Interferon alpha inhibits antigen-specific production of proinflammatory cytokines and enhances antigen-specific transforming growth factor beta production in antigen-induced arthritis.
Interferon alpha (IFN-α) has a complex role in autoimmunity, in that it may both enhance and prevent inflammation. We have previously shown that the presence of IFN-α at sensitization protects against subsequent antigen-triggered arthritis. To understand this tolerogenic mechanism, we performed a descriptive, hypothesis-generating study of cellular and humoral responses associated with IFN-α-mediated protection against arthritis. Arthritis was evaluated at day 28 in mice given a subcutaneous injection of methylated bovine serum albumin (mBSA), together with Freund adjuvant and 0 to 5,000 U IFN-α at days 1 and 7, followed by intraarticular injection of mBSA alone at day 21. The effect of IFN-α on mBSA-specific IgG1, IgG2a, IgG2b, IgA, and IgE was evaluated by enzyme-linked immunosorbent assay (ELISA). Cytokines in circulation and in ex vivo cultures on mBSA restimulation was evaluated with ELISA and Luminex, and the identity of cytokine-producing cells by fluorescence-activated cell sorting (FACS) analysis. Administration of IFN-α protected mice from arthritis in a dose-dependent manner but had no effect on antigen-specific antibody levels. However, IFN-α did inhibit the initial increase of IL-6, IL-10, IL-12, and TNF, and the recall response induced by intraarticular mBSA challenge of IL-1β, IL-10, IL-12, TNF, IFN-γ, and IL-17 in serum. IFN-α decreased both macrophage and CD4+ T cell-derived IFN-γ production, whereas IL-17 was decreased only in CD4+ T cells. Ex vivo, in mBSA-restimulated spleen and lymph node cell cultures, the inhibitory effect of in vivo administration of IFN-α on proinflammatory cytokine production was clearly apparent, but had a time limit. An earlier macrophage-derived, and stronger activation of the antiinflammatory cytokine transforming growth factor beta (TGF-β) was observed in IFN-α-treated animals, combined with an increase in CD4+ T cells producing TGF-β when arthritis was triggered by mBSA (day 21). Presence of IFN-α at immunizations also prevented the reduction in TGF-β production, which was induced by the intraarticular mBSA injection triggering arthritis in control animals. Administration of IFN-α has a profound effect on the cellular response to mBSA plus adjuvant, but does not affect antigen-specific Ig production. By including IFN-α at immunizations, spleen and lymph node cells inhibit their repertoire of antigen-induced proinflammatory cytokines while enhancing antiinflammatory TGF-β production, first in macrophages, and later also in CD4+ T cells. On intraarticular antigen challenge, this antiinflammatory state is reenforced, manifested as inhibition of proinflammatory recall responses and preservation of TGF-β levels. This may explain why IFN-α protects against antigen-induced arthritis.


Filtering of neural signals by focused attention in the monkey prefrontal cortex.
Prefrontal cortex is thought to be important in attention and awareness. Here we recorded the activity of prefrontal neurons in monkeys carrying out a focused attention task. Having directed attention to one location, monkeys monitored a stream of visual objects, awaiting a predefined target. Although neurons rarely discriminated between one non-target and another, they commonly discriminated between targets and non-targets. From the onset of the visual response, this target/non-target discrimination was effectively eliminated when the same objects appeared at an unattended location in the opposite visual hemifield. The results show that, in prefrontal cortex, filtering of ignored locations is strong, early and spatially global. Such filtering may be important in blindness to unattended signals--a conspicuous aspect of human selective attention.


Role of musculoskeletal disorders as concurrent chronic conditions: are they underestimated in the discharge?
Differences in recording concurrent chronic conditions (CCs) could change the weight of the influence of the different CCs on health status. To determine the role of musculoskeletal disorders as concurrent CCs. Cross-sectional study. Discharged patients with CCs were selected by random stratification. Sociodemographic and clinical variables, health status (SF-36), type and number of self-reported and hospital discharge summary CCs were recorded. Relationships were analyzed using linear regression models. In the 227 patients included, mean self-reported CCs were 6.22 (SD 3.37) and mean hospital discharge CCs were 3.1 (SD 1.95). The most-frequent self-reported CCs were hypertension 48.4%, back pain 40.4%, neck pain 39.6%, and musculoskeletal disorders 38.4%. The most frequent discharge CCs were arterial hypertensive disease 38.3%, all cancers 22.0%, ischemic heart diseases, and angina 15.9%. Musculoskeletal disorders had the greatest negative influence on SF-36 dimensions (p < 0.05). Musculoskeletal disorders were underestimated in the discharge summary in comparison with those reported by patients.


Circadian oscillations of molecular clock components in the cerebellar cortex of the rat.
The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum.


Cumulative live birth rates in low-prognosis women.
Do cumulative live birth rates (CLBRs) over multiple IVF/ICSI cycles confirm the low prognosis in women stratified according to the POSEIDON criteria? The CLBR of low-prognosis women is ~56% over 18 months of IVF/ICSI treatment and varies between the POSEIDON groups, which is primarily attributable to the impact of female age. The POSEIDON group recently proposed a new stratification for low-prognosis women in IVF/ICSI treatment, with the aim to define more homogenous populations for clinical trials and stimulate a patient-tailored therapeutic approach. These new criteria combine qualitative and quantitative parameters to create four groups of low-prognosis women with supposedly similar biologic characteristics. This study analyzed the data of a Dutch multicenter observational cohort study including 551 low-prognosis women, aged <44 years, who initiated IVF/ICSI treatment between 2011 and 2014 and were treated with a fixed FSH dose of 150 IU/day in the first treatment cycle. Low-prognosis women were categorized into one of the POSEIDON groups based on their age (younger or older than 35 years), anti-Müllerian hormone (AMH) level (above or below 0.96 ng/ml), and the ovarian response (poor or suboptimal) in their first cycle of standard stimulation. The primary outcome was the CLBR over multiple complete IVF/ICSI cycles, including all subsequent fresh and frozen-thawed embryo transfers, within 18 months of treatment. Cumulative incidence curves were obtained using an optimistic and a conservative analytic approach. The CLBR of the low-prognosis women was on average ~56% over 18 months of IVF/ICSI treatment. Younger unexpected poor (n = 38) and suboptimal (n = 179) responders had a CLBR of ~65% and ~68%, respectively, and younger expected poor responders (n = 65) had a CLBR of ~59%. The CLBR of older unexpected poor (n = 41) and suboptimal responders (n = 102) was ~42% and ~54%, respectively, and of older expected poor responders (n = 126) ~39%. For comparison, the CLBR of younger (n = 164) and older (n = 78) normal responders with an adequate ovarian reserve was ~72% and ~58% over 18 months of treatment, respectively. No large differences were observed in the number of fresh treatment cycles between the POSEIDON groups, with an average of two fresh cycles per woman within 18 months of follow-up. Small numbers in some (sub)groups reduced the precision of the estimates. However, our findings provide the first relevant indication of the CLBR of low-prognosis women in the POSEIDON groups. Small FSH dose adjustments between cycles were allowed, inducing therapeutic disparity. Yet, this is in accordance with current daily practice and increases the generalizability of our findings. The CLBRs vary between the POSEIDON groups. This heterogeneity is primarily determined by a woman's age, reflecting the importance of oocyte quality. In younger women, current IVF/ICSI treatment reaches relatively high CLBR over multiple complete cycles, despite reduced quantitative parameters. In older women, the CLBR remains relatively low over multiple complete cycles, due to the co-occurring decline in quantitative and qualitative parameters. As no effective interventions exist to counteract this decline, clinical management currently relies on proper counselling. No external funds were obtained for this study. J.A.L. is supported by a Research Fellowship grant and received an unrestricted personal grant from Merck BV. S.C.O., T.C.v.T., and H.L.T. received an unrestricted personal grant from Merck BV. C.B.L. received research grants from Merck, Ferring, and Guerbet. K.F. received unrestricted research grants from Merck Serono, Ferring, and GoodLife. She also received fees for lectures and consultancy from Ferring and GoodLife. A.H. declares that the Department of Obstetrics and Gynaecology, University Medical Centre Groningen received an unrestricted research grant from Ferring Pharmaceuticals BV, the Netherlands. J.S.E.L. has received unrestricted research grants from Ferring, Zon-MW, and The Dutch Heart Association. He also received travel grants and consultancy fees from Danone, Euroscreen, Ferring, AnshLabs, and Titus Healthcare. B.W.J.M. is supported by an National Health and Medical Research Council Practitioner Fellowship (GNT1082548) and reports consultancy work for ObsEva, Merck, and Guerbet. He also received a research grant from Merck BV and travel support from Guerbet. F.J.M.B. received monetary compensation as a member of the external advisory board for Merck Serono (the Netherlands) and Ferring Pharmaceuticals BV (the Netherlands) for advisory work for Gedeon Richter (Belgium) and Roche Diagnostics on automated AMH assay development, and for a research cooperation with Ansh Labs (USA). All other authors have nothing to declare. Not applicable.


