Haplotype resolved chromosome level genome assembly of Citrus australis reveals disease resistance and other citrus specific genes.
Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus. Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased, with the available genomes of varying accuracy and completeness. We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species; Citrus australis (round lime) using highly accurate PacBio HiFi long reads, complemented with Hi-C scaffolding. Hifiasm with Hi-C integrated assembly resulted in a 331 Mb genome of C. australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3 Mb and 98.8% genome assembly completeness (BUSCO). Repeat analysis showed that more than 50% of the genome contained interspersed repeats. Among them, LTR elements were the predominant type (21.0%), of which LTR Gypsy (9.8%) and LTR copia (7.7%) elements were the most abundant repeats. A total of 29 464 genes and 32 009 transcripts were identified in the genome. Of these, 28 222 CDS (25 753 genes) had BLAST hits and 21 401 CDS (75.8%) were annotated with at least one GO term. Citrus specific genes for antimicrobial peptides, defense, volatile compounds and acidity regulation were identified. The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2, 4, 7 and 8. This chromosome scale, and haplotype resolved C. australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.


Race- and sex-related differences in retinal thickness and foveal pit morphology.
To examine sex- and race-associated differences in macular thickness and foveal pit morphology by using spectral-domain optical coherence tomography (SD-OCT). One hundred eighty eyes of 90 healthy patients (43 women, 47 men) underwent retinal imaging with spectral-domain OCT. The lateral scale of each macular volume scan was corrected for individual differences in axial length by ocular biometry. From these corrected volumes, Early Treatment Diabetic Retinopathy Study (ETDRS) grids of retinal thickness were generated and compared between the groups. Foveal morphology was measured with previously described algorithms. Compared with the Caucasians, the Africans and African Americans had reduced central subfield thickness. Central subfield thickness was also reduced in the women compared with the men, although the women also showed significant thinning in parafoveal regions. There was no difference between the sexes in foveal pit morphology; however, the Africans/African Americans had significantly deeper and broader foveal pits than the Caucasians. Previous studies have reported race- and sex-associated differences in macular thickness, and the inference has been that these differences represent similar anatomic features. However, the data on pit morphology collected in the present study reveal an important and significant variation. Between the sexes, the differences are due to global variability in retinal thickness, whereas the variation in thickness observed between the races appears to be driven by differences in foveal pit morphology. These differences have important implications for the use of SD-OCT in detecting and diagnosing retinal disease.


Clomipramine in the treatment of patients with obsessive-compulsive disorder. The Clomipramine Collaborative Study Group.
Two double-blind studies at 21 centers evaluated the therapeutic efficacy, safety, and tolerability of up to 300 mg/d of clomipramine hydrochloride or an equivalent number of placebo capsules in the treatment of 520 patients with obsessive-compulsive disorder, of whom 239 had had the disorder for at least 2 years (study 1) and 281 had been ill for at least 1 year (study 2). On the two principal measures of the severity of the disorder, ie, the Yale-Brown Obsessive Compulsive Scale and the National Institute of Mental Health Global Obsessive Compulsive Scale, clomipramine was significantly more effective than placebo in both studies. The mean reduction in the Yale-Brown Obsessive Compulsive Scale score at the end of 10 weeks of treatment was 38% and 44% in studies 1 and 2, respectively, for the clomipramine-treated patients and 3% and 5% for the placebo-treated patients. The drug was also found to be superior on the basis of the physicians' and patients' evaluations of global therapeutic change. The most frequently observed adverse effects during clomipramine therapy were those typically associated with tricyclic antidepressant drugs. Although uncommon, the occurrence of seizures and elevated aminotransferase values are potentially serious side effects of clomipramine. Clomipramine was generally well tolerated and was effective in reducing obsessive and compulsive symptoms.


Response to an iron fortification programme in relation to vitamin A status in 6-12-year-old school children.
Plasma retinol and indices of iron status were measured in 148 school children (6-12 years) receiving a soup fortified with iron and vitamin C for a period of 15 weeks. The most significant change in serum iron (P = 0.0005) and transferrin saturation (P = 0.0002) was seen in subjects with plasma retinol > or = 40 micrograms/dl, while subjects with plasma retinol < 20 micrograms/dl showed no response. Serum ferritin improved most in the retinol categories < 40 micrograms/dl, suggesting that the absorption of iron was not impaired by marginal vitamin A status, but that it was rather the mobilisation of iron from stores that was affected. Changes in vitamin A status correlated positively and significantly with changes in serum iron (r = 0.37; P = 0.0001) transferrin saturation (r = 0.27; P = 0.004) and haemoglobin (r = 0.21; P = 0.03), but negatively with serum ferritin (r = -0.28; P = 0.003). The presence of marginal vitamin A deficiency in a community may limit the effectiveness of an iron intervention programme and vitamin A status should therefore also be considered when such programmes are planned.


Robust inference of positive selection from recombining coding sequences.
Accurate detection of positive Darwinian selection can provide important insights to researchers investigating the evolution of pathogens. However, many pathogens (particularly viruses) undergo frequent recombination and the phylogenetic methods commonly applied to detect positive selection have been shown to give misleading results when applied to recombining sequences. We propose a method that makes maximum likelihood inference of positive selection robust to the presence of recombination. This is achieved by allowing tree topologies and branch lengths to change across detected recombination breakpoints. Further improvements are obtained by allowing synonymous substitution rates to vary across sites. Using simulation we show that, even for extreme cases where recombination causes standard methods to reach false positive rates >90%, the proposed method decreases the false positive rate to acceptable levels while retaining high power. We applied the method to two HIV-1 datasets for which we have previously found that inference of positive selection is invalid owing to high rates of recombination. In one of these (env gene) we still detected positive selection using the proposed method, while in the other (gag gene) we found no significant evidence of positive selection. A HyPhy batch language implementation of the proposed methods and the HIV-1 datasets analysed are available at http://www.cbio.uct.ac.za/pub_support/bioinf06. The HyPhy package is available at http://www.hyphy.org, and it is planned that the proposed methods will be included in the next distribution. RDP2 is available at http://darwin.uvigo.es/rdp/rdp.html


The reproductive performance of the Mupli beetle, Luprops tristis, in relation to leaf age of the para rubber tree, Hevea brasiliensis.
An analysis of host plant leaf age preferences and phenology studies led to the predictions that tender rubber plant leaves are essential for the completion of the life cycle of the Mupli beetle, Luprops tristis Fabricius (Coleoptera: Tenebrionidae) and that low tender leaf availability during the post-dormancy stage will limit the beetle population. Analyses of the effects of feeding the beetles leaves of various ages, nitrogen (N) content, and moisture content on fecundity and the duration of post-dormancy survival were carried out. The results showed that tender leaf availability during the post-dormancy phase of L. tristis is a critical factor that determines the survival of L. tristis adults and the subsequent generation. The control of powdery mildew ( Odium hevea) disease-mediated premature leaf fall in rubber plantations may regulate the beetle population. A peak in fecundity during the early phase of post-dormancy is proposed as an adaptive mechanism of L. tristis to synchronize egg production and feeding with tender leaf availability in rubber plantations. Variations in nutrient levels and moisture content between deciduous rubber tree leaves of different ages are attributed to the leaf nutrient resorption mechanism of senescing leaves. These results established that tender leaves with high N and moisture levels are essential for post-dormancy survival and that N influences fecundity. The results of the experiments could aid decision making regarding the population management and control of L. tristis in rubber plantations.


Nuclear magnetic resonance spectroscopy and pattern recognition analysis of the biochemical processes associated with the progression of and recovery from nephrotoxic lesions in the rat induced by mercury(II) chloride and 2-bromoethanamine.
Nephrotoxic lesions were induced in Fischer 344 rats using HgCl2, a proximal tubular toxin, and 2-bromoethanamine (BEA), a medullary toxin. Biochemical effects of these toxins on urinary composition were observed by high resolution 1H NMR spectroscopy over 9 days after dosing. The onset of, progression of, and recovery from the induced toxic lesions were also followed histopathologically and related to the perturbed urinary biochemistry. Urinary concentrations of 20 endogenous substances were measured simultaneously by NMR at eight time points, to provide a time-related 20-dimensional description of the urinary biochemistry for each rat. Principal components analysis and nonlinear mapping were used to reduce the biochemical parameter spaces for each rat to two or three dimensions for display and classification purposes. An investigation of alternative data-presentation methods was made, and taking interanimal means of the map coordinates at each time point yielded a novel type of metabolic trajectory diagram with which the biochemical abnormalities associated with the HgCl2 and BEA lesions could be related to the progression and recovery phases of the toxic lesions. The time-course trajectories showed characteristically different paths for each toxin. These trajectories allowed the time points at which there were maximum metabolic differences to be determined and provided the visualization of net movements of the treatment group populations in time in relation to interanimal variation. Control animal urine samples subjected to this analysis showed simple clustering, with no evidence of metabolic trajectory. The trajectory for BEA showed different routes for onset of and recovery from toxicity, whereas for HgCl2 the outward trajectory (onset) mapped a space similar to the inward trajectory (recovery phase). This suggests that the NMR-detectable biochemical abnormalities after mercury toxicity mainly reflect the proportions of functional cells lining the nephron, whereas the biochemical abnormalities associated with renal medullary insult probably relate to functional integrity. An examination has been made for those metabolites that are most responsible for defining the trajectories, i.e., the discrimination of renal cortical and medullary toxicity from each other and from controls. These discriminatory metabolites (using paired t test, p < 0.001) included valine, taurine, trimethylamine N-oxide, and glucose for HgCl2 and acetate, methylamine, dimethylamine, lactate, and creatine for BEA, whereas citrate, succinate, N-acetyl resonances from as yet unidentified metabolites, hippurate, alanine, and 2-oxoglutarate played an important role in defining the biochemically perturbed trajectory of both toxins.(ABSTRACT TRUNCATED AT 400 WORDS)


Plasmomechanical Resonators Based on Dimer Nanoantennas.
Nanomechanical resonators are highly suitable as sensors of minute forces, displacements, or masses. We realize a single plasmonic dimer antenna of subwavelength size, integrated with silicon nitride nanobeams. The sensitive dependence of the antenna response on the beam displacement creates a plasmomechanical system of deeply subwavelength size in all dimensions. We use it to demonstrate transduction of thermal vibrations to scattered light fields and discuss the noise properties and achievable coupling strengths in these systems.


