Adrenoleukodystrophy: phenotype, genetics, pathogenesis and therapy.
The occasion of the presentation of the eighth Gordon Holmes Lecture left me feeling both honoured and awed, as a result of my review of the Selected Papers of Gordon Holmes (Phillips CG: Selected Papers of Gordon Holmes, compiled and edited for the Guarantors of Brain. Oxford University Press, 1979), kindly presented to me by the sponsors of the meeting. This volume lists 174 publications produced over a 55-year period, and contains reprints of contributions to neuroanatomy, neuropathology, and to disorders that affected the adrenal cortex, the spinal cord, the cerebellum and the cerebral cortex. Yet I also feel a sense of sadness; the invitation to present the lecture came from the late Anita Harding who, such a short time before her illness, gave me personal guidance and encouragement. In this lecture I endeavour to follow the example of Gordon Holmes, namely the stepwise analysis of a clinical problem, first by observation of the patient, followed by the application of techniques that can clarify it, leading to new knowledge not only about the specific disorder, but also about the nervous system and human biology in general and, it is to be hoped, to more effective therapy.


Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients.
We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 x 10(5) CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, and P. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.


Long-haul optical transmission link using low-noise phase-sensitive amplifiers.
The capacity and reach of long-haul fiber optical communication systems is limited by in-line amplifier noise and fiber nonlinearities. Phase-sensitive amplifiers add 6 dB less noise than conventional phase-insensitive amplifiers, such as erbium-doped fiber amplifiers, and they can provide nonlinearity mitigation after each span. Realizing a long-haul transmission link with in-line phase-sensitive amplifiers providing simultaneous low-noise amplification and nonlinearity mitigation is challenging and to date no such transmission link has been demonstrated. Here, we demonstrate a multi-channel-compatible and modulation-format-independent long-haul transmission link with in-line phase-sensitive amplifiers. Compared to a link amplified by conventional erbium-doped fiber amplifiers, we demonstrate a reach improvement of 5.6 times at optimal launch powers with the phase-sensitively amplified link operating at a total accumulated nonlinear phase shift of 6.2 rad. The phase-sensitively amplified link transmits two data-carrying waves, thus occupying twice the bandwidth and propagating twice the total power compared to the phase-insensitively amplified link.


UV disinfection of wastewater flocs: the effect of secondary treatment conditions.
Activated sludge flocs that are carried to the final effluent can significantly decrease the effectiveness of ultraviolet (UV) disinfection of wastewater. This effect is detected in a typical UV dose-response curve, where at higher UV doses there is a decrease in the inactivation rate (tailing). In this study, the effect of activated sludge process conditions on the UV inactivation kinetics of flocs was investigated. The conditions compared were nitrifying vs. non-nitrifying vs. an enhanced biological nutrient removal-University of Cape Town (BNR-UCT) system. The results showed that the flocs generated in the BNR-UCT process were easier to disinfect. The final effluent from the BNR-UCT process also showed improved kinetics of inactivation and reached higher levels of disinfection. The nitrifying system's final effluent had a lower number of initial fecal coliforms, which contributed to reaching higher disinfection levels compared to the non-nitrifying system.


The spread of pathogens through trade in wildlife.
Discussions on diseases of wildlife have generally focused on two basic models: the effect of disease on wildlife, and the role that wildlife plays in diseases affecting people or domestic animal health, welfare, economics and trade. Traditionally, wildlife professionals and conservationists have focused on the former, while most human/animal health specialists have been concerned largely with the latter. Lately, the (re-)emergence of many high-profile infectious diseases in a world with ever-increasing globalisation has led to a more holistic approach in the assessment and mitigation of health risks involving wildlife (with a concurrent expansion of literature). In this paper, the authors review the role of wildlife in the ecology of infectious disease, the staggering magnitude of the movement of wild animals and products across international borders in trade, the pathways by which they move, and the growing body of risk assessments from a multitude of disciplines. Finally, they highlight existing recommendations and offer solutions for a collaborative way forward.


Activation of transcription factor NF-kappaB requires ELKS, an IkappaB kinase regulatory subunit.
The nuclear factor-kappa B (NF-kappaB) family of transcription factors plays a seminal role in inflammation, apoptosis, development, and cancer. Modulation of NF-kappaB-mediated gene expression in response to diverse signals is coordinated by the IkappaB kinase (IKK) complex. We identified ELKS, an essential regulatory subunit of the IKK complex. Silencing ELKS expression by RNA interference blocked induced expression of NF-kappaB target genes, including the NF-kappaB inhibitor IkappaBalpha and proinflammatory genes such as cyclo-oxygenase 2 and interleukin 8. These cells were also not protected from apoptosis in response to cytokines. ELKS likely functions by recruiting IkappaBalpha to the IKK complex and thus serves a regulatory function for IKK activation.


The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter.
The Rb-related p107 protein has been implicated as an important control element in proper cell cycle progression. The p107 protein is thought to restrict cellular proliferation in part through its interaction with the E2F family of transcription factors and is, therefore, a specific target for regulation by several DNA viruses. Here, we demonstrate that p107 protein levels are induced in a biphasic manner in human fibroblasts during productive infection by the human cytomegalovirus (HCMV). Expression patterns of p107 protein levels during HCMV infection of human embryonic lung cells (HELs) demonstrate a sustained induction from early to late times of infection. We also demonstrate that the HCMV immediate-early protein IE1-72 complexes in vivo with the p107 protein and that this interaction can be reconstituted in an in vitro system by using reticulocyte-translated protein. Our data demonstrate that the interaction between p107 and the IE1-72 protein occurs at times of infection that temporally match the second tier of p107 protein induction and the phosphorylation pattern of the IE1-72 protein. Furthermore, we show here that the ability of p107 to transcriptionally repress E2F-responsive promoters can be overcome by expression of the IE1-72 protein. This effect appears to be specific, since the IE1-72 protein is not capable of relieving Rb-mediated repression of an E2F-responsive promoter. Finally, our data demonstrate that HCMV infection can induce cellular proliferation in quiescent cells and that IE1-72 expression alone can, to a degree, drive a similar progression through the cell cycle. These data suggest that IE1-72-mediated transactivation of E2F-responsive promoters through alleviation of p107 transcriptional repression may play a key role in the cell cycle progression stimulated by HCMV infection.


Fuzzy Logic in neurosurgery: predicting poor outcomes after lumbar disk surgery in 501 consecutive patients.
Despite a lot of research into patient selection, a significant number of patients fail to benefit from surgery for symptomatic lumbar disk herniation. We have used Fuzzy Logic-based fuzzy inference system (FIS) for identifying patients unlikely to improve after disk surgery and explored FIS as a tool for surgical outcome prediction. Data of 501 patients were retrospectively reviewed for 54 independent variables. Sixteen variables were short-listed based on heuristics and were further classified into memberships with degrees of membership within each. A set of 11 rules was formed, and the rule base used individual membership degrees and their values mapped from the membership functions to perform Boolean Logical inference for a particular set of inputs. For each rule, a decision bar was generated that, when combined with the other rules in a similar way, constituted a decision surface. The FIS decisions were then based on calculating the centroid for the resulting decision surfaces and thresholding of actual centroid values. The results of FIS were then compared with eventual postoperative patient outcomes based on clinical follow-ups at 6 months to evaluate FIS as a predictor of poor outcome. Fuzzy inference system has a sensitivity of 88% and specificity of 86% in the prediction of patients most likely to have poor outcome after lumbosacral miscrodiskectomy. The test thus has a positive predictive value of 0.36 and a negative predictive value of 0.98. Fuzzy inference system is a sensitive method of predicting patients who will fail to improve with surgical intervention.


Delayed elastic effects in Zerodur at room temperature.
Continuous testing at room temperature of large optics made of Zerodur has revealed a delayed elastic effect under low stress levels during both load and recovery after removal. Using a high-performance mechanical profilometer, a delayed strain of the order of 1% is realized over a period of a few weeks. The time-dependent phenomenon is elastic and reversible, but must be accounted for in various applications of optical design.


Random texts do not exhibit the real Zipf's law-like rank distribution.
Zipf's law states that the relationship between the frequency of a word in a text and its rank (the most frequent word has rank , the 2nd most frequent word has rank ,...) is approximately linear when plotted on a double logarithmic scale. It has been argued that the law is not a relevant or useful property of language because simple random texts - constructed by concatenating random characters including blanks behaving as word delimiters - exhibit a Zipf's law-like word rank distribution. In this article, we examine the flaws of such putative good fits of random texts. We demonstrate - by means of three different statistical tests - that ranks derived from random texts and ranks derived from real texts are statistically inconsistent with the parameters employed to argue for such a good fit, even when the parameters are inferred from the target real text. Our findings are valid for both the simplest random texts composed of equally likely characters as well as more elaborate and realistic versions where character probabilities are borrowed from a real text. The good fit of random texts to real Zipf's law-like rank distributions has not yet been established. Therefore, we suggest that Zipf's law might in fact be a fundamental law in natural languages.


