Frequency-domain multiplexing system for in vivo diffuse light measurements of rapid cerebral hemodynamics.
A novel frequency-domain multiplexing system has been developed for in vivo measurements of rapid cerebral hemodynamics. The instrument operates in the frequency-domain with three optical wavelengths, six source positions, and two detectors. Frequency-division multiplexing was used to modulate three wavelengths (690, 786, and 830 nm) at slightly different frequencies around 70 MHz. The three laser output beams were combined and switched into different source positions by use of fast optical switches (switch time <10 ms). Three narrowband, in-phase and in-quadrature demodulators decode the modulated signals. Our full-frame-acquisition rate is 2.5 Hz, with flexibility for acquisition rates greater than 50 Hz with smaller detection areas. We evaluate the performance of the instrument with tissue phantoms, and then employ the system to measure in vivo cerebral blood oxygenation during forepaw stimulation of a rat's brain.


The blood supply of the calcaneal tendon.
The microvascular anatomy of the calcaneal tendon was investigated in cadaver tendons by injection of barium sulphate and indian ink and a quantitative study of intratendinous blood supply was made, using a computer-assisted image analysis system. There was a reduction in both the number and the mean relative area of vessels in the mid-section of the tendon. This area of reduced vascularity may be of significance in the pathogenesis of rupture.


Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development.
Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.


Effects of APOC3 Heterozygous Deficiency on Plasma Lipid and Lipoprotein Metabolism.
Objective- Apo (apolipoprotein) CIII inhibits lipoprotein lipase (LpL)-mediated lipolysis of VLDL (very-low-density lipoprotein) triglyceride (TG) and decreases hepatic uptake of VLDL remnants. The discovery that 5% of Lancaster Old Order Amish are heterozygous for the APOC3 R19X null mutation provided the opportunity to determine the effects of a naturally occurring reduction in apo CIII levels on the metabolism of atherogenic containing lipoproteins. Approach and Results- We conducted stable isotope studies of VLDL-TG and apoB100 in 5 individuals heterozygous for the null mutation APOC3 R19X (CT) and their unaffected (CC) siblings. Fractional clearance rates and production rates of VLDL-TG and apoB100 in VLDL, IDL (intermediate-density lipoprotein), LDL, apo CIII, and apo CII were determined. Affected (CT) individuals had 49% reduction in plasma apo CIII levels compared with CCs ( P<0.01) and reduced plasma levels of TG (35%, P<0.02), VLDL-TG (45%, P<0.02), and VLDL-apoB100 (36%, P<0.05). These changes were because of higher fractional clearance rates of VLDL-TG and VLDL-apoB100 with no differences in production rates. CTs had higher rates of the conversion of VLDL remnants to LDL compared with CCs. In contrast, rates of direct removal of VLDL remnants did not differ between the groups. As a result, the flux of apoB100 from VLDL to LDL was not reduced, and the plasma levels of LDL-cholesterol and LDL-apoB100 were not lower in the CT group. Apo CIII production rate was lower in CTs compared with CCs, whereas apo CII production rate was not different between the 2 groups. The fractional clearance rates of both apo CIII and apo CII were higher in CTs than CCs. Conclusions- These studies demonstrate that 50% reductions in plasma apo CIII, in otherwise healthy subjects, results in a significantly higher rate of conversion of VLDL to LDL, with little effect on direct hepatic uptake of VLDL. When put in the context of studies demonstrating significant protection from cardiovascular events in individuals with loss of function variants in the APOC3 gene, our results provide strong evidence that therapies which increase the efficiency of conversion of VLDL to LDL, thereby reducing remnant concentrations, should reduce the risk of cardiovascular disease.


Alcohol consumption and fecundability: prospective Danish cohort study.
To investigate to what extent alcohol consumption affects female fecundability. Prospective cohort study. Denmark, 1 June 2007 to 5 January 2016. 6120 female Danish residents, aged 21-45 years, in a stable relationship with a male partner, who were trying to conceive and not receiving fertility treatment. Alcohol consumption was self reported as beer (330 mL bottles), red or white wine (120 mL glasses), dessert wine (50 mL glasses), and spirits (20 mL) and categorized in standard servings per week (none, 1-3, 4-7, 8-13, and ≥14). Participants contributed menstrual cycles at risk until the report of pregnancy, start of fertility treatment, loss to follow-up, or end of observation (maximum 12 menstrual cycles). A proportional probability regression model was used to estimate fecundability ratios (cycle specific probability of conception among exposed women divided by that among unexposed women). 4210 (69%) participants achieved a pregnancy during follow-up. Median alcohol intake was 2.0 (interquartile range 0-3.5) servings per week. Compared with no alcohol consumption, the adjusted fecundability ratios for alcohol consumption of 1-3, 4-7, 8-13, and 14 or more servings per week were 0.97 (95% confidence interval 0.91 to 1.03), 1.01 (0.93 to 1.10), 1.01 (0.87 to 1.16) and 0.82 (0.60 to 1.12), respectively. Compared with no alcohol intake, the adjusted fecundability ratios for women who consumed only wine (≥3 servings), beer (≥3 servings), or spirits (≥2 servings) were 1.05 (0.91 to1.21), 0.92 (0.65 to 1.29), and 0.85 (0.61 to 1.17), respectively. The data did not distinguish between regular and binge drinking, which may be important if large amounts of alcohol are consumed during the fertile window. Consumption of less than 14 servings of alcohol per week seemed to have no discernible effect on fertility. No appreciable difference in fecundability was observed by level of consumption of beer and wine.


Intracluster anionic oligomerization of acrylic ester molecules initiated by electron transfer from an alkali metal atom.
Stabilities and intracluster reactions have been investigated by photoionization mass spectrometry for clusters composed of an alkali metal atom (M; Na and K) and acrylic ester molecules, CH(2)=CHCO(2)R, such as methyl acrylate (MA; R = CH(3)) and ethyl acrylate (EA; R = C(2)H(5)). The following two features are commonly observed in the photoionization mass spectra of M(CH(2)=CHCO(2)R)(n): (1) The ion with n = 3 is clearly observed as a magic number. (2) Fragmented cluster ions with the loss of ROH, [M(CH(2)=CHCO(2)R)(n) - ROH] are detected only for n = 3. These features are both explained by an intracluster oligomerization reaction initiated by electron transfer from the metal atoms. The magic number trimer is concluded to have the stable structure of cyclohexane derivatives as a result of oligomerization. The fragmentation reaction is explained by Dieckmann cyclization after anionic oligomerization to produce another isomer of the trimer. The intracluster electron transfer is also supported by theoretical calculation for Na(MA) based on density functional theory.


Protein complexes are under evolutionary selection to assemble via ordered pathways.
Is the order in which proteins assemble into complexes important for biological function? Here, we seek to address this by searching for evidence of evolutionary selection for ordered protein complex assembly. First, we experimentally characterize the assembly pathways of several heteromeric complexes and show that they can be simply predicted from their three-dimensional structures. Then, by mapping gene fusion events identified from fully sequenced genomes onto protein complex assembly pathways, we demonstrate evolutionary selection for conservation of assembly order. Furthermore, using structural and high-throughput interaction data, we show that fusion tends to optimize assembly by simplifying protein complex topologies. Finally, we observe protein structural constraints on the gene order of fusion that impact the potential for fusion to affect assembly. Together, these results reveal the intimate relationships among protein assembly, quaternary structure, and evolution and demonstrate on a genome-wide scale the biological importance of ordered assembly pathways.


Predictors of Recurrent Severe Hypoglycemia in Adults With Type 1 Diabetes and Impaired Awareness of Hypoglycemia During the HypoCOMPaSS Study.
The HypoCOMPaSS study was designed to test the hypothesis that successful avoidance of biochemical hypoglycemia without compromising overall glycemic control would restore sufficient hypoglycemia awareness to prevent recurrent severe hypoglycemia in the majority of participants with established type 1 diabetes. Before starting the study, we planned to investigate associations between baseline characteristics and recurrent severe hypoglycemia over 2 years' follow-up. A total of 96 adults with type 1 diabetes and impaired awareness of hypoglycemia participated in a 24-week 2 × 2 factorial randomized controlled trial comparing insulin delivery and glucose monitoring modalities, with the goal of rigorous biochemical hypoglycemia avoidance. The analysis included 71 participants who had experienced severe hypoglycemia in the 12-month prestudy with confirmed absence (complete responder) or presence (incomplete responder) of severe hypoglycemia over 24 months' follow-up. There were 43 (61%) complete responders and 28 (39%) incomplete responders experiencing mean ± SD 1.5 ± 1.0 severe hypoglycemia events/person-year. At 24 months, incomplete responders spent no more time with glucose ≤3 mmol/L (1.4 ± 2.1% vs. 3.0 ± 4.8% for complete responders; P = 0.26), with lower total daily insulin dose (0.45 vs. 0.58 units/24 h; P = 0.01) and greater impairment of hypoglycemia awareness (Clarke score: 3.8 ± 2.2 vs. 2.0 ± 1.9; P = 0.01). Baseline severe hypoglycemia rate (16.9 ± 16.3 vs. 6.4 ± 10.8 events/person-year; P = 0.002) and fear of hypoglycemia were higher in incomplete responders. Peripheral neuropathy was more prevalent in incomplete responders (11 [39%] vs. 2 [4.7%]; P < 0.001) with a trend toward increased autonomic neuropathy. Recurrent severe hypoglycemia was associated with higher preintervention severe hypoglycemia rate, fear of hypoglycemia, and concomitant neuropathy.