Phosphatidylinositol 3-kinase inhibition down-regulates survivin and facilitates TRAIL-mediated apoptosis in neuroblastomas.
Recent findings have correlated neuroblastoma development with aberration of apoptosis. In particular, increased levels of survivin, a member of the inhibitor of apoptosis protein (IAP) family, have been associated with increased resistance to apoptosis in neuroblastomas. The purpose of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt pathway can alter the expression of survivin and facilitate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in neuroblastoma cells. Human neuroblastoma cells (SK-N-SH, BE[2]C, LAN-1 and IMR-32) were treated with LY294002 or wortmannin, inhibitors of PI3K, for 24 hours. Transient transfection of a dominant negative PI3K expression plasmid, pCGNN-Deltap85, was performed to inhibit PI3K activation. Cells were treated with TRAIL, caspase-3, or pan-caspase inhibitors. RNase protection assay was performed to assess mRNA changes in IAP family members, including survivin. Western blot analysis was performed to measure changes in the levels of procaspases and survivin. Apoptosis was assessed by a DNA fragmentation ELISA assay. The authors found that survivin and cIAP1 mRNA as well as protein expression were decreased after PI3K inhibition. Combination treatment with LY294002 and TRAIL increased apoptosis of SK-N-SH cells compared with TRAIL alone; these results were further corroborated by complete inhibition of apoptosis by caspase-3 or pan-caspase inhibitor. The authors report that PI3K pathway inhibition down-regulates survivin expression and enhances TRAIL-mediated apoptosis in neuroblastomas. PI3K pathway may play a crucial role in neuroblastoma cell survival; therefore, treatment with inhibitors of PI3K may provide potential novel therapeutic options.


Microvascular endothelial dysfunction predicts the development of erectile dysfunction in men with coronary atherosclerosis without critical stenoses.
Erectile dysfunction (ED) is associated with an increased risk for cardiovascular disease, stroke, and all-cause mortality, independent of conventional cardiovascular risk factors. Coronary endothelial dysfunction is independently associated with ED in men with early coronary atherosclerosis. We aimed to investigate whether coronary microvascular dysfunction predicts development of ED in patients presenting with coronary atherosclerosis without critical stenoses. Coronary microvascular function was evaluated in 130 men with coronary atherosclerosis without critical stenoses by administration of intracoronary acetylcholine at the time of diagnostic study. After a mean follow-up of 8.4 years, patients were assessed for the development of ED by administration of a questionnaire. In all, 68 (50%) men had microvascular endothelial dysfunction at baseline; 35 (51%) men with microvascular endothelial dysfunction developed ED on follow-up compared with 19 (31%) men without microvascular endothelial dysfunction. Men who developed ED had a lower coronary blood flow response (% [INCREMENT]CBF) compared with men who did not develop ED, with mean±SD of 25.4±71.3 versus 81.7±120 (P=0.003). In univariate analysis, microvascular endothelial dysfunction was a predictor for the development of ED, with relative risk of 2.4 (1.2-4.9) (P=0.016). In multivariate logistic regression adjusting for traditional cardiovascular risk factors (age, hypertension, hyperlipidemia, diabetes, vascular disease, and family history of coronary artery disease), only microvascular endothelial dysfunction (P=0.027) and age (P=0.044) remained significant predictors of development of ED. Coronary microvascular dysfunction is a predictor of the development of ED in men with coronary atherosclerosis without critical stenoses. This study underscores the systemic involvement of the endothelial function in vascular disease.


Tight junctions at the blood brain barrier: physiological architecture and disease-associated dysregulation.
The Blood-brain barrier (BBB), present at the level of the endothelium of cerebral blood vessels, selectively restricts the blood-to-brain paracellular diffusion of compounds; it is mandatory for cerebral homeostasis and proper neuronal function. The barrier properties of these specialized endothelial cells notably depend on tight junctions (TJs) between adjacent cells: TJs are dynamic structures consisting of a number of transmembrane and membrane-associated cytoplasmic proteins, which are assembled in a multimolecular complex and acting as a platform for intracellular signaling. Although the structural composition of these complexes has been well described in the recent years, our knowledge about their functional regulation still remains fragmentary. Importantly, pericytes, embedded in the vascular basement membrane, and perivascular microglial cells, astrocytes and neurons contribute to the regulation of endothelial TJs and BBB function, altogether constituting the so-called neurovascular unit.The present review summarizes our current understanding of the structure and functional regulation of endothelial TJs at the BBB. Accumulating evidence points to a correlation between BBB dysfunction, alteration of TJ complexes and progression of a variety of CNS diseases, such as stroke, multiple sclerosis and brain tumors, as well as neurodegenerative diseases like Parkinson's and Alzheimer's diseases. Understanding how TJ integrity is controlled may thus help improve drug delivery across the BBB and the design of therapeutic strategies for neurological disorders.


A complex-polarization-propagator protocol for magneto-chiral axial dichroism and birefringence dispersion.
A computational protocol for magneto-chiral dichroism and magneto-chiral birefringence dispersion is presented within the framework of damped response theory, also known as complex polarization propagator theory, at the level of time-dependent Hartree-Fock and time-dependent density functional theory. Magneto-chiral dichroism and magneto-chiral birefringence spectra in the (resonant) frequency region below the first ionization threshold of R-methyloxirane and l-alanine are presented and compared with the corresponding results obtained for both the electronic circular dichroism and the magnetic circular dichroism. The additional information content yielded by the magneto-chiral phenomena, as well as their potential experimental detectability for the selected species, is discussed.


Cancer incidence among pesticide applicators exposed to trifluralin in the Agricultural Health Study.
Trifluralin, 2,6-dinitro-N,N-dipropyl-4-trifluoromethylaniline, is a 2,6-dinitro herbicide widely used to control annual grasses and broadleaf weeds in agricultural settings. The association between trifluralin use and common cancer incidence was evaluated among 50,127 private and commercial pesticide applicators in the Agricultural Health Study (AHS), a prospective cohort study of licensed pesticide applicators and their spouses in Iowa and North Carolina. Poisson regression was used to examine internal dose-response relationships, while controlling for important lifestyle factors and other agricultural exposures. Two metrics of exposure (lifetime days and intensity-weighted lifetime days) were used in exposure-response analyses with non-exposed applicators, as well as applicators in the lowest tertile of exposure, as reference groups. Incident cancers were identified through state tumor registries from enrollment in 1993 through 2002. Trifluralin exposure was not associated with cancer incidence overall among 51% of private and commercial applicators (n=25,712) who had used trifluralin. However, there was an excess of colon cancer in the exposure category of higher half of highest tertile (rate ratios (RR) of 1.76 (95% CI=1.05-2.95) using the non-exposed as a referent and 1.93 (95% CI=1.08-3.45) using those with the lowest tertile of exposure as the referent). There was also a non-significantly elevated risk for kidney cancer and bladder cancer in the highest exposure group, although only the kidney cancer finding was consistent across exposure metrics. Although there was a possible link between trifluralin exposure and colon cancer, small numbers and inconsistencies in dose-response and subgroup analyses indicate that this may be a chance finding.


