Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.


netNMF-sc: leveraging gene-gene interactions for imputation and dimensionality reduction in single-cell expression analysis.
Single-cell RNA-sequencing (scRNA-seq) enables high-throughput measurement of RNA expression in single cells. However, because of technical limitations, scRNA-seq data often contain zero counts for many transcripts in individual cells. These zero counts, or dropout events, complicate the analysis of scRNA-seq data using standard methods developed for bulk RNA-seq data. Current scRNA-seq analysis methods typically overcome dropout by combining information across cells in a lower-dimensional space, leveraging the observation that cells generally occupy a small number of RNA expression states. We introduce netNMF-sc, an algorithm for scRNA-seq analysis that leverages information across both cells and genes. netNMF-sc learns a low-dimensional representation of scRNA-seq transcript counts using network-regularized non-negative matrix factorization. The network regularization takes advantage of prior knowledge of gene-gene interactions, encouraging pairs of genes with known interactions to be nearby each other in the low-dimensional representation. The resulting matrix factorization imputes gene abundance for both zero and nonzero counts and can be used to cluster cells into meaningful subpopulations. We show that netNMF-sc outperforms existing methods at clustering cells and estimating gene-gene covariance using both simulated and real scRNA-seq data, with increasing advantages at higher dropout rates (e.g., >60%). We also show that the results from netNMF-sc are robust to variation in the input network, with more representative networks leading to greater performance gains.


An 80-kilodalton antigen from Histoplasma capsulatum that has homology to heat shock protein 70 induces cell-mediated immune responses and protection in mice.
An extract of the cell wall and cell membrane from Histoplasma capsulatum yeast cells was assayed by Western blot (immunoblot) for reactivity with two monoclonal antibodies to heat shock protein 70. Four bands with molecular masses of 80, 66, 54, and 32 kDa bound both antibodies. The 80-kDa protein was isolated, analyzed for homology to heat shock protein 70, and tested for antigenicity and immunogenicity in C57BL/6 mice. The 80-kDa protein reacted with monoclonal antibody to heat shock protein 70. Sera from mice immunized with the antigen recognized H. capsulatum heat shock protein 70. Moreover, the amino-terminal sequence of the 80-kDa protein revealed substantial homology with heat shock protein 70 from several species. The 80-kDa protein induced delayed-type hypersensitivity responses in mice immunized with either viable yeast cells or antigen. Splenocytes from mice immunized with yeast cells or with antigen responded in vitro to the 80-kDa antigen. Immunization of mice with the antigen enhanced host resistance against a sublethal inoculum of H. capsulatum yeast cells, but it did not reduce the mortality of mice given a lethal challenge of yeast cells. Thus, this antigen manifests homology with members of the heat shock protein 70 family. Furthermore, the 80-kDa protein elicits cellular immune responses to H. capsulatum, and it mediates protective immunity.


Evaluation and comparison of current fetal ultrasound image segmentation methods for biometric measurements: a grand challenge.
This paper presents the evaluation results of the methods submitted to Challenge US: Biometric Measurements from Fetal Ultrasound Images, a segmentation challenge held at the IEEE International Symposium on Biomedical Imaging 2012. The challenge was set to compare and evaluate current fetal ultrasound image segmentation methods. It consisted of automatically segmenting fetal anatomical structures to measure standard obstetric biometric parameters, from 2D fetal ultrasound images taken on fetuses at different gestational ages (21 weeks, 28 weeks, and 33 weeks) and with varying image quality to reflect data encountered in real clinical environments. Four independent sub-challenges were proposed, according to the objects of interest measured in clinical practice: abdomen, head, femur, and whole fetus. Five teams participated in the head sub-challenge and two teams in the femur sub-challenge, including one team who tackled both. Nobody attempted the abdomen and whole fetus sub-challenges. The challenge goals were two-fold and the participants were asked to submit the segmentation results as well as the measurements derived from the segmented objects. Extensive quantitative (region-based, distance-based, and Bland-Altman measurements) and qualitative evaluation was performed to compare the results from a representative selection of current methods submitted to the challenge. Several experts (three for the head sub-challenge and two for the femur sub-challenge), with different degrees of expertise, manually delineated the objects of interest to define the ground truth used within the evaluation framework. For the head sub-challenge, several groups produced results that could be potentially used in clinical settings, with comparable performance to manual delineations. The femur sub-challenge had inferior performance to the head sub-challenge due to the fact that it is a harder segmentation problem and that the techniques presented relied more on the femur's appearance.


A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments.
The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed.


Multi-scale biomechanical remodeling in aging and genetic mutant murine mitral valve leaflets: insights into Marfan syndrome.
Mitral valve degeneration is a key component of the pathophysiology of Marfan syndrome. The biomechanical consequences of aging and genetic mutation in mitral valves are poorly understood because of limited tools to study this in mouse models. Our aim was to determine the global biomechanical and local cell-matrix deformation relationships in the aging and Marfan related Fbn1 mutated murine mitral valve. To conduct this investigation, a novel stretching apparatus and gripping method was implemented to directly quantify both global tissue biomechanics and local cellular deformation and matrix fiber realignment in murine mitral valves. Excised mitral valve leaflets from wild-type and Fbn1 mutant mice from 2 weeks to 10 months in age were tested in circumferential orientation under continuous laser optical imaging. Mouse mitral valves stiffen with age, correlating with increases in collagen fraction and matrix fiber alignment. Fbn1 mutation resulted in significantly more compliant valves (modulus 1.34 ± 0.12 vs. 2.51 ± 0.31 MPa, respectively, P<.01) at 4 months, corresponding with an increase in proportion of GAGs and decrease in elastin fraction. Local cellular deformation and fiber alignment change linearly with global tissue stretch, and these slopes become more extreme with aging. In comparison, Fbn1 mutated valves have decoupled cellular deformation and fiber alignment with tissue stretch. Taken together, quantitative understanding of multi-scale murine planar tissue biomechanics is essential for establishing consequences of aging and genetic mutations. Decoupling of local cell-matrix deformation kinematics with global tissue stretch may be an important mechanism of normal and pathological biomechanical remodeling in valves.


Differential expression of members of the tumor necrosis factor alpha-related apoptosis-inducing ligand pathway in prostate cancer cells.
Androgen ablation therapy induces apoptosis only in androgen-sensitive prostate cancer cells; therefore, other cytotoxic drugs are being used to induce apoptosis in androgen-refractory cells. Mifepristone, an antiprogestin used individually or together with the antiestrogen Tamoxifen, has been recommended for induction of cell death and treatment of several hormonal cancers. However, little is known about the mechanism of action of these drugs in prostate cancer. Therefore, we investigated the effect of Mifepristone on the tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) pathway, a newly identified and very effective member of tumor necrosis factor-alpha family. Mifepristone and Tamoxifen induced significant expression of death receptors in prostate cancer cells in vitro and in xenografts. However, Mifepristone in combination with Tamoxifen did not increase prostate cancer cell death compared with their individual values. The involvement of the TRAIL pathway was further confirmed by the activation of caspase-8 in Mifepristone-treated cells. This was followed by truncation of Bid, confirming that Mifepristone activates the TRAIL pathway. This knowledge is being used to design a combination treatment of TRAIL and Mifepristone to induce significant apoptosis in prostate cancer cells.


High blood pressure and the kidney: the forgotten contribution of William Senhouse Kirkes.
The realization of the key role for raised intra-arterial pressure as a pathogenetic agent in hypertension is usually credited to Ludwig Traube, but Traube in his writings gives credit for the idea to a little-known English doctor, William Senhouse Kirkes (1822-1864). Kirkes' main interest was in cardiology and vascular disease, and he gave the first account of embolism from vegetations in infective endocarditis in 1852. Three years later, he published a study of apoplexy in Bright's disease, in which he pointed clearly to the role of raised intra-arterial tension in the causation of arterial disease, a point that had eluded Bright, Johnson, and other contemporaries. Kirkes died at the age of only 42 while working on a book summarizing his work on cardiology and renal disease, and the neglect of his contribution probably resulted from his early death. We have traced his life history from the few available records; as a boy, Kirkes was apprenticed to become a surgeon and only later trained as a physician. We place his contributions within the setting of the development during the 19th century of understanding of the relationship between the kidney, vascular disease, and high blood pressure.