Myopathy With SQSTM1 and TIA1 Variants: Clinical and Pathological Features.
The aim of this study is to identify the molecular defect of three unrelated individuals with late-onset predominant distal myopathy; to describe the spectrum of phenotype resulting from the contributing role of two variants in genes located on two different chromosomes; and to highlight the underappreciated complex forms of genetic myopathies. Clinical and laboratory data of three unrelated probands with predominantly distal weakness manifesting in the sixth-seventh decade of life, and available affected and unaffected family members were reviewed. Next-generation sequencing panel, whole exome sequencing, and targeted analyses of family members were performed to elucidate the genetic etiology of the myopathy. Genetic analyses detected two contributing variants located on different chromosomes in three unrelated probands: a heterozygous pathogenic mutation in SQSTM1 (c.1175C>T, p.Pro392Leu) and a heterozygous variant in TIA1 (c.1070A>G, p.Asn357Ser). The affected fraternal twin of one proband also carries both variants, while the unaffected family members harbor one or none. Two unrelated probands (family 1, II.3, and family 3, II.1) have a distal myopathy with rimmed vacuoles that manifested with index extensor weakness; the other proband (family 2, I.1) has myofibrillar myopathy manifesting with hypercapnic respiratory insufficiency and distal weakness. The findings indicate that all the affected individuals have a myopathy associated with both variants in SQSTM1 and TIA1, respectively, suggesting that the two variants determine the phenotype and likely functionally interact. We speculate that the TIA1 variant is a modifier of the SQSTM1 mutation. We identify the combination of SQSTM1 and TIA1 variants as a novel genetic defect associated with myofibrillar myopathy and suggest to consider sequencing both genes in the molecular investigation of myopathy with rimmed vacuoles and myofibrillar myopathy although additional studies are needed to investigate the digenic nature of the disease.


The role of NK cells in the autoimmune thyroid disease-associated pregnancy loss.
Pregnancy loss is a frequent event. Autoimmune thyroid disorders and altered natural killer (NK) cell functions are two distinct risk factors, which independently could induce adverse pregnancy outcome. Thyroid autoimmunity has been an object of increased attention by investigators in the context of pregnancy loss. Peripheral NK cells and uNK cells comprise distinct cell populations in terms of phenotype and function but they play an important role in the course of a normal human pregnancy via several potential functions. In autoimmune thyroid diseases, several abnormalities of killer cell activity have been described. The functional defects involving NK maturation and/or functional activation observed in Graves' disease patients could independently influence the reproductive outcome. This suggestion needs extensive investigation and could be important for the therapeutical approach in preventing pregnancy loss in patients with thyroid autoimmunity.


Brain damage after open heart surgery in patients with acute cardioembolic stroke.
We evaluated 14 patients with acute cardiogenic embolism who underwent open heart surgery soon after the onset to determine the cerebral and cardiac factors that influence neurologic outcome. The mean interval from onset of cerebral embolism to surgery was 5.3 (range 1-16) days. Five of the 14 patients had vegetations from infective endocarditis (including prosthetic valve endocarditis) as embolic sources, eight had intracardiac thrombi, and one had atrial myxoma. The diagnosed site of infarction before surgery was based on computed tomographic and/or angiographic findings. Of the 14 patients, four had infarcts due to major artery occlusion, seven due to cortical branch occlusion, and two due to perforating artery occlusion; one patient presented with a transient ischemic attack without computed tomographic abnormalities. Ten patients (71%) showed no clinical aggravation after open heart surgery; however, two patients died of massive cerebral hemorrhage, one died of deterioration of brain edema, and another became comatose from midbrain hemorrhage immediately after surgery. The four patients with clinical aggravation comprised three with septic embolism and one with aseptic occlusion of a major artery. From these results, infective endocarditis and a large infarct appear to be possible aggravating factors when patients with recent cerebral embolism undergo open heart surgery.


Distribution of Chlamydia trachomatis genotypes in neonatal conjunctivitis in Hungary.
The objective of the present study was to determine the frequency and age distribution of different Chlamydia trachomatis (CT) genotypes causing ophthalmia neonatorum (ON) in Hungary. Using CT specific PCR, we tested 76 conjunctival samples from symptomatic infants up to 3 months old in the National Centre for Epidemiology, Budapest between 2008 and 2016. CT tested positive in 30 of 76 conjunctival samples (39.5 %). The sequencing of the positive samples was successful in every case but one, and resulted in 48 % dominance for genotype E (14/29), followed by 24 % for genotype G (7/29), 10 % for J (3/29), 6.9 % for K and F (2/29), and 3.4 % for H (1/29). CT must still be regarded as a common pathogen causing ON in Hungary. Routine screening and treatment of pregnant women can be recommended to prevent these conditions. Chronic ON cases can be reduced by early diagnosis. Further research is needed to explain the dominance of genotypes E and G.


Virtual staining of colon cancer tissue by label-free Raman micro-spectroscopy.
The great capability of the label-free classification of tissue via vibrational spectroscopy, like Raman or infrared imaging, is shown in numerous publications (review: Diem et al., J. Biophotonics, 2013, 6, 855-886). Herein, we present a new approach, virtual staining, that improves the Raman spectral histopathology (SHP) images of colorectal cancer tissue by combining the integrated Raman intensity image in the C-H stretching region (2800-3050 cm-1) with the pseudo-colour Raman image. This allows the display of fine structures such as the filamentous composition of muscle tissue. The morphology of the virtually stained images is in agreement with the gold standard in medical diagnosis, the haematoxylin-eosin staining. The virtual staining image also represents the whole biochemical fingerprint, and several tissue components including carcinoma were identified automatically with high sensitivity and specificity. For fast tissue classifications, a similar approach was applied on coherent anti-Stokes Raman scattering (CARS) spectral data that is faster and therefore potentially more suitable for clinical applications.


Early cave art and ancient DNA record the origin of European bison.
The two living species of bison (European and American) are among the few terrestrial megafauna to have survived the late Pleistocene extinctions. Despite the extensive bovid fossil record in Eurasia, the evolutionary history of the European bison (or wisent, Bison bonasus) before the Holocene (<11.7 thousand years ago (kya)) remains a mystery. We use complete ancient mitochondrial genomes and genome-wide nuclear DNA surveys to reveal that the wisent is the product of hybridization between the extinct steppe bison (Bison priscus) and ancestors of modern cattle (aurochs, Bos primigenius) before 120 kya, and contains up to 10% aurochs genomic ancestry. Although undetected within the fossil record, ancestors of the wisent have alternated ecological dominance with steppe bison in association with major environmental shifts since at least 55 kya. Early cave artists recorded distinct morphological forms consistent with these replacement events, around the Last Glacial Maximum (LGM, ∼21-18 kya).


Orthogonal Genetic Systems.
Xenobiology is an emerging area of synthetic biology that aims to safeguard genetically engineered cells by storing synthetic biology information in xeno-nucleic acid polymers (XNAs). Critical to the success of this effort is the need to establish cellular systems that can maintain an XNA chromosome in actively dividing cells. This viewpoint discusses the structural parameters of the nucleic acid backbone that should be considered when designing an orthogonal genetic system that can replicate without interference from the endogenous genome. In addition to practical value, these studies have the potential to provide new fundamental insight into the structure and function properties of unnatural nucleic acid polymers.


Pharmacological agents to reduce readmissions in bipolar disorder.
It is well recognized that medications have an important role to play in preventing relapse in bipolar disorder. The impact these treatments have on rates of admission to hospital in particular has been less well studied. We combined data on hospitalization from 11 randomized controlled trials in a network meta-analysis. We found that the published evidence demonstrates significant reductions in admission rates compared to placebo from lithium (risk ratio (RR) 0.44, 95% confidence interval (CI) 0.32-0.59), valproate (RR 0.50, 95% CI 0.28-0.90), a combination of lithium and valproate (RR 0.50, 95% CI 0.28-0.90), carbamazepine (RR 0.46, 95% CI 0.29-0.73) and olanzapine (RR 0.27, 95% CI 0.16-0.43). The evidence base contributing to these estimates remains fairly small, leading to broad confidence intervals for estimates of effect. More precise estimates could be obtained if unpublished outcomes data from other trials in this area became available. Several pharmacological treatments appear to be effective at reducing the need for hospital admission in people with bipolar disorder.


An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates.
Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.


Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling.
MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFalpha (tumour necrosis factor alpha). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the 'MLK3 protease', reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.


