless heterogeneity was moderate or substantial, in which case we used a random-effects model. We used Review Manager 5 to analyse data. We assessed the certainty of evidence for individual outcomes using the GRADE approach. We found 36 randomised controlled studies comparing lithium with placebo, one of 12 drugs, or electroconvulsive therapy for treatment of acute mania. Studies included male and female participants (n = 4220), of all ages, who all fitted criteria for a manic episode within the context of a diagnosis of bipolar disorder.Risk of bias was variable; 12 studies had a high risk of bias in one domain and 27 gave inadequate information on randomisation leading to an 'unclear' rating for selection bias.Lithium versus placeboHigh-certainty evidence found that lithium was an effective treatment for acute mania and was more effective than placebo at inducing a response (OR 2.13, 95% confidence interval (CI) 1.73 to 2.63; participants = 1707; studies = 6; I2 = 16%; high-certainty evidence), or remission (OR 2.16, 95% CI 1.73 to 2.69; participants = 1597; studies = 5; I2 = 21%; high-certainty evidence).Lithium was more likely than placebo to cause tremor (OR 3.25, 95% CI 2.10 to 5.04; participants = 1241; studies = 6; I2 = 0%; high-certainty evidence), and somnolence (OR 2.28, 95% CI 1.46 to 3.58; participants = 1351; studies = 7; I2 = 0%; high-certainty evidence).There was insufficient evidence to determine the effect of lithium for all-cause dropouts (OR 0.76; 95% CI 0.46 to 1.25; participants = 1353; studies = 7; I2 = 75%; moderate-certainty evidence), and weight gain (OR 1.48, 95% CI 0.56 to 3.92; participants = 735, studies = 3; I2= 51%; moderate-certainty evidence).Lithium versus antipsychotics or mood stabilisersFor the outcome of inducing a response, there was only very low-certainty evidence regarding lithium compared to haloperidol (MD -2.40, 95% CI -6.31 to 1.50; participants = 80; studies = 3; I2 = 95%), quetiapine (OR 0.66, 95% CI 0.28 to 1.55; participants = 335; studies = 2; I2 = 71%), and carbamazepine (SMD 0.21, 95% CI -0.18 to 0.60; participants = 102; studies = 3; I2 = 0%).Lithium was probably less likely to induce a response than olanzapine (OR 0.44, 95% CI 0.20 to 0.94; participants = 180; studies = 2; I2 = 0%; moderate-certainty evidence).Lithium may be less likely to induce a response than risperidone (MD 7.28, 95% CI 5.22 to 9.34; participants = 241; studies = 3; I2 = 49%; low-certainty evidence).There was no evidence of a difference between lithium and valproate (OR 1.22, 95% CI 0.87 to 1.70; participants = 607; studies = 5; I2 = 22%; moderate-certainty evidence).There was moderate-certainty evidence that lithium was more effective than topiramate at treating acute mania (OR 2.28, 95% CI 1.63 to 3.20; participants = 660; studies = 1).Data on adverse events for these comparisons contained too few studies to provide high-certainty evidence. This systematic review indicates that lithium is more effective than placebo as a treatment for acute mania but increases the risk for somnolence and tremor. Limited evidence suggests little or no difference between lithium and other mood stabilisers (valproate, carbamazepine) or antipsychotics (risperidone, quetiapine, haloperidol). Olanzapine may be an exception, as it is probably slightly more effective than lithium. There is uncertain evidence that risperidone may also be more effective than lithium. Lithium is probably more effective at treating acute mania than topiramate. When compared to placebo, lithium was more likely to cause adverse events. However, when compared to other drugs, too few studies provided data on adverse effects to provide high-certainty evidence. More, rigorously designed, large-scale studies are needed to definitively conclude if lithium is superior to other interventions in treating acute mania.


Side chain-specific 11/9-helix propensity of α/β-peptides with alternating residue types.
The 11/9-helix is among the most stable and non-traditional helical structures for α/β-peptides with alternating residue types. The effect of side chain groups of α-residues and β3-residues on the 11/9-helix propensity was examined under various solvent conditions. An α-amino acid residue with one of the four representative side chain groups was incorporated into the central position of an α/β-pentapeptide backbone. A β-branched valine residue did not show any destabilizing effect. α,α-Dimethylsubstituted Aib residue was tolerated under nonpolar conditions, but did not promote 11/9-helical folding. The oligomer with a glycine residue did not show 11/9-helical folding under polar solvent conditions. The single unmatched stereochemistry of d-alanine was deleterious to 11/9-helical folding. Replacement of a cyclic β-residue with an acyclic β3-residue in the 11/9-helical structure had a slight destabilizing effect, which could be compensated by a longer peptide sequence with more cyclic β-residues. These results provide a guidance for incorporating functional groups into an 11/9-helical α/β-peptide backbone to design functional oligomers.


Potent proteasome inhibitors derived from the unnatural cis-cyclopropane isomer of Belactosin A: synthesis, biological activity, and mode of action.
The natural product belactosin A (1) with a trans-cyclopropane structure is a useful prototype compound for developing potent proteasome (core particle, CP) inhibitors. To date, 1 and its analogues are the only CP ligands that bind to both the nonprimed S1 pocket as well as the primed substrate binding channel; however, these molecules harbor a high IC50 value of more than 1 μM. We have performed structure-activity relationship studies, thereby elucidating unnatural cis-cyclopropane derivatives of 1 that exhibit high potency to primarily block the chymotrypsin-like active site of the human constitutive (cCP) and immunoproteasome (iCP). The most active compound 3e reversibly inhibits cCP and iCP similarly with an IC50 of 5.7 nM. X-ray crystallographic analysis of the yeast proteasome in complex with 3e revealed that the ligand is accommodated predominantly into the primed substrate binding channel and covalently binds to the active site threonine residue via its β-lactone ring-opening.


Cognitive and emotional impairments in adults with attention-deficit/hyperactivity disorder and cocaine use.
Attention-deficit/hyperactivity disorder (ADHD) is an important modulator of cognitive and social functioning in cocaine addiction but it is unclear whether ADHD symptoms and cocaine use display mutually aggravating interaction effects on cognition, social functioning, and depressive symptoms. Therefore, we investigated the interaction of cocaine use and adult ADHD on social and non-social cognition and depressive symptoms. Twenty-four cocaine users with (CU+ADHD) and 30 without ADHD (CU-ADHD), 29 cocaine-naïve ADHD patients, and 40 cocaine-naïve healthy controls underwent comprehensive neuropsychological testing including assessment of social cognition (cognitive/emotional empathy and Theory-of-Mind). Additionally, depressive symptoms were measured with the Beck Depression Inventory. The effect size of global cognitive impairment was largest in CU+ADHD (d=1.22 vs. controls) followed by CU-ADHD (d=0.74), and cocaine-naïve ADHD patients (d=0.33). A similar pattern appeared regarding depressive symptoms (CU+ADHD: d=1.47; CU-ADHD: d=0.49, ADHD: d=0.34). In the measures of Theory-of-Mind (CU+ADHD: d=0.76; CU-ADHD: d=0.06, ADHD: d=0.01) and cognitive empathy (CU+ADHD: d=0.80; CU-ADHD: d=0.39, ADHD: d=-0.11) only CU+ADHD showed moderate to large impairments. Moreover, two-way analyses of covariance revealed a significant interaction effect of the factors ADHD and cocaine use on depressive symptoms (p<0.05) and Theory-of-Mind (p<0.05) but not on global cognitive performance (p=0.64). When occurring together, cognitive impairments associated with both ADHD and cocaine use are largely additive, whereas both factors seem to mutually potentiate one another with respect to mood and mental perspective-taking disturbances. Given the high comorbidity between ADHD and cocaine use, longitudinal studies are needed to investigate the origin of these potentiated impairments.


