Pathways between Socioeconomic Disadvantage and Childhood Growth in the Scottish Longitudinal Study, 1991-2001.
Socioeconomically disadvantaged children are more likely to be of shorter stature and overweight, leading to greater risk of obesity in adulthood. Disentangling the mediatory pathways between socioeconomic disadvantage and childhood size may help in the development of appropriate policies aimed at reducing these health inequalities. We aimed to elucidate the putative mediatory role of birth weight using a representative sample of the Scottish population born 1991-2001 (n = 16,628). Estimated height and overweight/obesity at age 4.5 years were related to three measures of socioeconomic disadvantage (mother's education, Scottish Index of Multiple Deprivation, synthetic weekly income). Mediation was examined using two approaches: a 'traditional' mediation analysis and a counterfactual-based mediation analysis. Both analyses identified a negative effect of each measure of socioeconomic disadvantage on height, mediated to some extent by birth weight, and a positive 'direct effect' of mother's education and Scottish Index of Multiple Deprivation on overweight/obesity, which was partly counterbalanced by a negative 'indirect effect'. The extent of mediation estimated when adopting the traditional approach was greater than when adopting the counterfactual-based approach because of inappropriate handling of intermediate confounding in the former. Our findings suggest that higher birth weight in more disadvantaged groups is associated with reduced social inequalities in height but also with increased inequalities in overweight/obesity.


Risk Factors for Treatment Failure and Recurrence after Metronidazole Treatment for Clostridium difficile-associated Diarrhea.
The incidence of treatment failure or recurrence of Clostridium difficile-associated diarrhea (CDAD) following metronidazole treatment has increased recently. We studied the treatment failure, recurrence rate, and risk factors predictive of treatment failure and recurrence after metronidazole treatment for CDAD. We retrospectively identified consecutive patients who were admitted and treated for CDAD at a single tertiary institution in Korea over a recent 10-year period (i.e., 1998-2008). Metronidazole was administered as the initial treatment to 111 of 117 patients (94.9%) with CDAD. Fourteen patients (12.6%) had no clinical response to the metronidazole treatment, and in 13 patients (13.4%) CDAD recurred after successful metronidazole treatment. Diabetes mellitus (p=0.014) and sepsis (p=0.002) were independent risk factors for metronidazole treatment failure. Patients who had received surgery within 1 month before CDAD developed were more likely to experience a recurrence after metronidazole treatment (p=0.032). Vancomycin exhibited a higher response rate after treatment failure, and metronidazole showed a reasonable response rate in the treatment of recurrence. Treatment failure and recurrence rates increased with time after metronidazole treatment for CDAD over the 10-year study period. Our data suggest that diabetes mellitus and sepsis are independent risk factors for metronidazole treatment failure, and that operation history within 1 month of development of CDAD is a predictor of a recurrence after metronidazole treatment.


Effect of fucoidan extracted from mozuku on experimental cartilaginous tissue injury.
We investigated the effect of fucoidan, a sulfated polysaccharide, on acceleration of healing of experimental cartilage injury in a rabbit model. An injured cartilage model was surgically created by introduction of three holes, one in the articular cartilage of the medial trochlea and two in the trochlear sulcus of the distal femur. Rabbits in three experimental groups (F groups) were orally administered fucoidan of seven different molecular weights (8, 50, 146, 239, 330, 400, or 1000 kD) for 3 weeks by screening. Control (C group) rabbits were provided water ad libitum. After the experimental period, macroscopic examination showed that the degree of filling in the fucoidan group was higher than that in the C group. Histologically, the holes were filled by collagen fiber and fibroblasts in the C group, and by chondroblasts and fibroblasts in the F groups. Image analysis of Alcian blue- and safranin O-stained F-group specimens showed increased production of glycosaminoglycans (GAGs) and proteoglycans (PGs), respectively. Some injured holes were well repaired both macroscopically and microscopically and were filled with cartilage tissues; cartilage matrices such as PGs and GAGs were produced in groups F 50, F 146, and F 239. Thus, fucoidan administration enhanced morphologically healing of cartilage injury.


Different Severity and Severity Predictors in Early-Onset and Late-Onset Asthma: A Taiwanese Population-Based Study.
Late-onset asthma has been shown to be more severe than early-onset asthma in clinic-based studies. However, population-based studies are scarce, and the predictors of severity have been less studied. To determine asthma severity and severity predictors regarding age at onset. A cross-sectional questionnaire survey was conducted among parents of children from 94 schools in Taiwan in 2004. Asthma severity was defined as short-acting β2-agonist (SABA), inhaled corticosteroid (ICS) and health care use in the last year. Information on age at onset, demographics, heredity and home exposure was collected. Ordered logistic or logistic regression was used for determining the associations between risk factors and severity. Participants aged 26-50 years were included, resulting in 21,057 (67.8%) participants. Among them, 449 reported ever having had physician-diagnosed asthma, and 381 of those subjects answered the question on age at asthma onset. The risks of rescue SABA, ICS and health care use were generally higher among late-onset (13-50 years) than early-onset (0-12 years) asthmatics. Use of SABA and health care increased from childhood-onset, adolescent- or young adult-onset to adult-onset asthma. Allergic rhinitis was positively associated with SABA use (OR, 9.08; 95% CI, 1.06-77.99) and ICS use (OR, 5.08; 95% CI, 1.47-17.52) in early-onset asthma. Dehumidifier use was negatively associated with SABA use (OR, 0.50; 95% CI, 0.29-0.87) and ICS use (OR, 0.38; 95% CI, 0.19-0.78) in late-onset asthma. In adults, late-onset asthma was more severe than early-onset asthma. Severity, as indicated by SABA and ICS use, was positively associated with allergic rhinitis in early-onset asthma and negatively associated with dehumidifier use in late-onset asthma.


Bilateral patching in retinal detachment: fluid mechanics and retinal "settling".
When a patient suffers a retinal detachment and surgery is delayed, it is known clinically that bilaterally patching the patient may allow the retina to partially reattach or "settle." Although this procedure has been performed since the 1860s, there is still debate as to how such a maneuver facilitates the reattachment of the retina. Finite element calculations using commercially available analysis software are used to elucidate the influence of reduction in eye movement caused by bilateral patching on the flow of subretinal fluid in a physical model of retinal detachment. It was found that by coupling fluid mechanics with structural mechanics, a physically consistent explanation of increased retinal detachment with eye movements can be found in the case of traction on the retinal hole. Large eye movements increase vitreous traction and detachment forces on the edge of the retinal hole, creating a subretinal vacuum and facilitating increased subretinal fluid. Alternative models, in which intraocular fluid flow is redirected into the subretinal space, are not consistent with these simulations. The results of these simulations explain the physical principles behind bilateral patching and provide insight that can be used clinically. In particular, as is known clinically, bilateral patching may facilitate a decrease in the height of a retinal detachment. The results described here provide a description of a physical mechanism underlying this technique. The findings of this study may aid in deciding whether to bilaterally patch patients and in counseling patients on pre- and postoperative care.


Simultaneous immunohistochemical detection of gangliosides and neuronal markers in paraformaldehyde-fixed nervous tissues by acetone etching.
A need for identifying ganglioside-positive cells with neuronal markers prompted us to establish a reliable method for double or triple immunostaining nervous tissues. Perfusion fixation with paraformaldehyde is typically performed for the routine immunostaining of various neuronal markers but is not suitable for immunostaining gangliosides. Acetone fixation of fresh cryosections is frequently used for ganglioside immunodetection; thus, we tested the effect of acetone treatment for unmasking the antigen epitope of gangliosides (acetone etching) on sections of paraformaldehyde-fixed nervous tissue from rats. Acetone etching significantly retrieved ganglioside immunoreactivity while preserving the immunoreactivity of neuronal markers. Various combinations of gangliosides and neuronal markers could be double-stained by the immunoenzyme method or triple-stained by the immunofluorescence method. This new method may provide additional information regarding the relationship between gangliosides and various neuronal markers from routinely paraformaldehyde-fixed nervous tissues, both freshly prepared specimens and those stocked in the laboratory.


Molecular markers of dihydroartemisinin-piperaquine resistance in northwestern Thailand.
Dihydroartemisinin-piperaquine (DHA-PPQ) combination therapy is the current first-line treatment for Plasmodium falciparum malaria in Thailand. Since its introduction in 2015, resistance to this drug combination has emerged in the eastern part of the Greater Mekong Subregion including the eastern part of Thailand near Cambodia. This study aimed to assess whether the resistance genotypes have arisen the western part of country. Fifty-seven P. falciparum-infected blood samples were collected in Tak province of northwestern Thailand between 2013 and 2019. Resistance to DHA was examined through the single nucleotide polymorphisms (SNPs) of kelch13. PPQ resistance was examined through the copy number plasmepsin-2 and the SNPs of Pfcrt. Among the samples whose kelch13 were successfully sequenced, approximately half (31/55; 56%) had mutation associated with artemisinin resistance, including G533S (23/55; 42%), C580Y (6/55; 11%), and G538V (2/55; 4%). During the study period, G533S mutation appeared and increased from 20% (4/20) in 2014 to 100% (9/9) in 2019. No plasmepsin-2 gene amplification was observed, but one sample (1/54) had the Pfcrt F145I mutation previously implicated in PPQ resistance. Kelch13 mutation was common in Tak Province in 2013-2019. A new mutation G533S emerged in 2014 and rose to dominance in 2019. PPQ resistance marker Pfcrt F145I was also detected in 2019. Continued surveillance of treatment efficacy and drug resistance markers is warranted.