Dr(a-) polymorphism of decay accelerating factor. Biochemical, functional, and molecular characterization and production of allele-specific transfectants.
The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on decay accelerating factor (DAF), a glycosyl-phosphatidylinositol-anchored membrane protein that protects host cells from complement-mediated damage. We studied the rare inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface expression of DAF but normal levels of several other glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression, Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-) phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change. This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I restriction fragment length polymorphism. We created transfected Chinese hamster ovary cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed the specificity of anti-Dra alloantisera. The allele-specific transfectants could form the basis of a new serological approach to immunohematology.


Self-association of organic solutes in solution: a NEXAFS study of aqueous imidazole.
N K-edge near-edge X-ray absorption fine-structure (NEXAFS) spectra of imidazole in concentrated aqueous solutions have been acquired. The NEXAFS spectra of the solution species differ significantly from those of imidazole monomers in the gas phase and in the solid state of imidazole, demonstrating the strong sensitivity of NEXAFS to the local chemical and structural environment. In a concentration range from 0.5 to 8.2 mol L(-1) the NEXAFS spectrum of aqueous imidazole does not change strongly, confirming previous suggestions that imidazole self-associates are already present at concentrations more dilute than the range investigated here. We show that various types of electronic structure calculations (Gaussian, StoBe, CASTEP) provide a consistent and complete interpretation of all features in the gas phase and solid state spectra based on ground state electronic structure. This suggests that such computational modelling of experimental NEXAFS will permit an incisive analysis of the molecular interactions of organic solutes in solutions. It is confirmed that microhydrated clusters with a single imidazole molecule are poor models of imidazole in aqueous solution. Our analysis indicates that models including both a hydrogen-bonded network of hydrate molecules, and imidazole-imidazole interactions, are necessary to explain the electronic structure evident in the NEXAFS spectra.


Can a patent extracranial-intracranial bypass provoke the conversion of an intracranial arterial stenosis to a symptomatic occlusion?
Although extracranial-intracranial (EC-IC) artery anastomosis seems to result in symptomatic improvement in certain types of cerebrovascular ischemic disease, this procedure can also be associated with significant morbidity, some of which paradoxically may be the direct result of a patent bypass. A review of the last 51/2 years at the Oregon Health Sciences University shows that 50 patients underwent 51 superficial temporal artery to middle cerebral artery bypass procedures with an angiographic patency rate of 91%. Of the 50 patients, 17 had intracranial stenotic lesions of either the middle cerebral artery (7 patients) or the internal carotid artery (10 patients). One patient had posterior cerebral artery stenosis. Five of the 18 patients with stenosis and a patent bypass developed a symptomatic occlusion of the stenotic lesion within 30 days after the anastomosis. It is hypothesized that the EC-IC bypass may have contributed to the occlusion in some of these patients by causing a change in the hemodynamic state. Possibly the bypass reversed the direction of flow distal to the stenosis to result in stasis and subsequent occlusion at the site of the stenosis or in some other manner affected hemostasis (i.e., the coagulation cascade).


Congenital generalized lipodystrophy in Taiwan.
Congenital generalized lipodystrophy (CGL) is a rare disorder characterized by scarce adipose tissue. This disease is distributed worldwide, but little is known about these patients in the Chinese population. Here, we delineate the phenotype and prognosis of CGL in our cohort. Patients diagnosed with CGL from 8 medical centers were reviewed. The initial presentation, laboratory findings, and molecular testing were retrospectively analyzed. A total of 16 patients were analyzed, and the current median age was 3.5 years (range, 9 months-17.5 years). In all patients, molecular results confirmed BSCL2 mutation. c.782dupG (p.Ile262Hisfs*12) was the most common genotype identified. All patients had triangular faces and muscular hypertrophy. In addition, 75% presented with hepatomegaly, 19% had cardiomegaly, and 44% exhibited acanthosis nigricans. Developmental delay was noted in 5 out of 9 patients (56%) with a median developmental quotient (DQ)/intelligence quotient (IQ) of 61. Thirteen patients (81.3%) had high triglyceride levels. Eight patients received leptin analysis, and 7 of them (88%) had low leptin levels. One patient exclusively received a lipid-lowering drug, 4 patients were exclusively placed on a fat-restricted diet, 5 patients were administered combination therapy, and 5 patients received no treatment. Three patients (19%) who developed diabetes mellitus received both oral hypoglycemic agents and insulin. Three patients (19%) experienced loss of ambulation and died prematurely. Our findings highlight the uniqueness of the genotype and phenotype in our cohort. Further long-term surveillance for comorbidities is necessary for early detection and management of these patients.


Analysis of new psychoactive substances in oral fluids by means of microextraction by packed sorbent followed by ultra-high-performance liquid chromatography-tandem mass spectrometry.
In recent years, new drugs, commonly known as new psychoactive substances (NPS), appeared on the market, which include, among others, synthetic cannabinoids, cathinones, and tryptamine analogs of psilocin. The aim of this work was to develop and validate a new method for simultaneous screening and quantification of 31 NPS in oral fluid by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The chosen target analytes represented different chemical and toxicological NPS classes, such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites. The procedure involved a rapid sample preparation based on protein precipitation followed by clean-up utilizing microextraction by packed sorbent (MEPS); the quantitative analysis was performed by UHPLC-MS/MS. The MEPS clean-up, regardless of non-quantitative recoveries for some analytes, provided an effective removal of interfering compounds, as demonstrated by reduced matrix effects found at different concentrations for all the analytes. The validation protocol, based on SWGTOX guidelines, demonstrated the suitability of the proposed method for quantitative analysis: linearity range ranged over 3 or 4 orders of magnitude; precision and accuracy tests gave RSD% values below 25%, and accuracy ranged from 85.9% to 107%, accomplishing SWGTOX requirements. Limits of detection (LODs) ranged between 0.005 ng/mL and 0.850 ng/mL and limits of quantification (LOQs) from 0.015 to 2.600 ng/mL.


Molecular mapping of developing dorsal horn-enriched genes by microarray and dorsal/ventral subtractive screening.
The dorsal horn of the spinal cord consists of distinct laminae that serve as a pivotal region for relaying a variety of somatosensory signals such as temperature, pain, and touch. The molecular mechanisms underlying the development of the dorsal horn are poorly understood. To define a molecular map of the dorsal horn circuit, we have profiled dorsal horn-enriched (DHE) gene expression in dorsal spinal cords on embryonic day 15.5 (E15.5) by genome-wide microarray and smart subtractive screening based on polymerase chain reaction (PCR). High-throughput in situ hybridization (ISH) was carried out to validate the expression of 379 genes in the developing dorsal spinal cord. A total of 113 DHE genes were identified, of which 59% show lamina-specific expression patterns. Most lamina-specific genes were expressed across at least two laminae, however. About 32% of all DHE genes are transcription factors, which represent the largest percentage of the group of all DHE functional classifications. Importantly, several individual lamina-specific transcription factors such c-Maf, Rora, and Satb1 are identified for the first time. Epistasis studies revealed several putative effectors of known DHE transcription factors such as Drg11, Tlx3(Rnx), and Lmx1b. These effector genes, including Grp, Trpc3, Pcp4, and Enc1, have been implicated in synaptic transmission, calcium homeostasis, and structural function and thus may have similar roles in the dorsal horn. The identification of a large number of DHE genes, especially those that are lamina specific, lays a foundation for future studies on the molecular machinery that controls the development of the dorsal horn and on functional differences of these distinct laminae in the dorsal spinal cord.


Sulpha allergy in lupus patients: a clinical perspective.
Systemic lupus erythematosus is a chronic, relapsing autoimmune disease that can affect multiple organ systems. An increased prevalence of drug allergy has been reported in lupus patients compared with the general population. Using a cohort of 417 lupus patients, we found a history of sulpha allergy in 27.3% of patients. European-American lupus patients with sulpha allergy are about two times more likely to suffer from lymphopenia, two times more likely to have anti-Ro autoantibody, and four times less likely to have anti-nRNP antibodies compared with lupus patients without a reported sulpha allergy (P = 0.0075, 0.025, and 0.032, respectively). In African-American lupus patients, a history of sulpha allergy was associated with over three times increased odds of developing pericarditis (P = 0.005).