An economic history of Medicare part C.
Twenty-five years ago, private insurance plans were introduced into the Medicare program with the stated dual aims of (1) giving beneficiaries a choice of health insurance plans beyond the fee-for-service Medicare program and (2) transferring to the Medicare program the efficiencies and cost savings achieved by managed care in the private sector. In this article we review the economic history of Medicare Part C, known today as Medicare Advantage, focusing on the impact of major changes in the program's structure and of plan payment methods on trends in the availability of private plans, plan enrollment, and Medicare spending. Additionally, we compare the experience of Medicare Advantage and of employer-sponsored health insurance with managed care over the same time period. Beneficiaries' access to private plans has been inconsistent over the program's history, with higher plan payments resulting in greater choice and enrollment and vice versa. But Medicare Advantage generally has cost more than the traditional Medicare program, an overpayment that has increased in recent years. Major changes in Medicare Advantage's payment rules are needed in order to simultaneously encourage the participation of private plans, the provision of high-quality care, and to save Medicare money.


Facile synthesis of Ag-ZnO hybrid nanospindles for highly efficient photocatalytic degradation of methyl orange.
Highly photocatalytically active Ag nanoparticle decorated ZnO nanospindles were synthesized by a facile wet chemical method. The structural and optical properties of the as-synthesized materials were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and high resolution TEM (HRTEM) with energy dispersive X-ray spectroscopy, UV-visible absorption spectroscopy and Raman spectroscopy. The photocatalytic activity of these nanostructures was evaluated by analyzing sunlight driven degradation of methyl orange (MO) dye and it was observed that Ag nanoparticle modified ZnO nanospindles show significantly enhanced photocatalytic activity for degradation of MO, as compared to ZnO nanospindles. We attribute the observed enhanced photocatalytic activity of Ag nanoparticle decorated ZnO nanospindles to their improved sunlight utilization efficiency and the efficient suppression of recombination of photogenerated charge carriers due to the electron scavenging action of Ag nanoparticles and the interfacial electron transfers due to the Schottky junction between Ag nanoparticles and ZnO nanospindles.


Nuclear actin: to polymerize or not to polymerize.
The form and function of actin in the nucleus have been enigmatic for over 30 years. Recently actin has been assigned numerous functional roles in the nucleus, but its form remains a mystery. The intricate relationship between actin form and function in the cytoplasm implies that understanding the structural properties of nuclear actin is elementary to fully understanding its function. In this issue, McDonald et al. (p. 541) use fluorescence recovery after photobleaching (FRAP) to tackle the question of whether nuclear actin exists as monomers or polymers.


Breast Cancer Cell-Derived GM-CSF Licenses Regulatory Th2 Induction by Plasmacytoid Predendritic Cells in Aggressive Disease Subtypes.
Reciprocal interactions between tumor cells and their microenvironment vitally impact tumor progression. In this study, we show that GM-CSF produced by primary breast tumor cells induced the activation of plasmacytoid predendritic cells (pDC), a cell type critical to anti-viral immunity. pDC that expressed the GM-CSF receptor were increased in breast tumors compared with noninvolved adjacent breast tissue. Tumor-activated pDC acquired naïve CD4(+) T-cell stimulatory capacity and promoted a regulatory Th2 response. Finally, the concomitant increase of GM-CSF and pDC was significantly associated with relatively more aggressive breast cancer subtypes. Our results characterize the first tumor-derived factor that can activate pDC to promote a regulatory Th2 response, with implications for therapeutic targeting of a tumor-immune axis of growing recognition in its significance to cancer.


Investigation of the H reflexes, F waves and sympathetic skin response with electromyography (EMG) in patients with stroke and the determination of the relationship with functional capacity.
The purpose of the study was to evaluate the relationship between sympathetic skin responses (SSR), electrodiagnostic reflex activities (F wave, H reflex), and functional capacity in post-stroke patients. The study comprised 40 hemiplegia patients (mean age 57.8 ± 10.9 years) and 40 healthy volunteers (mean age 557 ± 85 years). In electrophysiological studies, SSR, F wave and H reflex were evaluated and for the functional capacities of patients, FIM scores and Brunnstrom stages were calculated. There was no statistical significant difference between SSR latency and amplitude in the hemiplegic and non-hemiplegic extremities of patients (p > 0.05). SSR latency values of patients were higher than those of controls (p < 0.05). Amplitude values of paretic arms were significantly lower than the control group extremities (p < 0.05). There was a significant correlation between SSR amplitude values and FIM scores. A significant increase in H max/M max amplitude rate was detected in the affected side (p < 0.05) and F-wave mean latency values of the affected side were found to be significantly lower in the control group (p < 0.05).


Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein.
TRIM28 regulates its target genes at both transcriptional and posttranscriptional levels. Here we report that a TRIM28-TWIST1-EMT axis exists in breast cancer cells and TRIM28 promotes breast cancer metastasis by stabilizing TWIST1 and subsequently enhancing EMT. We find that TRIM28 is highly expressed in both cancer cell lines and advanced breast cancer tissues, and the levels of TRIM28 and TWIST1 are positively correlated with the aggressiveness of breast carcinomas. Overexpression and depletion of TRIM28 up- and down-regulates the protein, but not the mRNA levels of TWIST1, respectively, suggesting that TRIM28 upregulates TWIST1 post-transcriptionally. Overexpression of TRIM28 in breast cancer cell line promotes cell migration and invasion. Knockdown of TRIM28 reduces the protein level of TWIST1 with concurrent upregulation of E-cadherin and downregulation of N-cadherin and consequently inhibits cell migration and invasion. Furthermore, Immunoprecipitation and GST pull-down assays demonstrated that TRIM28 interacts with TWIST1 directly and this interaction is presumed to protect TWIST1 from degradation. Our study revealed a novel mechanism in breast cancer cells that TRIM28 enhances metastasis by stabilizing TWIST1, suggesting that targeting TRIM28 could be an efficacious strategy in breast cancer treatment.


MR imaging of brain abscesses.
The MR images and CT scans of 14 patients with surgically verified pyogenic cerebral abscesses were reviewed. The MR findings correlated well with those seen on CT and were believed to be sufficiently characteristic to allow early and accurate diagnosis with MR alone. These features include (1) peripheral edema producing mild hypointensity on short TR/short TE and marked hyperintensity on long TR/intermediate to long TE scans; (2) central necrosis with abscess fluid hypointense relative to white matter and hyperintense relative to CSF on short TR/short TE scans and hyperintense relative to gray matter on long TR/intermediate to long TE scans (the fluid had concentric zones of varying intensity in seven cases, a finding not previously identified in other lesions); (3) extraparenchymal spread (intraventricular or subarachnoid), which was detected more easily on MR than on CT and was manifested by increased intensity relative to normal CSF on both short TR/short TE and long TR/intermediate TE scans; and (4) visualization of the abscess capsule, which was iso- to mildly hyperintense relative to brain on short TR/short TE scans and iso- to hypointense relative to white matter on long TR/intermediate to long TE scans. On the long TR scans, the relative hypointensity of the rim allowed for visualization of the typical morphologic features of the capsule, which in turn aided in differentiation of abscesses from other lesions (as it does on CT). To investigate the cause of the capsular intensity, pathologic studies of the capsules were reviewed when available (10 cases). Fibrosis was identified in all mature abscess capsules, but the combination of the intensities seen on short TR/short TE and long TR/intermediate to long TE scans as well as the temporal changes in intensity were believed to be incompatible with fibrosis as a cause of the capsular changes. Intensity patterns were suggestive of hemorrhage, but neither acute nor chronic hemorrhage was identified on routine H and E stains, while iron stain revealed scant hemorrhage in only two of the eight patients in whom these stains were used. We believe the capsular intensity (in particular the hypointense rims on long TR scans) may reflect paramagnetic T1, and to a greater extent T2, shortening, possibly due to the presence of heterogeneously distributed free radicals that are products of the respiratory burst produced by actively phagocytosing macrophages in the capsule wall. Distinctive MR features of pyogenic abscesses should afford early and accurate diagnosis.