Preliminary evidence regarding the hypothesis that the sex ratio at sexual maturity may affect longevity in men.
In human populations, variation in mate availability has been linked to various biological and social outcomes, but the possible effect of mate availability on health or survival has not been studied. Unbalanced sex ratios are a concern in many parts of the world, and their implications for the health and survival of the constituent individuals warrant careful investigation. We indexed mate availability with contextual sex ratios and investigated the hypothesis that the sex ratio at sexual maturity might be associated with long-term survival for men. Using two unique data sets of 7,683,462 and 4,183 men who were followed for more than 50 years, we found that men who reached their sexual maturity in an environment with higher sex ratios (i.e., higher proportions of reproductively ready men) appeared to suffer higher long-term mortality risks than those in an environment with lower sex ratios. Mate availability at sexual maturity may be linked via several biological and social mechanisms to long-term survival in men.


A gene-based association method for mapping traits using reference transcriptome data.
Genome-wide association studies (GWAS) have identified thousands of variants robustly associated with complex traits. However, the biological mechanisms underlying these associations are, in general, not well understood. We propose a gene-based association method called PrediXcan that directly tests the molecular mechanisms through which genetic variation affects phenotype. The approach estimates the component of gene expression determined by an individual's genetic profile and correlates 'imputed' gene expression with the phenotype under investigation to identify genes involved in the etiology of the phenotype. Genetically regulated gene expression is estimated using whole-genome tissue-dependent prediction models trained with reference transcriptome data sets. PrediXcan enjoys the benefits of gene-based approaches such as reduced multiple-testing burden and a principled approach to the design of follow-up experiments. Our results demonstrate that PrediXcan can detect known and new genes associated with disease traits and provide insights into the mechanism of these associations.


Implementation of the findings of a national enquiry into the misdiagnosis of miscarriage in the Republic of Ireland: impact on quality of clinical care.
To describe the findings of a national inquiry into cases of misdiagnosis of miscarriage in the Republic of Ireland and to report the results of implementation of the findings of the inquiry, including investment in new equipment and training, new national guidelines, and rigorous annual audit of early pregnancy units. Narrative description of the inquiry and its findings and results of a subsequent audit. Not applicable. Women with problems of bleeding and/or pain in early pregnancy who were erroneously diagnosed as having a nonviable intrauterine pregnancy. After two cases of misdiagnosis of miscarriage that were widely reported in the Republic of Ireland in June 2010, a Miscarriage Misdiagnosis Review Team was commissioned by the Irish Health Service Executive (HSE) to undertake a national review of other possible cases of misdiagnosis of miscarriage. The Review Team made a series of recommendations that were subsequently implemented in full. The results of the implementation of the findings of the Review Team have been the subject of three annual audits across the country. The main outcome measure was the occurrence of misdiagnosis of miscarriage in the Republic of Ireland before and after implementation of the findings of the Review Team. Twenty-four confirmed cases of misdiagnosis of miscarriage were identified, mostly occurring between 2005 and 2010. Analysis led to a series of recommendations by the Review Team, which were implemented in full by the HSE. Over € 3 million was provided to fund implementation; 26 high-quality gynecological ultrasound machines were purchased to reequip 19 units involved in provision of care to women with suspected miscarriage. There was further allocation of resources for new equipment and improvement in the management and staffing of early pregnancy units across Ireland, with each center now having a dedicated and properly staffed Early Pregnancy Assessment Unit. A national training program in the management of early pregnancy problems has been implemented, along with regular national meetings to discuss early pregnancy problems. National clinical guidelines on the diagnosis and management of miscarriage for implementation have been distributed to all hospitals. No cases of miscarriage misdiagnosis were identified in any of the three annual audits, suggesting that implementation of the findings of the review has been successful. We believe that this is the first report of national change in practice leading to improvement in clinical outcomes in the management of suspected miscarriage.


Randomized controlled trial of a collaborative care intervention to manage cancer-related symptoms: lessons learned.
Collaborative care interventions to treat depression have begun to be tested in settings outside of primary care. However, few studies have expanded the collaborative care model to other settings and targeted comorbid physical symptoms of depression. The aims of this report were to: (1) describe the design and methods of a trial testing the efficacy of a stepped collaborative care intervention designed to manage cancer-related symptoms and improve overall quality of life in patients diagnosed with hepatobiliary carcinoma; and (2) share the lessons learned during the design, implementation, and evaluation of the trial. The trial was a phase III randomized controlled trial testing the efficacy of a stepped collaborative care intervention to reduce depression, pain, and fatigue in patients diagnosed with advanced cancer. The intervention was compared to an enhanced usual care arm. The primary outcomes included the Center for Epidemiological Studies-Depression scale, Brief Pain Inventory, and Functional Assessment of Cancer Therapy (FACT)-Fatigue, and the FACT-Hepatobiliary. Sociodemographic and disease-specific characteristics were recorded from the medical record; Natural Killer cells and cytokines that are associated with these symptoms and with disease progression were assayed from serum. and Discussion The issues addressed include: (1) development of collaborative care in the context of oncology (e.g., timing of the intervention, tailoring of the intervention, ethical issues regarding randomization of patients, and changes in medical treatment over the course of the study); (2) use of a website by chronically ill populations (e.g., design and access to the website, development of the website and intervention, ethical issues associated with website development, website usage, and unanticipated costs associated with website development); (3) evaluation of the efficacy of intervention (e.g., patient preferences, proxy raters, changes in medical treatment, and inclusion of biomarkers as endpoints); and (4) analyses and interpretation of the intervention (e.g., confounding factors, dose and active ingredients, and risks and benefits of collaborative care interventions in chronically ill patients). The limitations to the study, although not fully realized at this time as the trial is ongoing, include: (1) heterogeneity of the diagnoses and treatments of participants; and (2) inclusion of caregivers as proxy raters but not as participants in the intervention. Collaborative care interventions to manage multiple symptoms in a tertiary cancer center are feasible. However, researchers designing and implementing interventions that are web-based, target multiple symptoms, and for oncology patients may benefit from previous experiences.


Schisandrin B inhibits cell growth and induces cellular apoptosis and autophagy in mouse hepatocytes and macrophages: implications for its hepatotoxicity.
A number of drugs and herbal compounds have been documented to cause hepatoxicity. Schisandrin B (Sch B) is an active dibenzocyclooctadiene isolated from Schisandrae fructus, with a wide array of pharmacological activities. However, the potential hepatotoxicity of Sch B is a major safety concern, and the underlying mechanism for Sch B-induced liver toxic effects is not fully elucidated. In the present study, we aimed to investigate the liver toxic effects and the molecular mechanisms of Sch B in mouse liver and macrophage cells. The results have shown that Sch B exhibits potent grow inhibitory, proapoptotic, and proautophagic effects in AML-12 and RAW 264.7 cells. Sch B markedly arrested cells in G1 phase in both cell lines, accompanied by the down-regulation of cyclin dependent kinase 2 (CDK2) and cyclin D1 and up-regulation of p27 Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl) B-cell lymphoma 2 (Bcl-2), but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax). Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP) in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Sch B for clinical use.


Delayed and differential effects of the economic crisis in Sweden in the 1990s on health-related exclusion from the labour market: a health equity assessment.
Many OECD countries are currently experiencing economic crisis and introducing counter-measures with unknown effects. To learn from previous experience, we explored whether there were delayed or differential effects of the Swedish recession in the 1990s and the government's response to it for people with limiting longstanding illness or disability (LLSI) from different socioeconomic groups (SEGs), by policy analysis and secondary data analysis of the Swedish Survey of Living Conditions (ULF) from 1978 to 2005. The government policy response involved cutting public expenditure, privatising some services and measures to boost private sector employment. There was a decline in overall employment rates from the early 1990s, particularly among men and women with LLSI and in lower SEGs. Public sector employment declined from 53 to 40 percent among women and from 23 to 14 percent among men. Private sector employment increased modestly for women (from 31 percent to 37 percent), and stayed stable at 59-60 percent among men. Following economic recovery, employment rates continued to decline among men and women with LLSI from manual SEGs, while the employment levels increased among most healthy men and women. There was a concomitant increase in rates of LLSI, sickness absence and rates of disability pension particularly among women in lower SEGs. The policy response to the 1990s economic crisis in Sweden had differential consequences, hitting the employment of women in the public sector, especially women with both LLSI and low socioeconomic status. The observed increase in disability pension rates, particularly among women with LLSI in lower SEGs, may be a delayed effect of the policy response to the economic crisis.