The integration of the "spirituality in medicine" curriculum into the osteopathic communication curriculum at Kansas City University of Medicine and Biosciences.
With grant funding from the John Templeton Spirituality and Medicine Curricular Award to the George Washington Institute for Spirituality and Health, faculty at Kansas City University of Medicine and Biosciences (KCUMB) developed the "Spirituality in Medicine" curriculum. In developing the curriculum, faculty took into consideration competencies required by the Association of American Medical Colleges and qualitative results from surveys of medical school applicants and enrolled students. Strategies for curriculum delivery included lectures, panel discussions, role-playing, and training in the use of a spirituality assessment tool. A majority of the 250 students who received the training in 2010-2011 were able to demonstrate the following competencies: (1) being sensitive to patients' spiritual and cultural needs, (2) assessing patients' and their own spiritual needs, (3) appropriately using chaplain services for patient care, and (4) understanding the effects of health disparities and ethical issues on patient care. Challenges to implementation included a reduction in chaplain availability due to the economic downturn, a lack of student exposure to direct patient care during shadowing, too little religious diversity among chaplains, and changes in assignment schedules. New competencies required by the National Board of Osteopathic Medical Examiners overlap with and help ensure sustainability of the Spirituality in Medicine curriculum. KCUMB leaders have incorporated the use of the spirituality assessment tool into other parts of the curriculum and into service experiences, and they have introduced a new elective in palliative care. Synergistic efforts by faculty leaders for this initiative were critical to the implementation of this curriculum.


Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer.
As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a potent open-source EZH2 inhibitor, JQEZ5, that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer. EZH2 overexpression induces murine lung cancers that are similar to human NSCLC with high EZH2 expression and low levels of phosphorylated AKT and ERK, implicating biomarkers for EZH2 inhibitor sensitivity. Our EZH2 inhibitor, JQEZ5, promotes regression of these tumors, revealing a potential role for anti-EZH2 therapy in lung cancer. Cancer Discov; 6(9); 1006-21. ©2016 AACR.See related commentary by Frankel et al., p. 949This article is highlighted in the In This Issue feature, p. 932.


An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophila endoderm.
GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. We have identified another endoderm-specific Drosophila GATA factor gene, dGATAe. Expression of dGATAe is first detected at stage 8 in the endoderm, and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of dGATAe, and misexpression of srp resulted in ectopic dGATAe expression. Embryos that either lacked dGATAe or were injected with double-stranded RNA (dsRNA) corresponding to dGATAe failed to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of dGATAe induced ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the dGATAe cDNA also induced endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and dGATAe.


Phase II randomized study of dacarbazine, carmustine, cisplatin and tamoxifen versus dacarbazine alone in advanced melanoma patients.
This randomized phase II trial was performed to define the activity and toxicity of the combination of dacarbazine (DTIC), carmustine (BCNU), cisplatin (DDP) and tamoxifen (DBDT regimen) versus DTIC alone in patients with metastatic melanoma. Sixty patients with metastatic melanoma were randomly assigned to receive BCNU 150 mg/m2 intravenously (i.v.) on day 1, cisplatin 25 mg/m2 i.v. daily on days 1 to 3, DTIC 220 mg/m2 i.v. daily on days 1 to 3 and tamoxifen 160 mg orally daily for 7 days prior to chemotherapy (DBDT arm; arm A). Treatment cycles were repeated every 28 days, while BCNU was given every two cycles. The DTIC arm (arm B) patients received DTIC alone 1200 mg/m2 i.v. on day 1, repeated every 21 days. Patients were evaluated every two cycles; responding patients continued the treatment for a maximum of 12 cycles. The overall response rate was 26% in the DBDT arm and 5% in the DTIC arm. Complete responses were 2.5% for DBDT and 0% for DTIC. The median progression-free survival and the median survival were 4 and 9 months, respectively for DBDT, and 2 and 7 months for DTIC. DBDT was associated with significant haematological toxicity: 33% of the patients experienced a grade III or IV neutropenia and 28% a grade III or IV thrombocytopenia. In conclusion, the overall response rate obtained with DBDT was greater than that obtained with DTIC alone; however, this combination increases toxicity with limited impact on overall survival.


Immunohistochemical evidence of local production of catecholamines in cells of the muscle origins at the lateral and medial humeral epicondyles: of importance for the development of tennis and golfer's elbow?
Tennis elbow (TE) is a painful condition affecting the common extensor origin at the lateral humeral epicondyle. Colour Doppler examination has shown increased blood flow at this site and the sensory, and sympathetic innervation patterns have been delineated. However, it is not known whether there is local production of catecholamines and/or acetylcholine in this tissue, which is the case in patellar and Achilles tendinopathies. To investigate the possible presence of local production of catecholamines and acetylcholine in non-neuronal cells (fibroblasts) in connective tissue at the muscle origin at the lateral humeral epicondyle in patients with TE. Immunohistochemical studies were performed on biopsies taken from the extensor origin in patients with TE and in pain-free controls. For reference purpose, biopsies from the flexor origin in patients with golfer's elbow (GE) were also studied. Seven patients with TE and four patients with GE. Six healthy asymptomatic individuals served as controls. Immunohistochemistry, using antibodies detecting synthesising enzymes for catecholamines (tyrosine hydroxylase; TH) and acetylcholine (choline acetyltransferase; ChAT). TH-like immunohistochemical reactions were seen in fibroblasts in four of the seven patients with TE and two of the four patients with GE. No such reactions were detected in controls (0/6). No ChAT reactions were seen in any of the investigated specimens. There is evidence of local, non-neuronal production of catecholamines, but not acetylcholine, in fibroblasts in the tissue at the muscle origin at the lateral and medial epicondyles in patients with TE and GE, respectively, which might have an influence on blood vessel regulation and pain mechanisms in these conditions.


Shrines in Central Italy conserve plant diversity and large trees.
Sacred natural sites (SNS) are instances of biocultural landscapes protected for spiritual motives. These sites frequently host important biological values in areas of Asia and Africa, where traditional resource management is still upheld by local communities. In contrast, the biodiversity value of SNS has hardly been quantitatively tested in Western contexts, where customs and traditions have relatively lost importance due to modernization and secularization. To assess whether SNS in Western contexts retain value for biodiversity, we studied plant species composition at 30 SNS in Central Italy and compared them with a paired set of similar but not sacred reference sites. We demonstrate that SNS are important for conserving stands of large trees and habitat heterogeneity across different land-cover types. Further, SNS harbor higher plant species richness and a more valuable plant species pool, and significantly contribute to diversity at the landscape scale. We suggest that these patterns are related not only to pre-existent features, but also to traditional management. Conservation of SNS should take into account these specificities, and their cultural as well as biological values, by supporting the continuation of traditional management practices.


Bioinformatic strategies for the analysis of genomic aberrations detected by targeted NGS panels with clinical application.
Molecular profiling of tumor samples has acquired importance in cancer research, but currently also plays an important role in the clinical management of cancer patients. Rapid identification of genomic aberrations improves diagnosis, prognosis and effective therapy selection. This can be attributed mainly to the development of next-generation sequencing (NGS) methods, especially targeted DNA panels. Such panels enable a relatively inexpensive and rapid analysis of various aberrations with clinical impact specific to particular diagnoses. In this review, we discuss the experimental approaches and bioinformatic strategies available for the development of an NGS panel for a reliable analysis of selected biomarkers. Compliance with defined analytical steps is crucial to ensure accurate and reproducible results. In addition, a careful validation procedure has to be performed before the application of NGS targeted assays in routine clinical practice. With more focus on bioinformatics, we emphasize the need for thorough pipeline validation and management in relation to the particular experimental setting as an integral part of the NGS method establishment. A robust and reproducible bioinformatic analysis running on powerful machines is essential for proper detection of genomic variants in clinical settings since distinguishing between experimental noise and real biological variants is fundamental. This review summarizes state-of-the-art bioinformatic solutions for careful detection of the SNV/Indels and CNVs for targeted sequencing resulting in translation of sequencing data into clinically relevant information. Finally, we share our experience with the development of a custom targeted NGS panel for an integrated analysis of biomarkers in lymphoproliferative disorders.


Detection of CMB-Cluster Lensing using Polarization Data from SPTpol.
We report the first detection of gravitational lensing due to galaxy clusters using only the polarization of the cosmic microwave background (CMB). The lensing signal is obtained using a new estimator that extracts the lensing dipole signature from stacked images formed by rotating the cluster-centered Stokes QU map cutouts along the direction of the locally measured background CMB polarization gradient. Using data from the SPTpol 500 deg^{2} survey at the locations of roughly 18 000 clusters with richness λ≥10 from the Dark Energy Survey (DES) Year-3 full galaxy cluster catalog, we detect lensing at 4.8σ. The mean stacked mass of the selected sample is found to be (1.43±0.40)×10^{14}M_{⊙} which is in good agreement with optical weak lensing based estimates using DES data and CMB-lensing based estimates using SPTpol temperature data. This measurement is a key first step for cluster cosmology with future low-noise CMB surveys, like CMB-S4, for which CMB polarization will be the primary channel for cluster lensing measurements.


Obatoclax overcomes resistance to cell death in aggressive thyroid carcinomas by countering Bcl2a1 and Mcl1 overexpression.
Poorly differentiated tumors of the thyroid gland (PDTC) are generally characterized by a poor prognosis due to their resistance to available therapeutic approaches. The relative rarity of these tumors is a major obstacle to our understanding of the molecular mechanisms leading to tumor aggressiveness and drug resistance, and consequently to the development of novel therapies. By simultaneously activating Kras and deleting p53 (Trp53) in thyroid follicular cells, we have generated a novel mouse model that develops papillary thyroid cancer invariably progressing to PDTC. In several cases, tumors further progress to anaplastic carcinomas. The poorly differentiated tumors are morphologically and functionally similar to their human counterparts and depend on MEK/ERK signaling for proliferation. Using primary carcinomas as well as carcinoma-derived cell lines, we also demonstrate that these tumors are intrinsically resistant to apoptosis due to high levels of expression of the Bcl2 family members, Bcl2a1 (Bcl2a1a) and Mcl1, and can be effectively targeted by Obatoclax, a small-molecule pan-inhibitor of the Bcl2 family. Furthermore, we show that Bcl2 family inhibition synergizes with MEK inhibition as well as with doxorubicin in inducing cell death. Thus, our studies in a novel, relevant mouse model have uncovered a promising druggable feature of aggressive thyroid cancers.


Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter.
Chloride plays a major role in cellular homeostasis by regulating the lumenal pH of intracellular organelles. We have described a pH-independent, fluorescent chloride reporter called Clensor that has successfully measured resting chloride in organelles of living cells. Here, we describe the rational design of Clensor. Clensor integrates a chloride sensitive fluorophore called 10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate (BAC) with the programmability, modularity and targetability available to nucleic acid scaffolds. We show that simple conjugation of BAC to a DNA backbone fails to yield a viable chloride-sensitive reporter. Fluorescence intensity and lifetime investigations on a series of BAC-functionalized structural variants yielded molecular insights that guided the rational design and successful realization of the chloride sensitive fluorescent reporter, Clensor. This study provides some general design principles that would aid the realization of diverse ion-sensitive nucleic acid reporters based on the sensing strategy of Clensor.


Patterns of Weekday and Weekend Sedentary Behavior Among Older Adults.
The purpose of this study was to compare estimates of sedentary time on weekdays vs. weekend days in older adults and determine if these patterns vary by measurement method. Older adults (N = 230, M = 83.5, SD = 6.5 years) living in retirement communities completed a questionnaire about sedentary behavior and wore an ActiGraph accelerometer for seven days. Participants engaged in 9.4 (SD = 1.5) hr per day of accelerometer-measured sedentary time, but self-reported engaging in 11.4 (SD = 4.9) hr per day. Men and older participants had more accelerometer-measured sedentary time than their counterparts. The difference between accelerometer-measured weekday and weekend sedentary time was nonsignificant. However, participants self-reported 1.1 hr per day more sedentary time on weekdays compared with weekend days. Findings suggest self-reported but not accelerometer-measured sedentary time should be investigated separately for weekdays and weekend days, and that self-reports may overestimate sedentary time in older adults.


Terminal care: the role of the general practitioner hospital.
A survey of all deaths occurring over a two-year period in a group practice population was carried out to assess the contribution of the local general practitioner hospital to terminal care overall. With the availability of the hospital, the general practitioners were able to provide a higher proportion of terminal care for their patients than in areas where general practitioners did not have access to hospital beds. This was particularly so in terminal care for patients dying of cancer.


The effects of protracted graphomotor tasks on tripod pinch strength and handwriting performance in children with dysgraphia.
To examine the impact of prolonged graphomotor tasks on tripod-pinch strength and on handwriting process and product measures of children with dysgraphia and typical peers. Participants were 51 children in third to fifth grades, divided into two groups: 23 children with dysgraphia and 28 typical peers, as determined by the Handwriting Proficiency Screening Questionnaire. The procedure included two sessions, with a 15-min break between sessions. In each session, the participants performed two tasks: the visual-motor control subtest of Bruininks-Oseretsky and a handwriting copying task, both performed on an electronic tablet as part of the Computerised Penmanship Evaluation Tool. Tripod pinch strength was evaluated before and after each session. Significantly lower tripod-pinch strength was found among children with dysgrphia in comparison to typically developed peers. This deterioration in tripod-pinch strength was associated with a significant deterioration in handwriting process and product measures along the protracted task. Pinch force that is required for mastering the handwriting tool needs to be considered in the evaluation process of children with dysgraphia. Combining the pinch factor with an evaluation of handwriting's process and product may supply a better insight about the child's deficits and assist in focusing treatment objectives.


Incidence and Risk Factors for Upper Extremity Climbing Injuries in Indoor Climbers.
The purpose of this study was to describe the prevalence, incidence and risk factors for climbing-related injuries of the upper extremities in recreational climbers. A total of 426 recreational climbers were recruited from indoor climbing halls. The baseline questionnaire included questions on potential risk factors for climbing injuries: personal factors, climbing-related factors and upper extremity injuries that had occurred in the previous 12 months. Follow-up questionnaires collected information on new injuries that occurred during the follow-up period. The incidence of climbing-related injuries during one-year follow-up was 42.4% with 13 injuries per 1000 h of climbing. The finger was the most frequently affected injury location (36.0%). The following risk factors were associated with the occurrence of upper extremity injuries: higher age (OR 1.03, 95% CI 1.01;1.05), performing a cooling-down (OR 2.02, 95% CI 1.28;3.18), climbing with campus board (OR 2.48, 95% CI 1.23;5.02), finger strength middle finger (OR 1.12, 95% CI 1.05;1.18) and previous injuries (OR 3.05, 95% CI 2.01;4.83). Climbing injuries of the upper body extremities are very common among recreational climbers in indoor halls and several risk factors can be identified that are related to a higher injury risk.


Evaluation of the skin test for hookworm as an epidemiological tool.
The usefulness of a skin test with larval Necator americanus antigen for assessment of hookworm prevalence was evaluated in an endemic area of Costa Rica. In comparison with standard coprologic techniques employed to survey the population, the skin test detected 83% of infections, showing a fairly satisfactory sensitivity. The overall specificity of the test was 50%, i.e., random. No correlation was found between skin reactivity and hookworm burden. The sensitivity of the test increased moderately with age, but its specificity decreased significantly at the same time. False positive reactions were more numerous among persons formerly infected with hookworm who had been negative for as long as 5 years. There was an indication of cross reactivity with intestinal nematodes other than hookworm. The test was used to detect hookworm infected persons in the community for selective treatment, in comparison with mass treatment of all the people in another village. The selective administration of an anthelminthic drug to only skin test positive persons did not achieve the same drop in community hookworm prevalence as did the indiscriminate treatment of the whole population.


Platelet functions in relation to diet and serum lipids in British farmers.
Coagulation and platelet aggregation induced by thrombin, ADP, adrenaline, and collagen were studied in three contrasted groups, each of 20 to 22 middle-aged male farmers. Serum lipids were similar in the three groups. In the west of Scotland group, however, platelet reactivity was significantly greater than in the east of Scotland. This was associated with a dietary intake, evaluated by three different techniques, higher in saturated fat but also lower in polyunsaturated fat and alcohol. Platelet function in the southern England group also correlated with dietary fats and in addition inversely with calcium intake. On an individual basis in the 63 farmers, all the platelet function tests were significantly correlated with the intake of saturated fat regulated by that of calcium and alcohol. The dietary effects on platelets appear to be mediated by the fatty acid composition of plasma lipids and of platelet phospholipids. In that fraction, the fatty acids 20:3 omega 9, 22:3 omega 9 and 20:4 were the most closely related to the platelet function tests. the trienoic acid 20:3 omega 9, identified with essential fatty acid deficiency, was also correlated with the intake of saturated fat and calcium. In this study, platelet functions were more dependent upon the dietary factors associated with coronary heart disease such as saturated fats, calcium, and alcohol than upon serum lipids.


Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has antibacterial and anti-biofilm activity against Streptococcus pyogenes in vitro.
Streptococcus pyogenes causes several infectious diseases such as tonsillitis, cellulitis, and streptococcal toxic shock syndrome. As antibiotics are used for the general treatment of S. pyogenes infection, cases of treatment failure due to drug-resistant bacteria have increased. Lonicera caerulea var. emphyllocalyx (LCE) has been used as a folk medicine in northern Japan (Hokkaido). In this study, we investigated the antibacterial effect of methanol extracts of the fruit, stem, and leaf of LCE (LCEEs) against S. pyogenes using disk diffusion assay. As LCEE (fruit) had the strongest antibacterial activity among the three LCEEs, we focused on functional analysis of antibacterial effects of LCEE (fruit). LCEE (fruit) suppressed the growth of S. pyogenes in a dose-dependent manner. Morphological analysis by transmission electron microscopy demonstrated that LCEE (fruit) damaged the shape of S. pyogenes. Microplate and confocal laser microscopy analysis showed that biofilm formation was also suppressed by LCEE (fruit) in a dose-dependent manner. To further evaluate the surface structure of these biofilms, we performed hydrophobic analysis, which demonstrated that LCEE (fruit) reduced the hydrophobicity of the bacterial surface structure. Our data demonstrated that LCEE (fruit) had anti-bacterial and anti-biofilm effects on S. pyogenes in vitro, suggesting that the direct anti-bacterial effects of the LCEE (fruit) may be useful for treatment of local S. pyogenes infection.