Effect of micellized natural (D-α-tocopherol) vs. synthetic (DL-α-tocopheryl acetate) vitamin E supplementation given to turkeys on oxidative status and breast meat quality characteristics.
This study evaluates the effect of vitamin E supplementation source (micellized natural vs. the synthetic form) and dosage (40, 80, or 120 mg/kg) on α-tocopherol concentration in plasma and muscle, antioxidant capacity, and breast meat quality in turkeys. Three hundred female turkeys were randomly selected at an average live weight 63.2 g±0.5 and distributed into 7 groups. One group (control) was fed a standard diet without vitamin E supplementation and the other 6 were given mixed diets supplemented with the natural (d-α-tocopherol) or synthetic (dl-α-tocopheryl acetate) form of vitamin E in 3 dosages (40, 80, or 120 mg/kg). Following 11 wk feeding, results showed that performance parameters were not modified either by source or dosage of vitamin E supplementation to the turkeys. Plasma and muscle α-tocopherol at d 9 of refrigerated storage were higher when turkeys were supplemented with the natural form at higher doses. Losses in the concentration of α-tocopherol in meat between the beginning and the end of the 9 d refrigerated storage were greater in the groups supplemented with the synthetic form of vitamin E compared to those receiving the natural supplementation. The relationship between plasma α-tocopherol and the Trolox equivalent antioxidant capacity followed a different trend depending on the vitamin E source. Intramuscular fat was not significantly affected by the vitamin E source supplementation; however the slope of the linear regression equation was lower for the natural form than for the synthetic form. Turkeys given the natural form had higher C18:1n-9 but lower C15:1, C17:1, C20:5n-3, and C22:6n-3 in breast muscle. Meat samples from turkeys supplemented with natural vitamin E had higher deoxymyoglobin at d 3, 6, and 9 and lower metmyoglobin at d 9 of refrigerated storage than those receiving the synthetic form. Dietary supplementation with medium doses (80 mg/kg) micellized d-α-tocopherol is an interesting feeding strategy for ensuring antioxidant status and improving meat quality.


Neuroanatomy predicts individual risk attitudes.
Over the course of the last decade a multitude of studies have investigated the relationship between neural activations and individual human decision-making. Here we asked whether the anatomical features of individual human brains could be used to predict the fundamental preferences of human choosers. To that end, we quantified the risk attitudes of human decision-makers using standard economic tools and quantified the gray matter cortical volume in all brain areas using standard neurobiological tools. Our whole-brain analysis revealed that the gray matter volume of a region in the right posterior parietal cortex was significantly predictive of individual risk attitudes. Participants with higher gray matter volume in this region exhibited less risk aversion. To test the robustness of this finding we examined a second group of participants and used econometric tools to test the ex ante hypothesis that gray matter volume in this area predicts individual risk attitudes. Our finding was confirmed in this second group. Our results, while being silent about causal relationships, identify what might be considered the first stable biomarker for financial risk-attitude. If these results, gathered in a population of midlife northeast American adults, hold in the general population, they will provide constraints on the possible neural mechanisms underlying risk attitudes. The results will also provide a simple measurement of risk attitudes that could be easily extracted from abundance of existing medical brain scans, and could potentially provide a characteristic distribution of these attitudes for policy makers.


Substrate-based platform for boosting the surface-enhanced Raman of plasmonic nanoparticles.
Metal nanoparticles allow for surface-enhanced Raman scattering (SERS), with applications including spectroscopy and highly-multiplexed biolabels. Despite advances in nanoparticles design nanoparticles, the SERS from these systems is still weak when compared with randomly roughened substrates, and this limits their efficacy for many applications. Here, we coherently boost the SERS signal of colloidally-synthesized silver nano-prisms over 50 × by using multilayer substrates. Theoretical calculations verify the enhancement, and uncover the near-field response. This points the way toward a versatile platform for greater SERS enhancement from nanoparticles.


Differential Regulation of Genes for Cyclic-di-GMP Metabolism Orchestrates Adaptive Changes During Rhizosphere Colonization by Pseudomonas fluorescens.
Bacteria belonging to the Pseudomonas genus are highly successful colonizers of the plant rhizosphere. The ability of different Pseudomonas species to live either commensal lifestyles or to act as agents of plant-growth promotion or disease is reflected in a large, highly flexible accessory genome. Nevertheless, adaptation to the plant environment involves a commonality of phenotypic outputs such as changes to motility, coupled with synthesis of nutrient uptake systems, stress-response molecules and adherence factors including exopolysaccharides. Cyclic-di-GMP (cdG) is a highly important second messenger involved in the integration of environmental signals with appropriate adaptive responses and is known to play a central role in mediating effective rhizosphere colonization. In this study, we examined the transcription of multiple, reportedly plant-upregulated cdG metabolism genes during colonization of the wheat rhizosphere by the plant-growth-promoting strain P. fluorescens SBW25. While transcription of the tested genes generally increased in the rhizosphere environment, we additionally observed a tightly orchestrated response to environmental cues, with a distinct transcriptional pattern seen for each gene throughout the colonization process. Extensive phenotypical analysis of deletion and overexpression strains was then conducted and used to propose cellular functions for individual cdG signaling genes. Finally, in-depth genetic analysis of an important rhizosphere colonization regulator revealed a link between cdG control of growth, motility and stress response, and the carbon sources available in the rhizosphere.


Health outcomes and costs of community mitigation strategies for an influenza pandemic in the United States.
The optimal community-level approach to control pandemic influenza is unknown. We estimated the health outcomes and costs of combinations of 4 social distancing strategies and 2 antiviral medication strategies to mitigate an influenza pandemic for a demographically typical US community. We used a social network, agent-based model to estimate strategy effectiveness and an economic model to estimate health resource use and costs. We used data from the literature to estimate clinical outcomes and health care utilization. At 1% influenza mortality, moderate infectivity (R(o) of 2.1 or greater), and 60% population compliance, the preferred strategy is adult and child social distancing, school closure, and antiviral treatment and prophylaxis. This strategy reduces the prevalence of cases in the population from 35% to 10%, averts 2480 cases per 10,000 population, costs $2700 per case averted, and costs $31,300 per quality-adjusted life-year gained, compared with the same strategy without school closure. The addition of school closure to adult and child social distancing and antiviral treatment and prophylaxis, if available, is not cost-effective for viral strains with low infectivity (R(o) of 1.6 and below) and low case fatality rates (below 1%). High population compliance lowers costs to society substantially when the pandemic strain is severe (R(o) of 2.1 or greater). Multilayered mitigation strategies that include adult and child social distancing, use of antivirals, and school closure are cost-effective for a moderate to severe pandemic. Choice of strategy should be driven by the severity of the pandemic, as defined by the case fatality rate and infectivity.


Associations of leisure-time sitting in cars with neighborhood walkability.
Too much sitting, including time spent sitting in cars, is associated with poor health outcomes. Identifying the built-environment attributes that may reduce vehicular sitting time can inform future initiatives linking the public health, urban design, and transportation sectors. Data collected in 2003-2004 from adult residents (n = 2521) of Adelaide, Australia were used. Logistic regression analyses examined associations of prolonged time spent sitting in cars during leisure time (30 min/day or more) with neighborhood walkability and its components (dwelling density; intersection density; land use mix; net retail area ratio). Lower overall walkability was significantly associated with a higher odds (OR = 1.43, 95% CI: 1.21-1.70) of spending prolonged time in cars. For analyses with walkability components, lower net retail area ratio, lower residential density, and lower intersection density were significantly associated with prolonged sitting in cars. This study found that residents of high walkable neighborhoods tended to spend less time sitting in cars. In particular, higher net retail area ratio, an indicator of tightly spaced commercial areas, was strongly associated with less time in cars. Policy and planning initiatives to reduce car use require further evidence, particularly on the influence of neighborhood retail areas.