An information retrieval service to support clinical decision-making at the point of care.
The information retrieval systems currently available in general practice, such as Medline, and web search engines are passive and relatively difficult to access during consultations. Emergent technologies, including the National Electronic Library for Health, offer opportunities for more active decision support. We examine the extent to which information retrieval could support primary care consultations by examining the impact of the new technology at different stages of the consultation. We advocate a system whereby professional organisations concerned with quality of care, such as the Royal College of General Practitioners, might contribute the the process.


Nutritional support in patients with cancer of the esophagus: impact on nutritional status, patient compliance to therapy, and survival.
The multimodal approach to patients with esophageal squamous cell carcinoma often includes polychemotherapy combined with radiation therapy. Cancer dysphagia and drug-related anorexia, mucositis and vomiting can all lead to malnutrition. The aim of this study was to analyze the impact of the administration of enteral nutrition (EN) on the patient's nutritional status, tolerance of chemotherapy and radiotherapy, and final oncological outcome. Fifty esophageal cancer patients who were to be submitted to chemotherapy (days 1-4 5-fluorouracil (FU) 1 g/m2/day and cisplatin (CDDP) 100 mg/m2/day 1) for two cycles plus radiotherapy (31 Gy) were referred to the Nutrition Support Unit prior to any therapy due to their malnourished status. Twenty-nine dysphagic patients received nutrition via tube (37 kcal/kg/day + 2.0 g proteins/kg/day for 34 days), while 21 others who were not dysphagic were given a standard oral diet (SD). The patients who received EN had a more severe weight loss than the SD patients (16.8% vs 12.8%, P <0.02). The dose of administered EN represented 86% of the planned support, and 70% of the nutritional therapy was administered in the home setting. Administration of EN support resulted in stable body weight and unchanged levels of visceral proteins, while SD patients had a decrease in body weight, total proteins and serum albumin (P <0.01). There was no difference between the two groups in terms of tolerance and response to cancer therapy, suitability for radical resection and median survival (9.5 months). EN in patients with cancer of the esophagus undergoing chemotherapy and radiotherapy is well tolerated, feasible even in the home setting, prevents further nutritional deterioration and achieves the same oncological results in dysphagic patients as those achieved in non-dysphagic patients.


Biopsy and re-biopsy in lung cancer: the oncologist requests and the role of endobronchial ultrasounds transbronchial needle aspiration.
As the leading cause of death worldwide, lung cancer has proven itself incurable in the advanced stages. For early stages, endobronchial ultrasounds transbronchial needle aspiration (EBUS-TBNA) is now considered the standard to assess mediastinal lymph node, to define the multimodality therapeutic approach. In recent years, EBUS-TBNA has extended its use also in the metastatic and locally recurrent disease. New molecules, with specific mutations that give resistance to current target therapies, have made re-biopsy at disease progression an important assessment, with therapeutic and clinical implication. Here we present the oncologist's point of view on EBUS-TBNA in the staging process, at recurrence and progression.


New perspectives for melanoma immunotherapy: role of IL-12.
Metastatic melanoma is a poor prognosis skin cancer. Since conventional treatments including surgery and chemotherapy often fail, novel therapeutic strategies are needed. In particular, identification of melanoma associated antigen has fostered the progress of both active (vaccines) and adoptive immunotherapy. Some promising results have been obtained, but most melanoma patients are not yet cured possibly because of different immune-escape mechanisms operated by tumor cells. Several studies have addressed the use of interleukin (IL)-12 for melanoma therapy due to its immunoregulatory function and anti-tumor activity mediated by stimulation of T and NK effector cells. Unfortunately, IL-12 has shown considerable toxicity. We [1] have recently demonstrated that IL-12 exerts a direct anti-tumor activity on murine B16 melanoma cells expressing a functional IL-12 receptor (R). In our model low levels of endogenous IL-12 reduced proliferation, increased apoptosis, and defective microvessel formation of tumor cells. This review summarizes information about melanoma immunotherapy and highlights a novel mechanism of IL-12-mediated anti-tumor activity based upon the direct effect of the cytokine on IL-12R(+) tumor cells. In this view, new therapeutic approaches may be planned including: i) pre-screening of melanoma patients for IL-12Rbeta2 expression to identify potential responders, ii) administration of small and less frequent doses of IL-12 to avoid toxicity and iii) targeting of IL-12 to IL-12R(+) tumor cells, such as local administration in patients with skin tumors or injection of IL-12 fused to an antibody specific to tumor cells.


Reduced morbidity after gradual discontinuation of lithium treatment for bipolar I and II disorders: a replication study.
The aim of this study was to verify reduction of early affective morbidity by gradual, rather than rapid, discontinuation of lithium treatment. For 78 patients with bipolar disorders, lithium treatment was discontinued either rapidly (over 1-14 days) or gradually (over 15-30 days). The effects of the two schedules were compared by survival analysis of time to first recurrence. Median time to recurrence was 5.6 times as long for gradual discontinuation (14.0 months) as for rapid discontinuation (2.5 months). The ratios of the median survival times for gradual and rapid discontinuation were similar in I and II subtypes and in depression and mania (4-6:1). The polarities of the episodes at onset and at first recurrence after lithium discontinuation were 83.6% concordant. These results independently confirm a reduction in morbid risk from slow discontinuation of lithium treatment for bipolar disorders.


Progress towards structural understanding of infectious sheep PrP-amyloid.
The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.


cDNA cloning and functional analysis of the p97 subunit of the 26S proteasome, a polypeptide identical to the type-1 tumor-necrosis-factor-receptor-associated protein-2/55.11.
Molecular cloning of cDNA for a new regulatory subunit, designated p97, of the human 26S proteasome showed that the polypeptide consists of 908 amino acid residues with a calculated molecular mass of 100184 Da and an isoelectric point of 4.94. Computer analysis showed that p97 is very similar to type-1 tumor-necrosis-factor (TNF)-receptor-associated protein (TRAP)-2 and 55.11, both of which were identified recently as binding proteins of the cytoplasmic domain of type-1 TNF receptor by yeast two-hybrid screening. This finding suggests that the 26S proteasome might serve as a mediator molecule in the TNF signaling pathway in cells. Computer-assisted similarity analysis also revealed the high sequence similarity of p97 with a yeast protein whose function is yet unknown, the gene for which is here termed NAS1 (non-ATPase subunit 1). Disruption of NAS1 resulted in several phenotypes, including lethality and temperature-sensitive growth, depending on the genetic background of the cells used. The human p97 cDNA suppressed the growth defect of nas1 disruptant cells, when expressed from single-copy or multi-copy vectors, indicating that p97 is functionally equivalent to yeast Nas1p. Culturing of the temperature-sensitive nas1 cells at the restrictive temperature promoted the accumulation polyubiquitinated cellular proteins, implying that the 26S proteasome requires a functional Nas1p subunit for ubiquitin-dependent proteolysis. These results indicate that p97/Nas1p plays an important regulatory role in the function of the 26S proteasome.


Measurements of neutron energy spectra from 7Li(p,n)7Be reaction with Bonner sphere spectrometer, Nested Neutron Spectrometer and ROSPEC.
Neutron spectrometry measurements were carried out at the McMaster Accelerator Laboratory (MAL), which is equipped with a 3-MV Van de Graaff-type accelerator. Protons were accelerated onto a thick natural lithium target inducing the (7)Li(p,n)(7)Be threshold reaction. Depending on the proton energy, slightly different poly-energetic neutron fields were produced. Neutron spectra were measured at two incident proton energies: 2.15 and 2.24 MeV, which produced poly-energetic neutrons with maximum kinetic energies of 401 and 511 keV, respectively. Measurements were performed at a distance of 1.5 m from the target in the forward direction with three different instruments: Bonner sphere spectrometer, Nested Neutron Spectrometer and ROtational proton recoil SPECtrometer.


Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.
The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)


APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies.
APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.


NeuralAct: A Tool to Visualize Electrocortical (ECoG) Activity on a Three-Dimensional Model of the Cortex.
Electrocorticography (ECoG) records neural signals directly from the surface of the cortex. Due to its high temporal and favorable spatial resolution, ECoG has emerged as a valuable new tool in acquiring cortical activity in cognitive and systems neuroscience. Many studies using ECoG visualized topographies of cortical activity or statistical tests on a three-dimensional model of the cortex, but a dedicated tool for this function has not yet been described. In this paper, we describe the NeuralAct package that serves this purpose. This package takes as input the 3D coordinates of the recording sensors, a cortical model in the same coordinate system (e.g., Talairach), and the activation data to be visualized at each sensor. It then aligns the sensor coordinates with the cortical model, convolves the activation data with a spatial kernel, and renders the resulting activations in color on the cortical model. The NeuralAct package can plot cortical activations of an individual subject as well as activations averaged over subjects. It is capable to render single images as well as sequences of images. The software runs under Matlab and is stable and robust. We here provide the tool and describe its visualization capabilities and procedures. The provided package contains thoroughly documented code and includes a simple demo that guides the researcher through the functionality of the tool.