Contact processes in crowded environments.
Periodically sheared colloids at low densities demonstrate a dynamical phase transition from an inactive to active phase as the strain amplitude is increased. The inactive phase consists of no collisions (contacts) between particles in the steady state limit, while in the active phase collisions persist. To investigate this system at higher densities, we construct and study a conserved-particle-number contact process with three-body interactions, which are potentially more likely than two-body interactions at higher densities. For example, consider one active (diffusing) particle colliding with two inactive (nondiffusing) particles such that they become active and consider spontaneous inactivation. In mean field, this system exhibits a continuous dynamical phase transition. Simulations on square lattices also indicate a continuous transition with exponents similar to those measured for the conserved lattice gas (CLG) model. In contrast, the three-body interaction requiring two active particles to activate one inactive particle exhibits a discontinuous transition. Finally, inspired by kinetically constrained models of the glass transition, we investigate the "caging effect" at even higher particle densities to look for a second dynamical phase transition back to an inactive phase. Square lattice simulations suggest a continuous transition with a new set of exponents differing from both the CLG model and what is known as directed percolation, indicating a potentially new universality class for a contact process with a conserved particle number.


Role of CD4+ lymphocytes in resistance to mucosal candidiasis.
The role of CD4+ lymphocytes in resistance of N:NIH(S) III bg/bg nu/+ mice to mucosal candidiasis was evaluated. Alimentary tract colonization with a pure culture of Candida albicans induced a population of lymphocytes in both the Peyer's patches and spleens of bg/bg nu/+ mice, but not bg/bg nu/nu mice, that proliferated and produced interleukin-2 (IL-2) in response to C. albicans antigens. The induction of candida-specific lymphocytes correlated with the clearance of C. albicans from the esophagus and tongue of resistant bg/bg nu/+ mice. Isogenic bg/bg nu/nu mice which do not develop candida-reactive lymphocytes were unable to clear C. albicans from their tongues and esophagi. Treatment of bg/bg nu/+ mice with anti-CD4+ monoclonal antibodies depleted their CD4+ lymphocytes and increased their susceptibility to mucosal candidiasis of the tongue and esophagus. In vivo treatment of bg/bg nu/+ mice with anti-IL-2, anti-gamma interferon (IFN-gamma), or both anti-IL-2 and anti-IFN-gamma monoclonal antibodies did not abrogate their resistance to mucosal candidiasis. Furthermore, treatment of C. albicans-susceptible bg/bg nu/nu mice with IFN-gamma and IL-2 did not protect them from mucosal candidiasis. Thus, CD4+ cells apparently play a critical role in resistance to mucosal candidiasis; however, we were unable to demonstrate a role for IL-2 and IFN-gamma in mediating resistance to mucosal candidiasis.


Use of an innovative, affordable, and open-source short message service-based tool to monitor malaria in remote areas of Uganda.
Quality health management requires timely and accurate data, and paper-based reporting does not fill this role adequately. The introduction of malaria rapid diagnostic tests and the availability of wireless communications present an opportunity to open direct data transmission and feedback between peripheral health workers and central managers. In November 2009, the Uganda Ministry of Health deployed a short message service-based reporting system in two districts. At a set-up cost of $100/health facility, local technician support of $ 400 per month, and a cost of $0.53/week/clinic, the SMS reporting system was started at more than 140 clinics. Positivity rates for rapid diagnostic tests and artemisinin combination therapy stock outs were 48% and 54% in Kabale and 71% and 54% in Gulu, among other reports, at more than 85% health facilities reporting weekly and without monetary incentives or additional supervision. The SMS-based reporting systems have potential to improve timeliness in reporting of specific, time-sensitive metrics at modest cost, while by-passing current bottlenecks in the flow of data. With the development of specific capacity to manage stock data at district level, the availability of timely data offers potential to address commodity distribution problems and reduce stock-outs.


Exploring treatment preferences facilitated recruitment to randomized controlled trials.
To explore how patients' treatment preferences were expressed and justified during recruitment to a randomized controlled trial (RCT) and how they influenced participation and treatment decisions. Qualitative analysis of audio recordings of recruitment appointments with 93 participants aged 51-70 years in a UK multicenter RCT of localized prostate cancer treatments. Treatment preferences at recruitment were more complex and dynamic than previously assumed. Most participants expressed views about treatments early in appointments, ranging on a continuum from hesitant to well-formed opinions. As recruiters elicited men's views and provided detailed evidence-based treatment and study information, some opted for their preference, but many became uncertain and open to RCT recruitment, often accepting a different treatment from their original "preference." Discussion of treatment preferences did not act as the expected barrier to recruitment but actively enabled many to express their concerns and reach an informed decision that often included RCT participation. Exploring treatment preferences and providing evidence-based information can improve levels of informed decision making and facilitate RCT participation. Treatment preferences should be reconceptualized from a barrier to recruitment to an integral part of the information exchange necessary for informed decision making about treatments and RCT participation.


Outcomes of Bedaquiline Treatment in Patients with Multidrug-Resistant Tuberculosis.
Bedaquiline is recommended by the World Health Organization for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). We pooled data from 5 cohorts of patients treated with bedaquiline in France, Georgia, Armenia, and South Africa and in a multicountry study. The rate of culture conversion to negative at 6 months (by the end of 6 months of treatment) was 78% (95% CI 73.5%-81.9%), and the treatment success rate was 65.8% (95% CI 59.9%-71.3%). Death rate was 11.7% (95% CI 7.0%-19.1%). Up to 91.1% (95% CI 82.2%-95.8%) of the patients experienced >1 adverse event, and 11.2% (95% CI 5.0%-23.2%) experienced a serious adverse event. Lung cavitations were consistently associated with unfavorable outcomes. The use of bedaquiline in MDR and XDR TB treatment regimens appears to be effective and safe across different settings, although the certainty of evidence was assessed as very low.


Nonlinear modelling of cancer: bridging the gap between cells and tumours.
Despite major scientific, medical and technological advances over the last few decades, a cure for cancer remains elusive. The disease initiation is complex, and including initiation and avascular growth, onset of hypoxia and acidosis due to accumulation of cells beyond normal physiological conditions, inducement of angiogenesis from the surrounding vasculature, tumour vascularization and further growth, and invasion of surrounding tissue and metastasis. Although the focus historically has been to study these events through experimental and clinical observations, mathematical modelling and simulation that enable analysis at multiple time and spatial scales have also complemented these efforts. Here, we provide an overview of this multiscale modelling focusing on the growth phase of tumours and bypassing the initial stage of tumourigenesis. While we briefly review discrete modelling, our focus is on the continuum approach. We limit the scope further by considering models of tumour progression that do not distinguish tumour cells by their age. We also do not consider immune system interactions nor do we describe models of therapy. We do discuss hybrid-modelling frameworks, where the tumour tissue is modelled using both discrete (cell-scale) and continuum (tumour-scale) elements, thus connecting the micrometre to the centimetre tumour scale. We review recent examples that incorporate experimental data into model parameters. We show that recent mathematical modelling predicts that transport limitations of cell nutrients, oxygen and growth factors may result in cell death that leads to morphological instability, providing a mechanism for invasion via tumour fingering and fragmentation. These conditions induce selection pressure for cell survivability, and may lead to additional genetic mutations. Mathematical modelling further shows that parameters that control the tumour mass shape also control its ability to invade. Thus, tumour morphology may serve as a predictor of invasiveness and treatment prognosis.


In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death.
In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.


Reliable and inexpensive colorimetric method for determining protein-bound tryptophan in maize kernels.
Biofortification programs in maize have led to the development of quality protein maize (QPM) with increased contents of the essential amino acids lysine and tryptophan, and increased nutritional value for protein deficient populations where maize is a staple food. Because multiple genetic systems control and modify the protein quality of QPM, tryptophan or lysine monitoring is required to maximize genetic gain in breeding programs. The objective of this work was to develop an accurate, reliable, and inexpensive method for tryptophan analysis in whole-grain maize flour to support QPM research efforts around the world. Tryptophan reacts with glyoxylic acid in the presence of sulfuric acid and ferric chloride, producing a colored compound that absorbs at 560 nm. A series of experiments varying the reagent concentrations, hydrolysis time, and length of the colorimetric reaction resulted in an optimized protocol which uses 0.1 M glyoxylic acid in 7 N sulfuric acid and 1.8 mM ferric chloride, and 30 min reaction time. This method produced stable and reproducible results for tryptophan concentration in whole-grain maize flour and was validated by comparison with data obtained using an acetic acid-based colorimetric procedure (r(2) = 0.80) and high pressure liquid chromatography (HPLC) (r(2) = 0.71). We describe adaptations that permit high throughput application of this tryptophan analysis method using a microplate platform.


The association between diet and serum concentrations of IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 in the European Prospective Investigation into Cancer and Nutrition.
Circulating concentrations of insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBP) have been associated with the risk of several types of cancer. Dietary correlates of IGF-I and IGFBPs are not yet well established. The objective of this study was to assess the association between dietary intake and serum concentrations of IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 in a cross-sectional analysis of 4,731 men and women taking part in the European Prospective Investigation into Cancer and Nutrition. Diet was assessed using country-specific validated dietary questionnaires. Serum concentrations of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-3 were measured, and the associations between diet and IGF-I and IGFBPs were assessed using multiple linear regression adjusting for sex, age, body mass index, smoking status, and alcohol and energy intake. Each 1 SD increment increase in total and dairy protein and calcium intake was associated with an increase in IGF-I concentration of 2.5%, 2.4%, and 3.3%, respectively (P for trend <0.001 for all) and a decrease in IGFBP-2 of 3.5%, 3.5%, and 5.4% (P for trend <0.001 for all), respectively. There were no significant associations between the intake of protein or calcium from nondairy sources and IGF-I. The results from this large cross-sectional analysis show that either the intake of dairy protein or calcium is an important dietary determinant of IGF-I and IGFBP-2 concentrations; however, we suggest that it is more likely to be protein from dairy products.


