Assessing salivary cortisol in studies of child development.
In a series of studies, we evaluated the susceptibility of radioimmunoassays (RIA) for saliva cortisol to interference effects caused by oral stimulants used to facilitate saliva collection in studies with children. When added directly to saliva samples, oral stimulants (drink mix crystals) artificially inflated estimated cortisol concentrations. The magnitude of the interference effect was concentration-dependent and more pronounced for some stimulants and RIA procedures than for others. Analysis of samples collected using oral stimulants from child and adult participants confirmed stimulant interference as an extraneous source of variability in measured saliva cortisol. Associations between serum and saliva cortisol and between saliva cortisol and "behavioral" variables were attenuated by stimulant interference. A survey of six large child studies estimated interference effects, indexed by low sample pH, to be present in 14.7% of the 1,148 total saliva samples, or 2%-54% (M = 22%) of samples within each study. Recommendations to minimize the impact of stimulant interference in studies involving salivary cortisol in the context of child health and development are outlined.


Lymphoepithelioid lymphoma (Lennert's lymphoma) is a monoclonal proliferation of helper/inducer T cells.
Lymphoepithelioid lymphoma (Lennert's lymphoma) was first described as a special variant of Hodgkin's disease. This lesion is characterized by a high percentage of epithelioid and T cells and rarely contains the classical Hodgkin's/Reed-Sternberg cells. Cytogenetic abnormalities indicate that Lennert's lymphoma is of T cell origin. In the present study, immunohistochemical investigation of four cases of Lennert's lymphoma revealed two major cell populations of T cells that predominantly express the helper-inducer phenotype and Ki-M6- and Ki-M8-positive macrophages and epithelioid cells. Double-staining experiments for the detection of cell surface antigens and the proliferation-associated antigen Ki67 showed that only the CD4-positive cells (helper-inducer T cells) were proliferating. Examination of the DNA of these Lennert's lymphoma samples also indicated that monoclonal rearrangement of the T cell receptor beta-chain genes has occurred, whereas the immunoglobulin heavy- and kappa-chain genes remained in germline configuration. Our results strongly suggest that Lennert's lymphoma is a CD4-positive T cell lymphoma.


Differentially expressed proteins of mesenchymal stem cells derived from human cord blood (hUCB) during osteogenic differentiation.
Multipotent mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) represent promising candidates for the development of future cellular therapy strategies. MSCs have been found to be able to differentiate into various tissues. One of the primary limitations in our understanding of the biology of human MSCs is the absence of prospective markers required for the monitoring of lineage-specific differentiation. hUCB-derived MSCs have been found to have significantly greater osteogenic potential. In this study, we focused on proteins that were differentially expressed during osteogenic differentiation of hUCB-MSCs. And we analyzed the protein expression inherent to osteogenic differentiation by two-dimensional gel electrophoresis, ESI-Q-TOF, and Western blotting. Eleven differentially expressed spots were observed between the two groups (before and after differentiation) on the 2-DE map. These might also be proved as useful cytosolic biomarker proteins for osteogenesis, and might be employed in quality control of osteoblasts in cell-therapy applications.


Analysis of MMP-dependent cell migration and invasion.
Analysing cell migration and invasion is of interest to many investigators as they mimic a part of physiological or pathological events. In this chapter, methods to analyse MMP-dependent 2D and 3D cell migration are described in detail. To study 2D cell migration, the phagokinetic track motility assay can be used. To study 3D cell migration, Matrigel invasion assay or microcarrier beads invasion assay can be used to obtain quantitative results.


Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum.
Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.


Magnetism and local molecular field.
Despite its somewhat naive simplicity, the method of the local molecular field has had undeniable success in satisfactorily explaining a large number of previously known facts and in opening the way for the discovery of new facts. Let us note, however, that all the structures that have been discussed above are collinear structures: on the average (in time) all the atomic magnets pointing in one or the opposite direction are parallel to a single direction. However, the local molecular field method can also be extended to noncollinear structures such as that of helimagnetism, which Yoshimori and Villain discovered independently in an absolutely unexpected manner; one can thus interpret phenomena in a remarkably simple and concrete manner. Nevertheless, the method can hardly be recommended for more complex structures such as the umbrella structure, which requires the decomposition of the principal crystal lattice into a large number of sublattices. Indeed, under these conditions an atom belonging to a given sublattice has only a very small number of neighbors (one or two) in each of the other sublattices, and the molecular field method, which consists in replacing the instantaneous action of an atom by that of an average atom, will be more likely to yield a correct result, the larger the number of atoms to which it is applied. Its correctness probably also increases as the atomic spin becomes larger. Independently of this problem, the method applied to a large number of sublattices completely loses its chief advantage, simplicity. The method also involves more insidious traps. If a judicious choice of parameters is made, the method can lead one to calculate curves and thermal variations of the spontaneous magnetization or paramagnetic susceptibility that coincide remarkably well with the experimental results, for example, to within a few thousandths. Under these conditions, one could expect that the elementary interaction energies deduced from these parameters would correspond to the actual values with the same accuracy. This is not so; errors of 10 to 20 percent and even greater are frequently made in this manner. A certain amount of caution thus becomes imperative. On the other hand, recourse to the local molecular field seems indispensable since more rigorous methods lead to insurmountable complications. Consider for example that the rigorous solution is not yet known for the simplest case, that of a simple cubic lattice with identical atoms of spin 1/2, and interactions reduced to those present between nearest-neighbor atoms. How then should one treat the case of garnets with 160 atoms in the unit cell, spins up to 5/2, and at least six different coupling constants? One must therefore be lenient toward the imperfections of the molecular field methods, considering the simplicity with which the successes recalled in the first few lines of these conclusions were obtained.


Substrate metabolism of isolated jejunal epithelium: conservation of three-carbon units.
This study characterizes the substrate metabolism of isolated jejunal epithelial cells. Utilization of substrates was assessed by spectrophotometric assay. Significant quantities of glucose, glutamine, and ketone bodies were consumed in a 1-h period; lactate and ammonia were produced. [U-14C]glucose was metabolized in this medium to approximately three moles of lactate per mole of CO2. The pattern of tricarboxylic acid (TCA) cycle metabolism was analyzed utilizing media containing different concentrations of potential metabolic substrates and trace quantities of [14C]- succinate. O2 consumption rates indicated that glutamine can serve as an energy source in the absence of other substrates. Relative 14CO2 production from [1,4-14C]succinate versus [2,3-14C]succinate, which estimates flux of TCA cycle intermediates to products other than CO2, was increased more than twofold when glutamine was the only major substrate available. Alanine was produced from TCA cycle intermediates. Analysis of the citrate labeling pattern in the presence of [2,3-14C] succinate suggested that carbon from the TCA cycle does not form a significant fraction of acetyl-CoA used for citrate synthesis and that glutamine carbon was not completely oxidized to CO2. These findings suggest that glucose and glutamine are converted to three-carbon compounds by the jejunal epithelium.


Tuning electric field aligned CNT architectures via chemistry, morphology, and sonication from micro to macroscopic scale.
Electric-field alignment of carbon nanotubes (CNT) is widely used to produce composite materials with anisotropic mechanical, electrical, and optical properties. Nevertheless, consistent results are difficult to achieve, and even under identical electric field conditions the resulting aligned morphologies can vary over μm to cm length scales. In order to improve reproducibility, this study addresses (1) how solution processing steps (oxidation, sonication) affect CNT properties, and (2) how CNT chemistry, morphology, and dispersion influence alignment. Aligned CNT were deposited onto PVDF membranes using a combination of electric-field alignment and vacuum-filtration. At each step in solution processing, the CNT chemistry (oxygen content) and morphology (length/diameter) were characterized and compared to the final aligned morphology. Well-dispersed CNT with high oxygen content (>8.5%O) yielded uniform membrane coatings and microscopically aligned CNT, whereas CNT with low oxygen CNT (<2.2%O) produced aligned bundles visible at a macroscopic level, but microscopically the individual CNT remained disordered. Based on regression analysis, CNT with larger mean length and diameter, smaller length and diameter variation, and higher oxygen content yielded increased electrical anisotropy, and bath sonication was slightly preferable to probe sonication for initial dispersion.


Comparison between indigenous and Western postnatal care practices in Mopani District, Limpopo Province, South Africa.
Postnatal care begins immediately after the expulsion of the placenta and continues for six to eight weeks post-delivery. High standard of care is required during the postnatal period because mothers and babies are at risk and vulnerable to complications related to postpartum haemorrhage and infections. Midwives and traditional birth attendants are responsible for the provision of postnatal care in different settings, such as clinics and hospitals, and homes. A qualitative, exploratory, descriptive and contextual research approach was followed in this study. Unstructured interviews were conducted with the traditional birth attendants. An integrated literature review was conducted to identify the Western postnatal care practices. Tesch's process was followed during data analysis. The following main categories were identified: similarities between indigenous and Western postnatal care practices, and differences between indigenous and Western postnatal care practices. Based on these findings, training of midwives and traditional birth attendants was recommended in order to empower them with knowledge and skills regarding the indigenous and Western postnatal care practices. It is evident that some indigenous postnatal care practices have adverse effects on the health of postnatal women and their newborn infants, but these are unknown to the traditional birth attendants. The employment of indigenous postnatal care practices by the traditional birth attendants is also influenced by their cultural beliefs, norms, values and attitudes. Therefore, there is an urgent need to train midwives and traditional birth attendants regarding the indigenous and Western postnatal care to improve the health of postnatal women and their babies.