Hydroxysafflor yellow A (HSYA) targets the NF-κB and MAPK pathways and ameliorates the development of osteoarthritis.
The inflammatory environment has been demonstrated to be strongly associated with the progression of osteoarthritis (OA). HSYA, the main active component in the medical and edible dual purpose plant safflower, has previously showed significant anti-inflammatory effects in several diseases. In the current study, the protective effects of HSYA in the inhibition of OA development and its underlying mechanism were examined by both in vitro and in vivo experiments. Our data indicated that interleukin-1 beta (IL-1β) induced over-production of pro-inflammatory cytokines, such as nitric oxide (NO) and prostaglandin E2 (PGE2); also, the expression of cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) were all inhibited by pretreatment with HSYA in a dose-dependent manner (2.5 to 40 μM). Furthermore, HSYA attenuated IL-1β-induced degradation of the extracellular matrix (ECM) by decreasing the expression of metalloproteinases (MMPs) and thrombospondin motifs 5 (ADAMTS5). Mechanistically, HSYA suppressed IL-1β-induced activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) cascades. Meanwhile, molecular docking studies revealed that HSYA has excellent binding abilities to p65, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK). In addition, the protective effects of HSYA were observed in a surgically induced mouse OA model. In summary, this study provides evidence that HSYA can be applied as a potential therapeutic agent in the treatment of OA.


Expression of adiponectin and its receptors is altered in epithelial ovarian tumors and ascites-derived ovarian cancer cell lines.
Recent evidence suggests that higher body mass index is associated with a modest increase in ovarian cancer risk. Reduced serum levels of adiponectin are correlated with obesity and increased cancer risk. The objectives of the present study are to determine if expressions of adiponectin and its receptors, AdipoR1 and AdipoR2, are altered in epithelial ovarian tumors and ascites-derived ovarian cancer cell lines and to determine if plasma adiponectin levels are altered in the chicken model of ovarian cancer. Adiponectin, AdipoR1, and AdipoR2 mRNA concentrations in ovaries and chicken ovarian cancer (COVCAR) cell lines were determined by quantitative real-time polymerase chain reaction analysis. Existence of adiponectin isoforms in the ovaries and COVCAR cells was identified by nondenaturing gel electrophoresis. Adiponectin, AdipoR1, and AdipoR2 protein amounts were determined by Western blot analysis. Plasma total adiponectin levels were determined by an enzyme immunoassay. Adiponectin, AdipoR1, and AdipoR2 mRNA concentrations were significantly lower in cancerous ovaries and COVCAR cell lines compared with normal ovaries and normal ovarian surface epithelial (NOSE) cells, respectively. Adiponectin in ovary and COVCAR cell lines appeared as a heavy-molecular-weight isoform that is greater than 720-kd mass. In addition, a lower-molecular-weight adiponectin isoform was found in COVCAR cells but not in NOSE cells. Adiponectin and AdipoR1 protein concentrations were not different in COVCAR cell lines compared with NOSE cells. However, AdipoR2 protein concentrations were significantly higher in cancerous ovaries but lower in COVCAR cell lines compared with normal ovaries and NOSE cells, respectively. Plasma adiponectin concentrations were not different in chickens that had ovarian carcinoma compared with control animals. Expression of adiponectin in ovarian tumors and in metastatic ovarian tumor cells is likely to affect cellular metabolism and proliferation through activating AdipoR1 and/or AdipoR2. Plasma adiponectin levels may not be predictive of advanced stages of ovarian tumor in the chicken model.


Activation of G-proteins in brain by endogenous and exogenous cannabinoids.
The biological response to cannabinoid agonist begins when the agonist-bound receptor activates G-protein G(alpha) subunits, thus initiating a cascade of signal transduction pathways. For this reason, information about cannabinoid receptors/G-protein coupling is critical to understand both the acute and chronic actions of cannabinoids. This review focuses on these mechanisms, predominantly examining the ability of cannabinoid agonists to activate G-proteins in brain with agonist-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding. Acute efficacies of cannabinoid agonists at the level of G-protein activation depend not only on the ability of the agonist to induce a high affinity state in G(alpha) for GTP, but also to induce a low affinity for GDP. When several agonists are compared, it is clear that cannabinoid agonists differ considerably in their efficacy. Both WIN 55212-2 and levonantradol are full agonists, while Delta(9)-tetrahydrocannabinol is a weak partial agonist. Of interest, anandamide and its stable analog methanandamide are partial agonists. Chronic treatment in vivo with cannabinoids produces significant tolerance to the physiological and behavioral effects of these drugs, and several studies have shown that this is accompanied by a significant loss in the ability of cannabinoid receptors to couple to G-proteins in brain. These effects vary across different brain regions and are usually (but not always) accompanied by loss of cannabinoid receptor binding. Although the relationship between cannabinoid receptor desensitization and tolerance has not yet been established, these mechanisms may represent events that lead to a loss of cannabinoid agonist response and development of tolerance.


Dispersant adsorption and viscoelasticity of alumina suspensions measured by quartz crystal microbalance with dissipation monitoring and in situ dynamic rheology.
Adsorption behavior and water content of adsorbed layers of four dispersants for aqueous ceramic processing were studied by quartz crystal microbalance with dissipation monitoring (QCM-D) on alumina surfaces. The dispersants were a poly(acrylic acid), a lignosulfonate, and two hydrophilic comb copolymers with nonionic polyoxyethylene chains of different molecular weights. A Voigt model was applied to analyze the viscoelastic behavior of the adsorbed dispersant layers. The results from QCM-D were compared with viscoelastic properties determined by in situ dynamic rheology measurements of highly concentrated alumina suspensions during slip casting. The QCM-D results showed that both the poly(acrylic acid) and the lignosulfonate adsorbed in low amounts and in a flat conformation, which generated thin, highly rigid layers less than 1 nm thick. The water content of these layers was found to be around 30% for the lignosulfonate and 35% for the poly(acrylic acid). High casting rate and strength in terms of storage modulus were observed in the final consolidate of the suspensions with the two polyelectrolytes. In contrast, the high molecular weight comb copolymer adsorbed in a less elastic layer with a thickness of about 6 nm, which is enough to provide steric stabilization. The viscous behavior of this layer was attributed to high water content, which was calculated to be around 90%. Such a water-rich layer gives a lubrication effect, which allows for reorientation of particles during the consolidation process, resulting in a high final strength of the ceramic material. During consolidation, the suspension showed a slow casting rate, most likely due to rearrangement facilitated by the lubricating layer. The short-chain comb copolymer adsorbed in a 1.5 nm thick, rigid layer and gave low final strength to the consolidated suspension. It is likely that the poor consolidation behavior is caused by flocculation due to insufficient stabilization of the dispersion.


COVID-19, coronavirus, SARS-CoV-2 and the small bowel.
Although SARS-CoV-2 may primarily enter the cells of the lungs, the small bowel may also be an important entry or interaction site, as the enterocytes are rich in angiotensin converting enzyme (ACE)-2 receptors. The initial gastrointestinal symptoms that appear early during the course of Covid-19 support this hypothesis. Furthermore, SARS-CoV virions are preferentially released apically and not at the basement of the airway cells. Thus, in the setting of a productive infection of conducting airway epithelia, the apically released SARS-CoV may be removed by mucociliary clearance and gain access to the GI tract via a luminal exposure. In addition, post-mortem studies of mice infected by SARS-CoV have demonstrated diffuse damage to the GI tract, with the small bowel showing signs of enterocyte desquamation, edema, small vessel dilation and lymphocyte infiltration, as well as mesenteric nodes with severe hemorrhage and necrosis. Finally, the small bowel is rich in furin, a serine protease which can separate the S-spike of the coronavirus into two "pinchers" (S1 and 2). The separation of the S-spike into S1 and S2 is essential for the attachment of the virion to both the ACE receptor and the cell membrane. In this special review, we describe the interaction of SARS-CoV-2 with the cell and enterocyte and its potential clinical implications.


[Autochthonous European dirofilariasis of the lung].
Two intrapulmonary round nodules were identified by x-ray examination in a 45-year old German woman during an examination conducted before her employment who had been complaining of pain in the right side of the thorax and of mild coughing irritation and tiredness for several months prior to the health check. Since the intraoperative frozen section did not show any malignancy, and atypical resection of the two involved segments was performed. Histological processing of the surgical specimens showed two spherical ischemic necroses surrounded by a connective tissue border with inflammatory infiltration. In the centre of the larger of the two necrotic lesions a parasite was seen intravascularly, which because of morphological and serological findings, was identified as a Dirofilaria. It must be assumed that the infection of this patient occurred during a stay in Corsica where these nematodes are endemic in several mammal hosts. The patient was therefore suffering from an autochthonous European dirofilariasis of the lung. Only two cases of this infection acquired in Europe have been reported so far.