Calcium, vitamin D, and risk for colorectal adenoma: dependency on vitamin D receptor BsmI polymorphism and nonsteroidal anti-inflammatory drug use?
Previous epidemiological studies have been inconclusive in demonstrating an inverse association among calcium, vitamin D, and risk for colorectal adenoma. The purpose of this analysis was to evaluate the associations among calcium and vitamin D and risk for incident, sporadic colorectal adenoma according to the vitamin D receptor BsmI polymorphism and nonsteroidal anti-inflammatory drug (NSAID) use. We analyzed data from a colonoscopy-based case-control study (n = 177 cases, 228 controls) conducted in North Carolina between 1995 and 1997. Adjusted odds ratios (ORs) comparing participants in the highest to those in the lowest tertiles of total calcium and vitamin D intakes were 0.64 [95% confidence interval (CI), 0.35-1.15], P(trend) = 0.14 and 0.69 (95% CI, 0.41-1.18), and P(trend) = 0.19, respectively. Adjusted ORs for those in the upper tertile of total calcium intake relative to those in the lower were 0.25 (95% CI, 0.08-0.80) among those who had a Bb genotype, 0.57 (95% CI, 0.18-1.82) among those who had a bb genotype, and 0.36 (95% CI, 0.15-0.85) among those who did not take NSAIDs. The ORs for the highest tertile of calcium intake was 0.05 (95% CI, 0.01-0.41), P(trend) < 0.01 among those who were Bb and did not take NSAIDs, and 0.16 (95% CI, 0.02-1.36), P(trend) = 0.47 among those who were bb and did not take NSAIDs. These data support the hypotheses that higher calcium intakes may decrease risk for colorectal neoplasms, and that such a relationship is more readily detectable among those who do not take NSAIDs, and may be strongest among those who have at least one vitamin D receptor BsmI b allele.


Variation in Use of Tympanostomy Tubes: Impact of Privately Owned Ambulatory Surgery Centers.
To determine whether privately owned ambulatory surgery centers (ASCs) increase pediatric tympanostomy tube use in their surrounding communities. We studied children <5 years of age who underwent outpatient tympanostomy tube placement in New York or Florida in 2010-2014. Data came from the Healthcare Cost and Utilization Project State Ambulatory Surgery Databases, which include all outpatient surgeries in these states. Population characteristics came from the US Census' American Community Survey. Weighted conditionally autoregressive models were used to assess the association between the zip code-level proportion of tympanostomy tube procedures performed in privately owned ASCs and the rate of tympanostomy tube use. In 2010-2014, 106 privately owned ASCs in Florida and 29 in New York performed tympanostomy tube placement in young children. After accounting for zip code-level urban/rural status, socioeconomic status (SES), and the proportion of residents of non-Hispanic white race, children residing in zip codes in the top tertile of privately owned ASC use in Florida had 52% greater tympanostomy tube use than children from zip codes in the bottom tertile (P < .001). In New York, high-SES zip codes with any use of privately owned ASCs had 2.6 times greater tympanostomy tube use than other high-SES zip codes (P < .001). This association was not present in low-SES areas. The presence of privately owned ASCs is associated with increased tympanostomy tube use in young children.


Microextraction sample preparation techniques in biomedical analysis.
Biologically active compounds are found in biological samples at relatively low concentration levels. The sample preparation of target compounds from biological, pharmaceutical, environmental, and food matrices is one of the most time-consuming steps in the analytical procedure. The microextraction techniques are dominant. Metabolomic studies also require application of proper analytical technique for the determination of endogenic metabolites present in biological matrix on trace concentration levels. Due to the reproducibility of data, precision, relatively low cost of the appropriate analysis, simplicity of the determination, and the possibility of direct combination of those techniques with other methods (combination types on-line and off-line), they have become the most widespread in routine determinations. Additionally, sample pretreatment procedures have to be more selective, cheap, quick, and environmentally friendly. This review summarizes the current achievements and applications of microextraction techniques. The main aim is to deal with the utilization of different types of sorbents for microextraction and emphasize the use of new synthesized sorbents as well as to bring together studies concerning the systematic approach to method development. This review is dedicated to the description of microextraction techniques and their application in biomedical analysis.


Lessons and implications from association studies and post-GWAS analyses of cervical cancer.
Cervical cancer has a heritable genetic component. A large number of genetic associations with cervical cancer have been reported in hypothesis-driven candidate gene studies, but many of these results are either inconsistent or have failed to be independently replicated. Genome-wide association studies (GWAS) have identified additional susceptibility loci previously not implicated in cervical cancer development, highlighting the power of genome-wide unbiased association analyses. Post-GWAS analyses including pathway-based analysis and functional characterization of associated variants have provided new insights into the pathogenesis of cervical cancer. In this review we summarize findings from candidate gene association studies, GWAS, and post-GWAS analyses of cervical cancer. We also discuss gaps in our understanding, possible clinical implications of the findings, and lessons for studies of other complex diseases.


High frequency photoacoustic imaging for in vivo visualizing blood flow of zebrafish heart.
A technique on high frame rate(28fps), high frequency co-registered ultrasound and photoacoustic imaging for visualizing zebrafish heart blood flow was demonstrated. This approach was achieved with a 40MHz light weight(0.38g) ring-type transducer, serving as the ultrasound transmitter and receiver, to allow an optic fiber, coupled with a 532nm laser, to be inserted into the hole. From the wire target study, axial resolutions of 38µm and 42µm were obtained for ultrasound and photoacoustic imaging, respectively. Carbon nanotubes were utilized as contrast agents to increase the flow signal level by 20dB in phantom studies, and zebrafish heart blood flow was successfully observed.


Dynamic dilution exponent in monodisperse entangled polymer solutions.
We study and model the linear viscoelastic properties of several entangled semi-dilute and concentrated solutions of linear chains of different molar masses and at different concentrations dissolved in their oligomers. We discuss the dilution effect of the oligomers on the entangled long chains. In particular, we investigate the influence of both concentration and molar mass on the value of the effective dynamic dilution exponent determined from the level of the storage plateau at low and intermediate frequencies. We show that the experimental results can be quantitatively explained by considering the tension re-equilibration process along the chains, in agreement with van Ruymbeke et al. (Macromol., 2014), i.e. by considering that the real dilution exponent α is always equal to 1, while larger values of the dilution exponent (1 < α < 1.3) found experimentally are attributed to the enhanced relaxation of the long chain extremities. Then we discuss the influence of the polymer concentration on the terminal relaxation time of the solutions and how this can be modelled by the enhanced contour length fluctuation process (CR-CLF). We point out that this larger dilution effect is not only a function of concentration but also depends on the molar mass of the chains. While the proposed approach successfully explains the viscoelastic properties of a large number of semi-dilute solutions of polymers in their own oligomers, important discrepancies are found for semi-dilute entangled polymers in small-molecule theta or good solvents. Possible explanations for the differences between these sample sets are proposed, based on the comparison of their viscoelastic behavior.


Type 2 diabetes impairs insulin receptor substrate-2-mediated phosphatidylinositol 3-kinase activity in primary macrophages to induce a state of cytokine resistance to IL-4 in association with overexpression of suppressor of cytokine signaling-3.
Chronic elevation of proinflammatory markers in type 2 diabetes (T2D) is well defined, but the role of anti-inflammatory cytokines in T2D is less clear. In this study, we report that normal IL-4-dependent elaboration of IL-1 receptor antagonist (IL-1RA) requires IRS-2-mediated PI3K activity in primary macrophages. We also show that macrophages isolated from obese/diabetic db/db mice have impaired IRS-2-mediated PI3K activity and constitutively overexpress suppressor of cytokine signaling (SOCS)-3, which impairs an important IL-4 anti-inflammatory function. Peritoneal proinflammatory cytokine levels were examined in diabese (db/db) mice, and IL-6 was found to be nearly 7-fold higher than in nondiabese (db/+) control mice. Resident peritoneal macrophages were isolated from db/db mice and were found to constitutively overexpress IL-6 and were unable to elaborate IL-1RA in response to IL-4-like db/+ mouse macrophages. Inhibition of PI3K with wortmannin or blockage of IRS-2/PI3K complex formation with a cell permeable IRS-2-derived tyrosine phosphopeptide inhibited IL-4-dependent IL-1RA production in db/+ macrophages. Examination of IL-4 signaling in db/db macrophages revealed that IL-4-dependent IRS-2/PI3K complex formation and IRS-2 tyrosine phosphorylation was reduced compared with db/+ macrophages. SOCS-3/IL-4 receptor complexes, however, were increased in db/db mouse macrophages compared with db/+ mice macrophages as was db/db mouse macrophage SOCS-3 expression. These results indicate that in the db/db mouse model of T2D, macrophage expression of SOCS-3 is increased, and impaired IL-4-dependent IRS-2/PI3K formation induces a state of IL-4 resistance that disrupts IL-4-dependent production of IL-1RA.


Reporting items for capillaroscopy in clinical research on musculoskeletal diseases: a systematic review and international Delphi consensus.
The level of detail included when describing nailfold videocapillaroscopy (NVC) methods varies among research studies, making interpretation and comparison of results challenging. The overarching objective of the present study was to seek consensus on the reporting standards in NVC methodology for clinical research in rheumatic diseases and to propose a pragmatic reporting checklist. Based on the items derived from a systematic review focused on this topic, a three-step web-based Delphi consensus on minimum reporting standards in NVC was performed among members of the European League against Rheumatism (EULAR) Study Group on Microcirculation in Rheumatic Diseases and the Scleroderma Clinical Trials Consortium. A total of 319 articles were selected by the systematic review, and 46 items were proposed in the Delphi process. This Delphi exercise was completed by 80 participants from 31 countries, including Australia and countries within Asia, Europe, North America and South America. Agreement was reached on items covering three main areas: patient preparation before NVC (15 items), device description (5 items) and examination details (13 items). Based on the available evidence, the description of NVC methods was highly heterogeneous in the identified studies and differed markedly on several items. A reporting checklist of 33 items, based on practical suggestions made (using a Delphi process) by international participants, has been developed to provide guidance to improve and standardize the NVC methodology to be applied in future clinical research studies.