Teaching clinical informatics to third-year medical students: negative results from two controlled trials.
Prior educational interventions to increase seeking evidence by medical students have been unsuccessful. We report two quasirandomized controlled trials to increase seeking of medical evidence by third-year medical students. In the first trial (1997-1998), we placed computers in clinical locations and taught their use in a 6-hour course. Based on negative results, we created SUMSearch, an Internet site that automates searching for medical evidence by simultaneous meta-searching of MEDLINE and other sites. In the second trial (1999-2000), we taught SUMSearch's use in a 5(1/2)-hour course. Both courses were taught during the medicine clerkship. For each trial, we surveyed the entire third-year class at 6 months, after half of the students had taken the course (intervention group). The students who had not received the intervention were the control group. We measured self-report of search frequency and satisfaction with search quality and speed. The proportion of all students who reported searching at least weekly for medical evidence significantly increased from 19% (1997-1998) to 42% (1999-2000). The proportion of all students who were satisfied with their search results increased significantly between study years. However, in neither study year did the interventions increase searching or satisfaction with results. Satisfaction with the speed of searching was 27% in 1999-2000. This did not increase between studies years and was not changed by the interventions. None of our interventions affected searching habits. Even with automated searching, students report low satisfaction with search speed. We are concerned that students using current strategies for seeking medical evidence will be less likely to seek and appraise original studies when they enter medical practice and have less time.


Prepartal standing behavior as a parameter for early detection of postpartal subclinical ketosis associated with inflammation and liver function biomarkers in peripartal dairy cows.
A degree of negative energy balance is commonly experienced by cows during early lactation. This physiological state, if pronounced or prolonged, leads to partial oxidation of nonesterified fatty acids as an energy source and, consequently, increasing blood β-hydroxybutyrate (BHB) concentrations and potentially development of ketosis in postpartal dairy cows. Twenty-four multiparous Holstein cows received a common prepartal and postpartal diet. Cows were fitted with an accelerometer mounted laterally on the distal left hind leg using vet wrap from -30 to 15 d relative to parturition. A retrospective analysis was performed using the postpartal BHB data at 8 time points from 0 to 15 d in milk measured with the Precision Xtra (Abbott Diabetes Care, Alameda, CA). Cows with an average blood BHB <1.4 mmol/L were designated nonketotic (NONKET; n = 12), and those with ≥1.4 mmol/L were designated ketotic (KET; n = 12). A total of 8 samples per cow were used for this analysis. Subsequent analyses of behavioral patterns and blood biomarkers were performed using this group effect. On average, blood BHB reached subclinical levels (1.4 ± 0.3 mmol/L; mean ± standard error of the mean) at 3 d postpartum for all cows in this study. Behavioral patterns were obtained from accelerometer data, and correlation analysis was performed between these behaviors such as standing and lying time from -30 to 3 d relative to parturition and blood BHB concentration at 3 d postpartum. The strongest correlation was obtained between standing time at 3 d before calving and blood BHB at 3 d postpartum. Dry matter intake was greater (ca. 3 kg/d) in NONKET cows than in KET cows. An interaction of group × time for milk yield resulted in an overall increase of 5.7 kg/d in NONKET cows in comparison with KET. The blood concentrations of biomarkers for liver function (γ-glutamyltransferase and glutamic-oxaloacetic transaminase), inflammation (IL-6), and metabolism (nonesterified fatty acids) were increased at various time points in KET cows in comparison with NONKET during the transition period. Overall, lower bilirubin in NONKET cows than in KET further confirmed an impaired liver function in the latter group of cows. Our findings revealed the potential for establishing correlations between prepartal behavioral patterns derived from accelerometer data and postpartal subclinical ketosis, and further confirming the latter by physiological alterations in biomarkers related to inflammation and liver function. Our data also indicate that cows with a predisposition to postpartal subclinical or clinical ketosis will remain standing for fewer hours during the days leading to parturition, which decreased DMI, and this condition was further reflected in lower milk yield.


Role of extrinsic and intrinsic nerves in hormonal induction of the migrating motor complex in the jejunum.
The mechanism of induction of the migrating motor complex (MMC) by neural or humoral agents and their role in the control of fasting motility are not well understood. Our aim was to determine the role of extrinsic and intrinsic nerves in mediating the induction of the MMC by motilin. Three groups of dogs were studied. Group I consisted of neurally intact control dogs. In group II, intrinsic neural continuity between the duodenum and the jejunum was interrupted by transection and reanastomosis of the distal duodenum. Dogs in group III underwent disruption of all intrinsic and extrinsic neural input to the entire jejunoileum. Serosal electrodes were sewn to duodenum and jejunum in all dogs. After a 2-week recovery, fasting myoelectric activity was recorded on four or more occasions. Motilin (0.1 microgram/kg iv) was given 30 min after a spontaneous duodenal phase III. In group I (controls), motilin induced a premature MMC, which originated in the duodenum and migrated along the small intestine. In group II (intrinsic neural disruption), motilin induced a premature MMC, which began simultaneously in the proximal duodenum and proximal jejunum. In group III (intrinsic and extrinsic neural disruption), motilin induced a premature MMC in the duodenum but not in the jejunum; rather, a short, nonmigrating burst of spike potentials occurred simultaneously in all jejunal electrodes. These observations suggest that extrinsic innervation is necessary for motilin to induce phase III activity in the jejunum. Extrinsic neural pathways appear to mediate motilin-induced MMC activity in the jejunum.


Human neuronal signaling and communication assays to assess functional neurotoxicity.
Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.


Breast cancer brain metastases express the sodium iodide symporter.
Breast cancer brain metastases are on the rise and their treatment is hampered by the limited entry and efficacy of anticancer drugs in this sanctuary. The sodium iodide symporter, NIS, actively transports iodide across the plasma membrane and is exploited clinically to deliver radioactive iodide into cells. As in thyroid cancers, NIS is expressed in many breast cancers including primary and metastatic tumors. In this study NIS expression was analyzed for the first time in 28 cases of breast cancer brain metastases using a polyclonal anti-NIS antibody directed against the terminal C-peptide of human NIS gene and immunohistochemical methods. Twenty-five tumors (84%) in this retrospective series were estrogen/progesterone receptor-negative and 15 (53.6%) were HER2+. Overall 21 (75%) cases and 80% of HER2 positive metastases were NIS positive. While the predominant pattern of NIS immunoreactivity is intracellular, plasma membrane immunopositivity was detected at least focally in 23.8% of NIS-positive samples. Altogether, these findings indicate that NIS expression is prevalent in breast cancer brain metastases and could have a therapeutic role via the delivery of radioactive iodide and selective ablation of tumor cells.


Tritherapy for human immunodeficiency virus infection does not modify replication of hepatitis C virus in coinfected subjects.
Triple antiretroviral therapy combining reverse transcriptase and protease inhibitors modifies the prognosis for human immunodeficiency virus (HIV) infection, with dramatic improvement in immune status. In an attempt to evaluate the impact of anti-HIV triple combination therapy on the course of hepatitis C virus (HCV)-related chronic hepatitis and on HCV replication, we studied the biological and virological characteristics of 22 HCV/HIV-coinfected patients who were given triple combination therapy. In comparison with baseline values, there was (1) a significant increase in the CD4 and CD8 cell counts and a decrease in the HIV RNA load and (2) no significant variation in aminotransferase activities or the HCV RNA load at 3, 6, or 9 months of tritherapy. Antiretroviral tritherapy seems to modify neither the biological activity of HCV-related chronic hepatitis nor the HCV load, despite immune restoration. Hepatic histopathologic analysis is warranted to assess the impact of immune restoration on liver lesions.


beta-catenin/TCF/Lef controls a differentiation-associated transcriptional program in renal epithelial progenitors.
In the embryonic kidney, progenitors in the metanephric mesenchyme differentiate into specialized renal epithelia in a defined sequence characterized by the formation of cellular aggregates, conversion into polarized epithelia and segmentation along a proximal-distal axis. This sequence is reiterated throughout renal development to generate nephrons. Here, we identify global transcriptional programs associated with epithelial differentiation utilizing an organ culture model of rat metanephric mesenchymal differentiation, which recapitulates the hallmarks of epithelialization in vivo in a synchronized rather than reiterative fashion. We observe activation of multiple putative targets of beta-catenin/TCF/Lef-dependent transcription coinciding with epithelial differentiation. We show in cultured explants that isolated activation of beta-catenin signaling in epithelial progenitors induces, in a TCF/Lef-dependent manner, a subset of the transcripts associated with epithelialization, including Pax8, cyclin D1 (Ccnd1) and Emx2. This is associated with anti-apoptotic and proliferative effects in epithelial progenitors, whereas cells with impaired TCF/Lef-dependent transcription are progressively depleted from the epithelial lineage. In vivo, TCF/Lef-responsive genes comprise a conserved transcriptional program in differentiating renal epithelial progenitors and beta-catenin-containing transcriptional complexes directly bind to their promoter regions. Thus, beta-catenin/TCF/Lef-mediated transcriptional events control a subset of the differentiation-associated transcriptional program and thereby participate in maintenance, expansion and stage progression of the epithelial lineage.