Rationally Designed Strigolactone Analogs as Antagonists of the D14 Receptor.
Strigolactones (SLs) are plant hormones that inhibit shoot branching and act as signals in communications with symbiotic fungi and parasitic weeds in the rhizosphere. SL signaling is mediated by DWARF14 (D14), which is an α/β-hydrolase that cleaves SLs into an ABC tricyclic lactone and a butenolide group (i.e. D-ring). This cleavage reaction (hydrolysis and dissociation) is important for inducing the interaction between D14 and its target proteins, including D3 and D53. In this study, a hydrolysis-resistant SL analog was predicted to inhibit the activation of the D14 receptor, thereby disrupting the SL signaling pathway. To test this prediction, carba-SL compounds, in which the ether oxygen of the D-ring or the phenol ether oxygen of the SL agonist (GR24 or 4-bromo debranone) was replaced with a methylene group, were synthesized as novel D14 antagonists. Subsequent biochemical and physiological studies indicated that carba-SLs blocked the interaction between D14 and D53 by inhibiting D14 hydrolytic activity. They also suppressed the SL-induced inhibition of rice tiller outgrowths. Additionally, carba-SLs antagonized the SL response in a Striga parasitic weed species. Structural analyses revealed that the D-ring of 7'-carba-4BD was hydrolyzed by D14 but did not dissociate from the 4BD skeleton. Thus, 7'-carba-4BD functioned as an antagonist rather than an agonist. Thus, the hydrolysis of the D-ring of SLs may be insufficient for activating the receptor. This study provides data relevant to designing SL receptor antagonists.


Frequency and associated factors for care giving among elderly patients visiting a teaching hospital in Karachi, Pakistan.
To study frequency and associated factors for care giving among elderly patients visiting a teaching hospital in Karachi, Pakistan. A cross sectional questionnaire-based study was conducted at the Community Health Centre (CHC), Aga Khan University Hospital (AKUH) Karachi, Pakistan from September to November 2009. All individuals, visiting the CHC and aged 65 years or above were interviewed after taking written informed consent. A total of 400 elderly completed the interview. Majority were females, 65-69 years age, More than half of the individuals ie: 227 (85%) had received Care Giver experience for assistance and among these 195(72%) had care provided by an immediate family member. A large proportion of them stated that their Care Givers managed to provide less than four hours in a day for care giving. Around 37% showed substantial improvement in their relationship with the care givers. About 70% of the respondents stated that the care provided by the Care Giver improved their quality of life. Elderly care is provided by majority of the family members resulting in increased satisfaction level, however small number still not satisfied due to unfulfilled need of these older people. This demands that efforts should be made to strengthen the family support by increasing awareness regarding elderly care and arranging support system by the government.


What can saliency models predict about eye movements? Spatial and sequential aspects of fixations during encoding and recognition.
Saliency map models account for a small but significant amount of the variance in where people fixate, but evaluating these models with natural stimuli has led to mixed results. In the present study, the eye movements of participants were recorded while they viewed color photographs of natural scenes in preparation for a memory test (encoding) and when recognizing them later. These eye movements were then compared to the predictions of a well defined saliency map model (L. Itti & C. Koch, 2000), in terms of both individual fixation locations and fixation sequences (scanpaths). The saliency model is a significantly better predictor of fixation location than random models that take into account bias toward central fixations, and this is the case at both encoding and recognition. However, similarity between scanpaths made at multiple viewings of the same stimulus suggests that repetitive scanpaths also contribute to where people look. Top-down recapitulation of scanpaths is a key prediction of scanpath theory (D. Noton & L. Stark, 1971), but it might also be explained by bottom-up guidance. The present data suggest that saliency cannot account for scanpaths and that incorporating these sequences could improve model predictions.


Prevalence of lacrimal gland prolapse in the functional blepharoplasty population.
Traditional functional blepharoplasty has focused on the excision of variable amounts of skin, muscle, and fat, with little attention given to the finding of the lacrimal gland prolapse (LGP). The reported incidence of LGP found on clinical examination, on patients of all ages presenting for blepharoplasty, is 15%. The author's experience with the procedure in the older age group (older than 60), is that this percentage is much lower than that found at surgery. To verify whether this is correct, the author evaluated lacrimal gland position intraoperatively in a group of patients undergoing functional upper blepharoplasty. In those patients who had LGP, the author routinely repositioned the gland and evaluated surgical outcomes. The author evaluated the presence of an intraoperatively displaced lacrimal gland in his functional blepharoplasty population over a 2.5-year period (2008-2010). Patients with a history of previous eyelid surgery, trauma, or who had concurrent ptosis or other eyelid malpositions were excluded from the study. When present, the degree of prolapse was graded as mild (0-2 mm), moderate (3-5 mm), or severe (6 mm or more). The author arbitrarily suture-repositioned the gland in all patients with 4 mm or more of prolapse. In cases with less prolapse, light cautery to the tip of the gland capsule and surrounding soft tissue allowed adequate repositioning without suture fixation. Pertinent patient demographics and postoperative complications were documented. Patients were seen consistently to 6 months after surgery with an average follow up of 12 months. Fifty-seven patients were included in the study. Thirty-four patients (60%) had some degree of LGP. Of these, 8 patients (24%) had mild prolapse, 23 patients (67%) had moderate prolapse, and 3 patients (9%) had severe prolapse. Nineteen patients (56%) with LGP had the gland suture-repositioned, and 15 patients (44%) received cautery to retro-place the gland. In one patient (3%), transient dry-eye symptoms developed after surgery. Otherwise, there were few benign and self-limiting postoperative complications, consisting of prolonged upper eyelid swelling and transient mild pain. Lacrimal gland prolapse is a common finding during functional upper blepharoplasty surgery and appears to be a normal involutional periorbital aging change. Most cases are moderate in degree (as defined in the article), and not associated with specific preoperative symptoms or complaints, except lateral hooding with concomitant visual field deficit. Repositioning the gland intraoperatively is generally complication free and typically not associated with increased morbidity or healing time. The clinical significance of LGP in the elderly undergoing blepharoplasty surgery is unknown and requires further study.


Neonatal adaptation in infants prenatally exposed to antidepressants--clinical monitoring using Neonatal Abstinence Score.
Intrauterine exposure to antidepressants may lead to neonatal symptoms from the central nervous system, respiratory system and gastrointestinal system. Finnegan score (Neonatal Abstinence Score, NAS) has routinely been used to assess infants exposed to antidepressants in utero. The purpose was to study neonatal maladaptation syndrome in infants exposed to selective serotonin reuptake inhibitors (SSRI) or serotonin-norepinephrine reuptake inhibitors (SNRI) in utero. Retrospective cohort study of women using antidepressants during pregnancy and their infants. Patients were identified from the electronic health record system at Karolinska University Hospital Huddinge containing pre-, peri- and postnatal information. Information was collected on maternal and infant health, social factors and pregnancy. NAS sheets were scrutinized. 220 women with reported 3rd trimester exposure to SSRIs or SNRIs and who gave birth between January 2007 and June 2009 were included. Seventy seven women (35%) used citalopram, 76 used (35%) sertraline, 34 (15%) fluoxetine and 33 (15%) other SSRI/SNRI. Twenty-nine infants (13%) were admitted to the neonatal ward, 19 were born prematurely. NAS was analyzed in 205 patients. Severe abstinence was defined as eight points or higher on at least two occasions (on a scale with maximum 40 points), mild abstinence as 4 points or higher on at least two occasions. Seven infants expressed signs of severe abstinence and 46 (22%) had mild abstinence symptoms. Hypoglycemia (plasma glucose <2.6 mmol/L) was found in 42 infants (19%). Severe abstinence in infants prenatally exposed to antidepressants was found to be rare (3%) in this study population, a slightly lower prevalence than reported in previous studies. Neonatal hypoglycemia in infants prenatally exposed to antidepressant may however be more common than previously described.


Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells.
The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells, including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus, this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia.


Withaferin A inhibits the proliferation of gastric cancer cells by inducing G2/M cell cycle arrest and apoptosis.
Human gastric adenocarcinoma (AGS) is one of the most common types of malignant tumor and the third-leading cause of tumor-associated mortality worldwide. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, exhibits antitumor activity in a variety of cancer models. However, to the best of our knowledge, the direct effect of WA on AGS cells has not previously been determined. The present study investigated the effects of WA on the proliferation and metastatic activity of AGS cells. WA exerted a dose-dependent cytotoxic effect on AGS cells. The effect was associated with cell cycle arrest at the G2/M phase and the expression of apoptotic proteins. Additionally, WA treatment resulted in a decrease in the migration and invasion ability of the AGS cells, as demonstrated using a wound healing assay and a Boyden chamber assay. These results indicate that WA directly inhibits the proliferation and metastatic activity of gastric cancer cells, and suggest that WA may be developed as a drug for the treatment of gastric cancer.