Controlled trial of Japanese acupuncture for chronic myofascial neck pain: assessment of specific and nonspecific effects of treatment.
This article examines the specific and nonspecific effects of Japanese acupuncture on chronic myofascial neck pain in a randomized single-blind trial. Forty-six patients were randomly assigned to receive relevant acupuncture, irrelevant acupuncture, or no-acupuncture control treatment consisting of nonsteroidal anti-inflammatory medication. The two acupuncture groups underwent comparable light shallow needling. The irrelevant acupuncture group received acupuncture at specific sites not relevant for cervical pain. The study measures included the McGill Pain Questionnaire-Short Form (SF-MPQ), the Short-Form Health Survey (SF-36), the Symptom Checklist 90-Revised (SCL-90-R), medication diary, and physiologic measures. The factors examined as predictors of outcome pain ratings were experience with, beliefs about, and knowledge of acupuncture before treatment; perceived efficacy, credibility, and logic of acupuncture; perceived competence of the acupuncturist; and painfulness of acupuncture. No differences were found among the three groups at baseline, except that the relevant acupuncture group reported having had more previous acupuncture treatments. No significant differences in terms of perceived credibility or perceived effectiveness of treatment were found between the two acupuncture groups. The relevant acupuncture group had significantly greater pre-/posttreatment differences in pain than the irrelevant acupuncture and control groups (p < .05). The nonspecific effects of confidence in the acupuncturist, willingness to try any treatment, mood, and physiologic effect of needling were not predictive of treatment outcome, whereas confidence in the treatment and past experiences with acupuncture did correlate significantly with a decrease in pain. Relevant acupuncture with heat contributes to modest pain reduction in persons with myofascial neck pain. Previous experience with and confidence in treatment help to predict benefit. Measurement of nonspecific effects of alternative therapy is recommended in future clinical trials.


Listen to the children: kids' impressions of Who Do You Tell ™.
School-based programs have been the central strategy to educate children about the serious nature of child sexual abuse in the hope of prevention or early intervention. Virtually all evaluations have been quantitative, seldom allowing children to directly comment on their experiences. This article presents the results of qualitative research with 116 students (51 boys and 65 girls) ranging from age 6 to 12 who had participated in the Who Do You Tell child sexual abuse education program. Ten focus groups were conducted with students in separate grades, from kindergarten through sixth grade, two to three months after program participation. The core themes that emerged from the focus group interviews are presented, as are implications for child sexual abuse prevention programs.


The evolutionary history of the common chloroplast genome of Arabidopsis thaliana and A. suecica.
The evolutionary history of the common chloroplast (cp) genome of the allotetraploid Arabidopsis suecica and its maternal parent A. thaliana was investigated by sequencing 50 fragments of cpDNA, resulting in 98 polymorphic sites. The variation in the A. suecica sample was small, in contrast to that of the A. thaliana sample. The time to the most recent common ancestor (T(MRCA)) of the A. suecica cp genome alone was estimated to be about one 37th of the T(MRCA) of both the A. thaliana and A. suecica cp genomes. This corresponds to A. suecica having a MRCA between 10 000 and 50 000 years ago, suggesting that the entire species originated during, or before, this period of time, although the estimates are sensitive to assumptions made about population size and mutation rate. The data was also consistent with the hypothesis of A. suecica being of single origin. Isolation-by-distance and population structure in A. thaliana depended upon the geographical scale analysed; isolation-by-distance was found to be weak on the global scale but locally pronounced. Within the genealogical cp tree of A. thaliana, there were indications that the root of the A. suecica species is located among accessions of A. thaliana that come primarily from central Europe. Selective neutrality of the cp genome could not be rejected, despite the fact that it contains several completely linked protein-coding genes.


Atypical object exploration in infants at-risk for autism during the first year of lifer.
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder usually diagnosed by the end of the second year of life. Early signs of ASD within the first year of life are still unclear. The main purpose of the present study was to compare object exploration skills between infants at-risk for ASD and typically developing (TD) infants to determine early markers for autism within the first year of life. Sixteen at-risk infants and 16 TD infants were longitudinally followed from 6 to 15 months of age during an object exploration task involving three objects with distinct size, shape, and texture, i.e., a long rattle, a rigid circular ball, and a soft circular koosh ball. All sessions were videotaped for coding of manual exploration (grasping and dropping), oral exploration (mouthing), and visual exploration (looking). We also obtained follow-up outcomes using various developmental questionnaires at 18 months and email follow-up on developmental delays/ASD diagnosis after the infants' second birthdays. Our results showed object-based differences in exploration patterns that extended across both groups. We also noticed context-dependent group differences for various exploratory behaviors across objects and ages. Specifically, at 6 months, at-risk infants showed less grasping of the rigid ball as well as less mouthing and greater looking at the rattle compared to TD infants. At 9 and 12 months, at-risk infants demonstrated significantly lower levels of purposeful dropping of all objects and greater looking at the rattle. Lastly, at 15 months, at-risk infants showed persistent mouthing of the rigid ball and rattle compared to TD infants. In addition, 10 out of 16 at-risk infants developed various motor, social, and language delays or ASD diagnosis at follow-up. Taken together, early context-dependent delays/abnormalities in object exploration could be markers for future developmental delays in infants at-risk for autism. Moreover, promoting early object experiences through socially embedded, free and structured play could have significant implications for multisystem development including perceptuo-motor, social communication, and cognitive development in at-risk infants.


Study rationale and design of ADVANCE: action in diabetes and vascular disease--preterax and diamicron MR controlled evaluation.
Patients with Type II (non-insulin-dependent) diabetes mellitus are at increased risk of macrovascular and microvascular disease, both of which are reduced by controlling raised blood pressure in hypertensive patients. Intensive glycaemic control has also been shown to reduce microvascular disease but the effects on macrovascular disease remain uncertain. This study will examine the hypotheses that lowering blood pressure with an ACE inhibitor-diuretic combination and intensively controlling gylcaemia with a sulphonylurea-based regimen in high-risk patients with Type II diabetes (both hypertensive and non-hypertensive) reduces the incidence of macrovascular and microvascular disease. The study is a 2 x 2 factorial randomised controlled trial that will include 10000 adults with Type II diabetes at high risk of vascular disease. Following 6 weeks on open label perindopril-indapamide combination, eligible patients are randomised to continued perindopril-indapamide or matching placebo, and to an intensive gliclazide MR-based glucose control regimen or usual guidelines-based therapy. Primary outcomes are, first, the composite of nonfatal stroke, non-fatal myocardial infarction or cardiovascular death and, second, the composite of new or worsening nephropathy or diabetic eye disease. The scheduled average duration of treatment and follow-up is 4.5 years. The study will be conducted in approximately 200 centres in Australasia, Asia, Europe and North America. ADVANCE is designed to provide reliable evidence on the balance of benefits and risks conferred by blood pressure lowering therapy and intensive glucose control therapy in high-risk diabetic patients, regardless of initial blood pressure or glucose concentrations.


Pilot study on the bioactivity of vitamin d in the skin after oral supplementation.
Laboratory studies suggest that vitamin D (VD) supplementation inhibits skin carcinogenesis. However, epidemiologic studies report mixed findings in the association between circulating VD levels and skin cancer risk. We conducted a clinical study to determine whether oral cholecalciferol supplementation would exert direct bioactivity in human skin through modulation of the VD receptor (VDR). We enrolled 25 individuals with serum 25-hydroxyvitamin-D levels <30 ng/mL and with skin photodamage to take 50,000 IU of cholecalciferol biweekly for 8 to 9 weeks. Then, we obtained baseline and end-of-study skin biopsies from photodamaged (PD) and photoprotected (PP) skin, and from benign nevi (BN) and tested for mRNA expression of VDR and cytochrome P450-24 (CYP24), and markers of keratinocytic differentiation. High-dose cholecalciferol supplementation significantly elevated circulating levels of 25-hydroxyvitamin-D (P < 0.0001) and 1,25-dihydroxyvitamin-D (P < 0.0001). VDR expression in PD- and PP-skin showed minimum changes after supplementation. CYP24 expression in PD- and PP-skin was increased after supplementation by 186%, P = 0.08, and 134%, P = 0.07, respectively. In BNs from 11 participants, a trend for higher VDR and CYP24 expression was observed (average of 20%, P = 0.08, and 544%, P = 0.09, respectively). Caspase-14 expression at the basal layer in PD skin samples was the only epidermal differentiation marker that was significantly increased (49%, P < 0.0001). High-dose cholecalciferol supplementation raised serum VD metabolite levels concurrently with CYP24 mRNA and caspase-14 levels in the skin. Our findings of significant variability in the range of VDR and CYP24 expression across study samples represent an important consideration in studies evaluating the role of VD as a skin cancer chemopreventive agent.