Thymocytes maintain immune activity through telomere elongation in rats under hypoxic conditions.
The main purpose of the present study was to investigate the change in thymocyte telomere length of rats exposed to different hypoxic conditions for different periods of time, as well as its effect on the immune system. A total of 110 male Wistar rats were randomly assigned to one of the three following groups: i) Sea level (SL) group, in which 10 rats were maintained at an altitude of 10 m; ii) moderate altitude (MA) group, in which 50 rats were maintained at an altitude of 2,260 m and then randomly sacrificed on days 1, 3, 7, 15 and 30 (n=10 each); and iii) simulated high altitude (SHA) group, in which 50 rats were maintained at a simulated altitude of 5,000 m, and then randomly sacrificed on days 1, 3, 7, 15 and 30 (n=10 each). The morphological changes of the thymus were observed, while the telomere length, the mRNA and protein expression levels of telomerase reverse transcriptase (TERT), and the peripheral blood lymphocyte count were measured. The results indicated that hypoxia induced morphological changes and apoptosis in thymocytes, as well as atrophy of the thymus tissue, and resulted in a significant increase in telomere length and TERT mRNA and protein expression levels. This effect appeared to be more pronounced in the SHA group compared with that in the MA group; however, no statistically significant changes were observed in the peripheral blood lymphocyte count. Based on these findings, the hypoxia-associated loss of thymic function appears to be only quantitative and not qualitative, and the thymus may be able to maintain its immune function even under hypoxic conditions.


Detection and mapping of mtDNA SNPs in Atlantic salmon using high throughput DNA sequencing.
Approximately half of the mitochondrial genome inherent within 546 individual Atlantic salmon (Salmo salar) derived from across the species' North Atlantic range, was selectively amplified with a novel combination of standard PCR and pyro-sequencing in a single run using 454 Titanium FLX technology (Roche, 454 Life Sciences). A unique combination of barcoded primers and a partitioned sequencing plate was employed to designate each sequence read to its original sample. The sequence reads were aligned according to the S. salar mitochondrial reference sequence (NC_001960.1), with the objective of identifying single nucleotide polymorphisms (SNPs). They were validated if they met with the following three stringent criteria: (i) sequence reads were produced from both DNA strands; (ii) SNPs were confirmed in a minimum of 90% of replicate sequence reads; and (iii) SNPs occurred in more than one individual. Pyrosequencing generated a total of 179,826,884 bp of data, and 10,765 of the total 10,920 S. salar sequences (98.6%) were assigned back to their original samples. The approach taken resulted in a total of 216 SNPs and 2 indels, which were validated and mapped onto the S. salar mitochondrial genome, including 107 SNPs and one indel not previously reported. An average of 27.3 sequence reads with a standard deviation of 11.7 supported each SNP per individual. The study generated a mitochondrial SNP panel from a large sample group across a broad geographical area, reducing the potential for ascertainment bias, which has hampered previous studies. The SNPs identified here validate those identified in previous studies, and also contribute additional potentially informative loci for the future study of phylogeography and evolution in the Atlantic salmon. The overall success experienced with this novel application of HT sequencing of targeted regions suggests that the same approach could be successfully applied for SNP mining in other species.


CORKSCREW1 defines a novel mechanism of domain specification in the maize shoot.
In higher plants, determinate leaf primordia arise in regular patterns on the flanks of the indeterminate shoot apical meristem (SAM). The acquisition of leaf form is then a gradual process, involving the specification and growth of distinct domains within the three leaf axes. The recessive corkscrew1 (cks1) mutation of maize (Zea mays) disrupts both leaf initiation patterns in the SAM and domain specification within the mediolateral and proximodistal leaf axes. Specifically, cks1 mutant leaves exhibit multiple midribs and leaf sheath tissue differentiates in the blade domain. Such perturbations are a common feature of maize mutants that ectopically accumulate KNOTTED1-like homeobox (KNOX) proteins in leaf tissue. Consistent with this observation, at least two knox genes are ectopically expressed in cks1 mutant leaves. However, ectopic KNOX proteins cannot be detected. We therefore propose that CKS1 primarily functions within the SAM to establish boundaries between meristematic and leaf zones. Loss of gene function disrupts boundary formation, impacts phyllotactic patterns, and leads to aspects of indeterminate growth within leaf primordia. Because these perturbations arise independently of ectopic KNOX activity, the cks1 mutation defines a novel component of the developmental machinery that facilitates leaf-versus-shoot development in maize.


Early biochemical signals arise from low affinity TCR-ligand reactions at the cell-cell interface.
The kinetics of acid release by a mixture of T cells and antigen presenting cells were measured with a microphysiometer during a brief exposure to antigenic peptides. We find that some of the early biochemical events that lead to cellular proliferation cause a specific increase in the rate of acid release. The duration of this increase in acid release reflects the life-time of the peptide-MHC complexes. Peptides that form long-lived complexes produce a response that is stable for more than an hour. Serial TCR engagement is suggested by the observation that the amplitude of this stable response can be rapidly shifted up or down with additional agonist peptide or with antibodies that block T cell receptor binding. Cells briefly exposed to a peptide that forms short-lived peptide-MHC complexes produce a response that decays rapidly as peptide is washed away. A quantitative analysis of the kinetics of this decay in acidification demonstrates that intercellular TCR-ligand reactions are rapid, reversible, and of low apparent affinity with < 20% of peptide-MHC ligand bound to a TCR at any one time. These results demonstrate that the fraction of peptide-MHC ligands bound to TCRs at the cell-cell interface is no higher than anticipated from the affinities observed in solution for isolated TCRs and ligands.


What is socio-ecological research delivering? A literature survey across 25 international LTSER platforms.
With an overarching goal of addressing global and regional sustainability challenges, Long Term Socio-Ecological Research Platforms (LTSER) aim to conduct place-based research, to collect and synthesize both environmental and socio-economic data, and to involve a broader stakeholder pool to set the research agenda. To date there have been few studies examining the output from LTSER platforms. In this study we enquire if the socio-ecological research from 25 self-selected LTSER platforms of the International Long-Term Ecological Research (ILTER) network has produced research products which fulfil the aims and ambitions of the paradigm shift from ecological to socio-ecological research envisaged at the turn of the century. In total we assessed 4983 publically available publications, of which 1112 were deemed relevant to the socio-ecological objectives of the platform. A series of 22 questions were scored for each publication, assessing relevance of responses in terms of the disciplinary focus of research, consideration of human health and well-being, degree of stakeholder engagement, and other relevant variables. The results reflected the diverse origins of the individual platforms and revealed a wide range in foci, temporal periods and quantity of output from participating platforms, supporting the premise that there is a growing trend in socio-ecological research at long-term monitoring platforms. Our review highlights the challenges of realizing the top-down goal to harmonize international network activities and objectives and the need for bottom-up, self-definition for research platforms. This provides support for increasing the consistency of LTSER research while preserving the diversity of regional experiences.


Quantifying the influence of measured and unmeasured individual differences on demography.
1. Demographic rates can vary not only with measured individual characters like age, sex and mass but also with unmeasured individual variables like behaviour, genes and health. 2. Predictions from population models that include measured individual characteristics often differ from models that exclude them. Similarly, unmeasured individual differences have the potential to impact predictions from population models. However, unmeasured individual differences are rarely included in population models. 3. We construct stage- and age-structured models (where stage is mass) of a roe deer population, which are parameterized from statistical functions that either include, or ignore, unmeasured individual differences. 4. We found that mass and age structures substantially impacted model parameters describing population dynamics, as did temporal environmental variation, while unmeasured individual differences impacted parameters describing population dynamics to a much smaller extent once individual heterogeneity related to mass and age has been included in the model. We discuss how our assumptions (unmeasured individual differences only in mean trait values) could have influenced our findings and under what circumstances unmeasured individual differences could have had a larger impact on population dynamics. 5. There are two reasons explaining the relative small influence of unmeasured individual differences on population dynamics in roe deer. First, individual body mass and age both capture a large amount of individual differences in roe deer. Second, in large populations of long-lived animals, the average quality of individuals (independent of age and mass) within the population is unlikely to show substantial variation over time, unless rapid evolution is occurring. So even though a population consisting of high-quality individuals would have much higher population growth rate than a population consisting of low-quality individuals, the probability of observing a population consisting only of high-quality individuals is small.


Children's multiple vitamins: overuse leads to overdose.
A suburban Ontario community hospital encountered 21 +/- 1 overdoses of children's multiple vitamins yearly between 1978 and 1981. Of these, 35% involved one particular cartoon character preparation. Parents were surveyed to determine whether this particular preparation resulted in a disproportionate number of accidental overdoses. However, the use of vitamins with a cartoon character format did not lead to a greater risk of overdose than did conventional preparations. Of the 1051 families who had given multiple vitamins to their children 34 (3%) had experienced an overdose. The number of vitamin preparations used by each family was the most powerful determinant of overdose (p less than 0.001). The risk of accidental overdose increased from 1.5% with one multiple-vitamin preparation to 8% with four or more preparations. Among all the variables examined, exposure was the most important element in the risk of multiple-vitamin overdose.