Appearance of β-dystroglycan precedes the formation of glio-vascular end-feet in developing rat brain.
Dystroglycan has an important role in binding of perivascular glial end-feet tothe basal lamina. Its β-subunit is localized in the glial end-feet. The investigation period lasted from E(embryonic day)12 to E20. Laminin and β-dystroglycan were detected by immunohistochemistry, the glial localization of the latter one was supported by electron microscopy. The immatureglial structures were visualized by the immunostaining of nestin. The β-dystroglycan immunoreactivity appeared at E16 following the laminin of basal lamina but preceding the perivascular processes of radial glia (E18) and astrocyte-like cells (E20). It occurred in cell bodies which attached to the vessels directly but not with vascular processes and end-feet. The presence of β-dystroglycan in such immature cells may promote their differentiation to perivascular astrocytes and influence the formation of the glio-vascular processes.


Metabolic alterations derived from absence of Two-Pore Channel 1 at cardiac level.
Two-pore channels (TPCs or TPCNs) are novel voltage-gated ion channels that have been postulated to act as Ca2+ and/or Na+ channels expressed exclusively in acidic organelles such as endosomes and lysosomes. TPCNs participate in the regulation of diverse biological processes and recently have been proposed to be involved in the pathophysiology of metabolic disorders such as obesity, fatty liver disease and type 2 diabetes mellitus. Due to the importance of these pathologies in the development of cardiovascular diseases, we aimed to study the possible role of two-pore channel 1 (TPCN1) in the regulation of cardiac metabolism. To explore the cardiac function of TPCN1, we developed proteomic approaches as 2-DE-MALDI-MS and LC-MALDI-MS in the cardiac left ventricle of TPCN1 KO and WT mice, and found alterations in several proteins implicated in glucose and fatty acid metabolism in TPCN1 KO vs. WT mice. The results confirmed the altered expression of HFABP, a key fatty acid transport protein, and of enolase and PGK1, the key enzymes in the glycolytic process. Finally, in vitro experiments performed in neonatal rat cardiomyocytes, in which TPCN1 was silenced using siRNAs, confirmed that the downregulation of TPCN1 gene expression increased 2-deoxy-D-[3H]-glucose uptake and GLUT4 mobilization into cell peripherals in cardiac cells. Our results are the first to suggest a potential role for TPCNs in cardiac metabolism regulation.


Antibiotic resistance plasmids spread among natural isolates of Escherichia coli in spite of CRISPR elements.
Clustered, regularly interspaced, short palindromic repeats (CRISPRs) are implicated in defence against foreign DNA in various archaeal and bacterial species. They have also been associated with a slower spread of antibiotic resistance. However, experimental and evolutionary studies raise doubts about the role of CRISPRs as a sort of immune system in Escherichia coli. We studied a collection of 263 natural E. coli isolates from human and animal hosts, representative of the phylogenetic and lifestyle diversity of the species and exhibiting various levels of plasmid-encoded antibiotic resistance. We characterized the strains in terms of CRISPRs, performed replicon typing of the plasmids and tested for class 1 integrons to explore the possible association between CRISPRs and the absence of plasmids and mobile antibiotic resistance determinants. We found no meaningful association between the presence/absence of the cas genes, reflecting the activity of the CRISPRs, and the presence of plasmids, integrons or antibiotic resistance. No CRISPR in the collection contained a spacer that matched an antibiotic resistance gene or element involved in antibiotic resistance gene mobilization, and 79.8% (210/263) of the strains lacked spacers matching sequences in the 2282 plasmid genomes available. Hence, E. coli CRISPRs do not seem to be efficient barriers to the spread of plasmids and antibiotic resistance, consistent with what has been reported for phages, and contrary to reports concerning other species.


Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure.
Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.


GaN-based ultraviolet light-emitting diodes with AuCl₃-doped graphene electrodes.
We demonstrate AuCl3-doped graphene transparent conductive electrodes integrated in GaN-based ultraviolet (UV) light-emitting diodes (LEDs) with an emission peak of 363 nm. AuCl3 doping was accomplished by dipping the graphene electrodes in 5, 10 and 20 mM concentrations of AuCl3 solutions. The effects of AuCl3 doping on graphene electrodes were investigated by current-voltage characteristics, sheet resistance, scanning electron microscope, optical transmittance, micro-Raman scattering and electroluminescence images. The optical transmittance was decreased with increasing the AuCl3 concentrations. However, the forward currents of UV LEDs with p-doped (5, 10 and 20 mM of AuCl3 solutions) graphene transparent conductive electrodes at a forward bias of 8 V were increased by ~48, 63 and 73%, respectively, which can be attributed to the reduction of sheet resistance and the increase of work function of the graphene. The performance of UV LEDs was drastically improved by AuCl3 doping of graphene transparent conductive electrodes.


A Longitudinal Investigation of Empathic Exchanges in Online Cancer Support Groups: Message Reception and Expression Effects on Patients' Psychosocial Health Outcomes.
Recent studies have devoted attention to the effects of both expression and reception in communication process. However, there remain both theoretical and methodological complexities concerning whether and under what condition message expression and reception play significant but different roles in explaining various psychosocial health outcomes. Relying on theoretical insights from the social support literature and methodological innovations offered by computational social science, this study aims to examine the effects of empathic exchanges on cancer patient's short- and long-term psychosocial health outcomes. Our findings suggest that both empathy expression and reception are crucial to attaining benefits for cancer patients, each predicting differential cognitive and affective health outcomes. Further, our finding supports the stress-buffering hypothesis such that empathy reception provides a beneficial effect for patients who experienced a higher degree of depression associated with their cancer diagnosis and follow-up treatments.


Microfracture in Hip Arthroscopy. Keep It Simple!
Despite all the advances in hip arthroscopy, microfracture is still the workhorse for treating focal and full-thickness cartilage lesions. The success of this treatment is owed to its reliability and simplicity. Given the structure of the hip joint, however, there are challenges to this procedure using a conventional microfracture pick. This note presents our current and preferred microfracture technique using a curve drill guide and flexible drill. This method offers greater range of access to different regions of the joint with ease, thus ensuring a reproducible and quicker procedure with less risk.


Mother's postnatal stress: an investigation of links to various factors during pregnancy and post-partum.
Higher levels of parental stress have long-term effects on children's health and could lead to dysfunction in the parent-child interaction. Different background factors can be predictors of high parental stress. The aim of this study was to examine parental stress among Swedish women and identify different factors linked to women's parental stress. About 702 women were recruited to a clinical study and followed up six months after birth. Data were collected by two questionnaires, and 279 women completed the Swedish Parental Stress Questionnaire (SPSQ). Less than very good mental health and depressive symptoms after birth were strongly associated with parental stress, and the strongest association was found between post-partum depressive symptoms and high levels of stress in the subscale Incompetence. Multiparity was associated with high stress in two subscales, and lower level of education was a protective factor for stress in nearly all subscales. Depressive symptoms and perceived poor mental health post-partum are the most important factors related to high parental stress. The results point to the importance of identifying and supporting mothers with depressive symptoms, since these women have both mental illness and increased stress.


Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure.
Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.


Effectors of Filamentous Plant Pathogens: Commonalities amid Diversity.
Fungi and oomycetes are filamentous microorganisms that include a diversity of highly developed pathogens of plants. These are sophisticated modulators of plant processes that secrete an arsenal of effector proteins to target multiple host cell compartments and enable parasitic infection. Genome sequencing revealed complex catalogues of effectors of filamentous pathogens, with some species harboring hundreds of effector genes. Although a large fraction of these effector genes encode secreted proteins with weak or no sequence similarity to known proteins, structural studies have revealed unexpected similarities amid the diversity. This article reviews progress in our understanding of effector structure and function in light of these new insights. We conclude that there is emerging evidence for multiple pathways of evolution of effectors of filamentous plant pathogens but that some families have probably expanded from a common ancestor by duplication and diversification. Conserved folds, such as the oomycete WY and the fungal MAX domains, are not predictive of the precise function of the effectors but serve as a chassis to support protein structural integrity while providing enough plasticity for the effectors to bind different host proteins and evolve unrelated activities inside host cells. Further effector evolution and diversification arise via short linear motifs, domain integration and duplications, and oligomerization.