Phylogenetic analysis of the 56 kDa protein genes of Orientia tsutsugamushi in Southwest Area of Korea.
This study was conducted to determine which genotypes were present in southwestern Korea. Nested polymerase chain reaction (PCR) and DNA sequence analysis targeting the Orientia tsutsugamushi-specific 56-kDa protein gene was performed with samples of blood and eschar. Of the 69 PCR-positive samples, 61 clustered with the Boryong previously isolated in Korea. CUH 4-6 had sequence homology of 100% with Kato, and CUH 4-3 had homology of 99.8% with Kato and formed the Kato cluster. CUH 4-57, CUH 4-31, CUH 4-142, and CUH 4-324 formed a Kawasaki cluster. CUH 4-271 had sequence homology of 100% with Jecheon and formed a Karp cluster. CUH 4-117 had homology of 99.8% with Neimeng-65, and Gilliam cluster. The most common genotype of O. tsutsugamushi in the southwestern part of Korea is the Boryong genotype. We also identified O. tsutsugamushi of the Kato, Neimeng-65 and Kawasaki genotypes, which had not been identified before in Korea.


Oral Challenge with Wild-Type Salmonella Typhi Induces Distinct Changes in B Cell Subsets in Individuals Who Develop Typhoid Disease.
A novel human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently established by the Oxford Vaccine Group. In this model, 104 CFU of Salmonella resulted in 65% of participants developing typhoid fever (referred here as typhoid diagnosis -TD-) 6-9 days post-challenge. TD was diagnosed in participants meeting clinical (oral temperature ≥38°C for ≥12h) and/or microbiological (S. Typhi bacteremia) endpoints. Changes in B cell subpopulations following S. Typhi challenge remain undefined. To address this issue, a subset of volunteers (6 TD and 4 who did not develop TD -NoTD-) was evaluated. Notable changes included reduction in the frequency of B cells (cells/ml) of TD volunteers during disease days and increase in plasmablasts (PB) during the recovery phase (>day 14). Additionally, a portion of PB of TD volunteers showed a significant increase in activation (CD40, CD21) and gut homing (integrin α4β7) molecules. Furthermore, all BM subsets of TD volunteers showed changes induced by S. Typhi infections such as a decrease in CD21 in switched memory (Sm) CD27+ and Sm CD27- cells as well as upregulation of CD40 in unswitched memory (Um) and Naïve cells. Furthermore, changes in the signaling profile of some BM subsets were identified after S. Typhi-LPS stimulation around time of disease. Notably, naïve cells of TD (compared to NoTD) volunteers showed a higher percentage of cells phosphorylating Akt suggesting enhanced survival of these cells. Interestingly, most these changes were temporally associated with disease onset. This is the first study to describe differences in B cell subsets directly related to clinical outcome following oral challenge with wild-type S. Typhi in humans.


Personal tumor antigens in blood malignancies: genomics-directed identification and targeting.
Hematological malignancies have long been at the forefront of the development of novel immune-based treatment strategies. The earliest successful efforts originated from the extensive body of work in the field of allogeneic hematopoietic stem cell transplantation. These efforts laid the foundation for the recent exciting era of cancer immunotherapy, which includes immune checkpoint blockade, personal neoantigen vaccines, and adoptive T cell transfer. At the heart of the specificity of these novel strategies is the recognition of target antigens presented by malignant cells to T cells. Here, we review the advances in systematic identification of minor histocompatibility antigens and neoantigens arising from personal somatic alterations or recurrent driver mutations. These exciting efforts pave the path for the implementation of personalized combinatorial cancer therapy.


Administrative Coding in Electronic Health Care Record-Based Research of NAFLD: An Expert Panel Consensus Statement.
Electronic health record (EHR)-based research allows the capture of large amounts of data, which is necessary in NAFLD, where the risk of clinical liver outcomes is generally low. The lack of consensus on which International Classification of Diseases (ICD) codes should be used as exposures and outcomes limits comparability and generalizability of results across studies. We aimed to establish consensus among a panel of experts on ICD codes that could become the reference standard and provide guidance around common methodological issues. Researchers with an interest in EHR-based NAFLD research were invited to collectively define which administrative codes are most appropriate for documenting exposures and outcomes. We used a modified Delphi approach to reach consensus on several commonly encountered methodological challenges in the field. After two rounds of revision, a high level of agreement (>67%) was reached on all items considered. Full consensus was achieved on a comprehensive list of administrative codes to be considered for inclusion and exclusion criteria in defining exposures and outcomes in EHR-based NAFLD research. We also provide suggestions on how to approach commonly encountered methodological issues and identify areas for future research. This expert panel consensus statement can help harmonize and improve generalizability of EHR-based NAFLD research.


Molecular detection of Toxoplasma gondii in natural surface water bodies in Poland.
The aim of this study was molecular detection of Toxoplasma gondii in 36 natural surface water bodies in Poland, including preliminary genotype identification and determination of co-occurrence of this parasite with other protozoa that have been detected in previous studies. The examined DNA samples were obtained before to detect Cryptosporidium, Giardia and free-living amoebae. Nested polymerase chain reaction (PCR) based on B1 gene and sequencing was performed for both confirmation of parasite presence in water and genotype identification. T. gondii DNA was detected in 19.4% (7/36) water bodies, while in the case of other studies, T. gondii prevalence ranged between 0% and over 56%. These differences may be caused by natural variations in T. gondii occurrence as well as different sample volumes and methods of sample processing or DNA isolation and detection. Two cases of double contamination were reported: T. gondii with Cryptosporidium parvum and T. gondii with potentially pathogenic Acanthamoeba T4 genotype, thus there is a possibility of mixed infection in humans after occasional contact with water. Obtained T. gondii strains were genetically identical or closely similar (99.8%) to RH strain representing genotype I, however, further examinations involving more loci will be conducted to identify the genotype.


A Double-Blinded, Randomized Comparison of Medetomidine-Tiletamine-Zolazepam and Dexmedetomidine-Tiletamine-Zolazepam Anesthesia in Free-Ranging Brown Bears (Ursus Arctos).
We compared anesthetic features, blood parameters, and physiological responses to either medetomidine-tiletamine-zolazepam or dexmedetomidine-tiletamine-zolazepam using a double-blinded, randomized experimental design during 40 anesthetic events of free-ranging brown bears (Ursus arctos) either captured by helicopter in Sweden or by culvert trap in Canada. Induction was smooth and predictable with both anesthetic protocols. Induction time, the need for supplemental drugs to sustain anesthesia, and capture-related stress were analyzed using generalized linear models, but anesthetic protocol did not differentially affect these variables. Arterial blood gases and acid-base status, and physiological responses were examined using linear mixed models. We documented acidemia (pH of arterial blood < 7.35), hypoxemia (partial pressure of arterial oxygen < 80 mmHg), and hypercapnia (partial pressure of arterial carbon dioxide ≥ 45 mmHg) with both protocols. Arterial pH and oxygen partial pressure were similar between groups with the latter improving markedly after oxygen supplementation (p < 0.001). We documented dose-dependent effects of both anesthetic protocols on induction time and arterial oxygen partial pressure. The partial pressure of arterial carbon dioxide increased as respiratory rate increased with medetomidine-tiletamine-zolazepam, but not with dexmedetomidine-tiletamine-zolazepam, demonstrating a differential drug effect. Differences in heart rate, respiratory rate, and rectal temperature among bears could not be attributed to the anesthetic protocol. Heart rate increased with increasing rectal temperature (p < 0.001) and ordinal day of capture (p = 0.002). Respiratory rate was significantly higher in bears captured by helicopter in Sweden than in bears captured by culvert trap in Canada (p < 0.001). Rectal temperature significantly decreased over time (p ≤ 0.05). Overall, we did not find any benefit of using dexmedetomidine-tiletamine-zolazepam instead of medetomidine-tiletamine-zolazepam in the anesthesia of brown bears. Both drug combinations appeared to be safe and reliable for the anesthesia of free-ranging brown bears captured by helicopter or by culvert trap.


Solving dielectric and plasmonic waveguide dispersion relations on a pocket calculator.
We present a robust iterative technique for solving complex transcendental dispersion equations routinely encountered in integrated optics. Our method especially befits the multilayer dielectric and plasmonic waveguides forming the basis structures for a host of contemporary nanophotonic devices. The solution algorithm ports seamlessly from the real to the complex domain--i.e., no extra complexity results when dealing with leaky structures or those with material/metal loss. Unlike several existing numerical approaches, our algorithm exhibits markedly-reduced sensitivity to the initial guess and allows for straightforward implementation on a pocket calculator.


Correction: Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi.
The version of this article published in BMC Genomics 2013, 14: 274, contains 9 unpublished genomes (Botryobasidium botryosum, Gymnopus luxurians, Hypholoma sublateritium, Jaapia argillacea, Hebeloma cylindrosporum, Conidiobolus coronatus, Laccaria amethystina, Paxillus involutus, and P. rubicundulus) downloaded from JGI website. In this correction, we removed these genomes after discussion with editors and data producers whom we should have contacted before downloading these genomes. Removing these data did not alter the principle results and conclusions of our original work. The relevant Figures 1, 2, 3, 4 and 6; and Table 1 have been revised. Additional files 1, 3, 4, and 5 were also revised. We would like to apologize for any confusion or inconvenience this may have caused. Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 94 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also revealed a complex history of lineage-specific expansions and attritions for the PL1 family. Our study provides insights into the variety and expansion of fungal CAZyme classes and revealed the relationship of CAZyme size and diversity with their nutritional strategy and host specificity.