Dosimetric evaluation of four-dimensional dose distributions of CyberKnife and volumetric-modulated arc radiotherapy in stereotactic body lung radiotherapy.
Advanced image-guided stereotatic body lung radiotherapy techniques using volumetric-modulated arc radiotherapy (VMAT) with four-dimensional cone-beam computed tomography (4D CBCT) and CyberKnife with real-time target tracking have been clinically implemented by different authors. However, dosimetric comparisons between these techniques are lacking. In this study, 4D CT scans of 14 patients were used to create VMAT and CyberKnife treatment plans using 4D dose calculations. The GTV and the organs at risk (OARs) were defined on the end-exhale images for CyberKnife planning and were then deformed to the midventilation images (MidV) for VMAT optimization. Direct 4D Monte Carlo dose optimizations were performed for CyberKnife (4D(CK)). Four-dimensional dose calculations were also applied to VMAT plans to generate the 4D dose distributions (4D(VMAT)) on the exhale images for direct comparisons with the 4D(CK) plans. 4D(CK) and 4D(VMAT) showed comparable target conformity (1.31 ± 0.13 vs. 1.39 ± 0.24, p = 0.05). GTV mean doses were significantly higher with 4D(CK). Statistical differences of dose volume metrics were not observed in the majority of OARs studied, except for esophagus, with 4D(VMAT) yielding marginally higher D1% than 4D(CK). The normal tissue volumes receiving 80%, 50%, and 30% of the prescription dose (V80%, V50%, and V30%) were higher with 4D(VMAT), whereas 4D(CK) yielded slightly higher V10% in posterior lesions than 4D(VMAT). VMAT resulted in much less monitor units and therefore greater delivery efficiency than CyberKnife. In general, it was possible to produce dosimetrically acceptable plans with both techniques. The selection of treatment modality should consider the dosimetric results as well as the patient's tolerance of the treatment process specific to the SBRT technique.


Epidemiological and genetic observations on human schistosomiasis in Kinshasa, Zaire.
A survey for Schistosoma intercalatum conducted in Kinshasa, Zaire, in September 1994 revealed a prevalence of 3.6% (n = 167). Three isolates of schistosomes were made by exposing Bulinus wrighti to miracidia hatched from eggs collected from 2 infected children. Characterization of the isolates by biochemical (isoenzymes of phosphoglucomutase), molecular (restriction fragment length polymorphism and randomly amplified polymorphic deoxyribonucleic acid analysis) and morphological (egg measurements) techniques confirmed the existence of an autochthonous transmission focus of S. intercalatum in Kinshasa. The study also provided evidence of the occurrence of natural hybridization between S. intercalatum and S. haematobium. No potential snail host for either species was found in the 2 rivers examined. Apart from Bu. globosus from Zambia and Bu. wrighti, snail infection experiments showed an incompatible relationship between the parasite isolates and snails belonging to the Bu. forskalii group, the Bu. iruncatus/Bu. tropicus complex, and the Bu. africanus group.


Conformational flexibility and biological activity of salmon calcitonin.
We have assessed the biological activity of salmon calcitonin I (sCT) using an in vivo biological assay of hypocalcemic activity in rats. The changes in biological activity observed are explained on the basis of changes in the conformational properties of the hormone analogues. Helical content in the presence and absence of lipids and detergents was assessed by using circular dichroism, and the section of the molecule that folds into a helix was predicted on the basis of the helix-coil transition theory of Mattice and co-workers. In the amino acid sequence of sCT, residue 8 is valine and residue 16 is leucine. The synthetic calcitonin derivatives [Gly8]sCT and [Ala16]sCT have higher biological activity than the native hormone although they have a lower helical content. The increased biological activity of these derivatives is ascribed to an increase in their conformational flexibility resulting from the substitution of amino acid residues with less bulky side chains and less tendency to form helical structures. The derivative [Met8]sCT has less substitution than sCT on the beta-carbon at position 8, but it has increased helix-forming potential in the region of residues 8-12. These two factors affect conformational flexibility in opposite ways, resulting in the biological activity of [Met8]sCT being slightly higher than that of sCT. However, increased conformational flexibility does not always increase biological activity. Substitution of the L-arginine at residue 24 for a D-arginine has little effect on the conformational properties or biological activity of sCT. However, [Gly8, D-Arg24]sCT is less active than sCT, [Gly8]sCT, or [D-Arg24]sCT.(ABSTRACT TRUNCATED AT 250 WORDS)


Mean Propulsive Velocity Is a Viable Method for Adjusting the Resistance-Training Load at Moderate Altitude.
We examined the viability of using mean propulsive velocity (MPV) to adjust the load in the countermovement jump (CMJ) at moderate altitude. Twenty-four volunteers were assigned to a 4-week power-oriented resistance training (RT) program in either normoxia (N, 690 m) or intermittent hypobaric hypoxia (IH, 2,320 m). The load was adjusted to maintain execution velocity of CMJ at 1m·s-1 of MPV. Relative peak power output (Prel), and percentage of velocity loss throughout the sets (VL) were determined for each session. The internal load was measured by the rating of perceived exertion (RPE). The absolute load lifted was higher in IH compared to N (75.6 ± 8.4 vs. 58.5 ± 12.3 kg P < 0.001). However, similar relative increases for both groups were found when comparing the final values (IH: 8.2%, P = 0.007; N: 9.8%, P = 0.03) with no changes in VL between groups (P = 0.36). Post-study Prel improved significantly only in IH (+7% W·kg-1, P = 0.002). Mean RPE was greater in IH vs. N (6.8 ± 1.5 vs. 5.6 ± 2, P < 0.001). The MPV seems to be a viable method for adjusting external load during RT at moderate altitude. However, given that RT at moderate altitude increases RPE, it is prudent to monitor internal load when using the MPV to best determine the actual physiological stress of the session.


Caffeic acid phenethyl ester inhibits alpha-melanocyte stimulating hormone-induced melanin synthesis through suppressing transactivation activity of microphthalmia-associated transcription factor.
Caffeic acid phenethyl ester (1), a natural compound found in various plants and propolis, is a well-known anti-inflammatory, immunomodulatory, and cytotoxic agent. The present study aimed to investigate the molecular events underlying the antimelanogenic activity of 1 in alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16-F10 melanoma cells. In this investigation, 1 effectively reduced α-MSH-stimulated melanin synthesis by suppressing expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2), although this compound did not directly inhibit tyrosinase enzyme activity. On the other hand, the expression and nuclear translocation of microphthalmia-associated transcription factor (MITF) as a key transcription factor for tyrosinase expression regulating melanogenesis were not affected by treatment with 1. The upstream signaling pathways including cAMP response element-binding protein (CREB), glycogen synthase kinase-3β (GSK-3β), and Akt for activation and expression of MITF were also not influenced by 1. Interestingly, 1 inhibited transcriptional activity of a tyrosinase promoter by suppressing the interaction of MITF protein with an M-box containing a CATGTG motif on the tyrosinase promoter. Given the important role of MITF in melanogenesis, suppression of 1 on the function of MITF to transactivate tyrosinase promoter may present a novel therapeutic approach to treat hyperpigmentation disorders.


Leptin in anorexia nervosa and bulimia nervosa: importance of assay technique and method of interpretation.
Studies of the role of leptin in patients with anorexia nervosa and bulimia nervosa have conflicted in their data and interpretation. Such differences may be a result of the assay methods used or the way results are compared with those from normal controls. To investigate these possibilities, we analyzed serum leptin levels in anorexic, bulimic, obese, and control individuals, thereby spanning the full range of human body weights, using three frequently employed commercial kits. Kits from Linco (St Louis, MO) and DSL (Webster, TX) employ a radioimmunoassay method, and the R&D Systems kit (Minneapolis, MN) uses an enzyme-linked immunosorbent assay. We found that the three kits provide results that are highly linearly correlated with each other and remarkably linearly related to percent ideal body weight (%IBW) over more than three orders of magnitude (Linco, r = 0.90; R&D, r = 0.87; DSL, r = 0.86). For very low leptin levels, the more sensitive kits from R&D and Linco appeared to give more reliable results. Measurement method does not appear to explain the literature conflicts. We found that patients with anorexia nervosa have serum leptin values that lie above the line extrapolated from the %IBW/leptin curve generated from analysis of all non-anorexic patients. Therefore, in anorexia nervosa, inappropriately high leptin levels for %IBW may contribute to a blunted physiologic response to underweight and consequent resistance to dietary treatment. By contrast, most bulimic patients have leptin levels significantly below those predicted from the same %IBW/leptin curve. The relative leptin deficiency in bulimic subjects may contribute to food-craving behavior. We propose that using the %IBW/ leptin curve can facilitate identification of true pathophysiologic abnormalities in eating-disordered individuals and provide a basis for the design of therapeutic interventions or monitoring of response to treatment.