Stiffness of the Gastrocnemius-Achilles Tendon Complex Between Amateur Basketball Players and the Non-athletic General Population.
Muscle and tendon stiffness are related to sports performance, tendinopathy, and tendon degeneration. However, the effects of habitual loading on muscle and tendon mechanical properties are unclear. Using amateur basketball players as examples, we investigated the effects of mechanical loading on the stiffness of the gastrocnemius-Achilles tendon (AT) complex in non-dominant and dominant lower limbs. Then, we evaluated the correlation between gastrocnemius and AT stiffness. Forty participants (20 amateur basketball players; 20 normal non-athletic persons) were recruited for this study. Stiffness of the gastrocnemius-AT complex was assessed using MyotonPRO at neutral position and 10° dorsiflexion of the ankle joint in participants from amateur basketball players and the non-athletic general population. Our results showed a greater stiffness of the gastrocnemius-AT complex in amateur basketball players than that in healthy non-athletic subjects at neutral position and 10° dorsiflexion of the ankle joint (P < 0.05). No significant difference in stiffness was found between the non-dominant and dominant lower limbs either in amateur basketball players or in generally healthy subjects (P > 0.05). A significant positive correlation was obtained between stiffness of the AT and medial gastrocnemius (MG) in amateur basketball players (neutral position: r = 0.726 and P = 0.001; dorsiflexion 10°: r = 0.687 and P = 0.001). The amateur basketball players exhibit significantly higher stiffness value in Achilles and gastrocnemius. This is possibly caused by repeated training effects. The symmetric stiffness of the AT and gastrocnemius exists both in amateur basketball players and generally healthy subjects. A significant correlation between the AT and the MG was found in amateur basketball players.


Need analysis for a new high acuity medical psychiatry unit: which patients are considered for admission?
The study aims were: to estimate the proportion of patients with an indication for admission to a new high acuity Medical Psychiatric Unit (MPU), to explore the reasons for MPU-admission according to different health disciplines, and to check for differences in patient characteristics. The results of this study are to be utilized in the proposed establishment of a high-acuity MPU in a University Medical Center. Such a unit currently does not exist at Erasmus MC. Hospital in-patients were included if they received psychiatric consultation from the Psychiatric Consultative Service (PCS). As part of the study protocol, psychiatrists, other medical specialists, and nurses determined the need for admission to the proposed MPU. Patient groups were compared with respect to diagnoses, socio-demographic characteristics and patient routing. One hundred and fifty-one patients were included, 43% had an indication for MPU-admission, for the other patients PCS involvement was sufficient. There was agreement on suicide attempts as a reason for MPU-admission. For psychiatrists, the need for further diagnostic evaluation was a common reason for MPU admission, while other medical specialists more often emphasized the need for safety measures. Patients with an unplanned hospital admission had a higher chance of MPU eligibility (OR = 2.72, 95% CI 1.10-6.70). The main psychiatric diagnoses of MPU-eligible patients were organic disorders (including delirium), mood disorders, and disorders related to substance abuse. The most common diagnoses found were similar to those in previous research on MPU populations. Different medical disciplines have different views on the advantages of MPUs, while all see the need for such facilities. The proposed MPU should be able to accommodate patients directly from the Emergency Unit, and the MPU should provide specialized diagnostic care in an extra safe environment.


Overexpression of c-jun, junB, or junD affects cell growth differently.
The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.


Mimicking the hierarchical functions of dentin collagen cross-links with plant derived phenols and phenolic acids.
Proanthocyanidins (PACs) are secondary plant metabolites that mediate nonenzymatic collagen cross-linking and enhance the properties of collagen based tissue, such as dentin. The extent and nature of cross-linking is influenced by the composition and specific chemical structure of the bioactive compounds present in certain PAC-rich extracts. This study investigated the effect of the molecular weight and stereochemistry of polyphenol compounds on two important properties of dentin, biomechanics, and biostability. For that, purified phenols, a phenolic acid, and some of its derivatives were selected: PAC dimers (A1, A2, B1, and B2) and a trimer (C1), gallic acid (Ga), its esters methyl-gallate (MGa) and propyl-gallate (PGa), and a pentagalloyl ester of glucose (PGG). Synergism was assessed by combining the most active PAC and gallic acid derivative. Mechanical properties of dentin organic matrix were determined by the modulus of elasticity obtained in a flexural test. Biostability was evaluated by the resistance to collagenase degradation. PACs significantly enhanced dentin mechanical properties and decreased collagen digestion. Among the gallic acid derivatives, only PGG had a significant enhancing effect. The lack of observed C1:PGG synergy indicates that both compounds have similar mechanisms of interaction with the dentin matrix. These findings reveal that the molecular weight of polyphenols have a determinant effect on their interaction with type I collagen and modulates the mechanism of cross-linking at the molecular, intermolecular, and inter-microfibrillar levels.


Development of multiorgan pathology in the wpk rat model of polycystic kidney disease.
Rodent models of polycystic kidney disease (PKD) have provided valuable insight into the cellular changes associated with cystogenesis in humans. The present study characterizes the morphology of renal and extrarenal pathology of autosomal recessive PKD induced by the wpk gene in Wistar rats. In wpk(-/-) rats, proximal tubule and collecting duct cysts develop in utero and eventually consume the kidney. Increased apoptosis, mitosis, and extracellular tenascin deposition parallel cyst development. Extrarenal pathology occurs in the immune system (thymic and splenic hypoplasia) and central nervous system (CNS; hypoplasia to agenesis of the corpus callosum with severe hydrocephalus). Severity of hydrocephalus varied inversely with size of the corpus callosum. In wpk(-/-) rats, the corpus callosum exhibits relatively few axons that cross the midline. This CNS pathology is similar to that described in three human renal cystic syndromes: orofaciodigital, genitopatellar, and cerebrorenal-digital syndromes. Collecting duct and ventricular ependymal cilia appear morphologically normal. To determine if rodent background strain and the presence of modifier genes affect severity of the disease, we crossed the Wistar-wpk rat with Brown Norway (BN) and Long Evan (LE) rats and found the degree of renal and cerebral pathology was diminished as evidenced by lower kidney weight as a percent of body weight and serum urea nitrogen concentration in cystic rats on LE or BN strains as well as less prominent cranial enlargement. Crosses with BN rats allowed us to localize the wpk gene on chromosome 5 very close to the D5Rat73 marker. The wpk gene lies within a chromosomal region known to harbor a PKD modifier locus. In summary, the types of renal and cerebral pathology seen in the Wistar wpk rat are a unique combination seen only in this rodent model.


Artificial intelligence to detect MYC translocation in slides of diffuse large B-cell lymphoma.
In patients with suspected lymphoma, the tissue biopsy provides lymphoma confirmation, classification, and prognostic factors, including genetic changes. We developed a deep learning algorithm to detect MYC rearrangement in scanned histological slides of diffuse large B-cell lymphoma. The H&E-stained slides of 287 cases from 11 hospitals were used for training and evaluation. The overall sensitivity to detect MYC rearrangement was 0.93 and the specificity 0.52, showing that prediction of MYC translocation based on morphology alone was possible in 93% of MYC-rearranged cases. This would allow a simple and fast prescreening, saving approximately 34% of genetic tests with the current algorithm.


Molecular adaptation during adaptive radiation in the Hawaiian endemic genus Schiedea.
"Explosive" adaptive radiations on islands remain one of the most puzzling evolutionary phenomena. The rate of phenotypic and ecological adaptations is extremely fast during such events, suggesting that many genes may be under fairly strong selection. However, no evidence for adaptation at the level of protein coding genes was found, so it has been suggested that selection may work mainly on regulatory elements. Here we report the first evidence that positive selection does operate at the level of protein coding genes during rapid adaptive radiations. We studied molecular adaptation in Hawaiian endemic plant genus Schiedea (Caryophyllaceae), which includes closely related species with a striking range of morphological and ecological forms, varying from rainforest vines to woody shrubs growing in desert-like conditions on cliffs. Given the remarkable difference in photosynthetic performance between Schiedea species from different habitats, we focused on the "photosynthetic" Rubisco enzyme, the efficiency of which is known to be a limiting step in plant photosynthesis. We demonstrate that the chloroplast rbcL gene, encoding the large subunit of Rubisco enzyme, evolved under strong positive selection in Schiedea. Adaptive amino acid changes occurred in functionally important regions of Rubisco that interact with Rubisco activase, a chaperone which promotes and maintains the catalytic activity of Rubisco. Interestingly, positive selection acting on the rbcL might have caused favorable cytotypes to spread across several Schiedea species. We report the first evidence for adaptive changes at the DNA and protein sequence level that may have been associated with the evolution of photosynthetic performance and colonization of new habitats during a recent adaptive radiation in an island plant genus. This illustrates how small changes at the molecular level may change ecological species performance and helps us to understand the molecular bases of extremely fast rate of adaptation during island adaptive radiations.