Characterization of Smoc-1 uncovers two transcript variants showing differential tissue and age specific expression in Bubalus bubalis.
Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCACCTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual increment in expression of variant-02. Immunohistochemically, Smoc-1 was localized in the basement membrane zones and extracellular matrices of various tissues. These data added to our understandings about the tissue, age and species specific functions of the Smoc-1. It also enabled us to demonstrate varying expression of the two transcript variants of Smoc-1 amongst different somatic tissues/gonads and ages, in spite of their identical coding frames. Pursuance of these variants for their roles in various disease phenotypes such as hepatocellular carcinoma and angiogenesis is envisaged to establish broader biological significance of this gene.


Genetic by environment interactions affect plant-soil linkages.
The role of plant intraspecific variation in plant-soil linkages is poorly understood, especially in the context of natural environmental variation, but has important implications in evolutionary ecology. We utilized three 18- to 21-year-old common gardens across an elevational gradient, planted with replicates of five Populus angustifolia genotypes each, to address the hypothesis that tree genotype (G), environment (E), and G × E interactions would affect soil carbon and nitrogen dynamics beneath individual trees. We found that soil nitrogen and carbon varied by over 50% and 62%, respectively, across all common garden environments. We found that plant leaf litter (but not root) traits vary by genotype and environment while soil nutrient pools demonstrated genotype, environment, and sometimes G × E interactions, while process rates (net N mineralization and net nitrification) demonstrated G × E interactions. Plasticity in tree growth and litter chemistry was significantly related to the variation in soil nutrient pools and processes across environments, reflecting tight plant-soil linkages. These data overall suggest that plant genetic variation can have differential affects on carbon storage and nitrogen cycling, with implications for understanding the role of genetic variation in plant-soil feedback as well as management plans for conservation and restoration of forest habitats with a changing climate.


Intravenous alteplase for stroke in those older than 80 years old.
Risks and benefits of intravenous thrombolysis for patients with stroke > 80 years of age are unclear. We examined outcomes and symptomatic intracerebral hemorrhage rates in ≤ 80- and > 80-year-old patients in the Safe Implementation of Treatment in Stroke International Stroke Thrombolysis Register. We compared mortality and independence (modified Rankin Scale 0 to 2) at 3 months and symptomatic intracerebral hemorrhage (per Safe Implementation of Treatment in Stroke ≥ 4-point deterioration in National Institutes of Health Stroke Scale within 36 hours and Type 2 parenchymal hemorrhage and per National Institute of Neurological Disorders and Stroke [any increase in National Institutes of Health Stroke Scale and any hemorrhage]) of 1831 patients > 80 years of age with 19 411 patients ≤ 80 years of age compliant with other European licensing criteria. The > 80-year-old group (median, 83 years) had more severe strokes (median National Institutes of Health Stroke Scale 14 versus 12), lower levels of prestroke independence (modified Rankin Scale 0 to 1, 82% versus 93%), and a larger proportion of females (59% versus 39%) compared with the younger group (68 years). Symptomatic intracerebral hemorrhage was not significantly increased after adjustment for other risk factors in those >80 years of age compared with those ≤ 80 years of age (per Safe Implementation of Treatment in Stroke 1.8% versus 1.7%, P = 0.70, adjusted OR, 0.90, 95% CI, 0.73 to 1.09; P = 0.28; per National Institute of Neurological Disorders and Stroke 9.5% versus 7.8%, P < 0.005, adjusted OR, 0.96, 95% CI, 0.87 to 1.06, P = 0.42). The patients >80 years of age had a higher mortality rate (30% versus 12%; P < 0.005; adjusted OR, 1.53; 95% CI, 1.43 to 1.65; P < 0.005) and reduced independence (35% versus 57%; P < 0.005; adjusted OR, 0.73; 95% CI, 0.68 to 0.78; P < 0.005). Selected patients with acute ischemic stroke > 80 years of age otherwise fulfilling the intravenous alteplase license criteria have a similar rate of symptomatic intracerebral hemorrhage compared with younger patients and are appropriate candidates for thrombolysis. The higher mortality and the poorer functional outcome are consistent with the overall worse prognosis seen in the natural history of this age group.


Humoral immune response to influenza A(H1N1)pdm2009 in patients with natural infection and in vaccine recipients in the 2009 pandemic.
The 2009 pandemic H1N1 mainly affected adolescents and children, and most of the elderly in Japan escaped clinical illness. To clarify the role of humoral immunity in the infection, the time kinetics of hemagglutination inhibition (HI), neutralization (NT), and IgG subclass antibody response directed against influenza A(H1N1)pdm2009 were analyzed in three consecutive specimens obtained from 51 young adults and children (group 1) who contracted pandemic influenza and from 74 pediatric clinic employees (group 2) inoculated with pandemic monovalent vaccine. In group 1 patients, 6 and 30 patients had lower HI and NT antibody in the acute phase respectively. Thereafter, HI and NT antibody titers increased fourfold or more in 50 patients with peak response in the third specimens obtained four weeks after the onset. IgG1 in 45 patients, IgG3 in 18 patients, and IgG4 in 29 patients showed elevated responses. Forty (54%) and 70 (95%) subjects in group 2 had positive HI and NT antibodies in the prevaccination samples, with increased antibody responses in the follow-up peaking in the second specimens. Forty of those vaccinated had increased IgG1 responses peaking in the third specimens, whereas elevated IgG3 was observed in 22 recipients with the highest level in the second samples. IgG4 did not show any increase in subjects in group 2. A few participants showed an IgG2 response in both groups. An immunologically naive population contracted influenza with apparent clinical symptoms. However, already primed subjects through subclinical infection elicited the unique pattern of IgG subclass responses by vaccination, which differed from those of naive populations.


Solving complex eigenvalue problems on a quantum annealer with applications to quantum scattering resonances.
Quantum computing is a new and rapidly evolving paradigm for solving chemistry problems. In previous work, we developed the Quantum Annealer Eigensolver (QAE) and applied it to the calculation of the vibrational spectrum of a molecule on the D-Wave quantum annealer. However, the original QAE methodology was applicable to real symmetric matrices only. For many physics and chemistry problems, the diagonalization of complex matrices is required. For example, the calculation of quantum scattering resonances can be formulated as a complex eigenvalue problem where the real part of the eigenvalue is the resonance energy and the imaginary part is proportional to the resonance width. In the present work, we generalize the QAE to treat complex matrices: first complex Hermitian matrices and then complex symmetric matrices. These generalizations are then used to compute a quantum scattering resonance state in a 1D model potential for O + O collisions. These calculations are performed using both a software (classical) annealer and hardware annealer (the D-Wave 2000Q). The results of the complex QAE are also benchmarked against a standard linear algebra library (LAPACK). This work presents the first numerical solution of a complex eigenvalue problem of any kind on a quantum annealer, and it is the first treatment of a quantum scattering resonance on any quantum device.


Systematic Quantum Mechanical Region Determination in QM/MM Simulation.
Hybrid quantum mechanical-molecular mechanical (QM/MM) simulations are widely used in enzyme simulation. Over ten convergence studies of QM/MM methods have revealed over the past several years that key energetic and structural properties approach asymptotic limits with only very large (ca. 500-1000 atom) QM regions. This slow convergence has been observed to be due in part to significant charge transfer between the core active site and the surrounding protein environment, which cannot be addressed by improvement of MM force fields or the embedding method employed within QM/MM. Given this slow convergence, it becomes essential to identify strategies for the most atom-economical determination of optimal QM regions and to gain insight into the crucial interactions captured only in large QM regions. Here, we extend and develop two methods for quantitative determination of QM regions. First, in the charge shift analysis (CSA) method, we probe the reorganization of electron density when core active site residues are removed completely, as determined by large-QM region QM/MM calculations. Second, we introduce the highly parallelizable Fukui shift analysis (FSA), which identifies how core/substrate frontier states are altered by the presence of an additional QM residue in smaller initial QM regions. We demonstrate that the FSA and CSA approaches are complementary and consistent on three test case enzymes: catechol O-methyltransferase, cytochrome P450cam, and hen eggwhite lysozyme. We also introduce validation strategies and test the sensitivities of the two methods to geometric structure, basis set size, and electronic structure methodology. Both methods represent promising approaches for the systematic, unbiased determination of quantum mechanical effects in enzymes and large systems that necessitate multiscale modeling.