Massage therapy for stress management: implications for nursing practice.
Unresolved stress has been shown to have numerous adverse effects on the body. A review of the literature has revealed 2 major themes: (1) research that argues that massage has a direct relationship with positive health outcomes and (2) research that stresses that although there are little or no measurable physiological changes that occur from massage, patients' perceptions of stress and anxiety were significantly reduced. The simple act of touch-focused care, even a simple 5-min hand or foot massage, can be useful in lowering a patient's perceived level of stress. Further research is necessary on the benefits and risks of implementing massage therapy in the hospital setting.


Relationship between History of Ischemic Stroke and All-Cause Mortality in Incident Dialysis Patients.
Few studies have focused on the association between history of ischemic stroke at predialysis stage and mortality after dialysis initiation. To examine whether history of stroke in incident dialysis patients is associated with mortality, including all-cause and cardiovascular (CV)-related mortality. The study database was derived from the Aichi Cohort Study of Prognosis in Patients Newly Initiated into Dialysis, a multicenter, prospective, cohort analysis. We classified patients into 2 groups according to their history of ischemic stroke and compared their outcomes. Propensity scores (PSs) represented the probability of being assigned to a group with or without a history of ischemic stroke. We defined the following outcomes: all-cause mortality; CV-related mortality; non-CV-related mortality; infection-related mortality; and stroke event after dialysis initiation. Factors contributing to the outcomes were examined using stepwise multivariate Cox proportional hazards analysis. All-cause mortality was significantly higher in the ischemic stroke group (log-rank test p < 0.001). All-cause, non-CV-related, and infection-related mortality and stroke event after dialysis initiation were significantly higher in the ischemic stroke group after PS matching (log-rank test: p < 0.001, <0.001, 0.002, and 0.002, respectively). History of ischemic stroke was associated with all-cause mortality in univariate analysis (hazard ratio [HR] 1.85, 95% CI 1.44-2.37). History of ischemic stroke before dialysis initiation was associated with all-cause mortality in multivariate analysis (HR 1.39, 95% CI 1.05-1.85). The present study revealed that history of ischemic stroke before dialysis initiation was associated with all-cause, non-CV-related, and infection-related mortality and stroke event after dialysis initiation during maintenance dialysis.


Reduction of N-glycolylneuraminic acid xenoantigen on human adipose tissue-derived stromal cells/mesenchymal stem cells leads to safer and more useful cell sources for various stem cell therapies.
Adipose tissue is an attractive source for somatic stem cell therapy. Currently, human adipose tissue-derived stromal cells/mesenchymal stem cells (hADSCs/MSCs) are cultured with fetal bovine serum (FBS). Recently, however, not only human embryonic stem cell lines cultured on mouse feeder cells but also bone marrow-derived human MSCs cultured with FBS were reported to express N-glycolylneuraminic acid (Neu5Gc) xenoantigen. Human serum contains high titers of natural preformed antibodies against Neu5Gc. We studied the presence of Neu5Gc on hADSCs/MSCs cultured with FBS and human immune response mediated by Neu5Gc. Our data indicated that hADSCs/MSCs cultured with FBS expressed Neu5Gc and that human natural preformed antibodies could bind to hADSCs/MSCs. However, hADSCs/MSCs express complement regulatory proteins such as CD46, CD55, and CD59 and are largely resistant to complement-mediated cytotoxicity. hADSCs/MSCs cultured with FBS could be injured by antibody-dependent cell-mediated cytotoxicity mechanism. Further, human monocyte-derived macrophages could phagocytose hADSCs/MSCs cultured with FBS and this phagocytic activity was increased in the presence of human serum. Culturing hADSCs/MSCs with heat-inactivated human serum for a week could markedly reduce Neu5Gc on hADSCs/MSCs and prevent immune responses mediated by Neu5Gc, such as binding of human natural preformed antibodies, antibody-dependent cell-mediated cytotoxicity, and phagocytosis. Adipogenic and osteogenic differentiation potentials of hADSCs/MSCs cultured with heat-inactivated human serum were not less than that of those cultured with FBS. For stem cell therapies based on hADSCs/MSCs, hADSCs/MSCs that presented Neu5Gc on their cell surfaces after exposure to FBS should be cleaned up to be rescued from xenogeneic rejection.


An internal thermal sensor controlling temperature preference in Drosophila.
Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.


A systematic review of adeno-associated virus gene therapies in neurology: the need for consistent safety monitoring of a promising treatment.
Adeno-associated virus (AAV) gene therapies are generating much excitement in the rare disease field, particularly for previously untreatable neurological conditions. Efficacy has been claimed for several gene therapy products and the number of trials is rapidly increasing. However, reports of severe treatment-related adverse reactions are emerging, including death. There is still insufficient knowledge about their aetiology, prevention and treatment. We therefore undertook to systematically review publicly available data on AAV gene therapies in order to collate existing information on both safety and efficacy. Here, we review emerging efficacy reports of these novel therapies, many of which show promise. We also collate an increasing number of adverse reactions. Overwhelmingly, these results make a case for unified reporting of adverse events. This is likely to be critical for improving the safety of these promising treatments.


The FtsK family of DNA translocases finds the ends of circles.
A global view of bacterial chromosome choreography during the cell cycle is emerging, highlighting as a next challenge the description of the molecular mechanisms and factors involved. Here, we review one such factor, the FtsK family of DNA translocases. FtsK is a powerful and fast translocase that reads chromosome polarity. It couples segregation of the chromosome with cell division and controls the last steps of segregation in time and space. The second model protein of the family SpoIIIE acts in the transfer of the Bacillus subtilis chromosome during sporulation. This review focuses on the molecular mechanisms used by FtsK and SpoIIIE to segregate chromosomes with emphasis on the latest advances and open questions.


Distinct aspects of frontal lobe structure mediate age-related differences in fluid intelligence and multitasking.
Ageing is characterized by declines on a variety of cognitive measures. These declines are often attributed to a general, unitary underlying cause, such as a reduction in executive function owing to atrophy of the prefrontal cortex. However, age-related changes are likely multifactorial, and the relationship between neural changes and cognitive measures is not well-understood. Here we address this in a large (N=567), population-based sample drawn from the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) data. We relate fluid intelligence and multitasking to multiple brain measures, including grey matter in various prefrontal regions and white matter integrity connecting those regions. We show that multitasking and fluid intelligence are separable cognitive abilities, with differential sensitivities to age, which are mediated by distinct neural subsystems that show different prediction in older versus younger individuals. These results suggest that prefrontal ageing is a manifold process demanding multifaceted models of neurocognitive ageing.


Theoretical analysis of mechanical displacement measurement using a multiple cavity mode transducer.
We present an optomechanical displacement transducer that relies on three cavity modes parametrically coupled to a mechanical oscillator and whose frequency spacing matches the mechanical resonance frequency. The additional resonances allow reaching the standard quantum limit at a substantially lower input power (compared to the case of a single cavity mode), as both sensitivity and quantum backaction are enhanced. Furthermore, it is shown that in the case of multiple cavity modes, coupling between the modes is induced via reservoir interaction, e.g., enabling quantum backaction noise cancellation. Experimental implementation of the schemes is discussed in both the optical and microwave domain.


Usefulness of cardiac arrest centers - extending lifesaving post-resuscitation therapies: the Arizona experience - .
The post-cardiac arrest syndrome is a complex, multisystems response to the global ischemia and reperfusion injury that occurs with the onset of cardiac arrest, its treatment (cardiopulmonary resuscitation) and the re-establishment of spontaneous circulation. Regionalization of post-cardiac arrest care, utilizing specified cardiac arrest centers (CACs), has been proposed as the best solution to providing optimal care for those successfully resuscitated after out-of-hospital cardiac arrest. A multidisciplinary team of intensive care specialists, including critical care/pulmonologists, cardiologists (general, interventional, and electrophysiology), neurologists, and physical medicine/rehabilitation experts, is crucial for such centers. Particular attention to the timely initiation of targeted temperature management and early coronary angiography/percutaneous coronary intervention is best provided by such CACs. A State-wide program of CACs was started in Arizona in 2007. This is a voluntary program, whereby medical centers agree to provide all resuscitated cardiac arrest patients brought to their facility with state-of-the-art post-resuscitation care, including targeted temperature management for comatose patients and strong consideration for emergent coronary angiography for all patients with a likely cardiac etiology for their cardiac arrest. Survival improved by more than 50% at facilities that became CACs with a commitment to provide aggressive post-resuscitation care to all such patients. Providing aggressive, post-resuscitation care is the next real opportunity to increase long-term survival for cardiac arrest patients.


Predictive methods for computational metalloenzyme redesign - a test case with carboxypeptidase A.
Computational metalloenzyme design is a multi-scale problem. It requires treating the metal coordination quantum mechanically, extensive sampling of the protein backbone, and additionally accounting for the polarization of the active site by both the metal cation and the surrounding protein (a phenomenon called electrostatic preorganization). We bring together a combination of theoretical methods that jointly offer these desired qualities: QM/DMD for mixed quantum-classical dynamic sampling, quantum theory of atoms in molecules (QTAIM) for the assessment of electrostatic preorganization, and Density Functional Theory (DFT) for mechanistic studies. Within this suite of principally different methods, there are both complementarity of capabilities and cross-validation. Using these methods, predictions can be made regarding the relative activities of related enzymes, as we show on the native Zn2+-dependent carboxypeptidase A (CPA), and its mutant proteins, which are hypothesized to hydrolyze modified substrates. For the native CPA, we replicated the catalytic mechanism and the rate in close agreement with the experiment, giving validity to the QM/DMD predicted structure, the DFT mechanism, and the QTAIM assessment of catalytic activity. For most sequences of the modified substrate and tried CPA mutants, substantially worsened activity is predicted. However, for the substrate mutant that contains Asp instead of Phe at the C-terminus, one CPA mutant exhibits a reasonable activity, as predicted across the theoretical methods. CPA is a well-studied system, and here it serves as a testing ground for the offered methods.