Inhibition of MEK and PI3K/mTOR suppresses tumor growth but does not cause tumor regression in patient-derived xenografts of RAS-mutant colorectal carcinomas.
Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti-EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC). Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab. At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment. The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses.


Antiviral drug discovery: broad-spectrum drugs from nature.
Covering: up to April 2014. The development of drugs with broad-spectrum antiviral activities is a long pursued goal in drug discovery. It has been shown that blocking co-opted host-factors abrogates the replication of many viruses, yet the development of such host-targeting drugs has been met with scepticism mainly due to toxicity issues and poor translation to in vivo models. With the advent of new and more powerful screening assays and prediction tools, the idea of a drug that can efficiently treat a wide range of viral infections by blocking specific host functions has re-bloomed. Here we critically review the state-of-the-art in broad-spectrum antiviral drug discovery. We discuss putative targets and treatment strategies, with particular focus on natural products as promising starting points for antiviral lead development.


Effectiveness of a smartphone application for weight loss compared with usual care in overweight primary care patients: a randomized, controlled trial.
Many smartphone applications (apps) for weight loss are available, but little is known about their effectiveness. To evaluate the effect of introducing primary care patients to a free smartphone app for weight loss. Randomized, controlled trial. (ClinicalTrials.gov: NCT01650337). 2 academic primary care clinics. 212 primary care patients with body mass index of 25 kg/m2 or greater. 6 months of usual care without (n = 107) or with (n = 105) assistance in downloading the MyFitnessPal app (MyFitnessPal). Weight loss at 6 months (primary outcome) and changes in systolic blood pressure and behaviors, frequency of app use, and satisfaction (secondary outcomes). After 6 months, weight change was minimal, with no difference between groups (mean between-group difference, -0.30 kg [95% CI, -1.50 to 0.95 kg]; P = 0.63). Change in systolic blood pressure also did not differ between groups (mean between-group difference, -1.7 mm Hg [CI, -7.1 to 3.8 mm Hg]; P = 0.55). Compared with patients in the control group, those in the intervention group increased use of a personal calorie goal (mean between-group difference, 2.0 d/wk [CI, 1.1 to 2.9 d/wk]; P < 0.001), although other self-reported behaviors did not differ between groups. Most users reported high satisfaction with MyFitnessPal, but logins decreased sharply after the first month. Despite being blinded to the name of the app, 14 control group participants (13%) used MyFitnessPal. In addition, 32% of intervention group participants and 19% of control group participants were lost to follow-up at 6 months. The app was given to patients by research assistants, not by physicians. Smartphone apps for weight loss may be useful for persons who are ready to self-monitor calories, but introducing a smartphone app is unlikely to produce substantial weight change for most patients. Robert Wood Johnson Foundation Clinical Scholars Program, National Institutes of Health/National Center for Advancing Translational Sciences for the UCLA Clinical and Translational Science Institute, and the Resource Centers for Minority Aging Research Center for Health Improvement of Minority Elderly under the National Institutes of Health/National Institute on Aging.


The influence of an insulin-like growth factor I gene promoter polymorphism on hip bone geometry and the risk of nonvertebral fracture in the elderly: the Rotterdam Study.
The absence of the wildtype allele of a promoter polymorphism of the IGF-I gene is associated with increased risk (1.5; 95% CI, 1.1-2.0) of fragility fracture in women (n = 4212) but not in men (n = 2799). An approximation of hip bone geometry (from DXA) suggested the polymorphism is associated with bone strength and stability in gender-specific ways. Previously, we found a CA-repeat promoter polymorphism in the insulin-like growth factor I (IGF-I) gene associated with IGF-I levels and BMD in postmenopausal women, but the relationship with fractures is unclear. In this large population-based study of elderly men and women, we examined the association between this IGF-I promoter polymorphism with parameters of bone geometry and the occurrence of fractures. Within the Rotterdam Study, a prospective population-based cohort, the IGF-I polymorphism was analyzed in relation to incident nonvertebral fractures in 2799 men and 4212 women followed on average for 8.6 years. Furthermore, we estimated structural parameters of hip bone geometry indirectly from DXA outputs of the femoral neck in 2372 men and 3114 women. We studied neck width, cortical thickness, and the cortical buckling ratio and the section modulus as indexes of bone stability and bending strength. Women heterozygotes and noncarriers of the allele had, respectively, 1.2 (95% CI, 1.0-1.5) and 1.5 (95% CI, 1.1-2.0) increased risk of having a fragility fracture at older age compared with homozygotes for the 192-bp allele (p trend = 0.0007). In men, fracture risk was not influenced by the polymorphism. Compared with homozygotes for the 192-bp allele, noncarrier males had approximately 1% narrower femoral necks and 2.2% lower section moduli (p trend < 0.05). Noncarrier females had 1.7% thinner cortices and 1.6% higher buckling ratios (p trend < 0.05) but no significant differences in femoral neck widths and section moduli. In women with low body mass index, genotype differences in bone strength (section modulus) and fracture risk were accentuated (p interaction = 0.05). The genotype-dependent differences in hip bone geometry did not fully explain the genotype-dependent differences in fracture risk. The CA-repeat promoter polymorphism in the IGF-I gene is associated with the risk for fragility fracture at old age in women and with bone structure in both genders.


Influence of non ionizing radiation of base stations on the activity of redox proteins in bovines.
The influence of electromagnetic fields on the health of humans and animals is still an intensively discussed and scientifically investigated issue (Prakt Tierarzt 11:15-20, 2003; Umwelt Medizin Gesellschaft 17:326-332, 2004; J Toxicol Environment Health, Part B 12:572-597, 2009). We are surrounded by numerous electromagnetic fields of variable strength, coming from electronic equipment and its power cords, from high-voltage power lines and from antennas for radio, television and mobile communication. Particularly the latter cause's controversy, as everyone likes to have good mobile reception at anytime and anywhere, whereas nobody wants to have such a basestation antenna in their proximity. In this experiment, the NIR has resulted in changes in the enzyme activities. Certain enzymes were disabled, others enabled by NIR. Furthermore, individual behavior patterns were observed. While certain cows reacted to NIR, others did not react at all, or even inversely. The present results coincide with the information from the literature, according to which NIR leads to changes in redox proteins, and that there are individuals who are sensitive to radiation and others that are not. However, the latter could not be distinctly attributed - there are cows that react clearly with one enzyme while they do not react with another enzyme at all, or even the inverse. The study approach of testing ten cows each ten times during three phases has proven to be appropriate. Future studies should however set the post-exposure phase later on.


Identification of kidney mesenchymal genes by a combination of microarray analysis and Sall1-GFP knockin mice.
SALL1, a causative gene for Townes-Brocks syndrome, encodes a zinc finger protein, and its mouse homolog (Sall1) is essential for metanephros development, as noted during gene targeting. In the embryonic kidney, Sall1 is expressed abundantly in mesenchyme-derived structures from condensed mesenchyme, S-, comma-shaped bodies, to renal tubules and podocytes. We generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescein-activated cell sorting. The GFP-positive population indeed expressed mesenchymal genes, while the negative population expressed genes in the ureteric bud. To systematically search for genes expressed in the mesenchyme-derived cells, we compared gene expression profiles in the GFP-positive and -negative populations using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development including Sall1, GDNF, Raldh2, Pax8 and FoxD1, and genes expressed abundantly in the metanephric mesenchyme such as Unc4.1, Six2, Osr-2 and PDGFc. We also found groups of genes including SSB-4, Smarcd3, micro-Crystallin, TRB-2, which are not known to be expressed in the metanephric mesenchyme. Therefore a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney.