Oral care and the risk of bloodstream infections in mechanically ventilated adults: A review.
Bacteraemia, defined as the presence of viable bacteria in the circulating blood can result in bloodstream infection, which is one of the most frequent and challenging hospital-acquired infections. Bacteraemia occurs in healthy populations with manipulation of the oral mucosa, including toothbrushing. Oral care is commonly administered to mechanically ventilated patients, it is important to determine whether this practice contributes to the incidence of bacteraemia. This paper reviews the literature on the link between the manipulation of the oral cavity and the development of bacteraemia in mechanically ventilated adults. Searches were conducted using Medline, CINAHL, and the Cochrane Library databases. Article inclusion criteria were (1) a focus on mechanical ventilation and critical illness, (2) human subjects, (3) adult subjects, and (4) publication in English (or available English translation). Nine articles met inclusion criteria and were critiqued. All relied upon clinical data as outcome measures; many were retrospective. The three organisms most often associated with nosocomial bloodstream infections were Staphylococcus aureus, coagulase negative staphylococci, and Enterococcus species. Establishing the origin of bacteraemia was problematic in most studies. Additional research is needed to understand the relationship of oral care practices to bacteraemia in mechanically ventilated adults.


The identity of crop pollinators helps target conservation for improved ecosystem services.
Insect pollinated mass flowering crops are becoming more widespread and there is a need to understand which insects are primarily responsible for the pollination of these crops so conservation measures can be appropriately targeted in the face of pollinator declines. This study used field surveys in conjunction with cage manipulations to identify the relative contributions of different pollinator taxa to the pollination of two widespread flowering crops, field beans and oilseed rape. Flower visiting pollinator communities observed in the field were distinct for each crop; while field beans were visited primarily by a few bumblebee species, multiple pollinator taxa visited oilseed, and the composition of this pollinator community was highly variable spatially and temporally. Neither pollinator community, however, appears to be meeting the demands of crops in our study regions. Cage manipulations showed that multiple taxa can effectively pollinate both oilseed and field beans, but bumblebees are particularly effective bean pollinators. Combining field observations and cage manipulations demonstrated that the pollination demands of these two mass flowering crops are highly contrasting, one would benefit from management to increase the abundance of some key taxa, whilst for the other, boosting overall pollinator abundance and diversity would be more appropriate. Our findings highlight the need for crop specific mitigation strategies that are targeted at conserving specific pollinator taxa (or group of taxa) that are both active and capable of crop pollination in order to reduce pollination deficits and meet the demands of future crop production.


Successful management by provocative angiography and endovascular stent of Ureteroarterial fistula in a patient with a long-term indwelling ureteral stent.
We present the clinical course of a ureteroiliac arterial fistula in a patient who had been managed by ureteral stenting for 8 years for severe ureteral stricture after abdominoperineal resection with pelvic irradiation for advanced rectal cancer. A multidisciplinary team approach including provocative angiography and an endovascular stent saved the life of the patient. Ureteroarterial fistula is a rare complication of a long-term indwelling ureteral stent that is potentially fatal unless a prompt diagnosis and adequate therapy are provided. Heightened awareness and a high index of suspicion for this condition are required to make an early diagnosis.


A simple isokinetic cycle for measurement of leg muscle function.
The measurement of net pedaling torque during isokinetic cycling allows for the evaluation of leg muscle strength and work capacity over fixed time intervals. However, the expense and difficulty of constructing an isokinetic cycle have limited the widespread application of this useful research tool. We have modified a simple commercially available isokinetic cycle that uses hydraulics to maintain pedaling velocity. A strain gauge on the flywheel axle strut measures the torsion on the strut caused by pedaling. To evaluate this device, seven healthy subjects (3 males and 4 females) were each tested twice at 60, 90, and 120 rpm for peak power during a 10-s sprint and at 100 rpm for total work performed during a 30-s sprint. These results were compared with predicted values for age, height, and sex developed on a more complicated isokinetic cycle. Subjects also performed a progressive cycle ergometry test. For the group, peak power was 97.30 +/- 12.64% of predicted (males 883.70 +/- 202.76 W; females 657.00 +/- 66.42 W) and work output was 107.70 +/- 15.75% of predicted (males 15.50 +/- 2.85 kJ; females 11.70 +/- 2.17 kJ), whereas maximal progressive exercise capacity was 126.40 +/- 25.84% (males 245.30 +/- 56.58 W; females 212.30 +/- 35.49 W). The relatively lower work values generated on this cycle (compared with the maximal progressive exercise capacity) can be attributed to the location of the strain gauge, resulting in measurement of effective work output on the flywheel.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypertension, admission blood pressure and perioperative cardiovascular risk.
We performed a retrospective case-control study to investigate hypertension and admission blood pressure as risk factors for postoperative cardiovascular death. We identified records of 76 patients who had died of a cardiovascular cause within 30 days of anaesthesia and elective surgery and 76 matched controls. From the records of each patient (case and control) we recorded the admission blood pressure and details of any history of hypertension. A pre-operative history of hypertension was strongly associated with perioperative cardiovascular death (p < 0.001 with one degree of freedom: odds ratio 4.14, 95% confidence intervals 1.63-11.69). There was no association between systolic or diastolic pressure at admission for operation and perioperative cardiovascular death. The mean admission systolic pressure of the cases was 145.5 mmHg (range 90-250 mmHg) and that of the controls was 146.5 mmHg (range 100-200 mmHg). The mean admission diastolic pressure of the cases was 83.2 mmHg (range 60-130 mmHg), and that of the controls was 84.5 mmHg (range 60-110 mmHg).


Thailand--lighting up a dark market: British American tobacco, sports sponsorship and the circumvention of legislation.
To examine how British American Tobacco (BAT) used sports sponsorship to circumvent restrictions on tobacco promotion in Thailand, both a key emerging market and a world leader in tobacco control. Analysis of previously confidential BAT company documents. Since its inception in 1987, BAT's sports sponsorship programme in Thailand has been politically sensitive and legally ambiguous. Given Thailand's ban on imported cigarettes, early events provided promotional support to smuggled brands. BAT's funding of local badminton, snooker, football and cricket tournaments generated substantial media coverage for its brands. After the General Agreement on Trade and Tariffs decision that obliged Thailand to open its cigarette market to imports, Thailand's 1992 tobacco control legislation established one of the world's most restrictive marketing environments. BAT's sponsorship strategy shifted to rallying and motorbike racing, using broadcasts of regional competitions to undermine national regulations. BAT sought to dominate individual sports and to shape media coverage to maximise brand awareness. An adversarial approach was adopted, testing the limits of legality and requiring active enforcement to secure compliance with legislation. The documents show the opportunities offered by sports sponsorship to tobacco companies amid increasing advertising restrictions. Before the 1992 tobacco control legislation, sponsored events in Thailand promoted international brands by combining global and local imagery. The subsequent strategy of "regionalisation as defensibility" reflected the capacity of international sport to transcend domestic restrictions. These transnational effects may be effectively dealt with via the Framework Convention on Tobacco Control, but will require the negotiation of a specific protocol.


Clinical Research That Matters: Designing Outcome-Based Research for Older Adults to Qualify for Systematic Reviews and Meta-Analyses.
Though older adults are more sensitive to the effects of medications than their younger counterparts, they are often excluded from manufacturer-based clinical studies. Practice-based research is a practical method to identify medication-related effects in older patients. This research also highlights the role of a pharmacist in improving care in this population. A single study rarely has strong enough evidence to change geriatric practice, unless it is a large-scale, multisite, randomized controlled trial that specifically targets older adults. It is important to design studies that may be used in systematic reviews or meta-analyses that build a stronger evidence base. Recent literature has documented a gap in advanced pharmacist training pertaining to research skills. In this paper, we hope to fill some of the educational gaps related to research in older adults. We define best practices when deciding on the type of study, inclusion and exclusion criteria, design of the intervention, how outcomes are measured, and how results are reported. Well-designed studies increase the pool of available data to further document the important role that pharmacists have in optimizing care of older patients.


Interpreting the Paramagnetic NMR Spectra of Potential Ru(III) Metallodrugs: Synergy between Experiment and Relativistic DFT Calculations.
Ruthenium-based compounds are potential candidates for use as anticancer metallodrugs. The central ruthenium atom can be in the oxidation state +2 (e.g., RAPTA, RAED) or +3 (e.g., NAMI, KP). In this study we focus on paramagnetic NAMI analogs of a general structure [4-R-pyH](+)trans-[Ru(III)Cl4(DMSO)(4-R-py)](-), where 4-R-py stands for a 4-substituted pyridine. As paramagnetic systems are generally considered difficult to characterize in detail by NMR spectroscopy, we performed a systematic structural and methodological NMR study of complexes containing variously substituted pyridines. The effect of the paramagnetic nature of these complexes on the (1)H and (13)C NMR chemical shifts was systematically investigated by temperature-dependent NMR experiments and density-functional theory (DFT) calculations. To understand the electronic factors influencing the orbital (δ(orb), temperature-independent) and paramagnetic (δ(para), temperature-dependent) contributions to the total NMR chemical shifts, a relativistic two-component DFT approach was used. The paramagnetic contributions to the (13)C NMR chemical shifts are correlated with the distribution of spin density in the ligand moiety and the (13)C isotropic hyperfine coupling constants, Aiso((13)C), for the individual carbon atoms. To analyze the mechanism of spin distribution in the ligand, the contributions of molecular spin-orbitals (MSOs) to the hyperfine coupling constants and the spatial distribution of the z-component of the spin density in the MSOs calculated at the relativistic four-component DFT level are discussed and rationalized. The significant effects of the substituent and the solvent on δ(para), particularly the contact contribution, are demonstrated. This work should contribute to further understanding of the link between the electronic structure and the NMR chemical shifts in open-shell systems, including the ruthenium-based metallodrugs investigated in this account.