One-pot radical polymerization of one inimer to form one-dimensional polymeric nanomaterials.
A one-pot polymerization strategy is put forward for producing 1D polymeric nanomaterials directly from a single inimer. An inimer bearing an NMP initiating site is polymerized in the presence of a RAFT CTA to form a pearl-necklace structure constituted of hyperbranched polymer "pearls". The obtained 1D nanorods show high regularity evidenced by the long-range order in small angle XRD patterns and the formation of liquid crystalline phases.


touché Is required for touch-evoked generator potentials within vertebrate sensory neurons.
The process by which light touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing channelrhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP-positive peripheral neurites. In wild-type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild-type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that cutaneous mechanoreceptors with generator potentials are necessary for responsiveness to light touch in zebrafish.


Comparison of bacteriological conversion and treatment outcomes among MDR-TB patients with and without diabetes in Mexico: Preliminary data.
Diabetes mellitus (DM) is a well-known risk factor for tuberculosis (TB). However, it is not known to what extent DM affects the outcome in patients with multidrug-resistant (MDR-TB) and extensively drug-resistant TB (XDR-TB) treated with second-line anti-TB drugs. The objective of this study was to compare the microbiological evolution (sputum smear and culture conversion) and final outcomes of MDR/XDR-TB patients with and without DM, managed at the national TB reference centre in Mexico City. Ninety patients were enrolled between 2010 and 2015: 73 with MDR-TB (81.1%), 11 with pre-XDR-TB (e.g. MDR-TB with additional resistance to one injectable drug or a fluoroquinolone, 12.2%) and 6 (6.7%) with XDR-TB. Out of these, 49 (54.4%) had DM and 42 (86%) were undergoing insulin treatment. No statistically significant differences were found in treatment outcomes comparing DM vs. non-DM MDR-TB cases: 18/32 (56.3%) of DM cases and 19/24 (79.2%) non DM patients achieved treatment success (p=0.07). The time to sputum smear and culture conversion was longer (although not statistically) in patients without DM, as follows: the mean (±SD) time to sputum smear conversion was 53.9 (±31.4) days in DM patients and 65.2 (±34.8) days in non-DM ones (p=0.15), while the time to culture conversion was 66.2 (±27.6) days for DM and 81.4 (±37.7) days for non-DM MDR-TB cases (p=0.06). The study results support the Mexican National TB programme to strengthen its collaboration with the DM programme, as an entry point for TB (and latent TB infection) screening and management.


Mechanisms of human immunodeficiency virus type 1 concerted integration related to strand transfer inhibition and drug resistance.
The "strand transfer inhibitors" of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3' OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3'-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.


Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage.
Neutrophil gelatinase-associated lipocalin (NGAL) is a protein belonging to the lipocalin superfamily initially found in activated neutrophils, in accordance with its role as an innate antibacterial factor. However, it subsequently was shown that many other types of cells, including in the kidney tubule, may produce NGAL in response to various injuries. The increase in NGAL production and release from tubular cells after harmful stimuli of various kinds may have self-defensive intent based on the activation of specific iron-dependent pathways, which in all probability also represent the mechanism through which NGAL promotes kidney growth and differentiation. NGAL levels predict the future appearance of acute kidney injury after treatments potentially detrimental to the kidney and even the acute worsening of unstable nephropathies. Furthermore, recent evidence also suggests that NGAL somehow may be involved in the pathophysiological process of chronic renal diseases, such as polycystic kidney disease and glomerulonephritis. NGAL levels clearly correlate with severity of renal impairment, probably expressing the degree of active damage underlying the chronic condition. For all these reasons, NGAL may become one of the most promising next-generation biomarkers in clinical nephrology and beyond.


The atmospheric chemistry of trace gases and particulate matter emitted by different land uses in Borneo.
We report measurements of atmospheric composition over a tropical rainforest and over a nearby oil palm plantation in Sabah, Borneo. The primary vegetation in each of the two landscapes emits very different amounts and kinds of volatile organic compounds (VOCs), resulting in distinctive VOC fingerprints in the atmospheric boundary layer for both landscapes. VOCs over the Borneo rainforest are dominated by isoprene and its oxidation products, with a significant additional contribution from monoterpenes. Rather than consuming the main atmospheric oxidant, OH, these high concentrations of VOCs appear to maintain OH, as has been observed previously over Amazonia. The boundary-layer characteristics and mixing ratios of VOCs observed over the Borneo rainforest are different to those measured previously over Amazonia. Compared with the Bornean rainforest, air over the oil palm plantation contains much more isoprene, monoterpenes are relatively less important, and the flower scent, estragole, is prominent. Concentrations of nitrogen oxides are greater above the agro-industrial oil palm landscape than over the rainforest, and this leads to changes in some secondary pollutant mixing ratios (but not, currently, differences in ozone). Secondary organic aerosol over both landscapes shows a significant contribution from isoprene. Primary biological aerosol dominates the super-micrometre aerosol over the rainforest and is likely to be sensitive to land-use change, since the fungal source of the bioaerosol is closely linked to above-ground biodiversity.


SYNTAX score based on coronary computed tomography angiography may have a prognostic value in patients with complex coronary artery disease: An observational study from a retrospective cohort.
The SYNergy between percutaneous coronary intervention with TAXus and cardiac surgery (SYNTAX) score is an invasive coronary angiography (ICA)-based score for quantifying the complexity of coronary artery disease (CAD). Although the SYNTAX score was originally developed based on ICA, recent publications have reported that coronary computed tomography angiography (CCTA) is a feasible modality for the estimation of the SYNTAX score.The aim of our study was to investigate the prognostic value of the SYNTAX score, based on CCTA for the prediction of major adverse cardiac and cerebrovascular events (MACCEs) in patients with complex CAD.The current study was approved by the institutional review board of our institution, and informed consent was waived for this retrospective cohort study. We included 251 patients (173 men, mean age 66.0 ± 9.29 years) who had complex CAD [3-vessel disease or left main (LM) disease] on CCTA. SYNTAX score was obtained on the basis of CCTA. Follow-up clinical outcome data regarding composite MACCEs were also obtained. Cox proportional hazards models were developed to predict the risk of MACCEs based on clinical variables, treatment, and computed tomography (CT)-SYNTAX scores.During the median follow-up period of 1517 days, there were 48 MACCEs. Univariate Cox hazards models demonstrated that MACCEs were associated with advanced age, low body mass index (BMI), and dyslipidemia (P < .2). In patients with LM disease, MACCEs were associated with a higher SYNTAX score. In patients with CT-SYNTAX score ≥23, patients who underwent coronary artery bypass graft surgery (CABG) and percutaneous coronary intervention had significantly lower hazard ratios than patients who were treated with medication alone. In multivariate Cox hazards model, advanced age, low BMI, and higher SYNTAX score showed an increased hazard ratio for MACCE, while treatment with CABG showed a lower hazard ratio (P < .2).On the basis of our results, CT-SYNTAX score can be a useful method for noninvasively predicting MACCEs in patients with complex CAD, especially in patients with LM disease.


High economic burden of caring for patients with suspected extraesophageal reflux.
Extraesophageal symptoms are common manifestations of gastroesophageal reflux disease (GERD). Lack of a definitive diagnostic or treatment standards complicate management, which often leads to multiple specialty consultations, procedures, pharmaceuticals and diagnostic tests. The aim of this study was to determine the economic burden associated with extraesophageal reflux (EER). Direct costs of evaluation were estimated for patients referred with symptoms attributed to EER between 2007 and 2011. Medicare payment for evaluation and management and pharmaceutical prices was used to calculate first year and overall costs of evaluating and treating extraesophageal symptoms attributed to reflux. Overall, 281 patients were studied (cough (50%), hoarseness (23%), globus/post-nasal drainage (15%), asthma (9%), and sore throat (3%)). Over a median (interquartile range) of 32 (16-46) months follow-up, patients had a mean (95% confidence interval) of 10.1 (9.4-10.9) consultations with specialists and underwent 6.4 (3-9) diagnostic procedures. Overall, the mean initial year direct cost was $5,438 per patient being evaluated for EER. Medical and non-medical components contributed $5,154 and $283. Of the overall cost, 52% were attributable to the use of proton pump inhibitors. During the initial year, direct costs were 5.6 times higher than those reported for typical GERD ($971). A total of 54% of patients reported improvement of symptoms. Overall cost per improved patient was $13,700. EER contributes substantially to health-care expenditures. In this cohort, the cost for initial year's evaluation and treatment of EER symptoms was quintuple that of typical GERD. Prescription costs and, in particular, proton pump inhibitors were the single greatest contributor to the cost of EER management.