Secukinumab in moderate-to-severe plaque psoriasis: a multi-center, retrospective, real-life study up to 52 weeks observation.
To evaluate efficacy and safety of the anti-IL-17 drug secukinumab in a real-life large cohort of patients with moderate-to-severe plaque psoriasis in Central Italy. Multicenter, retrospective study with an observation period of up to 52 weeks. Efficacy was assessed by Psoriasis Area and Severity Index (PASI) score; clinical and laboratory examinations were performed at baseline and at weeks 4, 12, 24, 36, and 52. A 90% and a 100% PASI score reduction (PASI90 and PASI100) were reported in 67.5% and 55% of patients at week 12, respectively. A rapid improvement of skin lesions was observed particularly in young patients and in patients naïve to biologics: at week 4, the achievement of PASI90 and PASI100 was higher in younger patients (odds ratio [OR] 0.95, and 0.95; p = 0.003, and 0.005, respectively); PASI90 was achieved by 42.0% of patients naïve to biologics and by 17.0% of patients with prior exposure to biologics (PBT) (OR 0.24; p = 0.001); and PASI100 was reached by 25.5% of naïve patients and 9.8% of PBT (OR 0.28; p = 0.015).The drug was well tolerated. Secukinumab was effective in this real-life analysis, with rapid clinical improvement and long-term maintenance of results.


Target of Rapamycin (TOR) Regulates Growth in Response to Nutritional Signals.
All organisms can respond to the availability of nutrients by regulating their metabolism, growth, and cell division. Central to the regulation of growth in response to nutrient availability is the target of rapamycin (TOR) signaling that is composed of two structurally distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). The TOR genes were first identified in yeast as target of rapamycin, a natural product of a soil bacterium, which proved beneficial as an immunosuppressive and anticancer drug and is currently being tested for a handful of other pathological conditions including diabetes, neurodegeneration, and age-related diseases. Studies of the TOR pathway unraveled a complex growth-regulating network. TOR regulates nutrient uptake, transcription, protein synthesis and degradation, as well as metabolic pathways, in a coordinated manner that ensures that cells grow or cease growth in response to nutrient availability. The identification of specific signals and mechanisms that stimulate TOR signaling is an active and exciting field of research that has already identified nitrogen and amino acids as key regulators of TORC1 activity. The signals, as well as the cellular functions of TORC2, are far less well understood. Additional open questions in the field concern the relationships between TORC1 and TORC2, as well as the links with other nutrient-responsive pathways. Here I review the main features of TORC1 and TORC2, with a particular focus on yeasts as model organisms.


Immune Responses to a Single Dose of the AZD1222/Covishield Vaccine at 16 Weeks in Individuals in Sri Lanka.
Due to limited access to vaccines, many countries have only administered a single dose of the AZD1222, whereas the dosage intervals have increased ≥4 wk. We sought to investigate the immunogenicity of a single dose of vaccine at ≥16 wk postimmunization. Severe acute respiratory syndrome coronavirus 2-specific Abs in 553 individuals and Abs to the receptor-binding domain of the Wuhan virus (wild-type) and the variants of concern, angiotensin-converting enzyme 2 receptor blocking Abs ex vivo and cultured IFN-γ T cell (Homo sapiens) responses and B cell (H. sapiens) ELISPOT responses, were investigated in a subcohort. The seropositivity rates in those >70 y of age (93.7%) was not significantly different compared with other age groups (97.7-98.2; Pearson χ2 = 7.8; p = 0.05). The Ab titers (Ab index) significantly declined (p < 0.0001) with increase in age. A total of 18 of 69 (26.1%) of individuals did not have angiotensin-converting enzyme 2 receptor-blocking Abs, whereas responses to the receptor-binding domain of wild-type (p = 0.03), B.1.1.7 (p = 0.04), and B.1.617.2 (p = 0.02) were significantly lower in those who were >60 y. Ex vivo IFN-γ T cell ELISPOT responses were seen in 10 of 66 (15.1%), whereas only a few expressed CD107a. However, >85% had a high frequency of cultured IFN-γ T cell ELISPOT responses and B cell ELISPOTs. Virus-specific Abs were maintained at ≥16 wk after receiving a single dose of AZD1222, although levels were lower to variants of concern, especially in older individuals. A single dose induced a high frequency of memory T and B cell responses.


MyD88 adaptor-like D96N is a naturally occurring loss-of-function variant of TIRAP.
Signals elicited by TLRs following the detection of microbes are integrated and diversified by a group of four cytoplasmic adaptor molecules featuring an evolutionarily conserved Toll/IL-1R signaling domain. Single nucleotide polymorphisms (SNPs) in TLRs and their adaptor molecules have been shown to influence susceptibility to a range of infectious and other diseases. The adaptor MyD88 adaptor-like (Mal)/Toll/IL-1R-containing adaptor protein is involved in TLR2 and 4 signal transduction by recruiting another adaptor molecule, MyD88, to the plasma membrane. In this study, we used naturally occurring variants of Mal as tools to study the molecular biology of Mal in more detail in cellular model systems and to thereby identify functionally interesting variants whose corresponding nonsynonymous SNPs might be of further epidemiological interest. Of seven reported variants for Mal, we found Mal D96N associated with reduced NF-kappaB signaling and cytokine production after overexpression in HEK293 and Huh-7 cells. The D96N mutation prevented Mal from recruiting its signaling partner MyD88 to the plasma membrane and altered posttranslational modification of Mal. These findings led us to investigate the frequency of heterozygosity for the corresponding SNP rs8177400 in a Caucasian case-control study on the etiology of lymphoma, a disease in which TLRs have been implicated. Although rs8177400 did not modify lymphoma risk in general, its frequency of heterozygosity was accurately determined to 0.97%. Our data add rs8177400 (D96N) to the list of functionally important variants of Mal and warrant further research into its immunological, epidemiological, and diagnostic relevance.


Protective effects of a natural product, curcumin, against amyloid β induced mitochondrial and synaptic toxicities in Alzheimer's disease.
The purpose of our study was to investigate the protective effects of a natural product-'curcumin'- in Alzheimer's disease (AD)-like neurons. Although much research has been done in AD, very little has been reported on the effects of curcumin on mitochondrial biogenesis, dynamics, function and synaptic activities. Therefore, the present study investigated the protective effects against amyloid β (Aβ) induced mitochondrial and synaptic toxicities. Using human neuroblastoma (SHSY5Y) cells, curcumin and Aβ, we studied the protective effects of curcumin against Aβ. Further, we also studied preventive (curcumin+Aβ) and intervention (Aβ+curcumin) effects of curcumin against Aβ in SHSY5Y cells. Using real time RT-PCR, immunoblotting and immunofluorescence analysis, we measured mRNA and protein levels of mitochondrial dynamics, mitochondrial biogenesis and synaptic genes. We also assessed mitochondrial function by measuring hydrogen peroxide, lipid peroxidation, cytochrome oxidase activity and mitochondrial ATP. Cell viability was studied using the MTT assay. Aβ was found to impair mitochondrial dynamics, reduce mitochondrial biogenesis and decrease synaptic activity and mitochondrial function. In contrast, curcumin enhanced mitochondrial fusion activity and reduced fission machinery, and increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in curcumin treated cells. Interestingly, curcumin pre- and post-treated cells incubated with Aβ showed reduced mitochondrial dysfunction, and maintained cell viability and mitochondrial dynamics, mitochondrial biogenesis and synaptic activity. Further, the protective effects of curcumin were stronger in pretreated SHSY5Y cells than in post-treated cells, indicating that curcumin works better in prevention than treatment in AD-like neurons. Our findings suggest that curcumin is a promising drug molecule to treat AD patients.


Metastases of spinal myxopapillary ependymoma: unique characteristics and clinical management.
OBJECTIVE Myxopapillary ependymoma is a benign WHO Grade I tumor most commonly located in the conus-cauda equina region of the spine. Although this tumor is defined by an overall excellent survival, dissemination throughout the whole neuraxis occurs frequently. The current study evaluated the clinical characteristics and significance of myxopapillary ependymoma metastases. METHODS Patients who underwent surgery from 2005 to 2015 for treatment of spinal myxopapillary ependymoma were included in the study. Charts were reviewed for primary tumor symptoms and initial treatment, local recurrence, response to salvage therapy, and presence and behavior of distant metastases. RESULTS Nineteen patients with spinal myxopapillary ependymoma were included in the study (52.6% female). The median age at first diagnosis was 32 years old (range 9-58 years old), and 26.3% were ≤ 18 years old. The median follow-up of all included patients was 48 months (range 6-456 months). Of the primary tumors, 84.2% were located in the lower thoracic or upper lumbar spine, spanning 1-3 levels in 94.7%. All patients underwent surgery for initial treatment: in 78.9% a gross-total resection (GTR) was achieved, with adjuvant radiation therapy (RT) in 20%. Of the 21.2% who underwent a subtotal resection (STR), 75% underwent postoperative RT. Tumor progression was noted in 26.3% of patients after a median 36-month follow-up (range 12-240 months). In 57.9% of patients, distant metastases were found, of which 36.4% were present at initial diagnosis. Further metastases occurred within a median of 20 months (range 2-360 months). Following a diagnosis of metastatic tumor, 72.7% did not show progression and no symptoms were observed during a median follow-up of 36 months (range 6-216 months). Metastases occurred in all parts of the neuroaxis, but were principally localized in the thoracic and sacral spine in 38.9% and 33.3%, respectively; the brain was involved in 11.1%. In 54.5%, more than 1 level was affected. Overall survival was 100% with an excellent clinical and neurological outcome in 78.9% of cases. CONCLUSIONS Metastatic dissemination within the CNS can be observed in many patients with myxopapillary ependymoma. GTR of the primary tumor should be the primary treatment goal, and additional RT is recommended after STR. For distant metastases of myxopapillary ependymoma without clinical manifestation, close clinical and MRI follow-up represents a sufficient strategy because most of the metastases remain asymptomatic and do not show progression over time. Additional resection or irradiation as salvage therapy would be recommended if metastases become symptomatic.