Pharmacokinetics and tissue concentrations of azithromycin in ball pythons (Python regius).
To determine pharmacokinetics and tissue concentrations of azithromycin in ball pythons (Python regius) after IV or oral administration of a single dose. 2 male and 5 female ball pythons. Using a crossover design, each snake was given a single dose of azithromycin (10 mg/kg) IV. After a 4-week washout period, each snake was given a single dose of azithromycin (10 mg/kg) orally. Blood samples were collected prior to dose administration and 1, 3, 6, 12, 24, 48, 72, and 96 hours after azithromycin administration. Azithromycin was quantitated by use of liquid chromatography-mass spectrometry. After IV administration, azithromycin had an apparent volume of distribution of 5.69 L/kg and a plasma clearance of 0.19 L/h/kg. Harmonic means for the terminal half-life were 17 hours following IV administration and 51 hours following oral administration. Mean residence times were 37 and 94 hours following IV and oral administration, respectively. Following oral administration, azithromycin had a peak plasma concentration (Cmax) of 1.04 microg/mL, a time to Cmax of 8.4 hours, and a prolonged mean absorption time of 57 hours. Mean oral bioavailability was 77%. Tissue concentrations ranged from 4 to 140 times the corresponding plasma concentration at 24 and 72 hours after azithromycin administration. Azithromycin is well absorbed and tolerated by ball pythons. On the basis of plasma pharmacokinetics and tissue concentration data, we suggest an azithromycin dosage in ball pythons of 10 mg/kg, orally, every 2 to 7 days, depending upon the site of infection and susceptibil ity of the infective organism.


Shedding light on interictal epileptic spikes: An in vivo study using fast optical signal and electrocorticography.
Interictal epileptic spikes (IESs), apart from being a key marker of epileptic neuronal networks, constitute a nice model of the widespread endogenous phenomenon of neuronal hypersynchronization. Many questions concerning the mechanisms that drive neurons to hypersynchronize remain unresolved, but synaptic as well as nonsynaptic events are likely to be involved. In this study, changes in optical properties of neural tissues were observed in rats with penicillin-induced IES using fast optical signal (FOS) concomitantly with electrocorticography (ECoG). In this study, near-infrared optical imaging was used with ECoG to investigate variations in the optical properties of cortical tissue directly associated with neuronal activity in 15 rats. FOS changes correspond to variations of scattered light from neuronal tissue when neurons are activated. To independently evaluate our method, a control experiment on somatosensory was designed and applied to seven different rats. Time-frequency analysis was also used to track variations of (de)synchronization concomitantly with changes in optical signals during IES. FOS responses revealed that changes in optical signals occurred 320 msec before to 370 msec after the IES peak. These changes started before any changes in ECoG signal. In addition, time-frequency domain electrocorticography revealed an alternating decrease-increase-decrease in the ECoG spectral power (pointing to desynchronization-synchronization-desynchronization), which occurred concomitantly with an increase-decrease-increase in relative optical signal during the IES. These results suggest a relationship between (de)synchronization and optical changes. These changes in the neuronal environment around IESs raise new questions about the mechanisms that induce changes in optical properties of neural tissues before the IES, which may provide suitable conditions for neuronal synchronization during IESs. FOS-ECoG constitutes a multimodal approach and opens new avenues to study the mechanisms of neuronal synchronization in the pathologic brain, which has clinical implications, at least in epilepsy.


The complexity and diversity of the Pathogenicity Locus in Clostridium difficile clade 5.
The symptoms of Clostridium difficile infection are caused by two closely related toxins, TcdA and TcdB, which are encoded by the 19.6 kb Pathogenicity Locus (PaLoc). The PaLoc is variably present among strains, and in this respect it resembles a mobile genetic element. The C. difficile population structure consists mainly of five phylogenetic clades designated 1-5. Certain genotypes of clade 5 are associated with recently emergent highly pathogenic strains causing human disease and animal infections. The aim of this study was to explore the evolutionary history of the PaLoc in C. difficile clade 5. Phylogenetic analyses and annotation of clade 5 PaLoc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. Comparison of the core genome and PaLoc phylogenies obtained for clade 5 and representatives of the other clades identified two distinct PaLoc acquisition events, one involving a toxin A(+)B(+) PaLoc variant and the other an A(-)B(+) variant. Although the exact mechanism of each PaLoc acquisition is unclear, evidence of possible homologous recombination with other clades and between clade 5 lineages was found within the PaLoc and adjacent regions. The generation of nontoxigenic variants by PaLoc loss via homologous recombination with PaLoc-negative members of other clades was suggested by analysis of cdu2, although none is likely to have occurred recently. A variant of the putative holin gene present in the clade 5 A(-)B(+) PaLoc was likely acquired via allelic exchange with an unknown element. Fine-scale phylogenetic analysis of C. difficile clade 5 revealed the extent of its genetic diversity, consistent with ancient evolutionary origins and a complex evolutionary history for the PaLoc.


Impact of stimulation duration and gonadotropin type on the incidence of premature progesterone elevation - a retrospective analysis of the Ensure data.
Elevated progesterone levels on the day of trigger negatively impact the outcome of assisted reproductive technique (ART) treatment and forced ovarian stimulation might be a cause of progesterone elevation during ovarian stimulation. To analyze the impact of forced and prolonged stimulation on the progesterone elevation, this data analysis from the Ensure study compared hormonal stimulation with corifollitropin alpha (CFA)-only with CFA plus recombinant (rec) follicle-stimulating hormone (FSH) after day 8 (CFA-plus group) of ovarian stimulation. In the Ensure study, 268 patients underwent ovarian stimulation with 100 µg CFA and 128 patients with recombinant FSH. A total of 35 patients (13.1%) from the CFA-arm received the hCG trigger after stimulation with CFA-only, 233 patients (86.9%) needed additional rec FSH from day 8 onwards to meet the criteria for trigger. Progesterone levels >0.8 ng/ml on the trigger day occurred in 90 patients (38.6%) from the CFA plus FSH group and only in one patient (2.8%) in the CFA-only group (p < .001). The ongoing pregnancy rate (OPR) was 31.4% (11/35) for patients in the CFA-only group and 24.5% (57/233) for patients CFA-plus group with additional recFSH after day 8 (p = .378). This set of data demonstrates that prolongation of stimulation in combination with intense stimulation leads to a statistically significant increased incidence of progesterone elevation on the day of trigger.


Neonatal malaria in the gambia.
Neonatal malaria was thought to be uncommon, even in malaria endemic areas. Neonates with febrile illness and / or related symptoms are often presumed to have neonatal septicaemia and examination of blood films for malaria parasites is rarely included in the initial work-up of these babies. To determine the existence, or otherwise, and the clinical features of malaria in neonates with clinical features suggestive of septicaemia. In a prospective study, all consecutive babies admitted in the neonatal unit of the Royal Victoria Hospital over an eighth month period with an initial diagnosis of neonatal septicaemia were screened for malaria in addition to an extensive sepsis work-up. Malaria parasitaemia was diagnosed on Giemsa stained blood smears. One hundred and five babies admitted in the neonatal unit of the Royal Victoria Hospital, The Gambia with an initial diagnosis of neonatal septicaemia were studied. Fourteen (13.3%) of these babies had positive blood smears for malaria parasites and bacterial cultures of their blood, cerebrospinal fluid and urine yielded no pathogen. Plasmodium falciparum was the only species identified in all the babies. A male preponderance was obtained with a male: female ratio of 2.5:1. Malaria occurred more commonly in babies aged more than one week, thus suggesting that acquired rather than congenital malaria was the more common form of neonatal malaria in the area. All the babies with malaria were admitted within the rainy season period. The principal clinical features observed in these babies were fever, refusal to feed, maternal fever in the last trimester, excessive crying, difficulty in breathing, vomiting, hepatomegaly, abdominal distension and irritability. A similar picture was observed among babies with proven neonatal septicaemia. In addition, depressed primitive reflexes was found to be more common in babies with septicaemia (p=0.01). These data show that neonatal malaria is not uncommon in The Gambia and that the clinical features are akin to those of neonatal septicaemia.


Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.
Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca(2+) indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca(2+) imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca(2+) using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca(2+) waves from the cytosol to the nucleus and changes in Ca(2+) concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.


Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation.
CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.