Seasonal Preservation Success of the Marine Dinoflagellate Coral Symbiont, Symbiodinium sp.
Coral reefs are some of the most diverse and productive ecosystems on the planet, but are threatened by global and local stressors, mandating the need for incorporating ex situ conservation practices. One approach that is highly protective is the development of genome resource banks that preserve the species and its genetic diversity. A critical component of the reef are the endosymbiotic algae, Symbiodinium sp., living within most coral that transfer energy-rich sugars to their hosts. Although Symbiodinium are maintained alive in culture collections around the world, the cryopreservation of these algae to prevent loss and genetic drift is not well-defined. This study examined the quantum yield physiology and freezing protocols that resulted in survival of Symbiodinium at 24 h post-thawing. Only the ultra-rapid procedure called vitrification resulted in success whereas conventional slow freezing protocols did not. We determined that success also depended on using a thin film of agar with embedded Symbiodinium on Cryotops, a process that yielded a post-thaw viability of >50% in extracted and vitrified Symbiodinium from Fungia scutaria, Pocillopora damicornis and Porites compressa. Additionally, there also was a seasonal influence on vitrification success as the best post-thaw survival of F. scutaria occurred in winter and spring compared to summer and fall (P < 0.05). These findings lay the foundation for developing a viable genome resource bank for the world's Symbiodinium that, in turn, will not only protect this critical element of coral functionality but serve as a resource for understanding the complexities of symbiosis, support selective breeding experiments to develop more thermally resilient strains of coral, and provide a 'gold-standard' genomics collection, allowing for full genomic sequencing of unique Symbiodinium strains.


Targeted Disruption of Lats1 and Lats2 in Mice Impairs Adrenal Cortex Development and Alters Adrenocortical Cell Fate.
It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells.


Prolylcarboxypeptidase (PRCP) as a new target for obesity treatment.
Recently, we serendipitously discovered that mice with the deficiency of the enzyme prolylcarboxypeptidase (PRCP) have elevated alpha-melanocyte-stimulating hormone (alpha-MSH) levels which lead to decreased food intake and weight loss. This suggests that PRCP is an endogenous inactivator of alpha-MSH and an appetite stimulant. Since a modest weight loss can have the most profound influence on reducing cardiovascular risk factors, the inhibitors of PRCP would be emerging as a possible alternative for pharmacotherapy in high-risk patients with obesity and obesity-related disorders. The discovery of a new biological activity of PRCP in the PRCP-deficient mice and studies of alpha-MSH function indicate the importance and complexity of the hypothalamic pro-opiomelanocortin (POMC) system in altering food intake. Identifying a role for PRCP in regulating alpha-MSH in the brain may be a critical step in enhancing our understanding of how the brain controls food intake and body weight. In light of recent findings, the potential role of PRCP in regulating fuel homeostasis is critically evaluated. Further studies of the role of PRCP in obesity are much needed.


E2F2 induces MCM4, CCNE2 and WHSC1 upregulation in ovarian cancer and predicts poor overall survival.
To explore the genes co-upregulated with E2F2 in ovarian cancer and their association with survival outcomes in ovarian cancer patients. The raw data of GDS3592 was downloaded from GEO datasets for reanalysis. The overlapping subset between the top 150 upregulated genes in ovarian cancer epithelial cells (CEPIs) and the E2F2 positively correlated genes (Pearson's r≥0.5) in ovarian cancer cohort in TCGA was identified. The association between E2F2, MCM4, CCNE2 and WHSC1 and overall survival (OS) and recurrence-free survival (RFS) in ovarian cancer patients were assessed using Kaplan-Meier plotter. E2F2 is a significantly upregulated transcription factor in CEPIs. MCM4, CCNE2, and WHSC1 are co-upregulated with E2F2 among the 308 ovarian cancer samples (Pearson's r=0.5159, 0.3963 and 0.4941 respectively). Enforced E2F2 expression significantly enhanced MCM4, CCNE2 and WHSC1 transcription in SKOV3 and A2780 cells. High E2F2 and CCNE2 expression are associated with worse OS (high E2F2, HR: 1.48, 95%CI: 1.17-1.85, p<0.01; high CCNE2, HR: 1.36, 95%CI: 1.15-1.6, p<0.01). High MCM expression might be associated with worse RFS at the margin of significance (HR: 1.18, 95%CI: 1.00-1.39, p=0.055). MCM4, CCNE2, and WHSC1 are co-upregulated with E2F2 in ovarian cancer. Enforced E2F2 expression significantly increased MCM4, CCNE2, and WHSC1 expression in ovarian cancer cells. High E2F2 and CCNE2 expression are associated with worse OS among ovarian cancer patients.


Preclinical, phase I and pharmacokinetic studies with the dimethyl phenyltriazene CB10-277.
Decarbazine is an imidazole dimethyltriazene with reproducible activity in patients with metastatic melanoma. CB10-277 is a phenyl dimethyltriazene which, like dacarbazine, requires metabolic activation to its corresponding monomethyl species for antitumour activity. In preclinical models (human melanoma xenografts and transplantable rodent tumours) CB10-277 showed a similar spectrum and level of activity when compared to dacarbazine. Pharmacokinetic studies were performed with CB10-277 in mice treated i.v. at the LD10 (750 mg m-2) and plasma analysed by HPLC. The parent drug area under the plasma concentration vs time curve (AUC) was 142 mM x minutes. Drug metabolism occurred as evidenced by the HPLC identification of the monomethyl species (AUC = 8 mM x minutes) as well as other metabolites. A Phase I trial using a short infusion with doses repeated every 21 days has been performed. Thirty-six patients received 80 courses over a dose range of 80-6,000 mg m-2. The dose limiting toxicity was nausea and vomiting which occurred in 80% of the evaluable courses > or = 900 mg m-2. The only other common side effect was a flushing or warm sensation, which occurred in over 75% of courses at > or = 1,350 mg m-2. There were no hemodynamic consequences. Responses occurred in patients with melanoma (one complete, two partial, one mixed/11), sarcoma (one mixed/6) and carcinoid (one partial/l). Pharmacokinetics were performed in 46 courses. The CB10-277 AUC increased linearly with dose (r = 0.9203, P < 0.001) up to 700 mM x minutes at 6,000 mg m-2). Evidence of CB10-277 metabolism was observed, as in mice, by detection of the monomethyl species and other metabolites. However, the plasma levels of the monomethyl species in patients (1.8 and 3.7 mM x minutes at 6,000 mg m-2) were less than those predicted from studies in mice. Despite this, antitumour activity in dacarbazine sensitive histologies was observed and additional studies with CB10-277 are recommended.


Proposed role of embedded dentin chips for the clinical failure of nickel-titanium rotary instruments.
Discarded ProFile and ProTaper nickel-titanium rotary instruments, with unknown history of clinical use, were obtained from graduate endodontic clinics at Ohio State University and University of Texas Health Science Center at Houston Dental Branch. These discarded instruments and as-received instruments of both types were examined with a scanning electron microscope to investigate effects of clinical use and causes of failure. For used ProTaper instruments, dentinal debris was wedged mostly in narrow, radial, land-type regions and less on convex flute surfaces. For used ProFile instruments, dentinal debris was wedged mostly in the metal rollover and on concave flute surfaces. Used instruments of both types exhibited widened machining grooves, and elongated and stretched roll-over. Dentin chips were wedged in surface micro-cracks that appeared to propagate from original machining flaws and widen during in vivo root canal preparation. From our observational study, wedged dentinal deposits seem to play a pivotal role for clinical failure of these instruments.


Optimisation of Milk Protein Top-Down Sequencing Using In-Source Collision-Induced Dissociation in the Maxis Quadrupole Time-of-Flight Mass Spectrometer.
Top-down sequencing in proteomics has come of age owing to continuous progress in LC-MS. With their high resolution and broad mass range, Quadrupole Time-of-Flight (Q-ToF) hybrid mass spectrometers equipped with electrospray ionisation source and tandem MS capability by collision-induced dissociation (CID) can be employed to analyse intact proteins and retrieve primary sequence information. To our knowledge, top-down proteomics methods with Q-ToF have only been evaluated using samples of relatively low complexity. Furthermore, the in-source CID (IS-CID) capability of Q-ToF instruments has been under-utilised. This study aimed at optimising top-down sequencing of intact milk proteins to achieve the greatest sequence coverage possible from samples of increasing complexity, assessed using nine known proteins. Eleven MS/MS methods varying in their IS-CID and conventional CID parameters were tested on individual and mixed protein standards as well as raw milk samples. Top-down sequencing results from the nine most abundant proteoforms of caseins, alpha-lactalbumin and beta-lactoglubulins were compared. Nine MS/MS methods achieved more than 70% sequence coverage overall to distinguish between allelic proteoforms, varying only by one or two amino acids. The optimal methods utilised IS-CID at low energy. This experiment demonstrates the utility of Q-ToF systems for top-down proteomics and that IS-CID could be more frequently employed.