Designing a Microfluidic Device with Integrated Ratiometric Oxygen Sensors for the Long-Term Control and Monitoring of Chronic and Cyclic Hypoxia.
Control of oxygen over cell cultures in vitro is a topic of considerable interest, as chronic and cyclic hypoxia can alter cell behaviour. Both static and transient hypoxic levels have been found to affect tumour cell behaviour; it is potentially valuable to include these effects in early, in vitro stages of drug screening. A barrier to their inclusion is that rates of transient hypoxia can be a few cycles/hour, which is difficult to reproduce in traditional in vitro cell culture environments due to long diffusion distances from control gases to the cells. We use a gas-permeable three-layer microfluidic device to achieve spatial and temporal oxygen control with biologically-relevant switching times. We measure the oxygen profiles with integrated, ratiometric optical oxygen sensors, demonstrate sensor and system stability over multi-day experiments, and characterize a pre-bleaching process to improve sensor stability. We show, with both finite-element modelling and experimental data, excellent control over the oxygen levels by the device, independent of fluid flow rate and oxygenation for the operating flow regime. We measure equilibration times of approximately 10 min, generate complex, time-varying oxygen profiles, and study the effects of oxygenated media flow rates on the measured oxygen levels. This device could form a useful tool for future long-term studies of cell behaviour under hypoxia.


Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site.
We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.


Transmission of varicella to a gravida via close contacts immunized with varicella-zoster vaccine. A case report.
Varicella-zoster is a highly contagious DNA virus, transmitted by direct contact and respiratory droplets. An attenuated live-virus vaccine has recently become available and is of value for susceptible, nonimmunized people. As with other attenuated vaccines, such as measles, mumps and rubella virus, there is no evidence of transmission by those immunized, and it is generally recognized that these vaccines can be given to the close contacts of pregnant women. A 32-year-old woman at 39 weeks of gestation presented with generalized pruritic vesicles and pustules. Diagnosis of primary varicella infection was made and confirmed by serologic studies. The patient denied recent or past exposure. The only significant history that the patient could recall was her exposure to her two children, who were vaccinated with the varicella-zoster vaccine eight days prior to her admission but were asymptomatic. This is the first report of a pregnant woman contracting the primary varicella infection from exposure to close contacts vaccinated with the varicella vaccine. It may not be as safe as previously thought for seronegative gravidas to be in close contact with people vaccinated with the varicella vaccine.


Gene activation-associated long noncoding RNAs function in mouse preimplantation development.
In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.


Undifferentiated (spindle cell) pancreatic carcinoma: a case report with osteochondroid differentiation.
Undifferentiated (spindle cell) carcinomas of the pancreas are rare anaplastic variants of pancreatic ductal adenocarcinoma with a frequency of 2% of pancreatic exocrine tumors. Their clinicopathological features are limited and obtained by few previously case reports. We report a case of undifferentiated pancreatic carcinoma with a rare focal osteochondroid differentiation. A sixty-six-year-old woman was admitted to our hospital for abdominal pain and nonspecific nausea for almost 40 days. Imaging studies revealed a well-defined cystic-solid mass with heterogeneous density involving the tail of the pancreas. We performed an en bloc distal pancreatectomy with splenectomy for radical excision, as well as regional lymphadenectomy. The resected specimen revealed a 4.0×5.0 cm exophytic clear-bordered neoplasm of the tail of the pancreas containing necrotic and calcified areas, without splenic invasion. The lymph node involvement was not detected (0/5) and the surgical margins were negative. Microscopy showed pleomorphism with giant cells, spindle-shaped cells with anaplasia, and osteochondroid differentiation. A diagnosis of undifferentiated (spindle cell) carcinoma of the pancreas with focal osteochondroid differentiation was made. The patient declined chemotherapy and extended lymphadenectomy. She suffered from liver and lymph nodes metastasis 9 months after surgery, and she subsequently died 4 months later due to high tumor burden. Undifferentiated pancreatic carcinoma with osteochondroid differentiation is rare but associated with extremely poor prognosis. It should be included in the differential diagnosis of pancreatic mass lesions.


Fluorescence chemosensing of meldonium using a cross-reactive sensor array.
In this paper, we report a fluorescent sensor array approach for the urinary detection of a prohibited substance in sports, meldonium. Four chemosensors with ethidium bromide scaffolds were employed in this method. The interaction between meldonium and chemosensors was investigated by different techniques, such as ultraviolet-visible absorption and fluorescence spectroscopy, nuclear magnetic resonance, and mass spectrometry. Molecular dynamics simulation was also used to elucidate and support the interaction mechanisms between meldonium and the chemosensors. Differential responses obtained from the sensor array enabled the qualitative and quantitative analyses of meldonium with low error values. This method was able to detect and quantify meldonium at the nM level, fulfilling the requirements of minimum performance defined by the World Anti-Doping Agency.


HMGB1-mediated apoptosis and autophagy in ischemic heart diseases.
Acute myocardial infarction (MI) and its consequences are the most common and lethal heart syndromes worldwide and represent a significant health problem. Following MI, apoptosis has been generally seen as the major contributor of the cardiomyocyte fate and of the resultant myocardial remodeling. However, in recent years, it has been discovered that, following MI, cardiomyocytes could activate autophagy in an attempt to protect themselves against ischemic stress and to preserve cardiac function. Although initially seen as two completely separate responses, recent works have highlighted the intertwined crosstalk between apoptosis and autophagy. Numerous researches have tried to unveil the mechanisms and the molecular players involved in this phenomenon and have identified in high-mobility group box 1 (HMGB1), a highly conserved non-histone nuclear protein with important roles in the heart, one of the major regulator. Thus, the aim of this mini review is to discuss how HMGB1 regulates these two responses in ischemic heart diseases. Indeed, a detailed understanding of the crosstalk between apoptosis and autophagy in these pathologies and how HMGB1 regulates them would be of tremendous help in developing novel therapeutic approaches aimed to promote cardiomyocyte survival and to diminish tissue injury following MI.


Activation of the lectin pathway by natural IgM in a model of ischemia/reperfusion injury.
Reperfusion of ischemic tissues elicits an acute inflammatory response involving serum complement, which is activated by circulating natural IgM specific to self-Ags exposed by ischemia. Recent reports demonstrating a role for the lectin pathway raise a question regarding the initial events in complement activation. To dissect the individual roles of natural IgM and lectin in activation of complement, mice bearing genetic deficiency in early complement, IgM, or mannan-binding lectin were characterized in a mesenteric model of ischemia reperfusion injury. The results reveal that IgM binds initially to ischemic Ag providing a binding site for mannan-binding lectin which subsequently leads to activation of complement and injury.


Tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomised, placebo-controlled phase 2 study.
Tivantinib (ARQ 197), a selective oral inhibitor of MET, has shown promising antitumour activity in hepatocellular carcinoma as monotherapy and in combination with sorafenib. We aimed to assess efficacy and safety of tivantinib for second-line treatment of advanced hepatocellular carcinoma. In this completed, multicentre, randomised, placebo-controlled, double-blind, phase 2 study, we enrolled patients with advanced hepatocellular carcinoma and Child-Pugh A cirrhosis who had progressed on or were unable to tolerate first-line systemic therapy. We randomly allocated patients 2:1 to receive tivantinib (360 mg twice-daily) or placebo until disease progression. The tivantinib dose was amended to 240 mg twice-daily because of high incidence of treatment-emergent grade 3 or worse neutropenia. Randomisation was done centrally by an interactive voice-response system, stratified by Eastern Cooperative Oncology Group performance status and vascular invasion. The primary endpoint was time to progression, according to independent radiological review in the intention-to-treat population. We assessed tumour samples for MET expression with immunohistochemistry (high expression was regarded as ≥2+ in ≥50% of tumour cells). This study is registered with ClinicalTrials.gov, number NCT00988741. 71 patients were randomly assigned to receive tivantinib (38 at 360 mg twice-daily and 33 at 240 mg twice-daily); 36 patients were randomly assigned to receive placebo. At the time of analysis, 46 (65%) patients in the tivantinib group and 26 (72%) of those in the placebo group had progressive disease. Time to progression was longer for patients treated with tivantinib (1·6 months [95% CI 1·4-2·8]) than placebo (1·4 months [1·4-1·5]; hazard ratio [HR] 0·64, 90% CI 0·43-0·94; p=0·04). For patients with MET-high tumours, median time to progression was longer with tivantinib than for those on placebo (2·7 months [95% CI 1·4-8·5] for 22 MET-high patients on tivantinib vs 1·4 months [1·4-1·6] for 15 MET-high patients on placebo; HR 0·43, 95% CI 0·19-0·97; p=0·03). The most common grade 3 or worse adverse events in the tivantinib group were neutropenia (ten patients [14%] vs none in the placebo group) and anaemia (eight [11%] vs none in the placebo group). Eight patients (21%) in the tivantinib 360 mg group had grade 3 or worse neutropenia compared with two (6%) patients in the 240 mg group. Four deaths related to tivantinib occurred from severe neutropenia. 24 (34%) patients in the tivantinib group and 14 (39%) patients in the placebo group had serious adverse events. Tivantinib could provide an option for second-line treatment of patients with advanced hepatocellular carcinoma and well-compensated liver cirrhosis, particularly for patients with MET-high tumours. Confirmation in a phase 3 trial is needed, with a starting dose of tivantinib 240 mg twice-daily. ArQule, Daiichi Sankyo (Daiichi Sankyo Group).