Impact of Pretransplantation (18)F-Fluorodeoxyglucose-Positron Emission Tomography on Survival Outcomes after T Cell-Depleted Allogeneic Transplantation for Hodgkin Lymphoma.
Pretransplant (18)F-fluorodeoxyglucose (FDG) positron emission tomography status is an important prognostic factor for outcomes after autologous stem cell transplantation (SCT) in Hodgkin lymphoma (HL), but its impact on outcomes after allogeneic SCT remains unclear. We retrospectively evaluated outcomes after T cell-depleted allogeneic SCT of 116 patients with nonprogressive HL according to pretransplant Deauville scores. Endpoints were overall survival (OS), progression-free survival (PFS), relapse rate (RR), and nonrelapse-related mortality (NRM). OS, PFS, and RR did not differ significantly between the Deauville 1 to 2 and Deauville 3 to 5 cohorts (OS: 77.5% versus 67.3%, P = .49; PFS: 59.4% versus 55.7%, P = .43; RR: 20.9% versus 22.6%, P = .28 at 4 years). Differences in PFS remained statistically nonsignificant when comparisons were made between Deauville 1 to 3 and Deauville 4 to 5 cohorts (60.9% versus 51.4%, P = .10), and RR remained very similar (21.5% versus 23.8%, P = .42). Multivariate analyses demonstrated trends toward significance for an effect of Deauville score on PFS (hazard ratio 1.82 for Deauville 4 to 5, P = .06) and for number of lines of prior therapy on OS (hazard ratio 2.34 for >5 lines, P = .10). The latter effect appeared to be driven by higher NRM rather than increased RR. Our findings suggest that Deauville score before allogeneic SCT in patients with nonprogressive HL has a relatively modest impact on survival outcomes in comparison with the impact in autologous SCT and that predictive values for the individual patient remain low, indicating that residual FDG-avid disease should not preclude allogeneic SCT. Furthermore, our findings bring into question the importance of attainment of metabolic complete response in this setting if it is at the expense of increasing NRM risk.


Prevalence of head lice and hygiene practices among women over twelve years of age in Sindh, Balochistan, and North West Frontier Province: National Health Survey of Pakistan, 1990-1994.
Head lice infestation is an infection of the scalp and skin which causes blood loss, discomfort, and social and psychological distress with the possibility of secondary bacterial infections occurring at scratch sites. In Pakistan, although some small scale studies have been conducted to investigate prevalence of head lice in school children and the general population, no population based estimates have been reported. The National Health Survey of Pakistan (NHSP 1990 - 94) was a nationally representative health examination survey of the Pakistani population. The NHSP is the first population based household survey to collect data on the prevalence of head lice in adult women in Pakistan. In this paper we use data from the NHSP to present an epidemiological profile of personal hygiene practices and head lice infestation among women aged 12 years or older in three provinces of Pakistan; Balochistan, Sindh and North West Frontier Province (NWFP). Overall about 7% women aged 12 years and older suffered from head lice infestation. Multivariable logistic regression analysis identified factors independently associated with presence of head lice. Age less than 16 years and crowding at home were associated with higher infestation-rates. The impact of household socio-economic status on infestation rates among women was different in urban and rural settings; urban women with low socio-economic status were more vulnerable than similar women in rural settings. Bathing infrequently in summer was associated with higher prevalence rates only in Sindh, possibly due to the fact that among the three provinces Sindh has a hotter and more humid summer. The results of our analysis of NHSP indicate high levels of head lice infestation among girls and women in the three Provinces. The epidemiological profile of hygienic practices of women indicated that NWFP and Balochistan as compared to Sindh, and rural as compared to urban areas were less developed with respect to access to water supply and soap for maintaining personal hygiene. Simple and cost-effective measures such as provision of water and soap, and improving awareness regarding maintaining personal hygiene can contribute significantly towards improving public health status of the women in Pakistan.


Theranostic two-dimensional superparamagnetic maghemite quantum structures for ROS-mediated cancer therapy.
In this work, size- and shape-controlled two-dimensional (2D) superparamagnetic maghemite (γ-Fe2O3) quantum flakes (MQFs) with high surface area and mesoporosity were prepared by facile hydrothermal synthesis for biological applications. These quantum flakes exhibited superparamagnetic behaviours over a wide temperature range of 75-950 K with high saturation magnetization of Ms - 23 emu g-1 and a lower coercivity of Hc - 6.1 Oe. MQFs also demonstrated a good colloidal stability and a positively charged flake surface. Selective toxicity dependent upon selective ROS scavenging/generation and cellular MQF uptake towards non-malignant human keratinocyte (HaCaT) and malignant melanoma (A357) and human breast cancer (MDA-MB 231) cell lines were witnessed. An increased ROS concentration resulted due to the peroxidase-like activity of MQFs in malignant cells. In contrast, ROS scavenging was observed in non-malignant cells due to dominant catalase-like activity. In vitro fluorescence properties added the diagnostic ability to the ambit of MQFs. Furthermore, the therapeutic efficiency could be significantly enhanced by the hyperthermic (25-47 °C) ability of MQF in cancerous cells. Our findings reveal the novel theranostic MQF structure with immense cancer therapeutic potential via augmentation of ROS generation by hyperthermia in a selective microenvironment.


Endocrine parameters, hormone receptors, and functions of the testicular interstitium and seminiferous epithelium in estradiol-immunized Ile-de-France rams.
The testicular response of Ile-de-France rams actively immunized against estradiol (E2) was evaluated during both the ovine nonbreeding season (spring) and breeding season (autumn). Plasma concentrations of LH, FSH and testosterone were elevated in E2-immunized rams during both spring and autumn when compared with BSA-immunized controls. Testis weights were significantly elevated by E2 immunization and were characterized by greater interstitial cell volume, including Leydig cells, blood and lymph vessels, greater seminiferous tubule length, and greater numbers of leptotene spermatocytes and round spermatids. Neither Sertoli cell number, Sertoli cell nuclear volume nor testicular FSH receptor number were affected by E2 immunization, but testis weight, Sertoli cell nuclear area, FSH receptor number and LH receptor number were significantly greater in autumn than in spring. A positive effect of E2 immunization on testicular LH receptors was evident in spring but not in autumn. Testicular androgen receptors were suppressed by E2 immunization but were not affected by season. It was concluded that E2 immunization results in moderate stimulation of the ovine testis to increase testosterone secretion and to enhance total daily spermatid production. This effect appears to result from a change in E2 negative feedback and increased pituitary gonadotropin secretion.


Viral discovery and sequence recovery using DNA microarrays.
Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.


Decadal trends and common dynamics of the bio-optical and thermal characteristics of the African Great Lakes.
The Great Lakes of East Africa are among the world's most important freshwater ecosystems. Despite their importance in providing vital resources and ecosystem services, the impact of regional and global environmental drivers on this lacustrine system remains only partially understood. We make a systematic comparison of the dynamics of the bio-optical and thermal properties of thirteen of the largest African lakes between 2002 and 2011. Lake surface temperatures had a positive trend in all Great Lakes outside the latitude of 0° to 8° south, while the dynamics of those lakes within this latitude range were highly sensitive to global inter-annual climate drivers (i.e. El Niño Southern Oscillation). Lake surface temperature dynamics in nearly all lakes were found to be sensitive to the latitudinal position of the Inter Tropical Convergence Zone. Phytoplankton dynamics varied considerably between lakes, with increasing and decreasing trends. Intra-lake differences in both surface temperature and phytoplankton dynamics occurred for many of the larger lakes. This inter-comparison of bio-optical and thermal dynamics provides new insights into the response of these ecosystems to global and regional drivers.


Acute myocardial infarction in a young woman with systemic lupus erythematosus.
A young woman was diagnosed with systemic lupus erythematosus at the age of 7 years and incurred an acute myocardial infarction at the age of 17 years. Her risk factors for coronary artery disease include hypertension, hypercholesterolemia, a relatively long disease duration, a fairly active disease as evidenced by the history of nephrotic syndrome and other organ system involvement, and a long history of prednisone use. It is difficult to determine the etiology of this patient's acute myocardial infarction without coronary artery histopathology, but aspects of her presentation (a history of virulent systemic lupus erythematosus, and the angiographic findings of ectasia and aneurysm) suggest that coronary arteritis was the etiology of her accelerated coronary artery disease and subsequent myocardial infarction. Acute myocardial infarction is an uncommon occurrence in premenopausal women less than 30 years old.35 These patients are typically found to have an associated systemic disease such as diabetes mellitus or familial hypercholesterolemia. Systemic lupus erythematosus is a less common systemic disease associated with premature coronary artery disease. Mechanisms of acute coronary syndromes in these patients include accelerated atherosclerosis, active coronary vasculitis, and/or vasospasm with superimposed thrombosis.


AHR, a novel acute hypoxia-response sequence, drives reporter gene expression under hypoxia in vitro and in vivo.
ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter-driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia-response sequence)-GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP-expressing vector under the control of CMV (cytomegalovirus promoter-GFP). We transduced AHR-GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV-GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR-GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR-GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia-activated gene expression system.


A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria.
Parasitic plants in the Orobanchaceae develop haustoria in response to contact with host roots or chemical haustoria-inducing factors. Experiments in this manuscript test the hypothesis that quinolic-inducing factors activate haustorium development via a signal mechanism initiated by redox cycling between quinone and hydroquinone states. Two cDNAs were previously isolated from roots of the parasitic plant Triphysaria versicolor that encode distinct quinone oxidoreductases. QR1 encodes a single-electron reducing NADPH quinone oxidoreductase similar to zeta-crystallin. The QR2 enzyme catalyzes two electron reductions typical of xenobiotic detoxification. QR1 and QR2 transcripts are upregulated in a primary response to chemical-inducing factors, but only QR1 was upregulated in response to host roots. RNA interference technology was used to reduce QR1 and QR2 transcripts in Triphysaria roots that were evaluated for their ability to form haustoria. There was a significant decrease in haustorium development in roots silenced for QR1 but not in roots silenced for QR2. The infrequent QR1 transgenic roots that did develop haustoria had levels of QR1 similar to those of nontransgenic roots. These experiments implicate QR1 as one of the earliest genes on the haustorium signal transduction pathway, encoding a quinone oxidoreductase necessary for the redox bioactivation of haustorial inducing factors.