Engineering plants for nematode resistance.
Biotechnology offers sustainable solutions to the problem of plant parasitic nematode control. There are several possible approaches for developing transgenic plants with improved nematode resistance; these include anti-invasion and migration strategies, feeding-cell attenuation, and antinematode feeding and development strategies. The essential elements of an effective control strategy are (a) genes that encode an antinematode effector protein, peptide or interfering RNA and (b) promoters that direct a specific pattern of expression for that effector. This review summarizes information on effectors that act directly against the nematode as well as those aimed at disrupting the nematode feeding site. We discuss patterns of promoter activity that could deliver expression of these effectors in a restricted and directed manner. Societal opposition to the technology of GM-nematode control is also discussed.


Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture-Based Isolates.
Hantaan virus (HTNV), identified in the striped field mouse (Apodemus agrarius), belongs to the genus Hantavirus of the family Bunyaviridae and contains tripartite RNA genomes, small (S), medium (M), and large (L) segments. HTNV is a major causative for hemorrhagic fever with renal syndrome (HFRS) with fatality rates ranging from 1% to 15% in the Republic of Korea (ROK) and China. Defining of HTNV whole-genome sequences and isolation of the infectious particle play a critical role in the characterization and preventive and therapeutic strategies of hantavirus outbreaks. Next-generation sequencing (NGS) provides an advanced tool for massive genomic sequencing of viruses. However, the isolation of viral infectious particles is a huge obstacle to investigate and develop anti-virals for hantaviruses. Here, we report 12 HTNV isolates from lung tissues of the striped field mouse in the highly HFRS-endemic areas. Sequence-independent, single-primer amplification (SISPA) NGS was attempted to recover the genomic sequences of HTNV isolates. The nucleotide sequence of HTNV S, M, and L segments were covered up to 99.4-100%, 97.5-100%, and 95.6-99.8%, respectively, based on the full length of the prototype HTNV 76-118. The whole-genome sequencing of HTNV isolates was accomplished by additional reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) PCR. In conclusion, this study will lead to the attempt and usage of SISPA NGS technologies to delineate the whole-genome sequence of hantaviruses, providing a new era of viral genomics for the surveillance, trace, and disease risk management of HFRS incidents.


A data-driven approach for quality assessment of radiologic interpretations.
Given the increasing emphasis on delivering high-quality, cost-efficient healthcare, improved methodologies are needed to measure the accuracy and utility of ordered diagnostic examinations in achieving the appropriate diagnosis. Here, we present a data-driven approach for performing automated quality assessment of radiologic interpretations using other clinical information (e.g., pathology) as a reference standard for individual radiologists, subspecialty sections, imaging modalities, and entire departments. Downstream diagnostic conclusions from the electronic medical record are utilized as "truth" to which upstream diagnoses generated by radiology are compared. The described system automatically extracts and compares patient medical data to characterize concordance between clinical sources. Initial results are presented in the context of breast imaging, matching 18 101 radiologic interpretations with 301 pathology diagnoses and achieving a precision and recall of 84% and 92%, respectively. The presented data-driven method highlights the challenges of integrating multiple data sources and the application of information extraction tools to facilitate healthcare quality improvement.


Building of neomycin-nucleobase-amino acid conjugates for the inhibition of oncogenic miRNAs biogenesis.
MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers, thus being oncogenic. The inhibition of oncogenic miRNAs (defined as the blocking of miRNAs' production or function) would find application in the therapy of different types of cancer in which these miRNAs are implicated. In this work, we describe the design and synthesis of new small-molecule RNA ligands with the aim of inhibiting Dicer-mediated processing of oncogenic miRNAs. One of the synthesized compound (4b) composed of the aminoglycoside neomycin conjugated to an artificial nucleobase and to amino acid histidine is able to selectively decrease miR-372 levels in gastric adenocarcinoma (AGS) cells and to restore the expression of the target LATS2 protein. This activity led to the inhibition of proliferation of these cells. The study of the interactions of 4b with pre-miR-372 allowed for the elucidation of the molecular mechanism of the conjugate, thus leading to new perspectives for the design of future inhibitors.


Cation fluxes cause plasma membrane depolarization involved in beta-glucan elicitor-signaling in soybean roots.
Inducible and specific ion fluxes on plasma membranes represent very early events during elicitation of plant cells. The hierarchy of such ion fluxes involved is still unknown. The effect of Phytophthora sojae-derived beta-glucan elicitors on the plasma membrane potential as well as on surface K+, Ca2+, and H+ fluxes has been investigated on soybean roots using ion-selective microelectrodes. Beta-Glucans with different degrees of polymerization transiently depolarized the plasma membrane. The elicitor concentration necessary for half-maximal depolarization closely resembled the corresponding binding affinities of soybean root membranes toward the respective beta-glucans. Upon repeated elicitor treatment, the root cells responded partially refractory, suggesting a complex responsiveness of the system. Within the root hair space, characteristic decreasing K(+)- and Ca(2+)-free concentrations were induced by the elicitors, probably causing depolarization through the influx of positive charges. Whereas K+ fluxes were inverted after passing the K+ equilibrium (Nernst-) potential, Ca2+ influx continued. No anion fluxes sufficient to account for charge compensation were observed under the same experimental conditions. K+ and Ca2+ fluxes as well as depolarization were inhibited by 100 microM or less of the Ca2+ antagonist La3+. Contrasting other systems, in soybean the main cause for elicitor-induced plasma membrane depolarization is the activation of cation instead of anion fluxes.


Enhancing the monitoring sensitivity of DOP-based OSNR monitors in high OSNR region using off-center narrow-band optical filtering.
Recently, OSNR monitoring based on the measurement of degree of polarization (DOP) has attracted much attention, thanks to its simplicity and high efficiency. However, the OSNR monitoring sensitivity is quite poor in the high OSNR region, resulting in high estimation error and narrow dynamic range. In this paper, we propose and experimentally demonstrate a narrow-band off-center optical filtering technique for DOP-based OSNR monitors. In 40-Gb/s RZ-OOK systems, OSNR monitoring sensitivity and DOP dynamic range are successfully enhanced to 3.14%/dB and 31.4%, respectively, in the high OSNR region (25~35dB/0.1nm).


Clinical presentation and outcomes of patients with biliary malignancies: the Aga Khan University experience.
Gallbladder cancer (GBC) is a common malignancy in our country; however, very limited data exist on this malignancy in Pakistan. This is a retrospective analysis of all the admitted patients diagnosed with GBC or cholangiocarcinoma in between 1st January 1995 to 31st December 2007. A total of 245 patients were admitted with diagnosis of GBC or cholangiocarcinoma. Sixty seven percent were females. Right hypochondrial pain (70.6%) and jaundice (49.8%) were the commonest symptoms, followed by nausea and vomiting (11.8%), weight loss (13%), fever (18.8%), anorexia (9.8%) and ascites (3.3%). Gallstones were seen in 132 (53.9%) patients. Pathological diagnosis was confirmed in 155 (63.2%) patients, adenocarcinoma (94.8%) being the predominant type. Metastasis was seen in 204 (83.3%) patients, with liver and abdominal lymph nodes being the frequent sites of metastasis. Most of the patients presented to the surgeons (42.9%) and gastroenterologists (35.9%) at their first visit. Only 89 (26.3%) patients were referred to medical oncologists and 42 (16.7%) of the patients actually received chemotherapy. The patients who received chemotherapy cisplatin and gemcitabine demonstrated partial responses (40%). Common bile duct stricture was seen in 78 patients and stenting was successful in 73 patients. Fourteen (5.7%) patients are alive to date, one is receiving chemotherapy, and another is alive with advanced disease while 10 patients had incidental diagnosis after surgery. Of all 53.9% of patients have died and 38% are lost to follow up. Most of the patients with biliary cancers present late with advanced disease at our referral tertiary care hospital. Minority of the patients received chemotherapy and most of responses were observed with cisplatin and gemcitabine combination or capecitbine based therapy.