Tumor-Induced Local and Systemic Impact on Blood Vessel Function.
Endothelial dysfunction plays a role in several processes that contribute to cancer-associated mortality. The vessel wall serves as a barrier for metastatic tumor cells, and the integrity and activation status of the endothelium serves as an important defense mechanism against metastasis. In addition, leukocytes, such as cytotoxic T-cells, have to travel across the vessel wall to enter the tumor tissue where they contribute to killing of cancer cells. Tumor cells can alter the characteristics of the endothelium by recruitment of leukocytes such as neutrophils and macrophages, which further stimulate inflammation and promote tumorigenesis. Recent findings also suggest that leukocyte-mediated effects on vascular function are not limited to the primary tumor or tissues that represent metastatic sites. Peripheral organs, such as kidney and heart, also display impaired vascular function in tumor-bearing individuals, potentially contributing to organ failure. Here, we discuss how vascular function is altered in malignant tissue and distant organs in individuals with cancer and how leukocytes function as potent mediators of these tumor-induced effects.


Randomized trial of hypofractionated external-beam radiotherapy for prostate cancer.
To determine if escalated radiation dose using hypofractionation significantly reduces biochemical and/or clinical disease failure (BCDF) in men treated primarily for prostate cancer. Between June 2002 and May 2006, men with favorable- to high-risk prostate cancer were randomly allocated to receive 76 Gy in 38 fractions at 2.0 Gy per fraction (conventional fractionation intensity-modulated radiation therapy [CIMRT]) versus 70.2 Gy in 26 fractions at 2.7 Gy per fraction (hypofractionated IMRT [HIMRT]); the latter was estimated to be equivalent to 84.4 Gy in 2.0 Gy fractions. High-risk patients received long-term androgen deprivation therapy (ADT), and some intermediate-risk patients received short-term ADT. The primary end point was the cumulative incidence of BCDF. Secondarily, toxicity was assessed. There were 303 assessable patients with a median follow-up of 68.4 months. No significant differences were seen between the treatment arms in terms of the distribution of patients by clinicopathologic or treatment-related (ADT use and length) factors. The 5-year rates of BCDF were 21.4% (95% CI, 14.8% to 28.7%) for CIMRT and 23.3% (95% CI, 16.4% to 31.0%) for HIMRT (P = .745). There were no statistically significant differences in late toxicity between the arms; however, in subgroup analysis, patients with compromised urinary function before enrollment had significantly worse urinary function after HIMRT. The hypofractionation regimen did not result in a significant reduction in BCDF; however, it is delivered in 2.5 fewer weeks. Men with compromised urinary function before treatment may not be ideal candidates for this approach.


Personality and problem gambling.
Matched groups of problem and social gamblers were compared in terms of their locus of control and their responses to the Myers Briggs Type Indicator. Contrary to previous studies no significant differences occurred on the locus of control measure. It was hypothesized that problem gamblers would be more extraverted and intuitive than social gamblers. However, none of the four scales on the Myers Briggs Type Indicator showed a significant difference between the groups. These findings and the results of earlier studies are compared and discussed. Earlier studies are difficult to interpret because of inappropriate or inconsistent controls. It is suggested that future personality studies of problem gamblers differentiate between different types of gambling and different types of problem gamblers.


Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.
The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.


Prevalence of antibodies to Japanese encephalitis virus among pigs in Bali and East Java, Indonesia, 2008.
Japanese encephalitis virus (JEV) is a fatal disease in Asia. Pigs are considered to be the effective amplifying host for JEV in the peridomestic environment. Bali Island and Java Island in Indonesia provide a model to assess the effect of pigs on JEV transmission, since the pig density is nearly 100-fold higher in Bali than Java, while the geographic and climatologic environments are equivalent in these areas. We surveyed antibodies to JEV among 123 pigs in Mengwi (Bali) and 96 pigs in Tulungagung (East Java) in 2008 by the hemagglutination-inhibition (HAI) test. Overall prevalences were 49% in Bali and 6% in Java, with a significant difference between them (P < 0.001). Monthly infection rates estimated from age-dependent antibody prevalences were 11% in Bali and 2% in Java. In addition, 2-mercaptoethanol-sensitive antibodies were found only from Bali samples. Further, the average HAI antibody titer obtained from positive samples was significantly higher in Bali (1:52) than Java (1:10; P < 0.001). These results indicated that JEV transmission in nature is more active in Bali than East Java.


How hierarchical is language use?
It is generally assumed that hierarchical phrase structure plays a central role in human language. However, considerations of simplicity and evolutionary continuity suggest that hierarchical structure should not be invoked too hastily. Indeed, recent neurophysiological, behavioural and computational studies show that sequential sentence structure has considerable explanatory power and that hierarchical processing is often not involved. In this paper, we review evidence from the recent literature supporting the hypothesis that sequential structure may be fundamental to the comprehension, production and acquisition of human language. Moreover, we provide a preliminary sketch outlining a non-hierarchical model of language use and discuss its implications and testable predictions. If linguistic phenomena can be explained by sequential rather than hierarchical structure, this will have considerable impact in a wide range of fields, such as linguistics, ethology, cognitive neuroscience, psychology and computer science.


Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis.
Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys(164) of the alpha subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys(164)alpha is involved in formation of the enzyme (E).farnesyl diphosphate (FPP).peptide ternary complex. Furthermore, mutation of Lys(164)alpha resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys(164)alpha, as well as the positioning of the charge, are important for overall catalysis by FTase.


Metabolic detoxification: implications for thresholds.
The fact that chemical carcinogenesis involves single, isolated, essentially irreversible molecular events as discrete steps, several of which must occur in a row to finally culminate in the development of a malignancy, rather suggests that an absolute threshold for chemical carcinogens may not exist. However, practical thresholds may exist due to saturable pathways involved in the metabolic processing, especially in the metabolic inactivation, of such compounds. An important example for such a pathway is the enzymatic hydrolysis of epoxides via epoxide hydrolases, a group of enzymes for which the catalytic mechanism has recently been established. These enzymes convert their substrates via the intermediate formation of a covalent enzyme-substrate complex. Interestingly, the formation of the intermediate proceeds faster by orders of magnitude than the subsequent hydrolysis, ie, the formation of the terminal product. Under normal circumstances, this does not pose a problem, since the microsomal epoxide hydrolase (mEH), the epoxide hydrolases with the best documented importance in the metabolism of carcinogens, is highly abundant in the liver, the organ with the highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme is favorable for the control of the steady-state level of a substrate epoxide and can keep it extremely low. However, once the mEH is titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to a sudden burst of the genotoxic effect of the noxious agent. This prediction of the computer simulation is nicely supported by experimental work. V79 Chinese hamster cells that we have genetically engineered to express human mEH at about the same level as that observed in human liver are completely protected from any measurable genotoxic effect of the model compound styrene oxide (STO) up to a dose of 100 microM in the cell culture medium (toxicokinetic threshold). In V79 cells that do not express mEH, STO leads to the formation of DNA strand breaks in a dose-dependent manner with no toxicokinetic threshold observable. Above 100 microM, the genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line. Thus, the saturable protection from STO-induced strand breaks by mEH represents a typical example of a practical threshold. However, it must be pointed out that even in the presence of protective amounts of mEH, a minute but definite level of STO is present that does not contribute sufficiently to the strand break formation to overcome the background noise of the detection procedure. As pointed out above, absolute thresholds probably do not exist in chemical carcinogenesis.


Synthesis and chemical diversity analysis of bicyclo[3.3.1]non-3-en-2-ones.
Functionalized bicyclo[3.3.1]non-3-en-2-ones are obtained from commercially available phenols by a hypervalent iodine oxidation, enone epoxidation, epoxide thiolysis, and intramolecular aldol reaction sequence. Reaction optimization studies identified room temperature as well as microwave-mediated procedures, providing moderate to good yields (57%-88%) in the thiophenol-mediated epoxide opening and intramolecular aldol reaction. In addition, the isolation of a key intermediate and in situ NMR studies supported the mechanistic hypothesis. The bicyclic ring products occupy novel chemical space according to ChemGPS and Chemaxon chemical diversity and cheminformatics analyses.


A family-based intervention for improving children's emotional problems through effects on maternal depressive symptoms.
This study focused on whether a brief family-based intervention for toddlers, the Family Check-Up (FCU), designed to address parent management skills and prevent early conduct problems, would have collateral effects on maternal depressive symptoms and subsequent child emotional problems. Parents with toddlers were recruited from the Women, Infants, and Children Nutritional Supplement Program based on the presence of socioeconomic, family, and child risk (N = 731). Families were randomly assigned to the FCU intervention or control group with yearly assessments beginning at child age 2. Maternal depressive symptoms were measured using the Center for Epidemiological Studies Depression Scale at child ages 2 and 3. Child internalizing problems were collected from primary caregivers, alternative caregivers, and teachers using the Child Behavior Checklist at ages 7.5 and 8.5. Structural equation models revealed that mothers in families randomly assigned to the FCU showed lower levels of depressive symptoms at child age 3, which in turn were related to lower levels of child depressed/withdrawal symptoms as reported by primary caregivers, alternative caregivers, and teacher at ages 7.5-8.5. Findings suggest that a brief, preventive intervention improving maternal depressive symptoms can have enduring effects on child emotional problems that are generalizable across contexts. As there is a growing emphasis for the use of evidence-based and cost-efficient interventions that can be delivered in multiple delivery settings serving low-income families and their children, clinicians and researchers welcome evidence that interventions can promote change in multiple problem areas. The FCU appears to hold such promise.