Snap-shot profilometry with the Empirical Mode Decomposition and a 3-layer color sensor.
Remote sensing finds more and more applications, from industrial control, to face recognition, not forgetting terrain surveying. This trend is well exemplified by fringe projection techniques, which enjoyed a considerable development in the recent years. In addition of high requirement in terms of measurement accuracy and spatial resolution, the end-users of full-field techniques show a growing interest for dynamic regimes. We report here what we believe to be the use for the first time of a CMOS 3-layer color sensor (Foveon X3) as the key element of a RGB fringe projection system, together with the processing specifically elaborated for this sensor. The 3-layer architecture allows the simultaneous recording of three phase-shifted fringe patterns and features the precious asset of an unambiguous relationship between the physical sensor pixel and the picture pixel and this for each color layer, on the contrary of common color sensor arrays (Bayer mosaic and tri-CCD). Due to the overlapping of the spectral responses of the layers, color transformation is mandatory to achieve the separation of the three phase-shifted RGB projected fringe patterns. In addition, we propose the use of the Empirical Mode Decomposition to equalize the non-uniform responses of the three layers. Although the conversion of the phase into a height is of primary importance in an actual measurement, it is not treated here, the literature being profuse on the central projection model.


The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution.
LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.


Effect of Cocoa and Its Flavonoids on Biomarkers of Inflammation: Studies of Cell Culture, Animals and Humans.
Chronic inflammation has been identified as a necessary step to mediate atherosclerosis and cardiovascular disease and as a relevant stage in the onset and progression of several types of cancer. Considerable attention has recently been focused on the identification of dietary bioactive compounds with anti-inflammatory activities as an alternative natural source for prevention of inflammation-associated diseases. The remarkable capacity of cocoa flavanols as antioxidants, as well as to modulate signaling pathways involved in cellular processes, such as inflammation, metabolism and proliferation, has encouraged research on this type of polyphenols as useful bioactive compounds for nutritional prevention of cardiovascular disease and cancer. Data from numerous studies suggest that cocoa and cocoa-derived flavanols can effectively modify the inflammatory process, and thus potentially provide a benefit to individuals with elevated risk factors for atherosclerosis/cardiovascular pathology and cancer. The present overview will focus on the most recent findings about the effects of cocoa, its main constituents and cocoa derivatives on selected biomarkers of the inflammatory process in cell culture, animal models and human cohorts.


Dynamics of saccadic adaptation: differences between athletes and nonathletes.
The aim of the study was to delineate differences in saccadic adaptation characteristics between a population of racquet sports athletes and nonathletes. Eye movements were recorded at 120 Hz using a video-based eye tracker (ELMAR 2020) in a sample of 27 athletes (varsity badminton and squash players) and 14 nonathletes (<3 hours/week participation in recreational sports). Responses to negative positional error and positive positional error were studied in two sessions on separate days. Negative positional errors were induced by displacing the stimuli backwards by 3 degrees from the initial target step (12 degrees). Likewise, positive positional errors were induced by displacing the stimuli forward by 3 degrees . Amplitude gains were calculated for trials before, during, and after the adaptation phase. The magnitude and the rate of change of saccadic adaptation were determined from the amplitude gains. Differences between the groups were compared using regression analysis. No significant differences were found between the two groups in the magnitude of saccadic adaptation, both for negative (athletes -60%, nonathletes -57%) and positive (athletes +26%, and nonathletes +27%) positional error. Racquet sports athletes showed a significantly faster rate of adaptation for the positive positional error. A significant difference was not observed in the rate of adaptation for the negative positional error. Racquet sports athletes and nonathletes adapt to positional error signals by similar amounts. However, racquet sports athletes respond to positive positional errors at a faster rate, suggesting that a strategic component or environmental influences (such as practice) may play a role in saccadic adaptation.


Can paternal preconceptional radiation account for the increase of leukaemia and non-Hodgkin's lymphoma in Seascale?
To determine if the excess of leukaemia and non-Hodgkin's lymphoma in Seascale is restricted to those born in the parish and whether it might be explained by the postulated relation with paternal preconceptional radiation. Comparison, separately for those born in the parish and those born elsewhere, of the numbers of these malignancies observed in Seascale with those expected on the basis of reference rates for England and Wales. Details of paternal radiation levels were sought for each case. The parish of Seascale in west Cumbria. Residents of Seascale below age 25 years in the years 1951-91. The observed and expected numbers of cases of leukaemia and non-Hodgkin's lymphoma within Seascale among those born there and among those born elsewhere. Also, the levels of any paternal preconceptional radiation associated with each case. A significant excess of leukaemia and non-Hodgkin's lymphoma at ages 0-24 was found in Seascale in those who were born there (ratio of observed to expected cases 8.6 and 20.2 respectively; p < 0.01). This also applied to those not born there (7.2 and 16.5; p < 0.01), a group often regarded as not showing an excess. The estimates were then conservatively recalculated so as to overestimate the risks among those born in Seascale and underestimate them among those born elsewhere. On this basis the six cases in those born in Seascale compare with 0.38 expected (15.8; p < 0.001), of which two were associated with paternal preconceptional life-time levels of 100 mSv or greater and three others with levels of 90-99 mSv. Among those born elsewhere, there were five cases (expected 0.74; ratio 6.7, p < 0.01), of which only one was associated with a high level of such radiation. Paternal preconceptional radiation cannot be the sole cause of the excess in Seascale since it will not explain the excess among those born outside Seascale. It follows that, unless two causes are to be postulated, any single cause must be a factor other than paternal preconceptional radiation. On this basis, the association found among those born there, if not partly due to chance, may reflect an indirect relation with the true cause. The recent hypothesis about such paternal radiation has originated in a subgroup of the excess cases that have aroused concern.


Interpretation of the ultraviolet-visible spectra of malaria parasite Plasmodium falciparum.
The absorption and scattering properties of three developmental stages of protozoan parasite Plasmodium falciparum were studied both experimentally and theoretically. Experimentally, the light attenuation and forward scattering from parasites extracted from host erythrocyte cultures were measured with UV-visible spectroscopy. The measured spectra were interpreted theoretically with a model based on the core-shell Mie theory in terms of the structural and compositional characteristics of the protozoa. The model accurately reproduced the features of the measured spectra of all developmental stages. The results show that realistic quantitative estimates of the parasite size, nucleotide, and hemozoin contents can be derived from the UV-visible spectroscopy measurements.


The effects of a rhythm and music-based therapy program and therapeutic riding in late recovery phase following stroke: a study protocol for a three-armed randomized controlled trial.
Stroke represents one of the most costly and long-term disabling conditions in adulthood worldwide and there is a need to determine the effectiveness of rehabilitation programs in the late phase after stroke. Limited scientific support exists for training incorporating rhythm and music as well as therapeutic riding and well-designed trials to determine the effectiveness of these treatment modalities are warranted. A single blinded three-armed randomized controlled trial is described with the aim to evaluate whether it is possible to improve the overall health status and functioning of individuals in the late phase of stroke (1-5 years after stroke) through a rhythm and music-based therapy program or therapeutic riding. About 120 individuals will be consecutively and randomly allocated to one of three groups: (T1) rhythm and music-based therapy program; (T2) therapeutic riding; or (T3) control group receiving the T1 training program a year later. Evaluation is conducted prior to and after the 12-week long intervention as well as three and six months later. The evaluation comprises a comprehensive functional and cognitive assessment (both qualitative and quantitative), and questionnaires. Based on the International classification of functioning, disability, and health (ICF), the outcome measures are classified into six comprehensive domains, with participation as the primary outcome measure assessed by the Stroke Impact Scale (SIS, version 2.0.). The secondary outcome measures are grouped within the following domains: body function, activity, environmental factors and personal factors. Life satisfaction and health related quality of life constitute an additional domain. A total of 84 participants were randomised and have completed the intervention. Recruitment proceeds and follow-up is on-going, trial results are expected in early 2014. This study will ascertain whether any of the two intervention programs can improve overall health status and functioning in the late phase of stroke. A positive outcome would increase the scientific basis for the use of such interventions in the late phase after stroke. Clinical Trials.gov Identifier: NCT01372059.