'Well London' and the benefits of participation: results of a qualitative study nested in a cluster randomised trial.
Well London is a multicomponent community engagement and coproduction programme designed to improve the health of Londoners living in socioeconomically deprived neighbourhoods. To evaluate outcomes of the Well London interventions, a cluster randomised trial (CRT) was conducted that included a longitudinal qualitative component, which is reported here. The aim is to explore in depth the nature of the benefits to residents and the processes by which these were achieved. The 1-year longitudinal qualitative study was nested within the CRT. Purposive sampling was used to select three intervention neighbourhoods in London and 61 individuals within these neighbourhoods. The interventions comprised activities focused on: healthy eating, physical exercise and mental health and well-being. Interviews were conducted at the inception and following completion of the Well London interventions to establish both if and how they had participated. Transcripts of the interviews were coded and analysed using Nvivo. Positive benefits relating to the formal outcomes of the CRT were reported, but only among those who participated in project activities. The extent of benefits experienced was influenced by factors relating to the physical and social characteristics of each neighbourhood. The highest levels of change occurred in the presence of: (1) social cohesion, not only pre-existing but also as facilitated by Well London activities; (2) personal and collective agency; (3) involvement and support of external organisations. Where the physical and social environment remained unchanged, there was less participation and fewer benefits. These findings show interaction between participation, well-being and agency, social interactions and cohesion and that this modulated any benefits described. Pathways to change were thus complex and variable, but personal well-being and local social cohesion emerged as important mediators of change.


White tea (Camellia sinensis) inhibits proliferation of the colon cancer cell line, HT-29, activates caspases and protects DNA of normal cells against oxidative damage.
Tea (Camellia sinensis) is one of the most consumed beverages in the world. White tea is made from the buds and young leaves of the tea plant which are steamed and dried, whilst undergoing minimal oxidation. The MTT assay was used to test the extract on the effect of the proliferation of the colorectal cancer cell line, HT-29. The extract inhibited the proliferation of HT-29 cells with an IC50 of 87μg/ml. The extract increased the levels of caspase-3, -8, and -9 activity in the cells. DNA damage in 3T3-L1 normal cells was detected by using the comet assay. The extract protected 3T3-L1 cells against H2O2-induced DNA damage. The results from this study show that white tea has antioxidant and antiproliferative effects against cancer cells, but protect normal cells against DNA damage. Regular intake of white tea can help to maintain good health and protect the body against disease.


Sleep restriction acutely impairs glucose tolerance in rats.
Chronic sleep curtailment in humans has been related to impairment of glucose metabolism. To better understand the underlying mechanisms, the purpose of the present study was to investigate the effect of acute sleep deprivation on glucose tolerance in rats. A group of rats was challenged by 4-h sleep deprivation in the early rest period, leading to prolonged (16 h) wakefulness. Another group of rats was allowed to sleep during the first 4 h of the light period and sleep deprived in the next 4 h. During treatment, food was withdrawn to avoid a postmeal rise in plasma glucose. An intravenous glucose tolerance test (IVGTT) was performed immediately after the sleep deprivation period. Sleep deprivation at both times of the day similarly impaired glucose tolerance and reduced the early-phase insulin responses to a glucose challenge. Basal concentrations of plasma glucose, insulin, and corticosterone remained unchanged after sleep deprivation. Throughout IVGTTs, plasma corticosterone concentrations were not different between the control and sleep-deprived group. Together, these results demonstrate that independent of time of day and sleep pressure, short sleep deprivation during the resting phase favors glucose intolerance in rats by attenuating the first-phase insulin response to a glucose load. In conclusion, this study highlights the acute adverse effects of only a short sleep restriction on glucose homeostasis.


Effect of dependent scattering on the optical properties of Intralipid tissue phantoms.
The calibration of optical tissue-simulating phantoms remains an open question in spite of the many techniques proposed for accurate measurements of optical properties. As a consequence, a reference phantom with well known optical properties is still missing. As a first step towards a reference phantom we have recently proposed to use dilutions of Intralipid 20%. In this paper we discuss a matter that is commonly ignored when dilutions are prepared, i.e., the possibility of deviations from the simple linear relationships between the optical properties of the dilution and the Intralipid concentration due to the effects of dependent scattering. The results of an experimental investigation showed that dependent scattering does not affect absorption. As for the reduced scattering coefficient the effect can be described adding a term proportional to the square of the concentration. However, for concentrations of interest for tissue optics deviations from linearity remain within about 2%. The experimental investigation also showed that the microphysical properties of Intralipid are not affected by dilution. These results show the possibility to easily obtain a liquid diffusive phantom whose optical properties are known with error smaller than about 1%. Due to the intrinsic limitations of the different techniques proposed for measuring the optical properties it seems difficult to obtain a similar accuracy for solid phantoms.


Effect of glycaemic control on complications following cardiac surgery: literature review.
No uniform consensus in the UK or Europe exists, for glycaemic management of patients with Diabetes or pre-diabetes undergoing cardiac surgery. [i] Determine the relationship between glycaemic control and cardiac surgical outcomes; [ii] Compare current vs gold standard management of patients with Diabetes or pre-diabetes undergoing cardiac surgery. Searches of MEDLINE, NHS Evidence and Web of Science databases were completed. Articles were limited to those in English, German and French. No date limit was enforced.13,232 articles were identified on initial literature review, and 50 relevant papers included in this review. No national standards for glycaemic control prior to cardiac surgery were identified. Upto 30% of cardiac surgical patients have undiagnosed Diabetes. Cardiac surgical patients without Diabetes with pre-operative hyperglycaemia have a 1 year mortality double that of patients with normoglyacemia, and equivalent to patients already diagnosed with Diabetes. Pre- and peri-operative hyperglycaemia is associated with worse outcomes. Evidence regarding tight glycaemic control vs moderate glycaemic control is conflicting. Tight control may be more effective in patients without Diabetes with pre-/peri-operative hyperglycaemia, and moderate control appears more effective in patients with pre-existing Diabetes. Patients with well controlled Diabetes may achieve comparable outcomes to patients without Diabetes with similar glycaemic control. Pre / peri-operative hyperglycaemia is associated with worse outcomes in both patients with, and without Diabetes undergoing CABG. This review supports the pre-operative screening, and optimisation of glycaemic control in patients undergoing cardiac surgery. Optimal glycaemic management remains unclear and clear guidelines are needed.


Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein.
Much data has accumulated supporting a proatherogenic role for oxidized low density lipoprotein (Ox-LDL). Micronutrient antioxidants such as alpha-tocopherol, the principal lipid-soluble antioxidant, assume potential significance because levels can be manipulated by dietary measures without resulting in side effects. Co-incubation of LDL in vitro with alpha-tocopherol inhibits its oxidative modification. Hence the effect of dietary supplementation with alpha-tocopherol on the time course of copper-catalyzed oxidation of LDL was tested in a randomized placebo-controlled single-blind study. Two groups of 12 male subjects were given either placebo or alpha-tocopherol (800 IU/day) for a period of 12 weeks. Alpha-tocopherol therapy did not result in any side effects or exert an adverse effect on the plasma lipid and lipoprotein profile. While the lipid standardized alpha-tocopherol levels were similar at baseline, the supplemented group had 3.3-fold and 4.4-fold higher levels compared to placebo at 6 and 12 weeks, respectively. In the 15 subjects in whom both plasma and LDL alpha-tocopherol levels were quantitated, there was a significant correlation (r = 0.79, P less than 0.0001). At baseline there were no significant differences in the time course curves of thiobarbituric acid-reacting substances (TBARS) activity or conjugated diene formation between the alpha-tocopherol and placebo groups. However, at both 6 and 12 weeks the mean levels of TBARS activity and conjugated diene formation were lower in the alpha-tocopherol group; the most significant differences were manifest at the 3-h time point. Also at both 6 and 12 weeks the mean rate of oxidation was lower in the alpha-tocopherol group.2+_


A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster.
The DNA polymerase-primase from Drosophila lacks 3'----5' exonuclease activity. However, a potent exonuclease can be detected after separating the 182-kDa polymerase subunit from the other three subunits of the enzyme (73, 60, and 50 kDa) by glycerol gradient sedimentation in the presence of 50% ethylene glycol. The exonuclease activity cosediments with the polymerase subunit, suggesting that the two activities reside in the same polypeptide. The 3'----5' exonuclease excises mismatched bases at the 3' termini of primed synthetic and natural DNA templates. Excision of a mispaired base at the 3' terminus occurs at a 10-fold greater rate than excision of the correctly paired base. When replication fidelity is measured by the bacteriophage phi X174 am3 reversion assay, the isolated polymerase subunit is at least 100-fold more accurate than either the intact polymerase-primase or a complex of the 182- and 73-kDa subunits. These results suggest that the 3'----5' exonuclease functions as a proofreading enzyme during Drosophila DNA replication in vitro and very likely in vivo.