Time-resolved diffuse optical spectroscopy of small tissue samples.
Time-resolved transmittance spectroscopy was performed in the wavelength range of 610 or 700 to 1050 nm on phantom parallelepipeds and bone tissue cubes of different sizes. The data were best fitted with solutions of the diffusion equation for a laterally infinite slab and for a parallelepiped to investigate how size and optical properties of the samples affect the results obtained with the two models. Monte Carlo simulations were also performed to support and help with the interpretation of the experimental data. The parallelepiped model performs much better than the infinite slab model for the estimate of the reduced scattering coefficient and, even more, the absorption coefficient. It can profitably be used to quantify the optical properties of biological tissue samples and to derive information such as tissue composition, when small volumes are involved.


Impact of APOE genotype on neuropathologic and neurochemical markers of Alzheimer disease.
The APOE epsilon4 allele has emerged as a major genetic factor for Alzheimer disease (AD), and its presence has been associated with an increase in beta-amyloid senile plaques (SPs) and neuritic plaques (NPs). Whether it affects neurofibrillary tangle (NFT) accumulation or cholinergic losses in AD remains controversial. In contrast, the epsilon2 allele has been reported to decrease the risk for AD. However, its effect on neuropathologic and neurochemical markers of disease is unclear. To investigate the relationship between APOE genotype and both pathologic severity and cholinergic dysfunction in AD. In an autopsy series of 296 patients with AD, APOE genotype was determined in blood or postmortem brain tissue. NPs and NFTs were counted in the midfrontal (MF), inferior parietal (IP), and superior temporal (ST) cortices and the hippocampus. Choline acetyltransferase (ChAT) activity was assessed in the MF, IP, and ST cortices. Compared with patients with no epsilon4 alleles, epsilon4 carriers (patients with either one or two epsilon4 alleles) did not differ significantly with regard to any pathologic or neurochemical measures, except for increased ST NPs. However, when cases were stratified into three groups according to the number of epsilon4 alleles, patients with two epsilon4 alleles had significantly more NPs and NFTs in all neocortical regions than those with either one or no epsilon4 alleles. The association of the epsilon4/4 genotype with neocortical pathologic severity remained significant even after adjusting for age at onset or age at death. In contrast, there were no significant group differences with regard to neocortical ChAT activity. When pathologic and neurochemical measures were compared between patients with the epsilon2 allele and those without, a strong relationship emerged between the epsilon2 allele and decreased NPs in all neocortical regions. The epsilon4 allele does not predict cholinergic decline in AD. Although the presence of a single epsilon4 allele appears to have no effect, the presence of two epsilon4 alleles is an important determinant of both NP and NFT accumulation. A putative protective role for the epsilon2 allele in AD may be mediated by reduced plaque burden.


Prochlorothrix hollandica PCC 9006: genomic properties of an axenic representative of the chlorophyll a/b-containing oxyphotobacteria.
Prochlorothrix hollandica is an oxygenic photosynthetic prokaryote that differs from the cyanobacteria in having chlorophyll a/b-protein complexes instead of phycobilisomes as major light-harvesting antennae. We report the isolation and culturing of an axenic strain of P. hollandica, available from the Pasteur Culture Collection of Cyanobacteria as strain PCC 9006. The strain has a mean DNA base composition of 51.6 +/- 0.1 mol% G+C and a genomic complexity of 3.37 +/- 0.17 x 10(9) daltons (5,505 kb). A reiterated DNA sequence represents approximately 4.4% of the genome. Restriction enzyme isoschizomers with different sensitivities to base methylation were used to demonstrate that most A residues in the sequence GATC are methylated in P. hollandica DNA and that this methylation increases with culture age. Furthermore, some C residues are methylated, although the specificity of the C methylation system does not match that of well-characterized C methylases. Nucleotide analysis showed that up to approximately 3.5% of both dA and dC residues are methylated in P. hollandica DNA.


Neutrophils dominate the immune cell composition in non-small cell lung cancer.
The response rate to immune checkpoint inhibitor therapy for non-small-cell lung cancer (NSCLC) is just 20%. To improve this figure, several early phase clinical trials combining novel immunotherapeutics with immune checkpoint blockade have been initiated. Unfortunately, these trials have been designed without a strong foundational knowledge of the immune landscape present in NSCLC. Here, we use a flow cytometry panel capable of measuring 51 immune cell populations to comprehensively identify the immune cell composition and function in NSCLC. The results show that the immune cell composition is fundamentally different in lung adenocarcinoma as compared with lung squamous cell carcinoma, and that neutrophils are the most prevalent immune cell type. Using T-cell receptor-β sequencing and tumour reactivity assays, we predict that tumour reactive T cells are frequently present in NSCLC. These results should help to guide the design of clinical trials and the direction of future research in this area.


Ivacaftor treatment of cystic fibrosis in children aged 12 to <24 months and with a CFTR gating mutation (ARRIVAL): a phase 3 single-arm study.
Ivacaftor is generally safe and effective in patients aged 2 years and older who have cystic fibrosis and specific CFTR mutations. We assessed its use in children aged 12 to <24 months. The ARRIVAL study is a phase 3, single-arm, two-part, multicentre study. Eligible children were aged 12 to <24 months at enrolment and had a confirmed diagnosis of cystic fibrosis and a CFTR gating mutation on at least one allele and could participate in one or both parts of the study. Children received 50 mg (bodyweight 7 to <14 kg) or 75 mg (bodyweight ≥14 to <25 kg) ivacaftor orally every 12 h. In study part A, children received ivacaftor for 3 days plus one morning. In study part B, children received 24 weeks of treatment. Children were enrolled into part A at seven sites in Australia (one site), the UK (one), and the USA (five) and into part B at 13 sites in Australia (two sites), Canada (one), the UK (three), and the USA (seven). Primary endpoints were pharmacokinetics (part A) and safety (parts A and B) in children who received at least one dose of ivacaftor. Secondary endpoints in part B were pharmacokinetics in children who received at least one dose of ivacaftor and absolute change from baseline in sweat chloride concentration. We also explored changes in growth parameters and markers of pancreatic function. This study is registered with ClinicalTrials.gov, number NCT02725567. Children aged 12 to <24 months were enrolled between Aug 25, 2016, and Nov 1, 2017. Seven children were enrolled in part A, of whom five received 50 mg and two received 75 mg ivacaftor. All completed treatment. Of 19 children enrolled in part B, including one from part A, all received 50 mg ivacaftor and 18 completed treatment (one withdrew because of difficulty with blood draws). All children received at least one dose of ivacaftor. Pharmacokinetics indicated exposure was similar to that in children aged 2 to <6 years and adults. No children discontinued because of adverse events or safety findings. In part A, three (43%) of seven children had treatment-emergent adverse events, all of which were mild and deemed not to be or unlikely to be related to ivacaftor. By 24 weeks in part B, treatment-emergent adverse events had been reported in 18 (95%) of 19 children, of which most were mild or moderate and the most frequent was cough (14 [74%] children). Two children in part B had four serious adverse events: one had constipation (possibly related to ivacaftor), distal intestinal obstruction syndrome, and eczema herpeticum, and one had persistent cough, all needing hospital admission. In five (28%) of 18 children aspartate or alanine aminotransferase concentrations rose to more than three times the upper limit of normal (to more than eight times in two children with concurrent infections). At week 24, the mean absolute change from baseline in sweat chloride concentration was -73·5 (SD 17·5) mmol/L. Growth parameters for age were normal at baseline and at week 24. At week 24, concentrations of faecal elastase-1 had increased and concentrations of immunoreactive trypsinogen had decreased from baseline. Mean serum lipase and amylase were raised at baseline and rapidly decreased after treatment was started. Ivacaftor was generally safe and well tolerated in children aged 12 to <24 months for up to 24 weeks and was associated with rapid and sustained reductions in sweat chloride concentrations. Improvements in biomarkers of pancreatic function suggest that ivacaftor preserves exocrine pancreatic function if started early. The study is continuing in infants younger than 12 months. Vertex Pharmaceuticals Incorporated.


Human muscular fetal cells: a potential cell source for muscular therapies.
Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.


Spontaneous mutagenesis in Csb(m/m)Ogg1⁻(/)⁻ mice is attenuated by dietary resveratrol.
Oxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are generated endogenously in apparently all living cells. The defect of the repair of 8-oxoG in Csb(m/m)Ogg1⁻(/)⁻ mice results in elevated basal levels of these lesions and increased frequencies of spontaneous mutations, which initiate tumorigenesis in the liver if cell proliferation is stimulated. Here, we describe that the phytoalexin resveratrol, applied either for 7 days per gavage (100 mg/kg body wt) or for 3-9 months in the diet (0.04% ad libitum), reduces the endogenous oxidative DNA base damage in the livers of the Csb(m/m)Ogg1⁻(/)⁻ mice by 20-30% (P < 0.01). A small but consistent effect is also observed in the wild-type animals. The spontaneous mutation frequencies determined in the lacI gene of BigBlue® Csb(m/m)Ogg1⁻(/)⁻ mice are concomitantly reduced by resveratrol to similar extents. Mechanistically, the protection is caused by an induction of the antioxidant defense system since (i) hepatocytes isolated from all resveratrol-treated animals were less susceptible to the generation of single-strand breaks and to cell killing by H₂O₂, (ii) messenger RNA levels of superoxide dismutases 1 and 2 (SOD1 and SOD2) heme oxygenase-1 and glutathione peroxidase were significantly upregulated after the short-term treatment and (iii) mutations primarily ascribed to the oxidative base modification 8-oxoG (G:C to T:A transversions) were more strongly suppressed than G:C to A:T transitions ascribed to spontaneous deamination. The results thus demonstrate that spontaneous somatic mutation rates resulting from endogenous oxidative DNA damage can be reduced by application of an exogenous agent.