Effect of Different Onset Thresholds on Isometric Midthigh Pull Force-Time Variables.
Dos'Santos, T, Jones, PA, Comfort, P, and Thomas, C. Effect of different onset thresholds on isometric midthigh pull force-time variables. J Strength Cond Res 31(12): 3463-3473, 2017-Various thresholds have been used to identify the onset of contraction during isometric midthigh pull (IMTP); however, no agreed onset threshold exists for this assessment. The purpose of this study was to compare relative body weight (BW) and arbitrary onset thresholds to a criterion onset threshold 5 SDs of BW for IMTP force-time variables; force at each threshold, peak force, time-specific force values (100, 150, and 200 ms), and rate of force development (RFD) during 0-100, 0-150, 0-200 ms. Academy rugby league players (n = 9, age: 18.5 ± 0.4 years; height: 1.82 ± 0.09 m; mass: 91.2 ± 13.1 kg) performed 2 IMTP trials on a force platform sampling at 1,000 Hz. The neutral force-time data pool (18 trials) was analyzed with five different thresholds and compared with criterion threshold to determine any variance in force-time variables. Five SDs of BW were significantly lower than 10% BW and 75 N for threshold force which led to significantly greater time-specific force values at 100 and 150 ms and unacceptable limits of agreements (LOA) for all force-time variables. No significant differences (p > 0.05) were observed between 2.5% and 5 SDs of BW; and between 5% and 5 SDs of BW for threshold force and all force-time variables with acceptable LOA. The 5 SDs of BW and 2.5% BW onset thresholds consistently resulted in the lowest values for threshold force, time-specific force values, and RFD, attributed to a lower onset bias. Therefore, scientists and practitioners are recommended to use a 5 SD of BW onset threshold for time-specific force values and RFD for accurate data because it accounts for signal noise during the weighing period. Subsequently, there is greater certainty that the onset of contraction identifies a true meaningful change in force, in contrast to relative BW thresholds.


Novel CFTR Mutations in Two Iranian Families with Severe Cystic Fibrosis.
Cystic fibrosis (CF) is a common autosomal recessive disorder that affects many body systems and is produced by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF is also the most frequently inherited disorder in the West. The aim of this study was to detect the mutations in the CFTR gene in two Iranian families with CF. After DNA extraction using the salting out method, a mutation panel consisting of 35 common mutations was tested by PCR, followed by reverse hybridization Strip Assay. To confirm the mutations, we have also performed Sanger sequencing for all 27 exons, intronic flanking regions, and 5' and 3' UTRs of the CFTR gene. Carrier testing in a spouse revealed a novel nonsense mutation in the CFTR gene (c.2777 T>A (p.L926X)) in exon 17 for husband and a previously described heterozygous splice site pathogenic mutation (c.1393-1G>A) in his wife. The other novel compound heterozygous missense mutation (c.3119 T>A (p.L1040H)), which was previously reported as nonsense c.3484C>T (p.R1162X) mutation, was found in exon 19 in patient screening. Two novel CFTR mutations in exons 17 and 19 are responsible for CF with severe phenotypes in two Iranian families. These two mutations supplement the mutation spectrum of CFTR and may contribute to a better understanding of CFTR protein function.


Perceived stress mediates the relationship between mindfulness and negative affect variability: A randomized controlled trial among middle-aged to older adults.
Despite the interest in mindfulness over the past 20 years, studies have only recently begun to examine mindfulness in older adults. The primary aim of this study was to evaluate pretreatment to post-treatment change in negative affect variability (NAV) following a mindfulness training among 134 mildly stressed, middle-aged to older adults. The secondary aim was to assess if the effects of mindfulness training on NAV would be partially explained by pretreatment to post-treatment reductions in perceived stress, a trend that would be congruent with several stress models. In this randomized control trial, participants were assigned to either a 6-week mindfulness meditation training programme or to a wait list control. Ecological momentary assessment, a data capturing technique that queries about present moment experiences in real time, captured NAV. Mixed-model ANOVAs and a path analysis were conducted. Participants in the mindfulness meditation training significantly reduced NAV when compared with wait list control participants. Further, there was a significant indirect group effect on reductions in NAV through change in perceived stress. Few studies have tested mechanisms of action, which connect changes that occur during mindfulness training with psychological outcomes in older adults. Understanding the mechanisms by which mindfulness enhances well-being may optimize interventions.


Effect of osmolarity of artificial tear drops on relief of dry eye symptoms: BJ6 and beyond.
Patients with clinically well defined keratoconjunctivitis sicca (KCS) participated in two trials of hypo-osmolar tear substitutes. The trials were double masked, single crossover studies with computer generated random order allocation and were conducted by postal questionnaire. In the first trial a hypo-osmolar formulation using polyvinylpyrrolidone and hydroxyethyl cellulose was compared with the patients' usual treatment (BJ6 or hypromellose) and found to be inferior, especially in respect of blurring and stickiness caused by higher viscosity. In the second trial hypo-osmolar preparations of BJ6 and hypromellose were compared with their iso-osmolar equivalents and found not to be significantly different in their effects. Hypo-osmolarity alone does not seem to guarantee relief of symptoms in KCS. Other factors including viscosity and colloid osmotic pressure may be more significant.


Measuring Adherence to Evidence-Based Clinical Practice Guidelines.
Dental Health Services Victoria publishes evidence-based clinical practice guidelines (CPGs) to assist public oral health practitioners to provide high-quality dental care. How well these CPGs are implemented into practice is unknown. The aim of this study was to assess adherence to selected CPGs. An electronic auditing tool was developed using clinical indicators derived for "stainless steel crown (SSC)," "restorative care for children under general anesthetic (GA)," and "direct restorative materials" CPG. Six trained dentists audited a random sample of 204 dental records of children aged 3-12 years from 2 major public dental agencies. In total, 319 material-based treatments were audited, comprising 170 resin composite, 81 glass ionomer cement, 64 SSC, and 4 amalgam restorations. Adherence to the current guidelines varied from 94% of the SSC to none of the amalgam treatments audited. Almost half (47%) of the resin composite restorations and 5% of glass ionomer cement restorations were nonadherent to the relevant guideline. Average adherence was up to 72% of cases. Clinicians need to consider recording the rationale upon which their professional judgment is based when they decide not to follow an appropriate CPG.


Diffusion tensor imaging for anatomical localization of cranial nerves and cranial nerve nuclei in pontine lesions: initial experiences with 3T-MRI.
With continuous refinement of neurosurgical techniques and higher resolution in neuroimaging, the management of pontine lesions is constantly improving. Among pontine structures with vital functions that are at risk of being damaged by surgical manipulation, cranial nerves (CN) and cranial nerve nuclei (CNN) such as CN V, VI, and VII are critical. Pre-operative localization of the intrapontine course of CN and CNN should be beneficial for surgical outcomes. Our objective was to accurately localize CN and CNN in patients with intra-axial lesions in the pons using diffusion tensor imaging (DTI) and estimate its input in surgical planning for avoiding unintended loss of their function during surgery. DTI of the pons obtained pre-operatively on a 3Tesla MR scanner was analyzed prospectively for the accurate localization of CN and CNN V, VI and VII in seven patients with intra-axial lesions in the pons. Anatomical sections in the pons were used to estimate abnormalities on color-coded fractional anisotropy maps. Imaging abnormalities were correlated with CN symptoms before and after surgery. The course of CN and the area of CNN were identified using DTI pre- and post-operatively. Clinical associations between post-operative improvements and the corresponding CN area of the pons were demonstrated. Our results suggest that pre- and post-operative DTI allows identification of key anatomical structures in the pons and enables estimation of their involvement by pathology. It may predict clinical outcome and help us to better understand the involvement of the intrinsic anatomy by pathological processes.