Investigating the performance of a novel pH and cathepsin B sensitive, stimulus-responsive nanoparticle for optimised sonodynamic therapy in prostate cancer.
Nano-formulations that are responsive to tumour-related and externally-applied stimuli can offer improved, site-specific antitumor effects, and can improve the efficacy of conventional therapeutic agents. Here, we describe the performance of a novel stimulus-responsive nanoparticulate platform for the targeted treatment of prostate cancer using sonodynamic therapy (SDT). The nanoparticles were prepared by self-assembly of poly(L-glutamic acid-L-tyrosine) co-polymer with hematoporphyrin. The nanoparticulate formulation was characterized with respect to particle size, morphology, surface charge and singlet oxygen production during ultrasound exposure. The response of the formulation to the presence of cathepsin B, a proteolytic enzyme that is overexpressed and secreted in the tumour microenvironment of many solid tumours, was assessed. Our results showed that digestion with cathepsin B led to nanoparticle size reduction. In the absence of ultrasound, the formulation exhibited greater toxicity at acidic pH than at physiological pH, using the human prostate cells lines LNCaP and PC3 as targets. Nanoparticle cellular uptake was enhanced at acidic pH - a condition that was also associated with greater cathepsin B production. Nanoparticles exhibited enhanced ultrasound-induced cytotoxicity against both prostate cancer cell lines. Subsequent proof-of-concept in vivo studies demonstrated that, when ectopic human xenograft LNCaP tumours in SCID mice were treated with SDT using the systemically-administered nanoparticulate formulation at a single dose, tumour volumes decreased by up to 64% within 24 h. No adverse effects were observed in the nanoparticle-treated mice and their body weight remained stable. The potential of this novel formulation to deliver safe and effective treatment of prostate cancer is discussed.


Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading.
To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor-mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.


Barriers, facilitators and views about next steps to implementing supports for evidence-informed decision-making in health systems: a qualitative study.
Mobilizing research evidence for daily decision-making is challenging for health system decision-makers. In a previous qualitative paper, we showed the current mix of supports that Canadian health-care organizations have in place and the ones that are perceived to be helpful to facilitate the use of research evidence in health system decision-making. Factors influencing the implementation of such supports remain poorly described in the literature. Identifying the barriers to and facilitators of different interventions is essential for implementation of effective, context-specific, supports for evidence-informed decision-making (EIDM) in health systems. The purpose of this study was to identify (a) barriers and facilitators to implementing supports for EIDM in Canadian health-care organizations, (b) views about emerging development of supports for EIDM, and (c) views about the priorities to bridge the gaps in the current mix of supports that these organizations have in place. This qualitative study was conducted in three types of health-care organizations (regional health authorities, hospitals, and primary care practices) in two Canadian provinces (Ontario and Quebec). Fifty-seven in-depth semi-structured telephone interviews were conducted with senior managers, library managers, and knowledge brokers from health-care organizations that have already undertaken strategic initiatives in knowledge translation. The interviews were taped, transcribed, and then analyzed thematically using NVivo 9 qualitative data analysis software. Limited resources (i.e., money or staff), time constraints, and negative attitudes (or resistance) toward change were the most frequently identified barriers to implementing supports for EIDM. Genuine interest from health system decision-makers, notably their willingness to invest money and resources and to create a knowledge translation culture over time in health-care organizations, was the most frequently identified facilitator to implementing supports for EIDM. The most frequently cited views about emerging development of supports for EIDM were implementing accessible and efficient systems to support the use of research in decision-making (e.g., documentation and reporting tools, communication tools, and decision support tools) and developing and implementing an infrastructure or position where the accountability for encouraging knowledge use lies. The most frequently stated priorities for bridging the gaps in the current mix of supports that these organizations have in place were implementing technical infrastructures to support research use and to ensure access to research evidence and establishing formal or informal ties to researchers and knowledge brokers outside the organization who can assist in EIDM. These results provide insights on the type of practical implementation imperatives involved in supporting EIDM.


SU-E-T-637: 4D-VMAT Vs. Gated VMAT in Lung Cancer SBRT.
To assess plan quality and treatment efficiency of 4D-VMAT and gated-VMAT in the treatment of non-small cell lung cancer using SBRT. Treatment planning software was developed in Matlab to simulate both 4D-VMAT and gated-VMAT on patients with stage I lung cancer and at least 1 cm of tumour motion. Gated-VMAT delivers radiation to the tumour during only a portion of the respiratory cycle and hence requires frequent start and stop motions of the gantry. In the 4D-VMAT algorithm, target and organ motion from the entire respiratory cycle is incorporated during optimization. Gantry moves continuously but delivery of each MLC aperture is synchronized to specific phases of target motion. All 4D-CT scan consisted of 10 phases and were acquired with the patients breathing freely. The SBRT fractionation scheme was 48 Gy in 4 fractions with at least 95% of the PTV receiving 100% of the prescription dose. For gated-VMAT, the PTV was derived from the ITV of the relevant respiratory phases plus a 5mm margin. In the 4D VMAT algorithm, the GTV was defined on a single phase and the PTV created with a 5mm margin. PTVs for the other respiratory phases were determined through 4D-image registration and deformation using a bspline transformation model. For both treatment deliveries, dose was accumulated on the maximum exhale phase and DVHs generated. Findings show gated-VMAT and 4D-VMAT deliveries resulted in maximum doses to most OARs far below SBRT protocol constraints. The 4D-VMAT beam on time is on average 8 min. Gated-VMAT will have similar beam on time but treatment time can more than double after accounting for 25 to 35 beam interruptions per arc. Gated-VMAT and 4D-VMAT were able to produce dosimetrically acceptable lung SBRT plans. The advantage of 4D-VMAT is the greater efficiency in treatment delivery.


Glycine and hyperammonemia: potential target for the treatment of hepatic encephalopathy.
Hepatic encephalopathy (HE) is a neuropsychiatric disorder caused by hepatic dysfunction. Numerous studies dictate that ammonia plays an important role in the pathogenesis of HE, and hyperammonemia can lead to alterations in amino acid homeostasis. Glutamine and glycine are both ammoniagenic amino acids that are increased in liver failure. Modulating the levels of glutamine and glycine has shown to reduce ammonia concentration in hyperammonemia. Ornithine Phenylacetate (OP) has consistently been shown to reduce arterial ammonia levels in liver failure by modulating glutamine levels. In addition to this, OP has also been found to modulate glycine concentration providing an additional ammonia removing effect. Data support that glycine also serves an important role in N-methyl D-aspartate (NMDA) receptor mediated neurotransmission in HE. This potential important role for glycine in the pathogenesis of HE merits further investigations.


Recent advances in the surgical treatment of differentiated thyroid cancer: a comprehensive review.
Differentiated thyroid cancers have become one of the fastest growing malignancies in the world. While surgery has remained the cornerstone of management of these tumors, the surgical approach has seen numerous innovations over the past few decades. The use of video-assistance and robotics has revolutionized thyroid surgery. This paper provides a comprehensive evaluation of the different approaches to thyroid surgery, the utility of prophylactic and therapeutic lymph node dissection, and evidence-based guidelines in the treatment of differentiated thyroid cancers. Minimally invasive video-ssisted thyroidectomy is both safe and effective in the hands of the trained surgeon and, in selected patient populations, has comparative perioperative morbidity and better cosmesis as compared to conventional open thyroidectomy. It is universally accepted that therapeutic central lymph node dissection should be performed when metastatic lymph nodes are identified on physical exam, ultrasound, or intraoperatively. In the absence of overt nodal metastasis, the role of elective prophylactic central lymph node dissection remains a matter of debate and prospective, randomized studies are warranted to evaluate the utility of this procedure.


Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.
Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors. Here, we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multisample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of AA nephropathy. LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of approximately 10×. Analysis at 3 to 9× coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern, whereas 83 cancer driver genes were enriched for recurrent nonsynonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding. LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas. By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures.


Phase separation in the outer membrane of Escherichia coli.
Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.


Exposure to Poverty and Productivity.
We study whether exposure to poverty can induce affective states that decrease productivity. In a controlled laboratory setting, we find that subjects randomly assigned to a treatment, in which they view a video featuring individuals that live in extreme poverty, exhibit lower subsequent productivity compared to subjects assigned to a control treatment. Questionnaire responses, as well as facial recognition software, provide quantitative measures of the affective state evoked by the two treatments. Subjects exposed to images of poverty experience a more negative affective state than those in the control treatment. Further analysis shows that individuals in a more positive emotional state exhibit less of a treatment effect. Also, those who exhibit greater attentiveness upon viewing the poverty video are less productive. The results are consistent with the notion that exposure to poverty can induce a psychological state in individuals that adversely affects productivity.


Fiber-optic coupling based on nonimaging expanded-beam optics.
We have fabricated and experimentally tested low-cost and mass-producible multimode fiber-optic couplers and connectors based on nonimaging beam-expanding optics and Liouville's theorem. Analysis indicates that a pair coupling loss of -0.25 dB can be achieved. Experimentally, we measured insertion losses as low as -0.38 dB. The beam expanders can be mass produced owing to the use of plastic injection-molding fabrication techniques and packaged in standard connector housings. This design is compatible with the fiber geometry and can yield highly stable coupling owing to its high tolerance for misalignments.