Tuning the nature and stability of self-assemblies formed by ester benzene 1,3,5-tricarboxamides: the crucial role played by the substituents.
As the benzene 1,3,5-tricarboxamide (BTA) moiety is commonly used as the central assembling unit for the construction of functionalized supramolecular architectures, strategies to tailor the nature and stability of BTA assemblies are needed. The assembly properties of a library of structurally simple BTAs derived from amino dodecyl esters (ester BTAs, 13 members) have been studied, either in the bulk or in cyclohexane solutions, by means of a series of analytical methods (NMR, DSC, POM, FT-IR, UV-Vis, CD, ITC, high-sensitivity DSC, SANS). Two types of hydrogen-bonded species have been identified and characterized: the expected amide-bonded helical rods (or stacks) that are structurally similar to those formed by BTAs with simple alkyl side chains (alkyl BTAs), and ester-bonded dimers in which the BTAs are connected by means of hydrogen bonds linking the amide N-H and the ester C[double bond, length as m-dash]O. MM/MD calculations coupled with simulations of CD spectra allow for the precise determination of the molecular arrangement and of the hydrogen bond pattern of these dimers. Our study points out the crucial influence of the substituent attached on the amino-ester α-carbon on the relative stability of the rod-like versus dimeric assemblies. By varying this substituent, one can precisely tune the nature of the dominant hydrogen-bonded species (stacks or dimers) in the neat compounds and in cyclohexane over a wide range of temperatures and concentrations. In the neat BTAs, stacks are stable up to 213 °C and dimers above 180 °C whilst in cyclohexane stacks form at c* > 3 × 10-5 M at 20 °C and dimers are stable up to 80 °C at 7 × 10-6 M. Ester BTAs that assemble into stacks form a liquid-crystalline phase and yield gels or viscous solutions in cyclohexane, demonstrating the importance of controlling the structure of these assemblies. Our systematic study of these structurally similar ester BTAs also allows for a better understanding of how a single atom or moiety can impact the nature and stability of BTA aggregates, which is of importance for the future development of functionalized BTA supramolecular polymers.


Pseudophakic Candida parapsilosis endophthalmitis with a consecutive keratitis.
We describe a case of Candida parapsilosis endophthalmitis with a consecutive keratitis after phacoemulsification and posterior chamber intraocular lens implantation in an otherwise healthy eye. Despite aggressive medical and surgical management during a 2-year period, multiple episodes recurred, with the development of an intracapsular plaque and an infectious nidus on the corneal endothelium 6 months after the initial presentation. After subtotal removal of the culture-positive capsule, intravitreal and topical amphotericin B, and oral fluconazole, the inflammation improved. However, the corneal endothelial plaque persisted with recurrent inflammation 2 months later, prompting debridement of the culture-positive plaque and further removal of the culture-negative capsular remnants and lens implant. The infection was quiescent for the subsequent 12-month period until recurrent intraocular inflammation developed with enlargement of the endothelial plaque. Culture of this plaque was again positive for C. parapsilosis. After debridement and intraocular and topical amphotericin B, the eye has now been quiescent for 13 months. This case demonstrates the development of a secondary keratitis in an eye affected by pseudophakic C. parapsilosis endophthalmitis, with the posterior cornea serving as a sanctuary site for the fungus despite aggressive management leading to recurrent infection 1 year after the clinical disease appeared to be quiescent.


Traumatic Events Are Associated with Diverse Psychological Symptoms in Typically-Developing Children.
Childhood traumatic events are significant risk factors for psychopathology according to adult retrospective research; however, few studies examine trauma exposure and psychological symptoms in pre-adolescent children. Typically-developing children, aged 9-12 years (N = 114), were recruited from the community and selected from the Developmental Chronnecto-Genomics (Dev-CoG) study examining child development. Children completed questionnaires about traumatic life events, posttraumatic stress, anxiety, depression, dissociation, anger, and internalizing and externalizing behaviors. Parents also completed internalizing and externalizing measures. The number of traumatic events significantly correlated with symptom severity across all child-report psychological measures, but surprisingly, trauma was not correlated with any parent-report scores. Follow-up analyses revealed a significant trauma effect for internalizing and externalizing behaviors according to child self-report, but not for parent-report measures. Results indicate that childhood trauma may be a non-specific risk factor for sub-clinical psychopathology in otherwise typically-developing children. Moreover, children appear to be the most appropriate reporters of their own psychological distress.


The relationship between male BMI and waist circumference on semen quality: data from the LIFE study.
What is the relationship between body size, physical activity and semen parameters among male partners of couples attempting to become pregnant? Overweight and obesity are associated with a higher prevalence of low ejaculate volume, sperm concentration and total sperm count. Higher BMI is associated with impaired semen parameters, while increasing waist circumference (WC) is also associated with impaired semen parameters in infertile men. Data from the Longitudinal Investigation of Fertility and the Environment (LIFE) Study were utilized. The LIFE study is a population-based prospective cohort of 501 couples attempting to conceive in two geographic areas (Texas and Michigan, USA) recruited in 2005-2009. Couples were recruited from four counties in Michigan and 12 counties in Texas to ensure a range of environmental exposures and lifestyle characteristics. In person interviews were conducted to ascertain demographic, health and reproductive histories followed by anthropometric assessment. We categorized BMI (kg/m(2)) as <25.0 (underweight and normal), 25.0-29.9 (overweight) 30.0-34.9 (obese, class I) and ≥35 (obese, class II) for analysis. Data were available for analysis in 468 men (93% participation), with a mean ± SD age of 31.8 ± 4.8 years, BMI of 29.8 ± 5.6 kg/m(2) and WC of 100.8 ± 14.2 cm. The majority of the cohort (82%) was overweight or obese with 58% reporting physical activity <1 time/week. The median sperm concentration for the men in the cohort was 60.2 M/ml with 8.6% having oligospermia (<15 M/ml). When examining semen parameters, ejaculate volume showed a linear decline with increasing BMI and WC (P < 0.01). Similarly, the total sperm count showed a negative linear association with WC (P < 0.01). No significant relationship was seen between body size (i.e. BMI or WC) and semen concentration, motility, vitality, morphology or DNA fragmentation index. The percentage of men with abnormal volume, concentration and total sperm increased with increasing body size (P < 0.05). No relationship between physical activity and semen parameters was identified. Our cohort was largely overweight and sedentary, which may result in limited external validity, i.e. generalizability. The lack of physical activity did preclude examination of exercise more frequently than once per week, thus our ability to examine more active individuals is limited. Body size (as measured by BMI or WC) is negatively associated with semen parameters with little influence of physical activity. Our findings are the first showing a relationship between WC and semen parameters in a sample of men without known infertility. Given the worldwide obesity epidemic, further study of the role of weight loss to improve semen parameters is warranted. Supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (Contracts #N01-HD-3-3355, N01-HD-3-3356 and N01-HD-3-3358). There are no competing interests.


Attention and sensory gain control: a peripheral visual process?
Attention-related sensory gain control in human extrastriate cortex is believed to improve the acuity of visual perception. Yet given wide variance in the spatial resolution of vision across the retina, it remains unclear whether sensory gain operates homogenously between foveal and nonfoveal retinotopic locations. To address this issue, we used event-related potentials (ERPs) in a variant of the canonical spatial attention task. Participants were cued to expect targets at either fixation (foveal targets) or at a location several degrees above fixation (parafoveal targets). At both target locations, manual reaction times were shorter for cued relative to uncued targets, indicating that attention was consistently oriented to the cued location. Nevertheless, attention-related increases in sensory-evoked cortical activity were only observed at the parafoveal target location, as measured by the amplitude of the lateral occipital P1 ERP component. A second experiment replicated this data pattern using targets with lower stimulus contrast, indicating that the absence of a P1 effect for foveal targets could not be attributed to a saturated P1 response under higher-contrast stimulus conditions. When considered in light of retinogeniculate projections to cortex showing systematic changes in their physiological organization beginning within a degree of visual angle of the fovea, our findings support the proposal that the strategic functions of visual attention may vary with the retinotopic location involved.