Measuring quality in community based housing support - the QPC-H instrument.
Purpose - The purpose of this paper is to test the psychometric properties and dimensionality of the instrument Quality in Psychiatric Care-Housing (QPC-H) and briefly describe the residents' perception of quality of housing support. Design/methodology/approach - A sample of 174 residents from 22 housing support services in nine Swedish municipalities participated in the study. Confirmatory factor analysis (CFA) revealed that the QPC-H consisted of six dimensions and had a factor structure largely corresponding to that found among other instruments in the Quality in Psychiatric Care (QPC) family of instruments. Findings - CFA revealed that the QPC-H consisted of six dimensions and had a factor structure largely corresponding to that found among other instruments in the QPC family of instruments. The internal consistency of the factors was acceptable except in the case of secure and secluded environment, probably due to few numbers of items. With this exception, the QPC-H shows adequate psychometric properties. Social implications - The residents' ratings of quality of housing service were generally high; the highest rating was for secluded environment and the lowest for participation. This dimension would thus seem to indicate an important area for improvement. Originality/value - The QPC-H includes important aspects of residents' assessment of quality of housing service and offers a simple and inexpensive way to evaluate housing support services from the residents' perspective.


Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan.
Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan. KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features. A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain. We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.


Comparison of transthoracic echocardiography with N-terminal pro-brain natriuretic Peptide as a tool for risk stratification of patients undergoing major noncardiac surgery.
The role of preoperative transthoracic echocardiography (TTE) for the risk stratification has not been well investigated yet. We compared the predictive power of TTE with N-terminal pro-brain natriuretic peptide (NT-proBNP), a representative biomarker that predicts perioperative cardiovascular risk, and investigated whether these tests have incremental value to the clinically determined risk. We evaluated the Revised Cardiac Risk Index (RCRI), TTE, and NT-proBNP in 1,923 noncardiac surgery cases. The primary endpoint was a perioperative major cardiovascular event (PMCE), which was defined by any single or combined event of secondary endpoints including myocardial infarction, development of pulmonary edema, or primary cardiovascular death within 30 days after surgery. All echocardiographic parameters including left ventricular ejection fraction, regional wall motion score index, and transmitral early diastolic velocity/tissue Doppler mitral annular early diastolic velocity (E/E') were predictive of PMCE (c-statistics=0.579±0.019 to 0.589±0.015), but none of these parameters were better than the clinically determined RCRI (c-statistics=0.594±0.019) and were inferior to NT-proBNP (c-statistics=0.748±0.019, p<0.001). The predictive power of RCRI {adjusted relative risk (RR)=1.4} could be improved by addition of echocardiographic parameters (adjusted RR=1.8, p<0.001), but not to that extent as by addition of NT-proBNP to RCRI (adjusted RR=3.7, p<0.001). TTE was modestly predictive of perioperative cardiovascular events but was not superior to NT-proBNP. Moreover, it did not have incremental value to the clinically determined risk. The results of our study did not support the use of routine echocardiography before noncardiac surgery.


Angled telescopic surgery, an approach for laryngeal diagnosis and surgery without suspension.
Many methods have been used successfully for the diagnosis and treatment of laryngeal diseases. Microscopic and, recently, telescopic surgery represent the state of the art in endoscopic laryngeal surgery but drawbacks are possible during their application. To keep the suspension apparatus adequately positioned, excessive force is sometimes placed on the upper teeth and tongue with the laryngoscope tube causing damage. Complications in relation to the pharynx, larynx and cardiovascular system have also been reported. In order to reduce complications resulting from the manipulation or stimulation of the upper aerodigestive tract and from torque forces on the upper teeth. We present a method of larynx surgery in which laryngeal suspension is not required. Technical report. We have devised a fiber-optic telescope with its 40mm distal portion deviated 60 degrees from the direction of the proximal portion. This angle was taken by measuring patients immediately before standard microlaryngeal surgery was performed. The surgical instruments have the same angle as the telescope, in order to work on the larynx. This technique provides an image that is not limited by the distal aperture of the laryngoscope and has an advantage in that magnification and illumination may be provided by changing the distance of the lesion from the tip of the instrument. we have operated on four patients with laryngeal diseases and have had no complications as a result of this approach. We feel that this technique gives us the freedom to view the lesions better and helps to minimize the drawbacks caused by laryngeal suspension.


Motivation to Address Self-Reported Hearing Problems in Adults With Normal Hearing Thresholds.
The purpose of this study was to compare the motivation to change in relation to hearing problems in adults with normal hearing thresholds but who report hearing problems and that of adults with a mild-to-moderate sensorineural hearing loss. Factors related to their motivation were also assessed. The motivation to change in relation to self-reported hearing problems was measured using the University of Rhode Island Change Assessment (McConnaughy, Prochaska, & Velicer, 1983). The relationship between objective and subjective measures and an adult's motivation was examined. The level of hearing handicap did not differ significantly between adults with normal hearing who reported problems hearing in background noise and adults who had a mild-to-moderate sensorineural hearing loss. Hearing handicap, personal distress, and minimization of hearing loss were factors significantly related to motivation. Age, degree of hearing loss, speech-in-noise scores, working memory, and extended high-frequency average thresholds were not significantly related to their motivation. Adults with normal hearing thresholds but self-reported hearing problems had the same level of hearing handicap and were equally motivated to take action for their hearing problems as age-matched adults with a mild-to-moderate sensorineural hearing loss. Hearing handicap, personal distress, and minimization of hearing loss were most strongly correlated with an individual's motivation to change.


Detection of parvovirus B19 capsid proteins in testicular tissues.
To detect B19 capsid proteins, VP1 and VP2, in testicular tissues, both normal and tumor, using immunohistochemistry. Samples of normal, fetal, and tumor testicular tissue (n = 31) and normal testicular DNA (n = 1) were tested for the presence of B19. Immunohistochemistry staining was used for the detection of viral capsid proteins VP1 and VP2. Polymerase chain reaction with 4 primer sets was used to test for the presence of B19 DNA in a normal testicular sample. B19 capsid protein VP1 and VP2 was detected by immunohistochemistry in 6 (85.7%) of 7 normal testicular samples and 17 (73.9%) of 23 tumor samples. The findings from a normal fetal testicular sample were equivocal. B19 DNA was detected in normal testicular DNA with 4 of the 4 primer sets used. In contrast to previous reports, B19 capsid proteins VP1 and VP2 have now been detected in both normal and tumor testicular tissue. The persistence of B19 in a diverse range of tissues, including the testes, requires more research into the molecular mechanisms by which B19 can enter these cells, as well as the possible etiologic roles in chronic diseases, including cancer.


Critical assessment of automated flow cytometry data analysis techniques.
Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis.


Viral kinetics suggests a reconciliation of the disparate observations of the modulation of claudin-1 expression on cells exposed to hepatitis C virus.
The tight junction protein claudin-1 (CLDN1) is necessary for hepatitis C virus (HCV) entry into target cells. Recent studies have made disparate observations of the modulation of the expression of CLDN1 on cells following infection by HCV. In one study, the mean CLDN1 expression on cells exposed to HCV declined, whereas in another study HCV infected cells showed increased CLDN1 expression compared to uninfected cells. Consequently, the role of HCV in modulating CLDN1 expression, and hence the frequency of cellular superinfection, remains unclear. Here, we present a possible reconciliation of these disparate observations. We hypothesized that viral kinetics and not necessarily HCV-induced receptor modulation underlies these disparate observations. To test this hypothesis, we constructed a mathematical model of viral kinetics in vitro that mimicked the above experiments. Model predictions provided good fits to the observed evolution of the distribution of CLDN1 expression on cells following exposure to HCV. Cells with higher CLDN1 expression were preferentially infected and outgrown by cells with lower CLDN1 expression, resulting in a decline of the mean CLDN1 expression with time. At the same time, because the susceptibility of cells to infection increased with CLDN1 expression, infected cells tended to have higher CLDN1 expression on average than uninfected cells. Our study thus presents an explanation of the disparate observations of CLDN1 expression following HCV infection and points to the importance of considering viral kinetics in future studies of receptor expression on cells exposed to HCV.


Long-lasting self-inhibition of neocortical interneurons mediated by endocannabinoids.
Neocortical GABA-containing interneurons form complex functional networks responsible for feedforward and feedback inhibition and for the generation of cortical oscillations associated with several behavioural functions. We previously reported that fast-spiking (FS), but not low-threshold-spiking (LTS), neocortical interneurons from rats generate a fast and precise self-inhibition mediated by inhibitory autaptic transmission. Here we show that LTS cells possess a different form of self-inhibition. LTS, but not FS, interneurons undergo a prominent hyperpolarization mediated by an increased K+-channel conductance. This self-induced inhibition lasts for many minutes, is dependent on an increase in intracellular [Ca2+] and is blocked by the cannabinoid receptor antagonist AM251, indicating that it is mediated by the autocrine release of endogenous cannabinoids. Endocannabinoid-mediated slow self-inhibition represents a powerful and long-lasting mechanism that alters the intrinsic excitability of LTS neurons, which selectively target the major site of excitatory connections onto pyramidal neurons; that is, their dendrites. Thus, modulation of LTS networks after their sustained firing will lead to long-lasting changes of glutamate-mediated synaptic strength in pyramidal neurons, with consequences during normal and pathophysiological cortical network activities.