Fatty acid patterns and risk of prostate cancer in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition.
Fatty acids in blood may be related to the risk of prostate cancer, but epidemiologic evidence is inconsistent. Blood fatty acids are correlated through shared food sources and common endogenous desaturation and elongation pathways. Studies of individual fatty acids cannot take this into account, but pattern analysis can. Treelet transform (TT) is a novel method that uses data correlation structures to derive sparse factors that explain variation. The objective was to gain further insight in the association between plasma fatty acids and risk of prostate cancer by applying TT to take data correlations into account. We reanalyzed previously published data from a case-control study of prostate cancer nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. TT was used to derive factors explaining the variation in 26 plasma phospholipid fatty acids of 962 incident prostate cancer cases matched to 1061 controls. Multiple imputation was used to deal with missing data in covariates. ORs of prostate cancer according to factor scores were determined by using multivariable conditional logistic regression. Four simple factors explained 38% of the variation in plasma fatty acids. A high score on a factor reflecting a long-chain n-3 PUFA pattern was associated with greater risk of prostate cancer (OR for highest compared with lowest quintile: 1.36; 95% CI: 0.99, 1.86; P-trend = 0.041). Pattern analyses using TT groupings of correlated fatty acids indicate that intake or metabolism of long-chain n-3 PUFAs may be relevant to prostate cancer etiology.


Effect of maternal calcium supplementation on offspring blood pressure in 5- to 10-y-old rural Gambian children.
Evidence suggests that increased maternal calcium intake during pregnancy may result in lower offspring blood pressure, prompting calls for more robust data in this field, particularly in settings of habitually low calcium intake. The objective was to investigate the effect of maternal calcium supplementation on blood pressure in offspring by recruiting children born after a randomized, double-blind, placebo-controlled trial of calcium supplementation during pregnancy. Children (n = 389) from a rural area of The Gambia (mean age: 7.4 ± 1.2 y; range: 5-10 y), whose mothers received a calcium supplement (1500 mg Ca/d from 20 wk of gestation until delivery) or placebo, were followed up in West Africa. Blood pressure was assessed under standardized conditions with use of the Omron 705IT automated oscillometric device (Morton Medical Ltd, London, United Kingdom), and anthropometric and body composition (bioelectrical impedance) measurements were also made. The analysis was restricted to 350 children born at term, which represented 64% of original trial births. There was no difference in systolic (adjusted mean difference: -0.04 mm Hg; 95% CI: -1.78, 1.69 mm Hg) or diastolic (adjusted mean difference: 0.25 mm Hg; 95% CI: -1.27, 1.77 mm Hg) blood pressure between children whose mothers had received calcium and those who received placebo. No interaction between childhood body mass index (in kg/m(2); mean: 14.0) and maternal calcium supplementation was observed in this study. Calcium supplementation in the second half of pregnancy in Gambian women with very low habitual calcium intakes may not result in lower offspring blood pressure at 5-10 y of age.


DNA damage tolerance: a double-edged sword guarding the genome.
Preservation of genome integrity is an essential process for cell homeostasis. During the course of life of a single cell, the genome is constantly damaged by endogenous and exogenous agents. To ensure genome stability, cells use a global signaling network, namely the DNA damage response (DDR) to sense and repair DNA damage. DDR senses different types of DNA damage and coordinates a response that includes activation of transcription, cell cycle control, DNA repair pathways, apoptosis, senescence, and cell death. Despite several repair mechanisms that repair different types of DNA lesions, it is likely that the replication machinery would still encounter lesions that are mis-repaired or not repaired. Replication of damaged genome would result in high frequency of fork collapse and genome instability. In this scenario, the cells employ the DNA damage tolerance (DDT) pathway that recruits a specialized low fidelity translesion synthesis (TLS) polymerase to bypass the lesions for repair at a later time point. Thus, DDT is not a repair pathway per se, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and preventing genome instability. Paradoxically, DDT process is also associated with increased mutagenesis, which can in turn drive the cell to cancer development. Thus, DDT process functions as a double-edged sword guarding the genome. In this review, we will discuss the replication stress induced DNA damage-signaling cascade, the stabilization and rescue of stalled replication forks by the DDT pathway and the effect of the DDT pathway on cancer.


Conditional RNA interference in vivo to study mutant p53 oncogenic gain of function on tumor malignancy.
Mutant p53 proteins are thought to have acquired a "gain of function" (GOF) activity that mainly contributes to tumor aggressiveness. Previously we reported that constitutive downregulation of mutant p53 by RNA interference reduces the tumorigenicity of cancer cells in an animal model; however, effects of adaptation to long-term mutant p53 inhibition could not be excluded. To address this point, mimicking more physiological conditions, we now describe the establishment of a lentiviral-based system for conditional interference with mutant p53 expression. In vivo studies assessed the efficacy of conditional RNA interference in inhibiting gain of function activity of mutant p53 proteins by reducing tumor growth ability. Moreover by using this system, microarray data were validated in vitro and in vivo and putative mutant p53 target genes that may contribute to its gain of function effects in cancer were identified. Results are confirmatory that depletion of mutant p53 protein impacts on tumor malignancy and validated the inducible lentiviral-based system as an efficient tool to study the gain of function activity of human tumor derived p53 mutants.


Clinical symptom presentation in suspected malingerers: an empirical investigation.
To empirically investigate the usefulness and validity of clinical presentation and recent history variables in the detection of malingered psychiatric disorder, 30 criminal defendants involved in forensic evaluations, who had a documented history of psychiatric hospitalization preceding their arrest on the instant offense (low risk of malingering group) and 30 defendants who complained of psychiatric difficulties but had no history of psychiatric hospitalization or treatment (suspected malingering group) were studied. Each subject's mental status was rated, blind to psychiatric history, diagnosis, and psycholegal opinions, on a Likert-like scale for the uncommon nature of their symptom presentation. In addition, the final outcome of the court cases, whether they were found competent to stand trial, not guilty by reason of insanity, or evidenced diminished capacity was determined by obtaining the court disposition in each case. Based on the unusual nature of their presentation, the defendants suspected of malingering were discriminated from the low risk of malingering defendants with a 90 percent rate of correct classification. Suspected malingerers were found to evidence current psychiatric presentations inconsistent with their recent Global Assessment of Functioning, unusual symptom presentation, and hallucinatory experiences rated as atypical for psychiatric disorder. A high proportion of suspected malingerers were found competent to proceed. The results are discussed in terms of the usefulness of clinical identification of malingering.


Models of care for co-morbid diabetes and chronic kidney disease.
Diabetes and chronic kidney disease (CKD) are two of the most prevalent co-morbid chronic diseases in Australia. The increasing complexity of multi-morbidity, and current gaps in health-care delivery for people with co-morbid diabetes and CKD, emphasize the need for better models of care for this population. Previously, proposed published models of care for co-morbid diabetes and CKD have not been co-designed with stake-holders or formally evaluated. Particular components of health-care shown to be effective in this population are interventions that: are structured, intensive and multifaceted (treating diabetes and multiple cardiovascular risk factors); involve multiple medical disciplines; improve self-management by the patient; and upskill primary health-care. Here we present an integrated patient-centred model of health-care delivery incorporating these components and co-designed with key stake-holders including specialist health professionals, general practitioners and Diabetes and Kidney Health Australia. The development of the model of care was informed by focus groups of patients and health-professionals; and semi-structured interviews of care-givers and health professionals. Other distinctives of this model of care are routine screening for psychological morbidity; patient-support through a phone advice line; and focused primary health-care support in the management of diabetes and CKD. Additionally, the model of care integrates with the patient-centred health-care home currently being rolled out by the Australian Department of Health. This model of care will be evaluated after implementation across two tertiary health services and their primary care catchment areas.