Fake anti-malarials: start with the facts.
This meeting report presents the key findings and discussion points of a 1-day meeting entitled 'Fake anti-malarials: start with the facts' held on 28th May 2015, in Geneva, Switzerland, to disseminate the findings of the artemisinin combination therapy consortium's drug quality programme. The teams purchased over 10,000 samples, using representative sampling approaches, from six malaria endemic countries: Equatorial Guinea (Bioko Island), Cambodia, Ghana, Nigeria, Rwanda and Tanzania. Laboratory analyses of these samples showed that falsified anti-malarials (<8 %) were found in just two of the countries, whilst substandard artemisinin-based combinations were present in all six countries and, artemisinin-based monotherapy tablets are still available in some places despite the fact that the WHO has urged regulatory authorities in malaria-endemic countries to take measures to halt the production and marketing of these oral monotherapies since 2007. This report summarizes the presentations that reviewed the public health impact of falsified and substandard drugs, sampling strategies, techniques for drug quality analysis, approaches to strengthen health systems capacity for the surveillance of drug quality, and the ensuing discussion points from the dissemination meeting.


The Effect of Thymoquinone on Apoptosis of SK-OV-3 Ovarian Cancer Cell by Regulation of Bcl-2 and Bax.
We aim to determine how cisplatin, thymoquinone (TQ), and combination regimen can affect apoptosis of cultured SK-OV-3 cells. We also want to explore the mechanism of these influences on the cells' apoptosis by Bcl-2 and Bax gene. Cell Counting Kit-8 assay was used to measure the viability of cultured ovarian cancer cells. Propidium iodide with flow cytometry was used in cell cycle analysis. Thereafter, we used fluorescein isothiocyanate-stained annexin V and propidium iodide to detect the effect of cisplatin, TQ, and combination regimen on apoptosis. Real-time PCR was used to measure the Bcl-2 and Bax levels. Western blotting was used to measure on protein expression levels of Bcl-2 and Bax. In Cell Counting Kit-8 assay, we found that inhibitory effect of TQ was even better than cisplatin, and combination regimen had best inhibitory effect on cell proliferation. In cell cycle analysis, we found all regimens had obvious effect to stop cell cycle in S phase. In apoptosis assay, we found that combination regimen was better to activate cell apoptosis than cisplatin alone. Combination regimen could decrease expression of Bcl-2 and increase expression of Bax more than cisplatin or TQ alone. Thymoquinone and cisplatin had comparable antitumoric effects on SK-OV-3 cells, and combination regimen was even better. Thymoquinone could also activate apoptosis by regulating Bcl-2 and Bax genes. These indicated potential advantage of TQ for ovarian cancer in clinical practice and suggested future clinical trials to confirm its effectiveness.


Superior long-term repopulating capacity of G-CSF+plerixafor-mobilized blood: implications for stem cell gene therapy by studies in the Hbb(th-3) mouse model.
High numbers of genetically modified hematopoietic stem cells (HSCs) equipped with enhanced engrafting potential are required for successful stem cell gene therapy. By using thalassemia as a model, we investigated the functional properties of hematopoietic stem and progenitor cells (HSPCs) from Hbb(th3)/45.2(+) mice after mobilization with G-CSF, plerixafor, or G-CSF+plerixafor and the engraftment kinetics of primed cells after competitive primary and noncompetitive secondary transplantation. G-CSF+plerixafor yielded the highest numbers of HSPCs, while G-CSF+plerixafor-mobilized Hbb(th3)/45.2(+) cells, either unmanipulated or transduced with a reporter vector, achieved faster hematologic reconstitution and higher levels of donor chimerism over all other types of mobilized cells, after competitive transplantation to B6.BoyJ/45.1(+) recipients. The engraftment benefit observed in the G-CSF+plerixafor group was attributed to the more primitive stem cell phenotype of G-CSF+plerixafor-LSK cells, characterized by higher CD150(+)/CD48 expression. Moreover, secondary G-CSF+plerixafor recipients displayed stable or even higher chimerism levels as compared with primary engrafted mice, thus maintaining or further improving engraftment levels over G-CSF- or plerixafor-secondary recipients. Plerixafor-primed cells displayed the lowest competiveness over all other mobilized cells after primary or secondary transplantation, probably because of the higher frequency of more actively proliferating LK cells. Overall, the higher HSC yields, the faster hematological recovery, and the superiority in long-term engraftment indicate G-CSF+plerixafor-mobilized blood as an optimal graft source, not only for thalassemia gene therapy, but also for stem cell gene therapy applications in general.


Genomic view of bipolar disorder revealed by whole genome sequencing in a genetic isolate.
Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.


Decreased intracellular calcium mediates the histamine H3-receptor-induced attenuation of norepinephrine exocytosis from cardiac sympathetic nerve endings.
Activation of presynatic histamine H(3) receptors (H(3)R) down-regulates norepinephrine exocytosis from cardiac sympathetic nerve terminals, in both normal and ischemic conditions. Analogous to the effects of alpha(2)-adrenoceptors, which also act prejunctionally to inhibit norepinephrine release, H(3)R-mediated antiexocytotic effects could result from a decreased Ca(2+) influx into nerve endings. We tested this hypothesis in sympathetic nerve terminals isolated from guinea pig heart (cardiac synaptosomes) and in a model human neuronal cell line (SH-SY5Y), which we stably transfected with human H(3)R cDNA (SH-SY5Y-H(3)). We found that reducing Ca(2+) influx in response to membrane depolarization by inhibiting N-type Ca(2+) channels with omega-conotoxin (omega-CTX) greatly attenuated the exocytosis of [(3)H]norepinephrine from both SH-SY5Y and SH-SY5Y-H(3) cells, as well as the exocytosis of endogenous norepinephrine from cardiac synaptosomes. Similar to omega-CTX, activation of H(3)R with the selective H(3)R-agonist imetit also reduced both the rise in intracellular Ca(2+) concentration (Ca(i)) and norepinephrine exocytosis in response to membrane depolarization. The selective H(3)R antagonist thioperamide prevented this effect of imetit. In the parent SH-SY5Y cells lacking H(3)R, imetit affected neither the rise in Ca(i) nor [(3)H]norepinephrine exocytosis, demonstrating that the presence of H(3)R is a prerequisite for a decrease in Ca(i) in response to imetit and that H(3)R activation modulates norepinephrine exocytosis by limiting the magnitude of the increase in Ca(i). Inasmuch as excessive norepinephrine exocytosis is a leading cause of cardiac dysfunction and arrhythmias during acute myocardial ischemia, attenuation of norepinephrine release by H(3)R agonists may offer a novel therapeutic approach to this condition.


Impact of a Meds to Beds Program on Re-presentation Rates in Medical and Surgical Patients at a Community Hospital.
While the benefits of bundled transitions of care services are understood, only a limited number of studies have analyzed the impact of a stand-alone bedside medication delivery service on repeat hospital encounters, and those published have reported mixed results. A retrospective analysis was conducted in medical and surgical patients at a large community hospital. Adult patients discharged from either the cardiology, medicine, pulmonary, orthopedic/spine surgery, or women's surgery unit and prescribed at least one new prescription upon discharge to home between September 2015 and March 2018 were included. The primary objective was to compare unplanned 30-day re-presentation rates in patients who received Meds to Beds services to those who did not. The secondary objective was to compare 30-day re-presentation rates by patient type. Re-presentation was defined as an inpatient admission, emergency department visit, or observational encounter for any diagnosis within the six-hospital health system. Chi-square and logistic regression tests were used to assess statistical significance, and the study was powered to detect a difference in the primary objective. A total of 45,546 patients were included. Of those, 4,286 received Meds to Beds services (Intervention Group, IG) while 41,260 patients did not (Control Group, CG). Overall 30-day re-presentation rate was not statistically different (15% IG versus 15.3% CG, OR 1.0; 95% CI 0.9-1.1; p = 0.76). However, the 30-day re-presentation rate was statistically lower for women's surgery (12.8% IG versus 15.6%, CG p = 0.03, NNT 36) and orthopedics/spine surgery patients (7.3% IG versus 10.2% CG, p < 0.01, NNT 34). While there was no statistically significant differences in overall 30-day re-presentation rates, there were significant reductions in two surgical patient subgroups. Avoidance of re-presentations and generation of prescription revenue outweighed program costs.


[Assessing the insecticide resistance of an Aedes aegypti strain in El Salvador].
To assess the level of insecticide susceptibility of a certain Aedes aegypti strain found in El Salvador and to explain the mechanisms for its resistance to temephos. An A. aegypti strain from the municipality of Soyapango, Department of San Salvador, El Salvador, was studied. Bioassays were used to determine the susceptibility of the larvae to the organophosphate insecticide temephos and to three pyrethroids (deltamethrin, lambda-cyhalothrin, and cypermethrin); and of adults to an organophosphate insecticide (chlorpyrifos). The resistance factor (RF50) with determined with respect to a reference susceptible strain (Rockefeller). The mechanism of temephos resistance was determined through the use of synergistic substances, biochemical assays for enzymatic activity, and polyacrylamide gel zymograms. The larvae of the strain studied proved highly resistant to temephos (RF50 = 24.16). Of the enzyme samples analyzed, only the esterase A4 was linked to the mechanism of temephos resistance. The adult mosquitoes were susceptible to lambda-cyhalothrin and chlorpyrifos; and resistance to deltamethrin and cypermethrin fell into the category needing further verification. Temephos resistance could reduce the efficiency of chemical control of the A. aegypti mosquito in El Salvador study area. Chlorpyrifos, lambda-cyhalothrin, and cypermethrin are good alternative insecticides for use in new efforts to control this vector.