The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of the hypertrophic phenotype in rat ventricular cardiomyocytes.
Adult mammalian ventricular cardiomyocytes are terminally differentiated cells that enlarge adaptively by hypertrophy. In this situation, genes normally expressed in the fetal ventricular cardiomyocyte (e.g. atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM) alpha-actin) are re-expressed, and there is transient expression of immediate early genes (e.g. c-fos). Using appropriate reporter plasmids, we studied the effects of transfection of the constitutively active or dominant negative mitogen-activated protein kinase kinase MEK1 on ANF, beta-MHC, and SkM alpha-actin promoter activities in cultured ventricular cardiomyocytes. ANF expression was stimulated (maximally 75-fold) by the hypertrophic agonist phenylephrine in a dose-dependent manner (EC50, 10 microM), and this stimulation was inhibited by dominant negative MEK1. Cotransfection of dominant negative MEK1 with a dominant negative mitogen-activated protein kinase (extracellular signal-regulated protein kinase (ERK2)) increased this inhibition. Transfection with constitutively active MEK1 constructs doubled ANF promoter activity. The additional cotransfection of wild-type ERK2 stimulated ANF promoter activity by about 5-fold. Expression of beta-MHC and SkM alpha-actin was also stimulated. Promoter activity regulated by activator protein-1 or c-fos serum response element consensus sequences was also increased. We conclude that the MEK1/ERK2 cascade may play a role in regulating gene expression during hypertrophy.


Ajuba family proteins link JNK to Hippo signaling.
Wounding, apoptosis, or infection can trigger a proliferative response in neighboring cells to replace damaged tissue. Studies in Drosophila have implicated c-Jun amino-terminal kinase (JNK)-dependent activation of Yorkie (Yki) as essential to regeneration-associated growth, as well as growth associated with neoplastic tumors. Yki is a transcriptional coactivator that is inhibited by Hippo signaling, a conserved pathway that regulates growth. We identified a conserved mechanism by which JNK regulated Hippo signaling. Genetic studies in Drosophila identified Jub (also known as Ajuba LIM protein) as required for JNK-mediated activation of Yki and showed that Jub contributed to wing regeneration after wounding and to tumor growth. Biochemical studies revealed that JNK promoted the phosphorylation of Ajuba family proteins in both Drosophila and mammalian cells. Binding studies in mammalian cells indicated that JNK increased binding between the Ajuba family proteins LIMD1 or WTIP and LATS1, a kinase within the Hippo pathway that inhibits the Yki homolog YAP. Moreover, JNK promoted binding of LIMD1 and LATS1 through direct phosphorylation of LIMD1. These results identify Ajuba family proteins as a conserved link between JNK and Hippo signaling, and imply that JNK increases Yki and YAP activity by promoting the binding of Ajuba family proteins to Warts and LATS.


Reward-Guided Learning with and without Causal Attribution.
When an organism receives a reward, it is crucial to know which of many candidate actions caused this reward. However, recent work suggests that learning is possible even when this most fundamental assumption is not met. We used novel reward-guided learning paradigms in two fMRI studies to show that humans deploy separable learning mechanisms that operate in parallel. While behavior was dominated by precise contingent learning, it also revealed hallmarks of noncontingent learning strategies. These learning mechanisms were separable behaviorally and neurally. Lateral orbitofrontal cortex supported contingent learning and reflected contingencies between outcomes and their causal choices. Amygdala responses around reward times related to statistical patterns of learning. Time-based heuristic mechanisms were related to activity in sensorimotor corticostriatal circuitry. Our data point to the existence of several learning mechanisms in the human brain, of which only one relies on applying known rules about the causal structure of the task.


Neural substrates of response-based sequence learning using fMRI.
Representation of sequential structure can occur with respect to the order of perceptual events or the order in which actions are linked. Neural correlates of sequence retrieval associated with the order of motor responses were identified in a variant of the serial reaction time task in which training occurred with a spatially incompatible mapping between stimuli and finger responses. After transfer to a spatially compatible version of the task, performance enhancements indicative of learning were only present in subjects required to make finger movements in the same order used during training. In contrast, a second group of subjects performed the compatible task using an identical sequence of stimuli (and different order of finger movements) as in training. They demonstrated no performance benefit, indicating that learning was response based. Analysis was restricted to subjects demonstrating low recall of the sequence structure to rule out effects of explicit awareness. The interaction of group (motor vs. perceptual transfer) with sequence retrieval (sequencing vs. rest) revealed significantly greater activation in the bilateral supplementary motor area, cingulate motor area, ventral premotor cortex, left caudate, and inferior parietal lobule for subjects in the motor group (illustrating successful sequence retrieval at the response level). Retrieval of sequential responses occurs within mesial motor areas and related motor planning areas.


Health Impacts of Active Transportation in Europe.
Policies that stimulate active transportation (walking and bicycling) have been related to heath benefits. This study aims to assess the potential health risks and benefits of promoting active transportation for commuting populations (age groups 16-64) in six European cities. We conducted a health impact assessment using two scenarios: increased cycling and increased walking. The primary outcome measure was all-cause mortality related to changes in physical activity level, exposure to fine particulate matter air pollution with a diameter <2.5 μm, as well as traffic fatalities in the cities of Barcelona, Basel, Copenhagen, Paris, Prague, and Warsaw. All scenarios produced health benefits in the six cities. An increase in bicycle trips to 35% of all trips (as in Copenhagen) produced the highest benefits among the different scenarios analysed in Warsaw 113 (76-163) annual deaths avoided, Prague 61 (29-104), Barcelona 37 (24-56), Paris 37 (18-64) and Basel 5 (3-9). An increase in walking trips to 50% of all trips (as in Paris) resulted in 19 (3-42) deaths avoided annually in Warsaw, 11(3-21) in Prague, 6 (4-9) in Basel, 3 (2-6) in Copenhagen and 3 (2-4) in Barcelona. The scenarios would also reduce carbon dioxide emissions in the six cities by 1,139 to 26,423 (metric tonnes per year). Policies to promote active transportation may produce health benefits, but these depend of the existing characteristics of the cities. Increased collaboration between health practitioners, transport specialists and urban planners will help to introduce the health perspective in transport policies and promote active transportation.


Clinical implementation of pharmacogenetics: more than one gene at a time.
Tricyclic antidepressant (TCA) clinical pharmacogenetic implementation guidelines for CYP2D6 and CYP2C19 genotypes highlight the importance of both genes. However, studies of the combined impact of the two genes are sparse, limiting the ability to make strong recommendations based on both genes. The warfarin pharmacogenetics literature highlights the strength of a multigenic approach for discovery and clinical implementation. For optimal impact and interpretation, investigators are encouraged to conduct studies in the context of previously well-defined pharmacogenetics markers.


Subgrouping of gliomas on the basis of genetic profiles.
Management of gliomas depends on histological diagnosis; there are, however, limitations to the systems presently used. Tumors in the same entity can have different clinical courses, especially when they are diagnosed as WHO grade II-III. Previous studies revealed that genetic subgrouping of gliomas provides useful information that could help establishment of treatment procedures on the basis of the genetic background of the tumors. Recently, the authors analyzed the chromosomal copy number aberrations (CNAs) of adult supratentorial gliomas by comparative genomic hybridization using microdissected tissue sections. The tumors were classified into subgroups according to chromosomal CNAs. WHO grade II-III gliomas contained a variety of genetic subgroups that correlated well with the clinical course. Of these, long progression-free survival was observed for tumors with +7q and those with -1p/19q, low-grade tumors of 2 major lineages, and, in our preliminary data, both were closely correlated with mutation of IDH1. Furthermore, in contrast with +7q tumors, the great majority of +7 or +7/-10q groups had wildtype IDH1. Genetic studies suggest that cytogenetic characterization may provide an additional classification system for gliomas, and new criteria could help to establish rational and objective means for analysis of treatment procedures.


Multi-agent reinforcement learning with approximate model learning for competitive games.
We propose a method for learning multi-agent policies to compete against multiple opponents. The method consists of recurrent neural network-based actor-critic networks and deterministic policy gradients that promote cooperation between agents by communication. The learning process does not require access to opponents' parameters or observations because the agents are trained separately from the opponents. The actor networks enable the agents to communicate using forward and backward paths while the critic network helps to train the actors by delivering them gradient signals based on their contribution to the global reward. Moreover, to address nonstationarity due to the evolving of other agents, we propose approximate model learning using auxiliary prediction networks for modeling the state transitions, reward function, and opponent behavior. In the test phase, we use competitive multi-agent environments to demonstrate by comparison the usefulness and superiority of the proposed method in terms of learning efficiency and goal achievements. The comparison results show that the proposed method outperforms the alternatives.