Functional analysis tools for post-translational modification: a post-translational modification database for analysis of proteins and metabolic pathways.
Post-translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high-resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post-Translational Modifications (FAT-PTM) database (https://bioinformatics.cse.unr.edu/fat-ptm/), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large-scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT-PTM database currently supports tools to visualize protein-centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein-centric metabolic pathways and groups of proteins that are co-modified by multiple PTMs. Overall, the FAT-PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.


In vitro evaluation of a 'stealth' adenoviral vector for targeted gene delivery to adult mammalian neurones.
Polymer coating of adenovirus type 5 (Ad5) particles produces a 'stealth' Ad5 (sAd5) that confers protection from immune recognition, blocks receptor-mediated uptake, and favours uptake into pinocytic cells. In mixed cultures of primary adult rat dorsal root ganglion neurones (DRGN), rat C6 glioma cells, A9 non-Coxsackie and Ad Receptor (CAR)- and CAR-expressing fibroblasts, reporter gene expression after sAd5 pinocytotic uptake was monitored using the green fluorescent protein (gfp) gene, and viral particle trafficking and polymer coat dismantling was followed using Yoyo-1 tagged Ad5 DNA and Texas Red (TR) to label the coat. sAd5.gfp was pinocytosed by significantly higher proportions of neurones, than other cells, but GFP was not expressed. The TR-labelled coat remained co-localised with tagged viral DNA within transfected DRGN, showing that sAd5 did not uncoat and viral DNA did not traffic to the nucleus. Noncoated Ad5 transduced non-neuronal DRG cells more efficiently than DRGN, whereas A9(CAR) cells were more significantly transduced than any other cell type. Retargeting of the sAd5.gfp with either fibroblast growth factor-2 or nerve growth factor (NGF) enhanced internalisation by DRGN into endocytic vesicles allowing uncoating and thus GFP expression. Retargeting with NGF resulted in significantly higher numbers of DRGN expressing GFP than non-neuronal DRG cells. These findings indicate that DRGN pinocytose atropic genetic particles at higher levels than non-neuronal DRG cells and the environment of pinocytic vesicles is not conducive to sAd5 uncoating and capsid dismantling, requiring reformulation of sAd5 with either a neurone specific ligand or a self-dismantling coat to target sAd5 transgene expression to neurones.


Duplicated leptin receptors in two species of eel bring new insights into the evolution of the leptin system in vertebrates.
Since its discovery in mammals as a key-hormone in reproduction and metabolism, leptin has been identified in an increasing number of tetrapods and teleosts. Tetrapods possess only one leptin gene, while most teleosts possess two leptin genes, as a result of the teleost third whole genome duplication event (3R). Leptin acts through a specific receptor (LEPR). In the European and Japanese eels, we identified two leptin genes, and for the first time in vertebrates, two LEPR genes. Synteny analyses indicated that eel LEPRa and LEPRb result from teleost 3R. LEPRb seems to have been lost in the teleost lineage shortly after the elopomorph divergence. Quantitative PCRs revealed a wide distribution of leptins and LEPRs in the European eel, including tissues involved in metabolism and reproduction. Noticeably, leptin1 was expressed in fat tissue, while leptin2 in the liver, reflecting subfunctionalization. Four-month fasting had no impact on the expression of leptins and LEPRs in control European eels. This might be related to the remarkable adaptation of silver eel metabolism to long-term fasting throughout the reproductive oceanic migration. In contrast, sexual maturation induced differential increases in the expression of leptins and LEPRs in the BPG-liver axis. Leptin2 was strikingly upregulated in the liver, the central organ of the reproductive metabolic challenge in teleosts. LEPRs were differentially regulated during sexual maturation, which may have contributed to the conservation of the duplicated LEPRs in this species. This suggests an ancient and positive role of the leptin system in the vertebrate reproductive function. This study brings new insights on the evolutionary history of the leptin system in vertebrates. Among extant vertebrates, the eel represents a unique case of duplicated leptins and leptin receptors as a result of 3R.


The Free-movement pattern Y-maze: A cross-species measure of working memory and executive function.
Numerous neurodegenerative and psychiatric disorders are associated with deficits in executive functions such as working memory and cognitive flexibility. Progress in developing effective treatments for disorders may benefit from targeting these cognitive impairments, the success of which is predicated on the development of animal models with validated behavioural assays. Zebrafish offer a promising model for studying complex brain disorders, but tasks assessing executive function are lacking. The Free-movement pattern (FMP) Y-maze combines aspects of the common Y-maze assay, which exploits the inherent motivation of an organism to explore an unknown environment, with analysis based on a series of sequential two-choice discriminations. We validate the task as a measure of working memory and executive function by comparing task performance parameters in adult zebrafish treated with a range of glutamatergic, cholinergic and dopaminergic drugs known to impair working memory and cognitive flexibility. We demonstrate the cross-species validity of the task by assessing performance parameters in adapted versions of the task for mice and Drosophila, and finally a virtual version in humans, and identify remarkable commonalities between vertebrate species' navigation of the maze. Together, our results demonstrate that the FMP Y-maze is a sensitive assay for assessing working memory and cognitive flexibility across species from invertebrates to humans, providing a simple and widely applicable behavioural assay with exceptional translational relevance.


Modulation of cell surface expression of nonactivated cholecystokinin receptors using bivalent ligand-induced internalization.
CCK(2) receptor antagonists potentiate pain relief by MOP receptor agonists. In an attempt to enhance this effect, we prepared bivalent ligands incorporating CCK(2) receptor antagonist and MOP receptor agonist pharmacophores. (9) Ligands with 16- to 22-atom spacers could simultaneously bind both receptors but provided no advantage in activity over individual ligands. We now examine the effect of these ligands on receptor internalization as a mechanism of receptor regulation. We prepared CHO cell lines expressing nonfluorescent halves (YN and YC) of yellow fluorescent protein attached to each receptor. Spatial approximation of constructs was needed to yield fluorescence. Monovalent MOP agonist 1 signaled normally and internalized the MOP receptor. Monovalent CCK(2) antagonist 2 did not stimulate receptor internalization. In the dual receptor-bearing cells, bivalent ligands 3a-c capable of simultaneously binding both receptors resulted in cell surface fluorescence and internalization of the fluorescent complex in a time- and temperature-dependent manner. Bivalent ligand 4 with spacer too short to occupy both receptors simultaneously yielded no signal. Receptor tethering with appropriate bivalent ligands can down-regulate signaling by moving a nonactivated receptor into the endocytic pathway.


Consumer preferences for household water treatment products in Andhra Pradesh, India.
Over 5 billion people worldwide are exposed to unsafe water. Given the obstacles to ensuring sustainable improvements in water supply infrastructure and the unhygienic handling of water after collection, household water treatment and storage (HWTS) products have been viewed as important mechanisms for increasing access to safe water. Although studies have shown that HWTS technologies can reduce the likelihood of diarrheal illness by about 30%, levels of adoption and continued use remain low. An understanding of household preferences for HWTS products can be used to create demand through effective product positioning and social marketing, and ultimately improve and ensure commercial sustainability and scalability of these products. However, there has been little systematic research on consumer preferences for HWTS products. This paper reports the results of the first state-of-the-art conjoint analysis study of HWTS products. In 2008, we conducted a conjoint analysis survey of a representative sample of households in Andhra Pradesh (AP), India to elicit and quantify household preferences for commercial HWTS products. Controlling for attribute non-attendance in an error components mixed logit model, the study results indicate that the most important features to respondents, in terms of the effect on utility, were the type of product, followed by the extent to which the product removes pathogens, the retail outlet and, the time required to treat 10 L. Holding all other product attributes constant, filters were preferred to combination products and chemical additives. Department stores and weekly markets were the most favorable sales outlets, followed by mobile salespeople. In general, households do not prefer to purchase HWTS products at local shops. Our results can inform the types of products and sales outlets that are likely to be successful in commercial HWTS markets in AP, as well as the influence of different pricing and financing strategies on product demand and uptake.


SHORT-TERM PERCEPTUAL TUNING TO TALKER CHARACTERISTICS.
When a listener encounters an unfamiliar talker, the ensuing perceptual accommodation to the unique characteristics of the talker has two aspects: (1) the listener assesses acoustic characteristics of speech to resolve the properties of the talker's sound production; and, (2) the listener appraises the talker's idiolect, subphonemic phonetic properties that compose the finest grain of linguistic production. A new study controlled a listener's exposure to determine whether the perceptual benefit rests on specific segmental experience. Effects of sentence exposure were measured using a spoken word identification task of Easy words (likely words drawn from sparse neighborhoods of less likely words) and Hard words (less likely words drawn from dense neighborhoods of more likely words). Recognition of words was facilitated by exposure to voiced obstruent consonants. Overall, these findings indicate that talker-specific perceptual tuning might depend more on exposure to phonemically marked consonants than to exposure distributed across the phoneme inventory.