Inhibition of p300/CBP by early B-cell factor.
Early B-cell factor (EBF) is a DNA binding protein required for early B-cell development. It activates transcription of several B-cell-specific genes, including the lambda5 gene, which encodes a protein necessary for signaling by the pre-B-cell receptor. In an effort to understand the mechanism by which EBF activates transcription, we examined its interaction with the coactivator protein p300/CBP. We found that two domains of EBF each bind the histone acetyltransferase (HAT)/CH3 domain of p300/CBP both in vitro and in vivo. Surprisingly, transcriptional activation by EBF was not sensitive to E1A, a potent p300/CBP inhibitor. In fact, overexpressed EBF mimicked E1A by severely repressing the activity of several other transcription factors, including E47, a protein that acts cooperatively with EBF to promote transcription of the lambda5 gene. This broad inhibitory profile correlated with EBF's ability to repress the HAT activity of p300/CBP in vivo and in vitro. However, such a repressed complex is not likely to form at the lambda5 promoter in vivo since (i) EBF could not bind p300/CBP and DNA simultaneously and (ii) the cooperativity imparted by E47 was sensitive to E1A. Our data reveal an intriguing inhibitory property of EBF-a property shared only by E1A, Twist, Pu.1, and the Hox family of homeodomain proteins-and suggest that E47 and EBF play distinct roles during lambda5 promoter activation.


EGCG inhibits proliferation, invasiveness and tumor growth by up-regulation of adhesion molecules, suppression of gelatinases activity, and induction of apoptosis in nasopharyngeal carcinoma cells.
(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC), yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and β-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK) phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and β-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.


Predicting Metastatic Risk of Gastrointestinal Stromal Tumors: Role of Cell Proliferation and Cell Cycle Regulatory Proteins.
Gastrointestinal stromal tumors (GIST) are a heterogeneous group of neoplasms whose biologic behavior is difficult to predict. The aim of this study is to evaluate the prognostic value in GIST of some oncoproteins involved in regulation of cell proliferation. Tumor size, mitosis, necrosis, and p53, c-myc, and bcl-2 protein expression of 32 GIST were studied. Proliferative index was assessed with Ki67. The 32 cases were grouped into the following clinical categories: (1) clinically benign (BN) were defined as disease-free survival greater than 3 years (n=10); (2) clinically malignant (MN) in which local recurrence or metastasis occurred regardless of the follow-up time (n=15); and (3) clinically indeterminate (ID) owing to follow-up <3 years without metastasis or local recurrence (n=seven). Discriminant analysis was used to allocate any tumor to one of the two prognostic groups (BN or MN). In univariate analysis all six factors studied above proved to be of significant prognostic value. Using a multivariate stepwise discriminant analysis to take into account the interrelationship between factors, we found that c-myc expression was the most important prognostic factor, followed, in order of statistical weight, by size and Ki67. These were combined to define a discriminant score ([10.75 x c-myc]+[0.39 x size]+[0.078 x Ki67]-15.54=score), which was capable of correctly identifying tumors in our series whose known clinical behavior was BN or MN in 92% of the cases. The classification score was applied subsequently to the seven clinically ID cases: Three (42.9%) were predicted as BN, and four (57.1%) were predicted as MN. Both expression of oncoprotein c-myc and the proliferative index provide prognostic information in GIST, in addition to morphologically established prognostic factors such as size. These factors in a discriminant analysis proved to be useful for the clinical classification of GIST into BN or MN and to predict the clinical outcome of clinically ID tumors. Int J Surg Pathol 8(2):133-144, 2000


Closely related bird species demonstrate flexibility between beak morphology and underlying developmental programs.
The astonishing variation in the shape and size of bird beaks reflects a wide range of dietary specializations that played an important role in avian diversification. Among Darwin's finches, ground finches (Geospiza spp.) have beaks that represent scaling variations of the same shape, which are generated by alterations in the signaling pathways that regulate growth of the two skeletal components of the beak: the prenasal cartilage (pnc) and the premaxillary bone (pmx). Whether this developmental mechanism is responsible for variation within groups of other closely related bird species, however, has remained unknown. Here, we report that the Caribbean bullfinches (Loxigilla spp.), which are closely related to Darwin's finches, have independently evolved beaks of a novel shape, different from Geospiza, but also varying from each other only in scaling. However, despite sharing the same beak shape, the signaling pathways and tissues patterning Loxigilla beaks differ among the three species. In Loxigilla noctis, as in Geospiza, the pnc develops first, shaped by Bmp4 and CaM signaling, followed by the development of the pmx, regulated by TGFβIIr, β-catenin, and Dkk3 signaling. In contrast, beak morphogenesis in Loxigilla violacea and Loxigilla portoricensis is generated almost exclusively by the pmx through a mechanism in which Ihh and Bmp4 synergize to promote expansion of bone tissue. Together, our results demonstrate high flexibility in the relationship between morphology and underlying developmental causes, where different developmental programs can generate identical shapes, and similar developmental programs can pattern different shapes.


The Farsi version of the Juvenile Arthritis Multidimensional Assessment Report (JAMAR).
The Juvenile Arthritis Multidimensional Assessment Report (JAMAR) is a new parent/patient reported outcome measure that enables a thorough assessment of the disease status in children with juvenile idiopathic arthritis (JIA). We report the results of the cross-cultural adaptation and validation of the parent and patient versions of the JAMAR in the Farsi language. The reading comprehension of the questionnaire was tested in 10 JIA parents and patients. Each participating centre was asked to collect demographic, clinical data and the JAMAR in 100 consecutive JIA patients or all consecutive patients seen in a 6-month period and to administer the JAMAR to 100 healthy children and their parents. The statistical validation phase explored descriptive statistics and the psychometric issues of the JAMAR: the three Likert assumptions, floor/ceiling effects, internal consistency, Cronbach's alpha, interscale correlations, test-retest reliability, and construct validity (convergent and discriminant validity). A total of 102 JIA patients (14.7% systemic JIA, 67.6% oligoarticular, 15.7% RF negative polyarthritis, 2.0% other categories) and 198 healthy children, were enrolled in three paediatric rheumatology centres. Notably, none of the enrolled JIA patients is affected with enthesitis-related arthritis or undifferentiated arthritis. The JAMAR components discriminated healthy subjects from JIA patients. All JAMAR components revealed satisfactory psychometric performances. In conclusion, the Farsi version of the JAMAR is a valid tool for the assessment of children with JIA and is suitable for use both in routine clinical practice and clinical research.


Variation in administrators' and clinicians' attitudes toward critical elements of an infection control program and the role of the infection control practitioner in New South Wales, Australia.
Debate remains over the core activities of infection control (IC) programs. Differences in stakeholder opinions must be considered if consensus panel guidelines and recommendations are to be broadly applied. This article describes a survey of administrators and clinicians employed in hospitals in New South Wales, Australia. Respondents self-reported their levels of agreement with affirmative statements regarding the role of the infection control practitioner (ICP) and the essential requirements and infrastructure of IC programs. The study population included administrators and clinicians in each public, private, and freestanding day hospital in New South Wales. Respondents reported the intensity of their agreement with 16 affirmative statements relating to IC program infrastructure and resources and the ICP's role and responsibilities. The overall response rate was 62.1% (587/945). Clinicians (349/587) and administrators (238/587) accounted for 59.5% and 40.5% of the response rate, respectively. Overall, administrators and clinicians reported greatest levels of agreement for those elements not requiring additional resources. The extent of divergence between administrators and clinicians is not so great that it can not be resolved. Our findings demonstrate the degree of administrator support that clinicians can expect for each element. We advocate better communication between clinicians and administrators in conjunction with objective strategic planning. Our findings provide a guide for ICPs to either establish or negotiate the core components of their IC program.


Characterization and subcellular localization of tomato ringspot nepovirus putative movement protein.
Sequence comparison of the tomato ringspot nepovirus (TomRSV) genome with related viruses suggested that the region of the RNA-2-encoded polyprotein immediately upstream of the coat protein may be involved in the cell-to-cell movement of the virus (Rott et al., 1991, J. Gen. Virol. 72, 1505-1514). To further study the role of this portion of the genome, monoclonal antibodies against the putative movement protein were raised. Western blots of plant extracts allowed the detection of a viral nonstructural protein of M(r) 45K present only in TomRSV-infected tissues. Immunogold-labeling studies revealed that in Nicotiana clevelandii the putative movement protein was found only in infected cells immediately adjacent to the necrotic tissue, and that it was associated with tubular structures containing virus-like particles present in or near the cell wall. This provides further evidence that this protein is involved in the cell-to-cell movement of the virus and that this movement might take place via the formation of tubular structures.