Modification of tubeless percutaneous nephrolithotomy with a ureteropelvic junction stent.
To introduce the ureteropelvic junction stent as a safe and effective modification to tubeless percutaneous nephrolithotomy for select patients to maintain antegrade access to the collecting system. From April 2014 to December 2015, 31 patients underwent modified tubeless percutaneous nephrolithotomy with ureteropelvic junction (UPJ) stent left in situ and an extraction string coming out the nephrostomy tract. Primary study endpoints included complications, emergency department visits, or re-admissions. Secondary endpoints were perioperative parameters including mean operative time, blood loss, length of stay, and time to stent removal. There were three Clavien grade III complications: one patient required exchange of her UPJ stent with a double-J stent due to distal ureteral obstruction and two patients required ureteroscopic retrieval of retained stents. Minor issues included one patient with stent discomfort and another who experienced a vasovagal response during stent removal. Patients stayed an average of 2.2 +/- 1.5 days, including six discharged same day. Of 31 patients, 30 were successfully drained by ureteropelvic junction stent. UPJ stent is a safe and effective modification to percutaneous nephrolithotomy to maintain antegrade access and minimize stent discomfort. Further studies should be performed to determine optimal candidate selection and quantify stent-related symptoms.


Surfactant proteins and thyroid transcription factor-1 in pulmonary and breast carcinomas.
Antibodies to the pulmonary epithelial cell-specific proteins surfactant proteins A and B (SP-A and SP-B) and to thyroid transcription factor-1 (TTF-1), a homeodomain nuclear transcription protein, were used as immunohistochemical markers to asses their ability to distinguish primary pulmonary non-small cell carcinomas (n = 57) from carcinomas of the breast (n = 51). SP-A, SP-B, and TTF-1 were detected in 49%, 53%, and 63% of non-small cell carcinomas, respectively. These three antibodies stained pulmonary adenocarcinomas in 54%, 63% and 76% of specimens, respectively. Squamous cell carcinomas rarely stained using these markers. Antibodies to SP-B and TTF-1 never stained any of the 51 breast carcinomas, whereas four of these tumors stained for SP-A. To better define the potential diagnostic value of these antibodies, 13 breast carcinomas metastatic to the lung were studied. Of the three antibodies tested, only TTF-1 seemed useful, because none of the 13 metastatic tumors showed immunoreactivity to this antibody, whereas six specimens (46%) showed reactivity for both SP-A and SP-B. To emphasize further the potential usefulness of antibodies to TTF-1, sections of adenocarcinomas of the colon (n = 18) and prostate (n = 9), renal cell carcinomas (n = 8), and epithelioid mesotheliomas (n = 4) were evaluated; none was positive. Only one of 66 gastric and one of eight endometrial adenocarcinomas showed focal positivity. These results demonstrate the usefulness of immunodetection of a pulmonary cell selective transcription protein (TTF-1) in the diagnosis of pulmonary adenocarcinoma, readily distinguishing breast carcinomas from primary pulmonary adenocarcinomas. In contrast, staining for SP-A and SP-B is of limited value, because there is an unacceptably high rate of cross-reactivity between breast carcinomas metastatic to the lung and primary pulmonary carcinomas. The latter finding illustrates and supports the fact that tumor marker phenotypes might differ in primary and secondary tissue sites.


Antibiotic gold: tethering of antimicrobial peptides to gold nanoparticles maintains conformational flexibility of peptides and improves trypsin susceptibility.
Peptide-coated nanoparticles are valuable tools for diverse biological applications, such as drug delivery, molecular recognition, and antimicrobial action. The functionalization of pre-fabricated nanoparticles with free peptides in solution is inefficient either due to aggregation of the particles or due to the poor ligand exchange reaction. Here, we present a one-pot synthesis for preparing gold nanoparticles with a homogeneous distribution that are covered in situ with cationic peptides in a site-selective manner via Cys-residue at the N-terminus. Five representative peptides were selected, which are known to perturb cellular membranes and exert their antimicrobial and/or cell penetrating activity by folding into amphiphilic α-helical structures. When tethered to the nanoparticles at a single site, all peptides were found to switch their conformation from unordered state (in aqueous buffers) to their functionally relevant α-helical conformation in the presence of model membranes, as shown by circular dichroism spectroscopy. The conjugated peptides also maintained the same antibacterial activity as in the free form. Most importantly, when tethered to the gold nanoparticles the peptides showed an enormous increase in stability against trypsin digestion compared to the free forms, leading to a dramatic improvement of their lifetimes and activities. These findings suggest that site-selective surface tethering of peptides to gold nanoparticles has several advantages: (i) it does not prevent the peptides from folding into their biologically active conformation, (ii) such conjugation protects the peptides against protease digestion, and (iii) this way it is possible to prepare stable, water soluble antimicrobial nanoparticles as promising antibacterial agents.


l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.
In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p < 0.05). The results presented herein demonstrate that l-glutamine supplemented with l-alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.


Prevalence of bullying and victimization among children in early elementary school: do family and school neighbourhood socioeconomic status matter?
Bullying and victimization are widespread phenomena in childhood and can have a serious impact on well-being. Children from families with a low socioeconomic background have an increased risk of this behaviour, but it is unknown whether socioeconomic status (SES) of school neighbourhoods is also related to bullying behaviour. Furthermore, as previous bullying research mainly focused on older children and adolescents, it remains unclear to what extent bullying and victimization affects the lives of younger children. The aim of this study is to examine the prevalence and socioeconomic disparities in bullying behaviour among young elementary school children. The study was part of a population-based survey in the Netherlands. Teacher reports of bullying behaviour and indicators of SES of families and schools were available for 6379 children aged 5-6 years. One-third of the children were involved in bullying, most of them as bullies (17%) or bully-victims (13%), and less as pure victims (4%). All indicators of low family SES and poor school neighbourhood SES were associated with an increased risk of being a bully or bully-victim. Parental educational level was the only indicator of SES related with victimization. The influence of school neighbourhood SES on bullying attenuated to statistical non-significance once adjusted for family SES. Bullying and victimization are already common problems in early elementary school. Children from socioeconomically disadvantaged families, rather than children visiting schools in disadvantaged neighbourhoods, have a particularly high risk of involvement in bullying. These findings suggest the need of timely bullying preventions and interventions that should have a special focus on children of families with a low socioeconomic background. Future studies are necessary to evaluate the effectiveness of such programs.


Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM).
Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1-195 of hnRNP A1 is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)(n). We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in K(d) from 88nM to 209nM and 316nM, respectively. The loss of these orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the base mobility, but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main-chain to base hydrogen bonding makes up an important aspect of ligand recognition (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated and (3) a conserved lysine position proximal to RNP-2 (K(106)-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.


Human quadriceps muscle mitochondria: a functional characterization.
Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in micromol O min(-1) g protein(-1) (25 degrees C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 micromol O min(-1) g protein(-1)). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 micromol ATP min(-1) g protein(-1)). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.


[Transient brain ischemia: NMDA receptor modulation and delayed neuronal death].
Transient global ischemia induces delayed neuronal death in certain cell types and brain regions while sparing cells in other areas. A key process through which oxygen-glucose deprivation triggers cell death is the excessive accumulation of the neurotransmitter glutamate leading to over excitation of neurons. In certain neurons this increase in glutamate will potentiate the NMDA type of glutamate receptor, which can then initiate cell death. This review provides an update of the neurophysiological, cellular and molecular mechanisms inducing post-ischemic plasticity of NMDA receptors, focusing on the sensitive CA1 pyramidal neurons in the hippocampus as compared to the relatively resistant neighboring CA3 neurons. Both a change in the equilibrium between protein tyrosine kinases/phosphatases and an increased density of surface NMDA receptors in response to ischemia may explain the selective vulnerability of specific cell types. Implications for the treatment of stroke and reasons for the failures of human clinical trials utilizing NMDA receptor antagonists are also discussed.


A comparison of behavioral and multiple mini-interview formats in physician assistant program admissions.
The interview remains a widely used tool in health professions program admissions. The purpose of this study was to compare the use of a behavioral interview format with the multiple mini-interview format in measuring desired noncognitive behaviors. This dual cohort, observational, comparative study used a polytomous rating-scale model to analyze the results from two homogeneous groups of physician assistant (PA) program applicants (total N = 176). One group (n = 93) participated in two 20-minute behavioral interviews conducted by two raters per interviewee. The behavioral format included questions related to past behaviors and performance as a way to identify latent professionalism characteristics. The second group (n = 83) completed ten separate 7-minute stations with one rater per station. Each of the mini-stations assigned a specific topic and/or task to be completed. The score distributions related to applicant performance and station difficulty were plotted using Rasch analysis software. The behavioral interview format and multiple mini-interview had similar model fit. The behavioral interview did not adequately measure differences in applicant characteristics. In contrast, the multiple mini-interview measured more variation in noncognitive traits and identified better matching of station difficulty and person ability. In this study the multiple mini-interview format was a more reliable admissions tool in detecting latent professionalism attributes among PA program applicants. The multiple mini-interview format appeared to measure professional potential and organizational fit better than the behavioral interview format. A larger study across several programs may provide additional support for these findings.