Synthesis, Optimization, and Performance Demonstration of Electrospun Carbon Nanofiber-Carbon Nanotube Composite Sorbents for Point-of-Use Water Treatment.
We developed an electrospun carbon nanofiber-carbon nanotube (CNF-CNT) composite with optimal sorption capacity and material strength for point-of-use (POU) water treatment. Synthesis variables including integration of multiwalled carbon nanotubes (CNTs) and macroporosity (via sublimation of phthalic acid), relative humidity (20 and 40%), and stabilization temperature (250 and 280 °C) were used to control nanofiber diameter and surface area (from electron microscopy and BET isotherms, respectively), surface composition (from XPS), and strength (from AFM nanoindentation and tensile strength tests). Composites were then evaluated using kinetic, isotherm, and pH-edge sorption experiments with sulfamethoxazole (log Kow = 0.89) and atrazine (log Kow = 2.61), representative micropollutants chosen for their different polarities. Although CNFs alone were poor sorbents, integration of CNTs and macroporosity achieved uptake comparable to granular activated carbon. Through reactivity comparisons with CNT dispersions, we propose that increasing macroporosity exposes the embedded CNTs, thereby enabling their role as the primary sorbent in nanofiber composites. Because the highest capacity sorbents lacked sufficient strength, our optimal formulation (polyacrylonitrile 8 wt %, CNT 2 wt %, phthalic acid 2.4 wt %; 40% relative humidity; 280 °C stabilization) represents a compromise between strength and performance. This optimized sorbent was tested with a mixture of ten organic micropollutants at environmentally relevant concentrations in a gravity-fed, flow-through filtration system, where removal trends suggest that both hydrophobic and specific binding interactions contribute to micropollutant uptake. Collectively, this work highlights the promise of CNF-CNT filters (e.g., mechanical strength, ability to harness CNT sorption capacity), while also prioritizing areas for future research and development (e.g., improved removal of highly polar micropollutants, sensitivity to interfering cosolutes).


The impact of education, country, race and ethnicity on the self-report of postpartum depression using the Edinburgh Postnatal Depression Scale.
Universal screening for postpartum depression is recommended in many countries. Knowledge of whether the disclosure of depressive symptoms in the postpartum period differs across cultures could improve detection and provide new insights into the pathogenesis. Moreover, it is a necessary step to evaluate the universal use of screening instruments in research and clinical practice. In the current study we sought to assess whether the Edinburgh Postnatal Depression Scale (EPDS), the most widely used screening tool for postpartum depression, measures the same underlying construct across cultural groups in a large international dataset. Ordinal regression and measurement invariance were used to explore the association between culture, operationalized as education, ethnicity/race and continent, and endorsement of depressive symptoms using the EPDS on 8209 new mothers from Europe and the USA. Education, but not ethnicity/race, influenced the reporting of postpartum depression [difference between robust comparative fit indexes (∆*CFI) 0.01), but not between European countries (∆*CFI < 0.01). Investigators and clinicians should be aware of the potential differences in expression of phenotype of postpartum depression that women of different educational backgrounds may manifest. The increasing cultural heterogeneity of societies together with the tendency towards globalization requires a culturally sensitive approach to patients, research and policies, that takes into account, beyond rhetoric, the context of a person's experiences and the context in which the research is conducted.


Warfarin affects acute inflammatory response induced by subcutaneous polyvinyl sponge implantation in rats.
Warfarin (WF) is an anticoagulant which also affects physiological processes other than hemostasis. Our previous investigations showed the effect of WF which gained access to the organism via skin on resting peripheral blood granulocytes. Based on these data, the aim of the present study was to examine whether WF could modulate the inflammatory processes as well. To this aim the effect of WF on the inflammatory response induced by subcutaneous sponge implantation in rats was examined. Warfarin-soaked polyvinyl sponges (WF-sponges) were implanted subcutaneously and cell infiltration into sponges, the levels of nitric oxide (NO) and inflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) production by sponge cells were measured as parameters of inflammation. T cell infiltration and cytokine interferon-γ (IFN-γ), interleukin-17 (IL-17) and interleukin-10 (IL-10) were measured at day 7 post implantation. Warfarin exerted both stimulatory and suppressive effects depending on the parameter examined. Flow cytometry of cells recovered from sponges showed higher numbers of granulocytes (HIS48+ cells) at days 1 and 3 post implantation and CD11b+ cells at day 1 compared to control sponges. Cells from WF-sponges had an increased NO production (Griess reaction) at days 1 and 7. In contrast, lower levels of TNF (measured by ELISA) production by cells recovered from WF-soaked sponges were found in the early (day one) phase of reaction with unchanged levels at other time points. While IL-6 production by cells recovered from WF-soaked sponges was decreased at day 1, it was increased at day 7. Higher T cell numbers were noted in WF sponges at day 7 post implantation, and recovered cells produced more IFN-γ and IL-17, while IL-10 production remained unchanged. Warfarin affects some of the parameters of inflammatory reaction induced by subcutaneous polyvinyl sponge implantation. Differential (both stimulatory as well as inhibitory) effects of WF on inflammatory response to sponge implants might affect the course and/or duration of this reaction.


A transcriptome map of perennial ryegrass (Lolium perenne L.).
Single nucleotide polymorphisms (SNPs) are increasingly becoming the DNA marker system of choice due to their prevalence in the genome and their ability to be used in highly multiplexed genotyping assays. Although needed in high numbers for genome-wide marker profiles and genomics-assisted breeding, a surprisingly low number of validated SNPs are currently available for perennial ryegrass. A perennial ryegrass unigene set representing 9,399 genes was used as a reference for the assembly of 802,156 high quality reads generated by 454 transcriptome sequencing and for in silico SNP discovery. Out of more than 15,433 SNPs in 1,778 unigenes fulfilling highly stringent assembly and detection parameters, a total of 768 SNP markers were selected for GoldenGate genotyping in 184 individuals of the perennial ryegrass mapping population VrnA, a population being previously evaluated for important agronomic traits. A total of 592 (77%) of the SNPs tested were successfully called with a cluster separation above 0.9. Of these, 509 (86%) genic SNP markers segregated in the VrnA mapping population, out of which 495 were assigned to map positions. The genetic linkage map presented here comprises a total of 838 DNA markers (767 gene-derived markers) and spans 750 centi Mogan (cM) with an average marker interval distance of less than 0.9 cM. Moreover, it locates 732 expressed genes involved in a broad range of molecular functions of different biological processes in the perennial ryegrass genome. Here, we present an efficient approach of using next generation sequencing (NGS) data for SNP discovery, and the successful design of a 768-plex Illumina GoldenGate genotyping assay in a complex genome. The ryegrass SNPs along with the corresponding transcribed sequences represent a milestone in the establishment of genetic and genomics resources available for this species and constitute a further step towards molecular breeding strategies. Moreover, the high density genetic linkage map predominantly based on gene-associated DNA markers provides an important tool for the assignment of candidate genes to quantitative trait loci (QTL), functional genomics and the integration of genetic and physical maps in perennial ryegrass, one of the most important temperate grassland species.


A Study of Zoledronic Acid as Neo-Adjuvant, Perioperative Therapy in Patients with Resectable Pancreatic Ductal Adenocarcinoma.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by abundant granulocytic myeloid-derived suppressor cells (G-MDSC = CD45+/Lin-/CD33+/CD11b+/CD15+), which infiltrate tumors and suppress anti-tumor immunity. We have previously demonstrated in a murine model of PDAC that zoledronic acid (ZA) depletes G-MDSC resulting in decreased tumor growth and improved survival. We report here the results of a phase 1 clinical trial (NCT00892242) using ZA as neo-adjuvant, perioperative therapy in patients with non-metastatic, resectable pancreatic adenocarcinoma. Eligible PDAC patients received ZA (4mg) IV 2 weeks prior to surgery. Patients then received 2 additional doses of ZA 4 weeks apart. Blood and bone marrow were obtained from patients prior to treatment with ZA and 3 months after surgery for analysis of G-MDSC by flow cytometry. Twenty-three patients received pre-operative ZA with at least 6 months of follow-up Only 15 PDAC patients had non-metastatic PDAC, which was amenable to resection. ZA was well tolerated, and all adverse events were grade 1 or 2. The most common adverse events were fatigue, abdominal pain/discomfort, anorexia, and arthralgia. Of resected PDAC patients treated with ZA, 1- and 2-year overall survival (OS) was 85.7% and 33.3%, respectively, with a median OS of 18 months. This group had a 1- and 2-year progression-free survival (PFS) of 26.9% and 8.9%, respectively, with a median PFS of 12 months. The prevalence of G-MDSC was unchanged in the blood and bone marrow of PDAC patients pre- and post-treatment with ZA. ZA is safe and well tolerated as neo-adjuvant, peri-operative therapy in PDAC patients. In this small study, we did not observe a difference in OS or PFS compared to historical controls. Also, there was no difference in the prevalence of G-MDSC in the blood and bone marrow of PDAC patients pre- and post-treatment with ZA.