Pancreatic cancer: French clinical practice guidelines for diagnosis, treatment and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO, ACHBT, AFC).
This document is a summary of the French intergroup guidelines regarding the management of pancreatic adenocarcinoma (PA), updated in July 2018. This collaborative work was produced under the auspices of all French medical and surgical societies involved in the management of PA. It is based on the previous guidelines, recent literature review and expert opinions. Recommendations were graded in three categories, according to the level of evidence. Over the last seven years, significant changes in PA management have been implemented in clinical practice. Imaging/staging: diffusion magnetic resonance imaging is useful before surgery to rule out small liver metastases. centralization of pancreatic surgery in expert centers is associated with a decreased postoperative mortality. Adjuvant chemotherapy: modified FOLFIRINOX in fit patients, or gemcitabine, or 5-FU, or gemcitabine plus capecitabine, to be discussed on a case-by-case basis. Locally advanced PA: no survival benefit of chemoradiotherapy. Metastatic PA: FOLFIRINOX and gemcitabine plus nab-paclitaxel combination are first-line standards in fit patients; second-line with 5FU/nal-IRI or 5FU/oxaliplatin combination after first-line gemcitabine. Guidelines for management of PA are continuously evolving and need to be regularly updated. This constant progress is made possible through clinical and translational research. However, as each individual case is particular, they cannot substitute to multidisciplinary tumor board discussion.


PTEN: tumour suppressor, multifunctional growth regulator and more.
The tumour suppressor gene PTEN is mutated in a wide range of human cancers at a frequency roughly comparable with p53. In addition, germline PTEN mutations are associated with several dominant growth disorders. The molecular and cellular basis of these disorders has been elucidated by detailed in vivo genetic analysis in model organisms, in particular the fruit fly and mouse. Studies in the fly have shown that PTEN's growth regulatory functions are primarily mediated via its lipid phosphatase activity, which specifically reduces the cellular levels of phosphatidylinositol 3,4,5-trisphosphate. This activity antagonizes the effects of activated PI3-kinase in the nutritionally controlled insulin receptor pathway, thereby reducing protein synthesis and restraining cell and organismal growth, while also regulating other biological processes, such as fertility and ageing. Remarkably, this range of functions appears to be conserved in all higher organisms. PTEN also plays a role as a specialized cytoskeletal regulator, which, for example, is involved in directional movement of some migratory cells and may be important in metastasis. Furthermore, conditional knockouts in the mouse have recently revealed functions for PTEN in other processes, such as cell type specification and cardiac muscle contractility. Genetic approaches have therefore revealed a surprising diversity of global and cell type-specific PTEN-regulated functions that appear to be primarily controlled by modulation of a single phosphoinositide. Together with evidence from studies in cell culture that suggests links between PTEN and other growth regulatory genes such as p53, these studies provide new insights into PTEN-linked disorders and are beginning to suggest potential clinical strategies to combat these and other diseases.


Importance-Driven Accessory Lights Design for Enhancing Local Shapes.
We introduce a semi-automatic lighting design method that deploys per-voxel accessory lights (fill and detail lights) to enhance local shapes, as well as to increase the perceptibility and visual saliency of an object. Our approach allows the user to manually design arbitrary lights in a scene for creating the desired feeling of emotion. The user designed lights are used as key lights and our approach automatically configures per-voxel accessory lights that preserve the user designed feeling of emotion. Per-voxel fill lights brighten the shadows and thus increase the perceptibility and visual saliency. Per-voxel detail lights enhance the visual cues for the local shape perception. Moreover, the revealed local shapes are controlled by the user employing an importance distribution. Similarly, the perceptibility and visual saliency are also controlled based on an importance distribution. Our perceptual measurement guarantees that the revealed local shapes are independent of the key lights. In addition, our method provides two control parameters, which adjust the fill and detail lights, to provide the user with additional flexibility in designing the expected lighting effect. The major contributions of this paper are the idea of using the importance distribution to control local shapes, the per-voxel accessory lights and the perceptual measurement.


Single molecule characterization of P-selectin/ligand binding.
P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.


A Practice-Based Evaluation of Distress Screening Protocol Adherence and Medical Service Utilization.
Background: This study examined the extent to which cancer programs demonstrated adherence to their own prescribed screening protocol, and whether adherence to that protocol was associated with medical service utilization. The hypothesis is that higher rates of service utilization are associated with lower rates of adherence to screening protocols. Methods: Oncology social workers at Commission on Cancer-accredited cancer programs reviewed electronic health records (EHRs) in their respective cancer programs during a 2-month period in 2014. Rates of overall adherence to a prescribed distress screening protocol were calculated based on documentation in the EHR that screening adherence and an appropriate clinical response had occurred. We examined documentation of emergency department (ED) use and hospitalization within 2 months after the screening visit. Results: Review of 8,409 EHRs across 55 cancer centers indicated that the overall adherence rate to screening protocols was 62.7%. The highest rates of adherence were observed in Community Cancer Programs (76.3%) and the lowest rates were in NCI-designated Cancer Centers (43.3%). Rates of medical service utilization were significantly higher than expected when overall protocol adherence was lacking. After controlling for patient and institutional characteristics, risk ratios for ED use (0.82) and hospitalization (0.81) suggest that when overall protocol adherence was documented, 18% to 19% fewer patients used these medical services. Conclusions: The observed associations between a mandated psychosocial care protocol and medical service utilization suggest opportunities for operational efficiencies and costs savings. Further investigations of protocol integrity, as well as the clinical care models by which psychosocial care is delivered, are warranted.


Targeting proteasomal pathways by dietary curcumin for cancer prevention and treatment.
Curcumin, a major component of the golden spice turmeric (Curcuma longa), has been linked with the prevention and treatment of a wide variety of cancers through modulation of multiple cell signaling pathways. Since the first report from our laboratory in 1995 that curcumin can inhibit activation of the proinflammatory transcription factor NF-κB by inhibiting the 26S proteasomal degradation of IκBα, an inhibitor of NF-κB, this yellow pigment has been shown to inhibit the protease activities of the proteasome. The carbonyl carbons of the curcumin molecule directly interact with the hydroxyl group of the amino-terminal threonine residue of the proteasomal CT-L subunit of 20S proteasome and cellular 26S proteasome. Curcumin is also a potent inhibitor of COP9 signalosome and associated kinases, casein kinase 2 and protein kinase D, all linked to the ubiquitin-proteasomal system (UPS). Curcumin can also directly inhibit ubiquitin isopeptidases, a family of deubiquitinases (DUBs) that salvage ubiquitin for reuse by the 26S proteasome system. The inhibition of this enzyme by curcumin is mediated through α,β-unsaturated ketone and two sterically accessible β-carbons. Regulation of the UPS pathway by curcumin has been linked to regulation of cancer-linked inflammatory proteins (such as COX-2 and iNOS), transcription factors (NF-κB, STAT3, Sp, AP-1, GADD153/CHOP, HIF-1α), growth factors (VEGF, HER2), apoptotic proteins (p53, Bcl-2, survivin, DNA topoisomerase II, HDAC2, p300, hTERT) and cell cycle proteins (cyclin D1, cyclin E, cyclin B, p21, p27) associated with the prevention and therapy of cancer. Interestingly, the effect of curcumin on 26S proteasome appears to be dose-dependent, as low doses (≥1 µM) increase proteasome activity whereas high doses (≤10 µM) inhibit the proteasome activity. In this review, we discuss in detail how modulation of these targets by curcumin is linked to prevention and treatment of cancer.


Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation.
Endocytic trafficking of signaling receptors is an important mechanism for limiting signal duration. Components of the Endosomal Sorting Complexes Required for Transport (ESCRT), which target ubiquitylated receptors to intra-lumenal vesicles (ILVs) of multivesicular bodies, are thought to terminate signaling by the epidermal growth factor receptor (EGFR) and direct it for lysosomal degradation. In a genetic screen for mutations that affect Drosophila eye development, we identified an allele of Vacuolar protein sorting 4 (Vps4), which encodes an AAA ATPase that interacts with the ESCRT-III complex to drive the final step of ILV formation. Photoreceptors are largely absent from Vps4 mutant clones in the eye disc, and even when cell death is genetically prevented, the mutant R8 photoreceptors that develop fail to recruit surrounding cells to differentiate as R1-R7 photoreceptors. This recruitment requires EGFR signaling, suggesting that loss of Vps4 disrupts the EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and other receptors accumulate in endosomes and EGFR target genes are not expressed; epistasis experiments place the function of Vps4 at the level of the receptor. Surprisingly, Vps4 is required for EGFR signaling even in the absence of Shibire, the Dynamin that internalizes EGFR from the plasma membrane. In ovarian follicle cells, in contrast, Vps4 does not affect EGFR signaling, although it is still essential for receptor degradation. Taken together, these findings indicate that Vps4 can promote EGFR activity through an endocytosis-independent mechanism.