Transcriptional pattern of a novel gene, expressed specifically after the point-of-no-return during sexualization, in planaria.
We have investigated sexualization of asexual worms in the planarian Dugesia ryukyuensis. During sexualization there is a point from which an animal cannot return to the asexual state (point-of-no-return). To isolate the genes related to the point-of-no-return, we performed differential screening and isolated one novel gene that was expressed specifically in yolk glands of the worms after the point-of-no-return and named it Dryg. It encoded 655 amino acids with a predicted molecular mass of 79 kDa. We performed a series of experiments using Dryg as a molecular marker in the yolk gland. At first, we monitored how the yolk gland was formed during sexualization. The expression in sexualizing worms at stage 3 is limited to a single type of cell that has characteristics of neoblasts, the totipotent somatic cells; however, the expression is observed in the yolk gland in sexualized worms. Furthermore, we monitored yolk glands for expression during regeneration. The original yolk glands seem to disappear after ablation, then new yolk glands appeared along the ventral nerve cords. Because this expression pattern looks like that of sexualizing worms at stage 3, we speculate that yolk gland cells may differentiate from neoblasts during regeneration as observed during sexualization.


Zn2+-triggered amide tautomerization produces a highly Zn2+-selective, cell-permeable, and ratiometric fluorescent sensor.
It is still a significant challenge to develop a Zn(2+)-selective fluorescent sensor with the ability to exclude the interference of some heavy and transition metal (HTM) ions such as Fe(2+), Co(2+), Ni(2+), Cu(2+), Cd(2+), and Hg(2+). Herein, we report a novel amide-containing receptor for Zn(2+), combined with a naphthalimide fluorophore, termed ZTRS. The fluorescence, absorption detection, NMR, and IR studies indicated that ZTRS bound Zn(2+) in an imidic acid tautomeric form of the amide/di-2-picolylamine receptor in aqueous solution, while most other HTM ions were bound to the sensor in an amide tautomeric form. Due to this differential binding mode, ZTRS showed excellent selectivity for Zn(2+) over most competitive HTM ions with an enhanced fluorescence (22-fold) as well as a red-shift in emission from 483 to 514 nm. Interestingly, the ZTRS/Cd(2+) complex showed an enhanced (21-fold) blue-shift in emission from 483 to 446 nm. Therefore, ZTRS discriminated in vitro and in vivo Zn(2+) and Cd(2+) with green and blue fluorescence, respectively. Due to the stronger affinity, Zn(2+) could be ratiometrically detected in vitro and in vivo with a large emission wavelength shift from 446 to 514 nm via a Cd(2+) displacement approach. ZTRS was also successfully used to image intracellular Zn(2+) ions in the presence of iron ions. Finally, we applied ZTRS to detect zinc ions during the development of living zebrafish embryos.


The triple classification of dry eye for practical clinical use.
"Dry Eye is a condition produced by the inadequate interrelation between lacrimal film and ocular surface epithelium, and is caused by quantitative and qualitative deficits in one or both of them. It can be produced by one or combined etiologic causes, affecting one or several of the secretions of the glands serving the ocular surface, and producing secondary manifestations of different grades of severity". Clinicians need a practical classification to face diagnosis, prognosis and treatment. Dry eyes have many etiologies and pathogenesis, different affectation of the various dacryoglands and ocular surface epithelium, and diverse grades of severity. The specialists in xero-dacryology must know these three parameters to evaluate any case of dry eye, and to establish an adequate treatment. To facilitate this, an open session in the 8th congress of the International Society of Dacryology and Dry Eye (Madrid, April, 2005) proposed modifying the Triple Classification of dry eye approved in the XIV congress of the European Society of Ophthalmology (Madrid, June, 2003). There was consensus of all conclusions. The following classification has been established: First, a classification of the etio-pathogenesis, distributed in ten groups: age-related, hormonal, pharmacologic, immunopathic, hyponutritional, dysgenic, infectious/inflammatory, traumatic, neurologic and tantalic. Second, a classification of the affected glands and tissues, which under the acronym of ALMEN includes the Aqueo-serousdeficient, Lipodeficient, Mucindeficient and Epitheliopatic dry eyes, and the Non dacryological affected exocrine glands (saliva, nasal secretion, tracheo-pharyngeal secretion, etc). And thirdly, a classification of severity, in three grades: Grade 1 or mild (symptoms without slitlamp signs), grade 2 or moderate (symptoms with reversible signs), and grade 3 or severe (symptoms with permanent signs).


Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias.
DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark that plays a critical role in the regulation of gene expression. Profiling DNA methylation patterns across the genome is vital to understand DNA methylation changes that occur during development and in disease phenotype. In this study, we compared two commonly used approaches to enrich for methylated DNA regions of the genome, namely methyl-DNA immunoprecipitation (MeDIP) that is based on enrichment with antibodies specific for 5'-methylcytosine (5MeC), and capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein to discover differentially methylated regions (DMRs) in cancer. The enriched methylated DNA fractions were interrogated on Affymetrix promoter tiling arrays and differentially methylated regions were identified. A detailed validation study of 42 regions was performed using Sequenom MassCLEAVE technique. This detailed analysis revealed that both enrichment techniques are sensitive for detecting DMRs and preferentially identified different CpG rich regions of the prostate cancer genome, with MeDIP commonly enriching for methylated regions with a low CpG density, while MBD capture favors regions of higher CpG density and identifies the greatest proportion of CpG islands. This is the first detailed validation report comparing different methylated DNA enrichment techniques for identifying regions of differential DNA methylation. Our study highlights the importance of understanding the nuances of the methods used for DNA genome-wide methylation analyses so that accurate interpretation of the biology is not overlooked.


Terms of debate: Consensus definitions to guide the scientific discourse on visual distraction.
Hypothesis-driven research rests on clearly articulated scientific theories. The building blocks for communicating these theories are scientific terms. Obviously, communication - and thus, scientific progress - is hampered if the meaning of these terms varies idiosyncratically across (sub)fields and even across individual researchers within the same subfield. We have formed an international group of experts representing various theoretical stances with the goal to homogenize the use of the terms that are most relevant to fundamental research on visual distraction in visual search. Our discussions revealed striking heterogeneity and we had to invest much time and effort to increase our mutual understanding of each other's use of central terms, which turned out to be strongly related to our respective theoretical positions. We present the outcomes of these discussions in a glossary and provide some context in several essays. Specifically, we explicate how central terms are used in the distraction literature and consensually sharpen their definitions in order to enable communication across theoretical standpoints. Where applicable, we also explain how the respective constructs can be measured. We believe that this novel type of adversarial collaboration can serve as a model for other fields of psychological research that strive to build a solid groundwork for theorizing and communicating by establishing a common language. For the field of visual distraction, the present paper should facilitate communication across theoretical standpoints and may serve as an introduction and reference text for newcomers.


Spectral characteristics of skin sympathetic nerve activity in heat-stressed humans.
Skin sympathetic nerve activity (SSNA) exhibits low- and high-frequency spectral components in normothermic subjects. However, spectral characteristics of SSNA in heat-stressed subjects are unknown. Because the main components of the integrated SSNA during heat stress (sudomotor/vasodilator activities) are different from those during normothermia and cooling (vasoconstrictor activity), we hypothesize that spectral characteristics of SSNA in heat-stressed subjects will be different from those in subjects subjected to normothermia or cooling. In 17 healthy subjects, SSNA, electrocardiogram, arterial blood pressure (via Finapres), respiratory activity, and skin blood flow were recorded during normothermia and heat stress. In 7 of the 17 subjects, these variables were also recorded during cooling. Spectral characteristics of integrated SSNA, R-R interval, beat-by-beat mean blood pressure, skin blood flow variability, and respiratory excursions were assessed. Heat stress and cooling significantly increased total SSNA. SSNA spectral power in the low-frequency (0.03-0.15 Hz), high-frequency (0.15-0.45 Hz), and very-high-frequency (0.45-2.5 Hz) regions was significantly elevated by heat stress and cooling. Interestingly, heat stress caused a greater relative increase of SSNA spectral power within the 0.45- to 2.5-Hz region than in the other spectral ranges; cooling did not show this effect. Differences in the SSNA spectral distribution between normothermia/cooling and heat stress may reflect different characteristics of central modulation of vasoconstrictor and sudomotor/vasodilator activities.


Sentinel lymph node biopsy accurately stages the regional lymph nodes for T1-T2 oral squamous cell carcinomas: results of a prospective multi-institutional trial.
The validity of sentinel lymph node biopsy (SLNB) for T1 or T2, clinically N0, oral cancer was tested by correlation of sentinel node pathologic status with that of nodes within the completion neck dissection. This prospective, cooperative group trial involved 25 institutions over a 3-year period. One hundred forty patients with invasive oral cancers, stage T1 and T2, N0 including 95 cancers of the tongue, 26 of the floor of mouth, and 19 other oral cancers were studied. The study excluded lesions with diameter smaller than 6 mm or minimal invasion. Imaging was used to exclude nonpalpable gross nodal disease. Patients underwent injection of the lesion with (99m)Tc-sulfur colloid, nuclear imaging, narrow-exposure SLNB, and completion selective neck dissection. The major end point was the negative-predictive value (NPV) of SLNB. In the 106 SLNBs, which were found to be pathologically and clinically node-negative by routine hematoxylin and eosin stain, 100 patients were found to have no other pathologically positive nodes, corresponding to a NPV of 94%. With additional sectioning and immunohistochemistry, NPV was improved to 96%. In the forty patients with proven cervical metastases, the true-positive rate was 90.2% and was superior for tongue tumors relative to floor of mouth. For T1 lesions, metastases were correctly identified in 100%. For T1 or T2 N0 oral squamous cell carcinoma, SLNB with step sectioning and immunohistochemistry, performed by surgeons of mixed experience levels, correctly predicted a pathologically negative neck in 96% of patients (NPV, 96%).


Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli.
Binding of the bactericidal/permeability increasing protein (BPI) of granulocytes to Escherichia coli promptly produces several discrete outer envelope alterations and growth arrest without major impairment of bacterial structure or biosynthetic capabilities, raising the question whether these early effects of BPI are sufficient to cause bacterial death. In this study, the bactericidal action of BPI was examined more closely. We have found that bovine or human serum albumin blocks bacterial killing without preventing BPI binding or an increase in outer membrane permeability. Moreover, addition of serum albumin after BPI results in growth resumption without displacement of bound BPI and without (early) repair of the envelope alterations. These effects are opposite to those produced by Mg2+ (80 mM), which displaces greater than 85% of bound BPI and rapidly initiates outer envelope repair without restoration of bacterial growth. The extent of rescue by serum albumin depends on the time and pH of preincubation of BPI with E. coli: e.g., for E. coli J5 treated with human BPI, t1/2 = 79 min at pH 7.4 and 10 min at pH 6.0. The serum albumin effects on BPI action are the same in wild-type E. coli and in a mutant strain lacking an activatable phospholipase, indicating that serum albumin does not act by sequestering membrane-damaging products of bacterial phospholipid hydrolysis. The progression from reversible to irreversible growth arrest, revealed by the subsequent addition of serum albumin at different times, is paralleled by a decrease in amino acid uptake and an increase in the permeability of the cytoplasmic membrane to o-nitrophenyl-beta-D-galactoside. These findings demonstrate at least two stages in the action of BPI: (a) an early, reversible, sublethal stage in which BPI has effects on the outer envelope and causes growth arrest, and (b) time- and pH-dependent progression to a lethal stage, apparently involving cytoplasmic membrane damage, possibly caused by penetration of a small subpopulation of BPI.


Design and construction of highly stable, protease-resistant chimeric avidins.
The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.


Evidence of Partial Migration in a Large Coastal Predator: Opportunistic Foraging and Reproduction as Key Drivers?
Understanding animal movement decisions that involve migration is critical for evaluating population connectivity, and thus persistence. Recent work on sharks has shown that often only a portion of the adult population will undertake migrations, while the rest may be resident in an area for long periods. Defining the extent to which adult sharks use specific habitats and their migratory behaviour is essential for assessing their risk of exposure to threats such as fishing and habitat degradation. The present study used acoustic telemetry to examine residency patterns and migratory behaviour of adult bull sharks (Carcharhinus leucas) along the East coast of Australia. Fifty-six VR2W acoustic receivers were used to monitor the movements of 33 bull sharks in the central Great Barrier Reef (GBR). Both males and females were detected year-round, but their abundance and residency peaked between September and December across years (2012-2014). High individual variability in reef use patterns was apparent, with some individuals leaving the array for long periods, whereas others (36%) exhibited medium (0.20-0.40) or high residency (> 0.50). A large portion of the population (51%) undertook migrations of up to 1,400 km to other coral reefs and/or inshore coastal habitats in Queensland and New South Wales. Most of these individuals (76%) were mature females, and the timing of migrations coincided with the austral summer (Dec-Feb). All migrating individuals (except one) returned to the central GBR, highlighting its importance as a potential foraging ground. Our findings suggest that adult bull sharks appear to be highly dependent on coral reef resources and provide evidence of partial migration, where only a portion of the female population undertook seasonal migrations potentially to give birth. Given that estuarine habitats face constant anthropogenic pressures, understanding partial migration and habitat connectivity of large coastal predators should be a priority for their management.


MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles.
MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells. Here, we have investigated: 1) new miR-24-3p targets; 2) the expression and the function of miR-24-3p in human vascular ECs; 3) the impact of miR-24-3p inhibition in the angiogenesis reparative response to limb ischemia in mice. Using bioinformatics target prediction platforms and 3'-UTR luciferase assays, we newly identified Notch1 and its Delta-like ligand 1 (Dll1) to be directly targeted by miR-24-3p. miR-24-3p was expressed in human ECs and pericytes cultured under normal conditions. Exposure to hypoxia increased miR-24-3p in ECs but not in pericytes. Transfection with a miR-24-3p precursor (pre-miR-24-3p) increased miR-24-3p expression in ECs, reducing the cell survival, proliferation, and angiogenic capacity. Opposite effects were caused by miR-24-3p inhibition. The anti-angiogenic action of miR-24-3p overexpression could be prevented by simultaneous adenovirus (Ad)-mediated delivery of constitutively active Notch intracellular domain (NICD) into cultured ECs. We next demonstrated that reduced Notch signalling contributes to the anti-angiogenic effect of miR-24-3p in vitro. In a mouse unilateral limb ischemia model, local miR-24-3p inhibition (by adenovirus-mediated miR-24-3p decoy delivery) restored endothelial Notch signalling and increased capillary density. However, the new vessels appeared disorganised and twisted, worsening post-ischemic blood perfusion recovery. To better understand the underpinning mechanisms, we widened the search for miR-24-3p target genes, identifying several contributors to vascular morphogenesis, such as several members of the Wingless (Wnt) signalling pathway, β-catenin signalling components, and VE-cadherin, which synergise to regulate angiogenesis, pericytes recruitment to neoformed capillaries, maturation, and stabilization of newly formed vessels. Among those, we next focussed on β-catenin to demonstrate that miR-24-3p inhibition reduces β-catenin expression in hypoxic ECs, which is accompanied by reduced adhesion of pericytes to ECs. In summary, miR-24-3p differentially targets several angiogenesis modulators and contributes to autonomous and non-autonomous EC crosstalk. In ischemic limbs, miR-24-3p inhibition increases the production of dysfunctional microvessels, impairing perfusion. Caution should be observed in therapeutic targeting of miR-24-3p.


Vagal stimulation attenuates sympathetic enhancement of left ventricular function.
We studied the effects of stimulation of the vagal and sympathetic cardiac nerves on left ventricular contraction and relaxation in 16 anesthetized dogs. In each experiment, we paced the ventricles at a fixed rate of 120 beats/min, and we kept the systemic arterial pressure constant. We used the maximum rates of left ventricular pressure rise (dP/dtmax) and fall (dP/dtmin) as our indexes of ventricular contraction and relaxation. Sympathetic nerve stimulation at a frequency of 6 Hz increased dP/dtmax by 802 mmHg/s and raised dP/dtmin by 847 mmHg/s above the control level. The effect of vagal nerve stimulation was directly dependent on the level of sympathetic stimulation. In the absence of sympathetic stimulation, vagal nerve stimulation at a frequency of 5 Hz decreased dP/dtmax by 187 mmHg/s and reduced dP/dtmin by 199 mmHg/s below the control level. When the sympathetic stimulation was 6 Hz, vagal nerve stimulation at 5 Hz decreased dP/dtmax by 513 mmHg/s and reduced dP/dtmin by 709 mmHg/s. Furthermore, propranolol, in doses of 1.5 mg/kg, abolished any significant vagal effect on ventricular contraction or relaxation. We conclude that combined sympathetic-vagal stimulation results in a substantial antagonistic interaction such that vagal stimulation significantly attenuates the sympathetic enhancement of left ventricular function.


Phase I clinical trial of the CYP17 inhibitor abiraterone acetate demonstrating clinical activity in patients with castration-resistant prostate cancer who received prior ketoconazole therapy.
Abiraterone acetate is a prodrug of abiraterone, a selective inhibitor of CYP17, the enzyme catalyst for two essential steps in androgen biosynthesis. In castration-resistant prostate cancers (CRPCs), extragonadal androgen sources may sustain tumor growth despite a castrate environment. This phase I dose-escalation study of abiraterone acetate evaluated safety, pharmacokinetics, and effects on steroidogenesis and prostate-specific antigen (PSA) levels in men with CPRC with or without prior ketoconazole therapy. Thirty-three men with chemotherapy-naïve progressive CRPC were enrolled. Nineteen patients (58%) had previously received ketoconazole for CRPC. Bone metastases were present in 70% of patients, and visceral involvement was present in 18%. Three patients (9%) had locally advanced disease without distant metastases. Fasted or fed cohorts received abiraterone acetate doses of 250, 500, 750, or 1,000 mg daily. Single-dose pharmacokinetic analyses were performed before continuous daily dosing. Adverse events were predominantly grade 1 or 2. No dose-limiting toxicities were observed. Hypertension (grade 3, 12%) and hypokalemia (grade 3, 6%; grade 4, 3%) were the most frequent serious toxicities and responded to medical management. Confirmed > or = 50% PSA declines at week 12 were seen in 18 (55%) of 33 patients, including nine (47%) of 19 patients with prior ketoconazole therapy and nine (64%) of 14 patients without prior ketoconazole therapy. Substantial declines in circulating androgens and increases in mineralocorticoids were seen with all doses. Abiraterone acetate was well tolerated and demonstrated activity in CRPC, including in patients previously treated with ketoconazole. Continued clinical study is warranted.