Influence of monoclonal anti-Ia like antibodies on activation of human lymphocytes.
Four monoclonal antibodies directed against human DR antigens were examined for their effects on lymphocyte activation in vitro. Two (DA6.121 and DA6.231) were serologically and biochemically indistinguishable and recognized a common determinant on the DR beta chain. One, DA6.164, reacted with an epitope probably on the beta chain of a subset of DR molecules while the fourth, DA6.147 was directed against an alpha chain determinant. All four produced measurable inhibition of lymphocyte activation. Responses to PHA or PWM were generally reduced by less than 30% and those to autochthonous or to allogeneic lymphoid cells by more than 30% some being completely abolished. DA6.121 and 231 were the most consistently effective antibodies and behaved in an identical manner. There was no additive effect when different monoclonal reagents were mixed. Reduction in the level of activation occurred when the responding population consisted almost entirely of T lymphocytes, suggesting that ADCC was not involved. The presence or absence of complement had a profound effect on the level of lymphocyte mitogenesis but responses were depressed by anti-DR monoclonals under both conditions. The results indicate that both the alpha and the beta chain of DR play some part in lymphocyte activation. It is suggested that the continuous regeneration of DR antigens on the surface of stimulator cells may be important in eliciting MLR.


Normalization of obesity-associated insulin resistance through immunotherapy.
Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4(+) T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor V(alpha) repertoires, suggesting antigen-specific expansion. CD4(+) T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic interferon-gamma (IFN-gamma)-secreting T helper type 1 (T(H)1) cells, overwhelming static numbers of T(H)2 (CD4(+)GATA-binding protein-3 (GATA-3)(+)) and regulatory forkhead box P3 (Foxp3)(+) T cells. CD4(+) (but not CD8(+)) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through T(H)2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD3-specific antibody or its F(ab')(2) fragment, reduces the predominance of T(H)1 cells over Foxp3(+) cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4(+) T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.


Principles for designing future regimens for multidrug-resistant tuberculosis.
Fewer than 20% of patients with multidrug-resistant (MDR) tuberculosis are receiving treatment and there is an urgent need to scale up treatment programmes. One of the biggest barriers to scale-up is the treatment regimen, which is lengthy, complex, ineffective, poorly tolerated and expensive. For the first time in over 50 years, new drugs have been developed specifically to treat tuberculosis, with bedaquiline and potentially delamanid expected to be available soon for treatment of MDR cases. However, if the new drugs are merely added to the current treatment regimen, the new regimen will be at least as lengthy, cumbersome and toxic as the existing one. There is an urgent need for strategy and evidence on how to maximize the potential of the new drugs to improve outcomes and shorten treatment. We devised eight key principles for designing future treatment regimens to ensure that, once they are proven safe in clinical trials, they will be clinically effective and programmatically practicable. Regimens should contain at least one new class of drug; be broadly applicable for use against MDR and extensively drug-resistant Mycobacterium tuberculosis complex strains; contain three to five effective drugs, each from a different drug class; be delivered orally; have a simple dosing schedule; have a good side-effect profile that allows limited monitoring; last a maximum of 6 months; and have minimal interaction with antiretrovirals. Following these principles will maximize the potential of new compounds and help to overcome the clinical and programmatic disadvantages and scale-up constraints that plague the current regimen.


Stochastic dynamics of genetic broadcasting networks.
The complex genetic programs of eukaryotic cells are often regulated by key transcription factors occupying or clearing out of a large number of genomic locations. Orchestrating the residence times of these factors is therefore important for the well organized functioning of a large network. The classic models of genetic switches sidestep this timing issue by assuming the binding of transcription factors to be governed entirely by thermodynamic protein-DNA affinities. Here we show that relying on passive thermodynamics and random release times can lead to a "time-scale crisis" for master genes that broadcast their signals to a large number of binding sites. We demonstrate that this time-scale crisis for clearance in a large broadcasting network can be resolved by actively regulating residence times through molecular stripping. We illustrate these ideas by studying a model of the stochastic dynamics of the genetic network of the central eukaryotic master regulator NFκB which broadcasts its signals to many downstream genes that regulate immune response, apoptosis, etc.


Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.
While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.


Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ.
Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription. For Salmonellae, one such regulatory system is PhoP-PhoQ, which regulates genes required for intracellular survival and resistance to cationic peptides. Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxymyristate. Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-alpha expression by adherent monocytes. Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues.


Preoperative correlation of intraoperative cortical mapping with magnetic resonance imaging landmarks to predict localization of the Broca area.
Broca identified the posterior third of the inferior frontal gyrus as a locus essential for the production of fluent speech. The authors have conducted this retrospective analysis in an attempt to find readily identifiable landmarks on magnetic resonance (MR) imaging that correspond to intraoperative cortical stimulation-induced speech arrest. These landmarks demonstrate novel structural-functional relationships that can be used preoperatively to predict the location of the Broca area. Using a neuronavigation system, sites where stimulation produced speech arrest (Broca area) were recorded in a consecutive series of patients undergoing awake tumor resections in the perisylvian territory of the dominant hemisphere. The authors reviewed 33 consecutive patients by projecting the MR imaging data sets and marking the site where the Broca area was identified. Sulcus topography was analyzed with respect to this site by scrolling into neighboring planes and classifying the frontal operculum into one of the four schemes of sulcus variability described by Ebeling, et al. The following categories of frontal opercula were found: 18 (69%) of 26 were Type I, eight (31%) of 26 were Type III, and seven cases eluded classification because of sulcal effacement. For patients with Type I anatomy, the Broca area was adjacent to, and distributed evenly around, the inferior precentral sulcus (IPS). Quantitatively, the site of speech arrest was located a mean of 2.4 +/- 0.25 cm from the anteroinferior aspect of the pars opercularis, where it abuts the subarachnoid space surrounding the apex of the pars triangularis. For all patients with Type III anatomy, the Broca area was adjacent to the accessory sulcus that lies immediately posterior to the IPS. In these patients the mean distance from the anterior inferior pars opercularis was 2.3 +/- 0.29 cm. The mean distance from the Broca area to the edge of the tumor for the 26 patients with clear sulcal anatomy was 1.29 +/- 0.12 cm. The results indicate a correlation between the structure of the frontal operculum as seen on MR imaging and the functional localization of speech arrest in the dominant hemisphere. Additionally, sulcal landmarks that can be used preoperatively to predict the location of the Broca area within the inferior frontal gyrus are described based on the patient population. This information will allow the surgeon to determine if an awake craniotomy is necessary to identify the Broca area when planning a surgical procedure near the dominant frontal operculum.


C/EBPδ gene targets in human keratinocytes.
C/EBPs are a family of B-Zip transcription factors--TFs--involved in the regulation of differentiation in several tissues. The two most studied members--C/EBPα and C/EBPβ--play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets--MafB and SOX2--affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation.


Positive current correlations associated with super-Poissonian shot noise.
We report on shot noise cross spectrum measurements in a beam splitter configuration. Electrons tunneling through potential barriers are incident on a beam splitter and scattered into two separate channels. Such a partition process introduces correlations between the fluctuations of the two currents. Our work has confirmed that the generally expected negative correlations resulted from sub-Poissonian electron sources. More interestingly, positive cross correlations associated with barriers exhibiting super-Poissonian shot noise have also been observed. We have found that both positive and negative correlations can be related to the noise properties of the electron source.


Caveolin-1 is a risk factor for postsurgery metastasis in preclinical melanoma models.
Melanomas are highly lethal skin tumours that are frequently treated by surgical resection. However, the efficacy of such procedures is often limited by tumour recurrence and metastasis. Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis. The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined. CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas. The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1-/- mice. Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models. A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery. Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days. In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident. Similar results were obtained when evaluating A375 cells in B6Rag1-/- mice. These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.


Breast tuberculosis in men: A systematic review.
Breast tuberculosis in male is a rarely reported and poorly described condition. To quantify the number of breast tuberculosis in men, to describe clinical presentation and to present the diagnostic and therapeutic procedures applied. A systematic review of the literature including reports published in English, Spanish and French until December 2017. The search yielded 26 cases of male breast tuberculosis, median age 56.5 years. Most presented with an isolated breast lump (89%), associated with axillary lymphadenitis (27.8%) and skin inflammation (33.3%). The most common constitutional symptoms were pain (64.7%) and fever (35.3%). Fine-needle aspiration cytology and culture were the most common diagnostic modality (61.5%). Standard anti-tuberculosis regimen was the main treatment, alone or accompanied or preceded by incision and drainage. The risk of breast tuberculosis in men appears to be low, but the condition can be difficult to diagnose and the diagnostic delays can be long. Overall prognosis is good following standard anti-tuberculosis regimen with or without incision/drainage.