Examination of Staphylococcus epidermidis biofilms using flow-cell technology.
A common in vitro method to study Staphylococcus epidermidis biofilm development is to allow the bacteria to attach and grow on a solid surface in the presence of a continuous flow of nutrients. Under these conditions, the bacteria progress through a series of developmental steps, ultimately forming a multicellular structure containing differentiated cell populations. The observation of the biofilm at various time-points throughout this process provides a glimpse of the temporal changes that occur. Furthermore, use of metabolic stains and fluorescent reporters provides insight into the physiologic and transcriptional changes that occur within a developing biofilm. Currently, there are multiple systems available to assess biofilm development, each with advantages and disadvantages depending on the questions being asked. In this chapter, we describe the use of two separate flow-cell systems used to evaluate the developmental characteristics of staphylococcal biofilms: the FC270 flow-cell system from BioSurface Technologies, Corp. and the BioFlux1000 microfluidic flow-cell system from Fluxion Bioscience, Inc.


Epidermal growth factor receptor and variant III targeted immunotherapy.
Immunotherapeutic approaches to cancer have shown remarkable promise. A critical barrier to successfully executing such immune-mediated interventions is the selection of safe yet immunogenic targets. As patient deaths have occurred when tumor-associated antigens shared by normal tissue have been targeted by strong cellular immunotherapeutic platforms, route of delivery, target selection and the immune-mediated approach undertaken must work together to maximize efficacy with safety. Selected tumor-specific targets can spare potential toxicity to normal tissue; however, they are far less common than tumor-associated antigens and may not be present on all patients. In the context of immunotherapy for high-grade glioma, 2 of the most prominently studied antigens are the tumor-associated epidermal growth factor receptor and its tumor-specific genetic deletion variant III. In this review, we will summarize the immune-mediated strategies employed against these targets as well as the caveats particular to these approaches.


Future opportunities and challenges in academic anesthesia in the United Kingdom: a model for maintaining the scientific edge.
This review outlines the methodology of a major report into academic strategy recently undertaken by the Royal College of Anaesthetists in the United Kingdom. Analyzing the factors that made the report's conclusions robust and workable provides lessons for other countries or healthcare systems faced with similar problems in academic anesthesia. The main themes covered by this review include: organization of healthcare and university systems, medical postgraduate training, and funding of research. In the UK, there exists a dual process: clinical service delivery and postgraduate clinical training [which are both the province of the National Health Service (NHS)], and research and undergraduate education (which are both the province of universities). Both NHS and universities are sponsored by the UK government. 'Professors' or 'heads of academic department' in the UK have no automatic responsibility over the activities of the majority of anesthetic consultants in their own hospitals. Anesthesia in the UK has never been a specific unit of assessment in the research granting process, such that anesthetic departments have received little external grant support compared with many other departments such as medicine or cardiology. As a result, anesthetic departments across the UK have either become entrenched as small departments or they have vanished through mergers. The review's main conclusions are: the creation of a central National Institute for Academic Anaesthesia to coordinate and implement academic strategy and funding; engaging with national pathways for the training of future academics; and suggestions for the future role for anesthetic specialist societies in academic strategy. These initiatives can radically transform the research environment in a positive way.


Protective effect of bilberry (Vaccinium myrtillus) against doxorubicin-induced oxidative cardiotoxicity in rats.
Doxorubicin (DOX) is a commonly used chemotherapeutic agent. It is associated with serious dose-limiting cardiotoxicity, which is at least partly caused by generation of reactive oxygen species (ROS). Supplementations with bilberries were effective in reducing oxidative stress in many tissue injuries due their high content of antioxidants. The present study investigated the potential protective effect of bilberry extract against DOX-induced cardiotoxicity in rats. Rats were treated orally with a methanolic extract of bilberry for 10 days. DOX was injected intraperitoneally on day 7. Twenty-four hours after the last bilberry administration, rats were subjected to ECG study. Blood was then withdrawn and cardiac tissues were dissected for assessment of oxidative stress and cardiac tissue injury. Cardiac tissues were also subjected to histopathological examination. Bilberry extract significantly inhibited DOX-provoked reduced glutathione depletion and accumulation of oxidized glutathione, malondialdehyde and protein carbonyls in cardiac tissues. This was accompanied by significant amelioration of reduced cardiac catalase, superoxide dismutase, and glutathione peroxidase activities; and increased cardiac myeloperoxidase activity in response to DOX challenge. Pretreatment with bilberry significantly guarded against DOX-induced increase in serum activities of lactate dehydrogenase, creatine phosphokinase and creatine kinase-MB, as well as the level of troponin I. Bilberry alleviated ECG changes in rats treated with DOX and attenuated its pathological changes. Bilberry protects against DOX-induced cardiotoxicity in rats. This can be attributed, at least in part, to its antioxidant activity.


Novel protein targeted therapy of metastatic melanoma.
Metastatic melanoma patients have a dismal prognosis with poor responsiveness to chemotherapy, radiation therapy and current immunotherapy regimens and a median survival of less than six months. Novel therapies directed at melanoma-selective molecular targets are urgently needed. Based on the frequent constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway in malignant melanomas and the selective inhibition of MAPK signaling by anthrax lethal factor which proteolytically cleaves MAPK kinases, anthrax lethal toxin may be a useful agent for patients with metastatic melanoma. Anthrax lethal toxin consists of two proteins--protective antigen and lethal factor. These two proteins have been separately produced in good yields and in high purity. The three-dimensional structures of these proteins have been solved, and their molecular mechanisms of cell binding and action determined. Preclinical studies with anthrax lethal toxin show sensitivity of malignant melanoma cell lines in tissue culture and anti-tumor efficacy in melanoma xenograft models. Additional studies to define the maximal tolerated doses and dose-limiting toxicity of anthrax lethal toxin in rodent and primate models should pave the way for phase I studies testing the efficacy of the anthrax lethal toxin in patients with metastatic melanoma.


A predictive model of bifunctional transcription factor signaling during embryonic tissue patterning.
Hedgehog signaling controls pattern formation in many vertebrate tissues. The downstream effectors of the pathway are the bifunctional Gli transcription factors, which, depending on hedgehog concentration, act as either transcriptional activators or repressors. Quantitatively understanding the interplay between Gli activator and repressor forms for patterning complex tissues is an open challenge. Here, we describe a reductionist mathematical model for how Gli activators and repressors are integrated in space and time to regulate transcriptional outputs of hedgehog signaling, using the pathway readouts Gli1 and Ptch1 as a model system. Spatially resolved measurements of absolute transcript numbers for these genes allow us to infer spatiotemporal variations of Gli activator and repressor levels. We validate our model by successfully predicting expression changes of Gli1 and Ptch1 in mutants at different developmental stages and in different tissues. Our results provide a starting point for understanding gene regulation by bifunctional transcription factors during mammalian development.