Activation of an ER-body-localized beta-glucosidase via a cytosolic binding partner in damaged tissues of Arabidopsis thaliana.
The ER body is an endoplasmic reticulum (ER)-derived organelle. Because ER bodies are induced by wounding and methyl jasmonate (MeJA) treatment in rosette leaves, they might be responsible for defense systems. Recently, we isolated nai1 mutants that have no ER body and showed that the levels of PYK10 and PBP1 (PYK10-binding protein 1: At3g16420) were decreased in nai1 mutants. PYK10 is a beta-glucosidase that is localized in ER bodies. PBP1 consists of two repeated regions, each of which is highly homologous to the alpha-chain of jacalin, a carbohydrate-binding protein (lectin) of Artocarpus integriforia. We show in this study that PYK10 has two forms, an active form and an inactive form. The amount of active form increased during incubation of root homogenate. On the other hand, PYK10 separated into soluble and insoluble forms. Active PYK10 molecules mainly occurred as the insoluble form and inactive PYK10 molecules remain soluble. This suggests that the activation of PYK10 needs polymerization. In homogenates of both a pbp1 mutant and the wild type, PYK10 becomes insoluble, while PYK10 activity in pbp1 is only half of that in the wild type. PBP1 has an ability to interact with PYK10. Nonetheless, PBP1 does not bind active PYK10. These results suggest that PBP1 has some effect on the activation of PYK10. In addition, PBP1 was found to have a different subcellular distribution from PYK10. PBP1 may act like a molecular chaperone that facilitates the correct polymerization of PYK10, when tissues are damaged and subcellular structures are destroyed by pests.


Teams, strategies and networks: developments in nutritional support; a personal perspective.
Chris Pennington was an archetypal team player, strategist and networker. Clinical nutritional support has progressed remarkably since the 1970s and it has been a privilege to work in this field over this period during which teamwork, strategy development and networking have been crucial. British experience has been characterised by groups of individuals of differing professions and specialties coming together to enable progress to be made. This approach was initially in the form of nutrition support teams orientated to patient-centred ward-based care, then as hospital strategic committees and the concept of the 'patient journey'. Indeed, the formation of the British Association for Parenteral and Enteral Nutrition (now known as BAPEN) in 1992 required the statesmanlike burying of jealousies as societies came together into a multiprofessional association. With the understanding that disease-related malnutrition was highly prevalent it became apparent that it must be managed on a broad and organised clinical front. In the Organisation of Food and Nutritional Support in Hospitals a group of professionals developed for BAPEN concepts of hospital-wide organisation to tackle malnutrition that were based on previous reports, both national and international, and were made easily accessible from the BAPEN website, especially the 'Malnutrition Universal Screening Tool' and the National Institute for Health and Clinical Excellence nutrition guidelines. The coming together of six national clinical societies to develop evidence-based consensus guidelines for intravenous saline therapy (also on the BAPEN website) has shown that BAPEN can catalyse opinion well beyond its own nutritional constituency. In England Chris Pennington's Scottish lead is being followed by developing a patient-centred strategic framework for a managed home parenteral nutrition and intestinal failure national network. In research, education or clinical practice the engines of progress have been teams, strategies and networks.


Postoperative management of carpometacarpal joint fracture dislocation of the hand: A case report.
Injury to the carpometacarpal joints is rare. The strong ligamentous attachments and carpal bone alignment readily resist displacement. To the authors' knowledge, there are no studies evaluating postoperative recovery regimens of carpometacarpal fracture dislocations. The present study describes a postoperative hand therapy regimen that used a novel carpometacarpal brace permitting early mobilization.


Interpretation of subjective symptoms in double-blind placebo-controlled food challenges - interobserver reliability.
Subjective symptoms after food challenges are difficult to interpret and no standard is available. We discuss a strategy for how to interpret a diary. Furthermore, the interobserver reliability is evaluated. Diaries for 32 patients with subjective symptoms were used. The diaries were re-evaluated with a predefined strategy by three independent observers. The proportion of positives was 21.9% among the old diagnoses, according to the new approach 34.4% (observers I and II) and 37.5% (observer III) were positive. The new approach had high interobserver reliability (97 and 100%). The proportion of positives depends on how subjective symptoms are interpreted. Interpretations of subjective symptoms in diaries could be made with high interobserver reliability.


Determination of Protein Structural Ensembles by Hybrid-Resolution SAXS Restrained Molecular Dynamics.
Small-angle X-ray scattering (SAXS) experiments provide low-resolution but valuable information about the dynamics of biomolecular systems, which could be ideally integrated into molecular dynamics (MD) simulations to accurately determine conformational ensembles of flexible proteins. The applicability of this strategy is hampered by the high computational cost required to calculate scattering intensities from three-dimensional structures. We previously presented a hybrid resolution method that makes atomistic SAXS-restrained MD simulation feasible by adopting a coarse-grained approach to efficiently back-calculate scattering intensities; here, we extend this technique, applying it in the framework of metainference with the aim to investigate the dynamical behavior of flexible biomolecules. The efficacy of the method is assessed on the K63-diubiquitin, showing that the inclusion of SAXS restraints is effective in generating a reliable conformational ensemble, improving the agreement with independent experimental data.


BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge.
Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform.


Long-term inhibition of angiotensin prevents reduction of periarterial innervation of calcitonin gene-related peptide (CGRP)-containing nerves in spontaneously hypertensive rats.
The aim of this study was to investigate age-related changes in the density of calcitonin gene-related peptide (CGRP)-containing nerve fibers in spontaneously hypertensive rats (SHR) and the effects of long-term inhibition of the renin-angiotensin system on these changes. The density of immunocytochemically stained nerve fibers in the mesenteric artery was quantified by computer-assisted image processing. An age-related decrease in the density of CGRP-like immunoreactive (LI)-containing nerve fivers but not neuropeptide Y (NPY)-LI-containing sympathetic nerve fibers was found in the mesenteric artery of SHR but not Wistar Kyoto rats (WKY). The density of NPY-LI-containing sympathetic nerve fibers was significantly greater in SHR than in WKY. SHR were treated for 7 weeks with angiotensin converting enzyme inhibitor (0.005% temocapril), angiotensin II type-1 (AT1) receptor antagonist (0.025% losartan) or vasodilator (0.01% hydralazine) in their drinking water. Each drug treatment significantly lowered the systolic blood pressure measured by tail-cuff method. Long-term treatment of SHR with temocapril and losartan significantly increased the density of CGRP-LI-containing nerve fibers in mesenteric arteries. However, the density after hydralazine treatment was similar to the level in non-treated SHR. The density of NPY-LI-containing nerve fibers was not increased by any of the drug treatments. These results suggest that long-term inhibition of the renin-angiotensin system in SHR prevents remodeling of CGRPergic nerve fibers and prevents the reduction of CGRPergic nerve function.


Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.
Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.


Integrating protein-protein interaction networks with gene-gene co-expression networks improves gene signatures for classifying breast cancer metastasis.
Multiple studies have illustrated that gene expression profiling of primary breast cancers throughout the final stages of tumor development can provide valuable markers for risk prediction of metastasis and disease sub typing. However, the identification of a biologically interpretable and universally shared set of markers proved to be difficult. Here, we propose a method for de novo grouping of genes by dissecting the protein-protein interaction network into disjoint sub networks using pair wise gene expression correlation measures. We show that the obtained sub networks are functionally coherent and are consistently identified when applied on a compendium composed of six different breast cancer studies. Application of the proposed method using different integration approaches underlines the robustness of the identified sub network related to cell cycle and identifies putative new sub network markers for metastasis related to cell-cell adhesion, the proteasome complex and JUN-FOS signalling. Although gene selection with the proposed method does not directly improve upon previously reported cross study classification performances, it shows great promises for applications in data integration and result interpretation.


Lung cancer and cigarette smoking in women: a multicenter case-control study in Europe.
The association between cigarette smoking and lung cancer risk in women was investigated within the framework of a case-control study in 9 centres from 6 European countries. Cases were 1,556 women up to 75 years of age with histologically confirmed primary lung cancer; 2, 450 controls with age distribution similar to cases were selected. The predominant cell type was adenocarcinoma (33.5%), with similar proportions for squamous-cell type (26.4%) and small-cell carcinoma (22.3%). Overall, smoking cigarettes at any time was associated with a 5-fold increase in lung cancer risk (odds ratio 5.21, 95% confidence interval 4.49-6.04); corresponding figures for current smoking habits were 8.94, 7.54-10.6. The association showed a dose-response relationship with duration of the habit and daily and cumulative lifetime smoking. A significant excess risk of 70% was associated with every 10 pack-years smoked. After 10 years of smoking cessation, the relative risk decreased to 20% compared to current smokers. The following characteristics were associated with a higher relative risk: inhalation of smoke, smoking non-filter cigarettes, smoking dark-type cigarettes and starting at young age. The association was observed for all major histological types, being the strongest for small-cell type carcinoma, followed by squamous-cell type and the lowest for adenocarcinoma. The proportion of lung-cancer cases in the population attributable to cigarette smoking ranged from 14% to 85%. We concluded that women share most features of the association between cigarette smoking and lung cancer observed in men.