Estimation and Inference for High Dimensional Generalized Linear Models: A Splitting and Smoothing Approach.
The focus of modern biomedical studies has gradually shifted to explanation and estimation of joint effects of high dimensional predictors on disease risks. Quantifying uncertainty in these estimates may provide valuable insight into prevention strategies or treatment decisions for both patients and physicians. High dimensional inference, including confidence intervals and hypothesis testing, has sparked much interest. While much work has been done in the linear regression setting, there is lack of literature on inference for high dimensional generalized linear models. We propose a novel and computationally feasible method, which accommodates a variety of outcome types, including normal, binomial, and Poisson data. We use a "splitting and smoothing" approach, which splits samples into two parts, performs variable selection using one part and conducts partial regression with the other part. Averaging the estimates over multiple random splits, we obtain the smoothed estimates, which are numerically stable. We show that the estimates are consistent, asymptotically normal, and construct confidence intervals with proper coverage probabilities for all predictors. We examine the finite sample performance of our method by comparing it with the existing methods and applying it to analyze a lung cancer cohort study.


Basis for the gain and subsequent dilution of epidermal pigmentation during human evolution: The barrier and metabolic conservation hypotheses revisited.
The evolution of human skin pigmentation must address both the initial evolution of intense epidermal pigmentation in hominins, and its subsequent dilution in modern humans. While many authorities believe that epidermal pigmentation evolved to protect against either ultraviolet B (UV-B) irradiation-induced mutagenesis or folic acid photolysis, we hypothesize that pigmentation augmented the epidermal barriers by shifting the UV-B dose-response curve from toxic to beneficial. Whereas erythemogenic UV-B doses produce apoptosis and cell death, suberythemogenic doses benefit permeability and antimicrobial function. Heavily melanized melanocytes acidify the outer epidermis and emit paracrine signals that augment barrier competence. Modern humans, residing in the cooler, wetter climes of south-central Europe and Asia, initially retained substantial pigmentation. While their outdoor lifestyles still permitted sufficient cutaneous vitamin D3 (VD3) synthesis, their marginal nutritional status, coupled with cold-induced caloric needs, selected for moderate pigment reductions that diverted limited nutritional resources towards more urgent priorities (=metabolic conservation). The further pigment-dilution that evolved as humans reached north-central Europe (i.e., northern France, Germany), likely facilitated cutaneous VD3 synthesis, while also supporting ongoing, nutritional requirements. But at still higher European latitudes where little UV-B breaches the atmosphere (i.e., present-day UK, Scandinavia, Baltic States), pigment dilution alone could not suffice. There, other nonpigment-related mutations evolved to facilitate VD3 production; for example, in the epidermal protein, filaggrin, resulting in reduced levels of its distal metabolite, trans-urocanic acid, a potent UV-B chromophore. Thus, changes in human pigmentation reflect a complex interplay between latitude, climate, diet, lifestyle, and shifting metabolic priorities.


Direct measurement of polyol pathway activity in the ocular lens.
A method to measure the polyol pathway metabolic flux in the intact rabbit lens by 13C nuclear magnetic resonance spectroscopy is described. In the lens exposed to 35.5 mM glucose, the polyol pathway accounts for 1/3 of the total glucose turnover. The high metabolic activity of the pathway suggests a significant alteration in the reduced to oxidized pyridine nucleotide ratio in the lens exposed to high glucose.


Vaginal estrogen for genitourinary syndrome of menopause: a systematic review.
To comprehensively review and critically assess the literature on vaginal estrogen and its alternatives for women with genitourinary syndrome of menopause and to provide clinical practice guidelines. MEDLINE and Cochrane databases were searched from inception to April 2013. We included randomized controlled trials and prospective comparative studies. Interventions and comparators included all commercially available vaginal estrogen products. Placebo, no treatment, systemic estrogen (all routes), and nonhormonal moisturizers and lubricants were included as comparators. We double-screened 1,805 abstracts, identifying 44 eligible studies. Discrepancies were adjudicated by a third reviewer. Studies were individually and collectively assessed for methodologic quality and strength of evidence. Studies were extracted for participant, intervention, comparator, and outcomes data, including patient-reported atrophy symptoms (eg, vaginal dryness, dyspareunia, dysuria, urgency, frequency, recurrent urinary tract infection (UTI), and urinary incontinence), objective signs of atrophy, urodynamic measures, endometrial effects, serum estradiol changes, and adverse events. Compared with placebo, vaginal estrogens improved dryness, dyspareunia, urinary urgency, frequency, and stress urinary incontinence (SUI) and urgency urinary incontinence (UUI). Urinary tract infection rates decreased. The various estrogen preparations had similar efficacy and safety; serum estradiol levels remained within postmenopausal norms for all except high-dose conjugated equine estrogen cream. Endometrial hyperplasia and adenocarcinoma were extremely rare among those receiving vaginal estrogen. Comparing vaginal estrogen with nonhormonal moisturizers, patients with two or more symptoms of vulvovaginal atrophy were substantially more improved using vaginal estrogens, but those with one or minor complaints had similar symptom resolution with either estrogen or nonhormonal moisturizer. All commercially available vaginal estrogens effectively relieve common vulvovaginal atrophy-related complaints and have additional utility in patients with urinary urgency, frequency or nocturia, SUI and UUI, and recurrent UTIs. Nonhormonal moisturizers are a beneficial alternative for those with few or minor atrophy-related symptoms and in patients at risk for estrogen-related neoplasia. PROSPERO International prospective register of systematic reviews, http://www.crd.york.ac.uk/PROSPERO/, CRD42013006656.


Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies.
Only a few autoantibodies that are more or less specific for RA have been described so far. The rheumatoid factor most often tested for is not very specific for RA, while the more specific antiperinuclear factor for several reasons is not routinely used as a serological parameter. Here we show that autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue, are specifically present in the sera of RA patients. Using several citrulline-containing peptide variants in ELISA, antibodies could be detected in 76% of RA sera with a specificity of 96%. Sera showed a remarkable variety in the reactivity pattern towards different citrulline-containing peptides. Affinity-purified antibodies were shown to be positive in the immunofluorescence-based antiperinuclear factor test, and in the so-called antikeratin antibody test, and were reactive towards filaggrin extracted from human epidermis. The specific nature of these antibodies and the presence of these antibodies early in disease, even before other disease manifestations occur, are indicative for a possible role of citrulline-containing epitopes in the pathogenesis of RA.


Phylloplane yeasts from Portugal: seven novel anamorphic species in the Tremellales lineage of the Hymenomycetes (Basidiomycota) producing orange-coloured colonies.
A survey of epiphytic yeasts on leaves of selected Mediterranean plant species collected at the 'Arrábida Natural Park' (Portugal) yielded about 850 isolates, mostly of basidiomycetous affinity. Amongst the basidiomycetes, 35 strains showed the following characteristics: production of orange-coloured colonies, ability to produce starch-like compounds, assimilation of D-glucuronic acid and/or inositol, inability to utilize nitrate, and formation of ballistoconidia by many of the isolates. This group of yeasts was assigned to the Tremellales lineage of the Hymenomycetes and was further characterised using a combination of conventional phenotypic identification tests with molecular methods, namely PCR fingerprinting and rDNA sequencing. Eight additional strains presumptively identified as Bullera armeniaca, B. crocea or Cryptococcus hungaricus were also studied. Twenty-eight strains could be assigned to or were phylogenetically related to recognised species of Dioszegia in the 'Luteolus clade', but the 15 remaining strains belonged to other clades within the Tremellales. Ten phylloplane isolates were identified as Dioszegia hungarica, one as D. aurantiaca, another as D. crocea and three others were ascribed to the recently described species D. zsoltii. Seven novel species, viz. Cryptococcus amylolyticus, C. armeniacus, C. cistialbidi, Dioszegia buhagiarii, D. catarinonii, D. fristingensis and D. takashimae, are proposed for the remaining strains that did not correspond to any of the hitherto recognised species.


Fluoxetine-induced pancreatic beta cell dysfunction: New insight into the benefits of folic acid in the treatment of depression.
Major depressive disorder is a common psychiatric illness with reported prevalence rates of 12-16% in persons aged 12 and over. Depression is also associated with a high risk of new onset of type 2 diabetes (T2D). This relationship between depression and diabetes may be related to depression itself and/or drugs prescribed. Importantly, the use of selective serotonin reuptake inhibitors (SSRIs), the most commonly prescribed class of antidepressants, increases the risk of developing T2D. However, the mechanism(s) underlying this association remains elusive. Here we examine the effects of the SSRI fluoxetine (Prozac®) on beta cell function utilizing INS-1E cells, a rat beta cell line, to elucidate the underlying molecular mechanisms. Fluoxetine treatment significantly reduced glucose stimulated insulin secretion (GSIS). This decreased beta cell function was concomitant with an increased production of reactive oxygen species and oxidative damage which may contribute to decreased mitochondrial electron transport chain enzyme (ETC) activity. Importantly the fluoxetine-induced deficits in beta cell function were prevented by the addition of the antioxidant folic acid. These studies were conducted in vitro; the in vivo relevance remains to be determined. These findings suggest that use of SSRI antidepressants may increase the risk of new-onset T2D by causing oxidative stress in pancreatic beta cells. However, folic acid supplementation in patients taking SSRIs may reduce the risk of new onset diabetes via protection of normal beta cell function.