Effectiveness of breast massage for the treatment of women with breastfeeding problems: a systematic review.
The aim of this systematic review was to identify the effectiveness of breast massage as a treatment for women with breastfeeding problems. More specifically, the objective was to identify if breast massage as an intervention led to less pain or increased milk supply, or assisted in a reduction or resolution of blocked ducts, breast engorgement and mastitis. Breastfeeding protects babies against many illnesses, and the health benefits for women have been well documented. However, breastfeeding rates steadily drop to approximately 15% by six months, which is the World Health Organization's recommended length of time for exclusive breastfeeding. Breastfeeding problems such as blocked ducts, breast engorgement and mastitis are major complications attributing to the decline in breastfeeding rates. Breast massage may relieve pain and resolve symptoms associated with conditions that contribute to discontinued breastfeeding. This review considered both experimental and epidemiological study designs and included breastfeeding women of any age, parity or geographical location. The types of interventions considered for inclusion were any type of breast massage that was offered to women for breastfeeding problems. Comparators included the usual care provided to women with breastfeeding problems. Primary outcomes of interest were an increase in breast milk supply, reduction of breast pain, and symptom resolution of blocked ducts, engorgement and mastitis. Secondary outcomes included duration of breastfeeding. Studies published from 1980 to 2017 in English and Japanese were considered for inclusion in this review. The databases searched with the majority of results included CINAHL, Cochrane Library, Embase, PubMed, Science Direct, Scopus and Web of Science. Search for unpublished studies included Google Scholar, ClinicalTrials.gov and ProQuest Dissertations and Theses. There were six studies included in this review: three randomized controlled trials and three quasi-experimental studies. There was considerable heterogeneity of study outcome measures, and the use of unvalidated tools in many of the studies led to the inability to pool the results. Furthermore, the heterogeneity of the interventions themselves coupled with small sample sizes for each study greatly decreased generalizability of the outcomes and reduced the overall effectiveness of the interventions. However, all included studies reported a reduction in pain regardless of the breast massage technique used. Overall, varying types of breast massage were helpful in reducing immediate pain and resolving symptoms. Overall, different types of breast massage were reported as effective in reducing immediate pain for the participants. However, the lack of detailed explanation of the breast massage technique and the extensive training needed to undertake the breast massage decrease the ability to replicate the results. These outcomes may be useful for healthcare professionals caring for women with breastfeeding problems. Future research needs include validating a universal measurement tool for breastfeeding problems and the need for more robust randomized controlled trials, particularly in vulnerable groups such as mothers of preterm infants. Longer follow-up periods are also suggested to establish if breast massage impacts breastfeeding duration.


Vitamin D deficiency promotes growth of MCF-7 human breast cancer in a rodent model of osteosclerotic bone metastasis.
Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice. The effects of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on proliferation and apoptosis of MCF-7 breast cancer cells, and changes in the expression of genes within the vitamin D metabolic pathway (VDR, 1α- and 24-hydroxylase) were examined in vitro. MCF-7 breast cancer cells were injected intra-tibially into vitamin D deficient and vitamin D sufficient mice co-treated with and without osteoprotegerin (OPG). The development of tumor-related lesions was monitored via serial X-ray analysis. Tumor burden and indices of proliferation and apoptosis were determined by histology along with markers of bone turnover and serum intact PTH levels. In vitro, MCF-7 cells expressed critical genes for vitamin D signalling and metabolism. Treatment with 1,25(OH)(2)D(3) inhibited cell growth and proliferation, and increased apoptosis. In vivo, osteosclerotic lesions developed faster and were larger at endpoint in the tibiae of vitamin D deficient mice compared to vitamin D sufficient mice (1.49±0.08 mm(2) versus 1.68±0.15 mm(2), P<0.05). Tumor area was increased by 55.8% in vitamin D deficient mice (0.81±0.13 mm(2) versus 0.52±0.11 mm(2) in vitamin D sufficient mice). OPG treatment inhibited bone turnover and caused an increase in PTH levels, while tumor burden was reduced by 90.4% in vitamin D sufficient mice and by 92.6% in vitamin D deficient mice. Tumor mitotic activity was increased in the tibiae of vitamin D deficient mice and apoptosis was decreased, consistent with faster growth. Vitamin D deficiency enhances both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors.


Oxysterol mixture in hypercholesterolemia-relevant proportion causes oxidative stress-dependent eryptosis.
Oxysterol activity on the erythrocyte (RBC) programmed cell death (eryptosis) had not been studied yet. Effects of an oxysterol mixture in hyper-cholesterolemic-relevant proportion, and of individual compounds, were investigated on RBCs from healthy humans. Membrane phosphatidylserine (PS) externalization, calcium entry, ROS production, amino-phospholipid translocase (APLT) activity were evaluated by cytofluorimetric assays, cell volume from forward scatter. Prostaglandin PGE2 was measured by ELISA; GSH-adducts and lipoperoxides by spectrophotometry. Involvement of protein kinase C and caspase was investigated by inhibitors staurosporin, calphostin C, and Z-DEVD-FMK, respectively. Oxysterols caused PS externalization and cell shrinkage, associated with PGE2release, opening of PGE2-dependent calcium channels, ROS production, GSH depletion, membrane lipid oxidation. Addition of antioxidants prevented Ca(2+) influx and eryptosis. Calcium removal prevented cell shrinkage, with small effect (-20%) on the PS exposure, whereas ROS generation was unaltered. Either in the presence or absence of calcium i) oxysterols inhibited APLT, ii) staurosporin, calphostin C, Z-DEVD-FMK blunted and iii) antioxidants fully prevented the oxysterol-induced PS externalization. Only 7-ketocholesterol and cholestan-3β,5α,6β-triol were individually active. Eryptosis was observed in RBCs isolated after ex vivo spiking of human whole blood with the oxysterol mixture. Oxysterols induce an oxidative stress-dependent eryptosis, involving calcium-independent mechanisms. Eryptotic activity of oxysterols may be relevant in vivo.


Cytokines and angiogenesis in the corpus luteum.
In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.


A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis.
Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA. 1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied. We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10-4). This variant was also significant after Bonferroni correction. These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.


Analysis of reentrant two-mirror nonplanar ring laser cavity.
A rigorous analysis is performed on the reentrant nonplanar ring laser cavity constructed by a Herriott-type multipass cell. Since the cavity is highly nonplanar, the angle between the incident planes at each reflection becomes different from that of the image rotation angles. The beam rotation, astigmatism, and spherical aberration are considered to obtain a self-consistent solution of the Gaussian beam. It turns out that spherical aberration is an important issue for this nonplanar resonator. Without taking into account the spherical aberration, a stable resonator would be difficult to realize. Using a self-consistent Gaussian beam propagation method, the laser beam characteristics are solved analytically. The results are compared with that of the 2 x 2 ABCD method.


Unilateral adrenalectomy can be an alternative therapy for infantile onset Cushing' s syndrome caused by ACTH-independent macronodular adrenal hyperplasia with McCune-Albright syndrome.
We report herein the case of a 1-year-old boy with McCune-Albright syndrome (MAS) who presented with infantile-onset Cushing' s syndrome caused by ACTH independent macronodular adrenal hyperplasia (AIMAH). Abdominal CT, MRI, and adrenal scintigraphy with (131)I-adosterol identified bilateral adrenal involvement with the left adrenal gland being larger and functionally more active. Unilateral adrenalectomy of the left gland was performed and ameliorated many clinical symptoms, such as Cushingoid appearance and height restriction, and it also normalized many endocrinological data, such as diurnal rhythms of ACTH and cortisol, ACTH and cortisol responses to CRH, and urinary 24 hr free cortisol. Glucocorticoid was replaced for the first 1 year and 6 months after the operation. One adrenal crisis episode occurred at 3 weeks after the operation, but none have occurred since. These results suggest that unilateral adrenalectomy of the larger gland can be an alternative therapy for infantile onset Cushing' s syndrome caused by AIMAH with MAS, when asymmetric involvement is evident and the smaller gland is not markedly enlarged.


Isolated cutaneous sarcoidosis: a new insight into the old entity.
Cutaneous lesions are a part of common manifestation in multi-systemic sarcoidosis and skin involvement occurs in 20% to 35% of the patients with systemic disease. An exclusive cutaneous involvement is rare and it is reported in about 4%-5% of the patients of sarcoidosis. These lesions have been classified into specific and non-specific, depending on the presence of non- caseating granulomas, on histopathologic studies. In the present study, we are reporting two cases of isolated cutaneous sarcoidosis without systemic manifestations, which is a rare presentation. Sarcoidosis will continue to challenge even the most experienced clinicians with respect to a high degree of suspicion and further investigations for diagnosing and treating this disorder.


Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor.
Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1–TM3 (G31–R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1–TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1–TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1–TM3 in 50% TFE:water have been achieved.