Phase I clinical trial of a monoclonal antibody against CD40-ligand (IDEC-131) in patients with systemic lupus erythematosus.
To investigate the safety and pharmacology of a humanized monoclonal antibody against CD40-ligand (IDEC-131) in patients with systemic lupus erythematosus (SLE). Cohorts of 3 to 5 patients with symptomatic lupus each received 0.05, 0.25, 1.0, 5.0, or 15.0 mg/kg of IDEC-131 as a single intravenous infusion. Patients were followed for 3 months to evaluate toxicity and pharmacokinetics. This phase I, single dose, dose-escalating study was conducted in 23 patients at a single institution. All patients experienced at least 1 adverse event (AE) during a 3 month followup period, although 58 AE in 17 patients were considered possibly or probably related or of unknown relationship to treatment. No dose relationship in the distribution of AE was apparent. No infusion related cytokine-release syndrome was observed; no infusions were interrupted, and all patients completed treatment. Eight mild (grade 1 or 2) infections were reported in 8 patients. All infections were considered unrelated to drug administration and all resolved uneventfully. No patient developed detectable antibodies to IDEC-131. Flow cytometry revealed no apparent treatment related depletion of lymphocyte subsets. Pharmacokinetic analysis indicated that the maximum serum concentration and the area under the concentration curve of IDEC-131 were proportional to the dose administered. At doses between 1.0 and 15.0 mg/kg, the serum half-life ranged from 299 to 320 h. Efficacy was not formally evaluated in this single dose study. IDEC-131 (humanized Mab against CD40L) administered in a single intravenous infusion at doses of 0.05-15.0 mg/kg is safe and well tolerated in patients with SLE.


Epidemiology of Chikungunya Virus in Bahia, Brazil, 2014-2015.
Chikungunya is an emerging arbovirus that is characterized into four lineages. One of these, the Asian genotype, has spread rapidly in the Americas after its introduction in the Saint Martin island in October 2013. Unexpectedly, a new lineage, the East-Central-South African genotype, was introduced from Angola in the end of May 2014 in Feira de Santana (FSA), the second largest city in Bahia state, Brazil, where over 5,500 cases have now been reported. Number weekly cases of clinically confirmed CHIKV in FSA were analysed alongside with urban district of residence of CHIKV cases reported between June 2014 and October collected from the municipality's surveillance network. The number of cases per week from June 2014 until September 2015 reveals two distinct transmission waves. The first wave ignited in June and transmission ceased by December 2014. However, a second transmission wave started in January and peaked in May 2015, 8 months after the first wave peak, and this time in phase with Dengue virus and Zika virus transmission, which ceased when minimum temperature dropped to approximately 15°C. We find that shorter travelling times from the district where the outbreak first emerged to other urban districts of FSA were strongly associated with incidence in each district in 2014 (R(2)).


Ellipsosomes: organelles containing a cytochrome-like pigment in the retinal cones of certain fishes.
Ellipsosomes are dense spherical bodies containing a very large concentration of a heme pigment spectroscopically resembling pure cytochrome c. They are located at the outer ends of the inner segments of the cones of certain fishes. Although, superficially, they resemble the similarly located oil droplets in the cones of birds and reptiles, their ultrastructure and staining properties resemble those of the neighboring mitochondria. However, like the oil droplets, they may serve as intracellular color filters.


Stroke, dementia, and drug delivery.
Stroke and dementia represent a major health burden for elderly subjects as they are associated with significant morbidity and mortality. The rates of stroke and dementia are progressively increasing due to the ageing population in most westernized countries. Therefore, both these conditions represent a major therapeutic target. However, the therapeutic options available for the management of stroke and dementia remain largely unsatisfactory, the main reason being the difficulty in transferring the results obtained in animal and in vitro studies to the clinical setting. This review focuses on the recent advances in pathophysiology and treatment of these conditions and future directions for research. Moreover, the technique of functional magnetic resonance imaging is discussed in detail as a tool to assess the effects of therapeutic agents on the central nervous system and monitor the progression of diseases. Finally, an overview of the issue of drug delivery into the central nervous system is presented.


Effect of caloric density of a meal on lower oesophageal sphincter motility and gastro-oesophageal reflux in healthy subjects.
Patients with gastro-oesophageal reflux disease are advised to avoid the ingestion of large meals. In healthy volunteers, a relationship between the amount of postprandial gastro-oesophageal reflux and the volume of a liquid meal has been demonstrated. To evaluate whether the amount of postprandial gastro-oesophageal reflux is also related to the calorie content of a meal, a second parameter that will be reduced by avoidance of the ingestion of large meals. Twelve healthy volunteers (six female, 19-31 years) received two solid-liquid meals with either 842 kcal (solid 582 kcal, liquid 260 kcal) or 582 kcal (31% reduction) in a randomized order. The nutritional components (10% fat, 76% carbohydrates, 14% protein) and the volume of the meals were identical in both meals. The lower oesophageal sphincter pressure was measured continuously in the first postprandial hour with a Dent sleeve, and pH-metry was performed for 3 h postprandially with a glass electrode in the distal oesophagus. Blinded to the type of ingested meal, we calculated the mean lower oesophageal sphincter pressure, the frequency of transient lower oesophageal sphincter relaxations, the number of reflux episodes, and the fraction of time for which pH < 4. A similar decrease in lower oesophageal sphincter pressure was observed after ingestion of the high calorie meal (median 10.9 mmHg, range 4.8-16.7 mmHg) and low calorie meal (median 9.9 mmHg, range 3.9-18.4 mmHg). No difference in the number of transient lower oesophageal sphincter relaxations (high calorie: median 9 per hour, range 5-13 per hour; low calorie: median 7 per hour, range 0-14 per hour) and of reflux episodes (high calorie: median 12 in 3 h, range 3-22 in 3 h; low calorie: median 12 in 3 h, range 3-30 in 3 h) was registered after intake of both types of meal. Additionally, no difference was identified regarding the fraction of time for which pH < 4 between the high calorie (mean 2.3%, 0.2-23.7%) and low calorie meal (3.3%, 0.5-17.8%). Reducing the caloric density of a meal neither influences postprandial lower oesophageal sphincter pressure nor decreases gastro-oesophageal reflux in healthy volunteers. Thus, the amount of gastro-oesophageal reflux induced by ingestion of a meal seems to depend on the volume but not on the caloric density of a meal.


An exemplar of naturalistic inquiry in general practice research.
Background Before beginning any research project, novice researchers must consider which methodological approach will best address their research questions. The paucity of literature describing a practical application of naturalistic inquiry adds to the difficulty they may experience. Aim To provide a practical example of how naturalistic inquiry was applied to a qualitative study exploring collaboration between registered nurses and general practitioners working in Australian general practice. Discussion Naturalistic inquiry is not without its critics and limitations. However, by applying the axioms and operational characteristics of naturalistic inquiry, the authors captured a detailed 'snapshot' of collaboration in general practice in the time and context that it occurred. Conclusion Using qualitative methods, naturalistic inquiry provides the scope to construct a comprehensive and contextual understanding of a phenomenon. No individual positivist paradigm could provide the level of detail achieved in a naturalistic inquiry. Implications for practice This paper presents a practical example of naturalistic inquiry for the novice researcher. It shows that naturalistic inquiry is appropriate when the researcher seeks a rich and contextual understanding of a phenomenon as it exists in its natural setting.


Food sources of phyto-oestrogens and their precursors in Europe.
Phyto-oestrogens are dietary components found in some plants, which act in vivo like weak oestrogens. They may reduce the risk of some degenerative diseases moderated by oestrogen, including breast cancer and osteoporosis. The most widely studied are the isoflavones genistein and daidzein from soyabeans, but lignans may be more prevalent in the European diet. Soya foods have traditionally been consumed in the Orient for millennia, and are now widely available to European consumers. Levels of isoflavone in soyabeans from published literature vary between 560 and 3810 mg/kg, depending on variety and growing conditions. Soya protein concentrates and isolates derived from soyabeans contain 466-615 mg isoflavones/kg. Traditional soya milk, bean curds, bean sprouts, etc. contain 13 to 2030 mg isoflavone/kg, depending on the starting raw material and final water content. Fermented foods have a different isoflavone conjugate profile, which may be important in absorption and metabolism. Soya analogues of European foods include dairy and meat products, which contain 38 to 3000 mg total isoflavones/kg, depending the source of soya and dilution with other ingredients. A wide range of foods contain low levels of soya-derived isoflavones, but such foods do not make a significant contribution to mean intakes in Europe. Flaxseed is by far the richest source of lignan precursors. However, foods such as cereal brans, legumes and some vegetables are a more important source in the diets of Europeans because they are more widely consumed. For similar reasons, compared with soya isoflavones, lignans may be a more important source of phyto-oestrogens in the diets of Europeans.


Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys.
Preclinical studies of viral vector-based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys.


Neuronal correlates of goal-based motor selection in the prefrontal cortex.
Choosing an action that leads to a desired goal requires an understanding of the linkages between actions and their outcomes. We investigated neural mechanisms of such goal-based action selection. We trained monkeys on a task in which the relation between visual cues, action types, and reward conditions changed regularly, such that the monkeys selected their actions based on anticipated reward conditions. A significant number of neurons in the medial prefrontal cortex were activated, after cue presentation and before motor execution, only by particular action-reward combinations. This prefrontal activity is likely to underlie goal-based action selection.


Development and validation of a screener based on interRAI assessments to measure informal caregiver wellbeing in the community.
Informal caregivers are invaluable partners of the health care system. However, their caring responsibilities often affect their psychological wellbeing and ability to continue in their role. It is of paramount importance to easily identify caregivers that would benefit from immediate assistance. In this nonexperimental cohort study, a cross-sectional analysis was conducted among 362 informal caregivers (mean age 64.1 years, SD ± 13.1) caring for persons with high care needs (mean age 78.6 years, SD ± 15.0). Caregivers were interviewed using an interRAI-based self-reported survey with 82 items covering characteristics of caregivers including key aspects of wellbeing. A factor analysis identified items in the caregiver survey dealing with subjective wellbeing that were compared against other wellbeing measures. A screener, called Caregiver Wellbeing Index (CWBI), consisting of four items with response scores ranging from 0 to 2 was created. The CWBI was validated in a follow-up study in which 1020 screeners were completed by informal caregivers of home care clients. Clinical assessments of the care recipients (n = 262) and information on long-term care home (LTCH) admission (n = 176) were linked to the screener dataset. The association between the CWBI scores and caregiver and care recipient characteristics were assessed using logistic regression models and chi-square tests. The reliability of CWBI was also measured. The CWBI scores ranging from zero to eight were split in four 'wellbeing' levels (excellent, good, fair, poor). In the validation study, fair/poor psychological wellbeing was strongly associated with caregiver reports of inability to continue in their role; conflict with family; or feelings of distress, anger, or depression (P < 0.0001). Caregivers caring for a care recipient that presented changes in behavior, cognition, and mood were more likely to present fair/poor wellbeing (P < 0.0001). Additionally, caregivers with high CWBI scores (poor wellbeing) were also more likely to provide care for someone who was admitted to a LTCH (OR 3.52, CI 1.32-9.34) after controlling for care recipient and caregiver characteristics. The Cronbach alpha value 0.89 indicated high reliability. The CWBI is a valid screener that can easily identify caregivers that might benefit from further assessment and interventions.


Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution.
Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.


Morphological Indexes: Can They Predict Lupus Nephritis Outcomes? A Retrospective Study.
Lupus nephritis is a serious complication of systemic lupus erythematosus. Currently, therapy is guided by findings in the renal biopsy, following the International Society of Nephrology / Renal Pathology Society classification. Austin and Hill's histomorphological indexes are not routinely obtained. In this retrospective single-centre study, we aimed to analyze the importance and applicability of the different morphological indexes in predicting response to treatment and prognosis. Patients with kidney biopsy demonstrating lupus nephritis from the 2010 - 2016 period were included. We analyzed their demographic data, comorbidities, clinical presentation and laboratorial evaluation at the time of renal biopsy. We evaluated the following outcomes: clinical remission, renal function and proteinuria at end of follow-up. Histologic analysis was performed using the International Society of Nephrology / Renal Pathology Society classification and the morphological indexes described by Austin (Activity and Chronicity) and Hill. Univariate and multivariate statistical analysis was performed using STATA software. We analyzed 46 biopsy-proven lupus nephritis cases, with a median follow-up of 31.9 (13.2 - 45.6) months. Based on biopsy findings, 35 patients were started on immunosuppressive therapy. We observed that Class IV patients had, at presentation, lower estimated glomerular filtration rate (67.3 vs 94.6 mL/min; p = 0.02), higher proteinuria (4.26 vs 2.37 g/24 hours; p = 0.02) and a non-significantly higher C3 consumption (58.9 vs 77.4 mg/dL; p = 0.06). We did not observe correlations between International Society of Nephrology / Renal Pathology Society classification and the outcomes at the end of follow-up. In contrast, both the Hill biopsy index and Austin's Chronicity index were correlated with renal function and proteinuria at the end of follow-up. Austin's Activity index correlated with the immunological findings (C3, C4 and anti-dsDNA) at presentation. Because clinical activity poorly correlates with histologic activity, histological findings are fundamental when assessing patients with suspected lupus nephritis. The most recent International Society of Nephrology / Renal Pathology Society report supports the European League Against Rheumatism guidelines, encouraging the adoption of histomorphological indexes when evaluating lupus nephritis. Our data, showing a correlation between the renal outcomes and the indexes described by Austin and Hill, supports this view. The histomorphological indexes in lupus nephritis are easily obtainable, can predict renal outcomes and may help in the management of such patients.


Outcomes of nerve reconstruction for radial nerve injuries based on the level of injury in 244 operative cases.
We report a single surgeon series of 244 patients with radial nerve injuries who had nerve repair, neurolysis, or nerve graft over a 17-year period. 44 patients had a Level I or infraclavicular injury, 37 had a Level II injury within the spiral groove of the humerus, 104 had a Level III injury from the lateral arm to antebrachial fossa and 64 had a Level IV injury affecting the posterior interosseous nerve. Nerve grafting was used most frequently in all groups, and was the only method of reconstruction for level II injury. At 21.5 months follow up, Level IV injuries had significantly better outcome of finger and thumb extension, while wrist extension recovered in at least 80% of the patients irrespective of the level of injury. The radial nerve recovered better if repaired or reconstructed within 5 months of injury.


Effects of 2.45 GHz electromagnetic fields with a wide range of SARs on bacterial and HPRT gene mutations.
Present day use of mobile phones is ubiquitous. This causes some concern for human health due to exposure to high-frequency electromagnetic fields (HFEMF) from mobile phones. Consequently, we have examined the effects of 2.45 GHz electromagnetic fields on bacterial mutations and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations. Using the Ames test, bacteria were exposed to HFEMF for 30 min at specific absorption rates (SARs) from 5 to 200 W/kg. In all strains, there was no significant difference in the frequency of revertant colonies between sham exposure and HFEMF-exposed groups. In examination of mutations of the HPRT gene, Chinese hamster ovary (CHO)-K1 cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. We detected a combination effect of simultaneous exposure to HFEMF and bleomycin at the respective SARs. A statistically significant difference was observed between the cells exposed to HFEMF at the SAR of 200 W/kg. Cells treated with the combination of HFEMF at SARs from 50 to 200 W/kg and bleomycin exhibited increased HPRT mutations. As the exposure to HFEMF induced an increase in temperature, these increases of mutation frequency may be a result of activation of bleomycin by heat. We consider that the increase of mutation frequency may be due to a thermal effect.


Seroreactivity for spotted fever rickettsiae and co-infections with other tick-borne agents among habitants in central and southern Sweden.
Patients seeking medical care with erythema migrans or flu-like symptoms after suspected or observed tick bite in the southeast of Sweden and previously investigated for Borrelia spp. and/or Anaplasma sp. were retrospectively examined for serological evidence of rickettsial infection (Study 1). Twenty of 206 patients had IgG and/or IgM antibodies to Rickettsia spp. equal to or higher than the cut-off titre of 1:64. Seven of these 20 patients showed seroconversion indicative of recent or current infection and 13 patients had titres compatible with past infection, of which five patients were judged as probable infection. Of 19 patients with medical records, 11 were positive for Borrelia spp. as well, and for Anaplasma sp., one was judged as positive. Five of the 19 patients had antibodies against all three pathogens. Erythema migrans or rash was observed at all combinations of seroreactivity, with symptoms including fever, muscle pain, headache and respiratory problems. The results were compared by screening an additional 159 patients (Study 2) primarily sampled for the analysis of Borrelia spp. or Mycoplasma pneumoniae. Sixteen of these patients were seroreactive for Rickettsia spp., of which five were judged as recent or current infection. Symptoms of arthritis, fever, cough and rash were predominant. In 80 blood donors without clinical symptoms, approximately 1 % were seroreactive for Rickettsia spp., interpreted as past infection. The study shows that both single and co-infections do occur, which illustrate the complexity in the clinical picture and a need for further studies to fully understand how these patients should best be treated.