A prospective study of pyrogenic reactions in hemodialysis patients using bicarbonate dialysis fluids filtered to remove bacteria and endotoxin.
Pyrogenic reactions (PR) are a well-recognized complication of hemodialysis and have been associated with dialyzer reuse, high-flux dialysis, and bicarbonate dialysate. However, the roles of bacteria and endotoxin in dialysate for producing PR are not well defined. To determine the effect of removing most bacteria and endotoxin from the dialysate on the incidence of PR, a cohort of chronic hemodialysis patients receiving high-flux, high-efficiency, or conventional hemodialysis at three centers with bicarbonate dialysis fluids that had been filtered with a polysulfone high-flux hemodialyzer was prospectively studied. Unfiltered bicarbonate concentrate had median bacterial and endotoxin concentrations of 479,000 CFU/mL and 39,800 pg/mL, respectively. After filtration of the bicarbonate concentrate at the central proportioner, dialysate had a median 9.2 CFU/mL of bacteria and 17.8 pg/mL of endotoxin. Dialysate filtered at individual proportioning dialysis machines had a median 0.001 CFU/mL of bacteria and 0.19 pg/mL of endotoxin. Nine PR were identified among 303 patients after 28,007 hemodialysis treatments (0.3 PR/1,000 treatments). The rate of PR was similar for the three hemodialysis treatment modalities and for first-use compared with reused dialyzers. Although the PR rate in this study was lower (P = 0.046) than the PR rate of a previous study with unfiltered dialysis fluids (0.7 PR/1,000 treatments), it represents a difference of only 10 PR in over 28,000 treatments. It was concluded that filtration of hemodialysis fluids is efficacious in removing bacterial and endotoxin contamination and can result in a lower incidence of PR in patients receiving high-flux, high-efficiency, or conventional hemodialysis.


The MDS Mortality Risk Index: The evolution of a method for predicting 6-month mortality in nursing home residents.
Accurate prognosis is vital to the initiation of advance care planning particularly in a vulnerable, at risk population such as care home residents. The aim of this paper is to report on the revision and simplification of the MDS Mortality Rating Index (MMRI) for use in clinical practice to predict the probability of death in six months for care home residents. The design was a secondary analysis of a US Minimum Data Set (MDS) for long term care residents using regression analysis to identify predictors of mortality within six months. Using twelve easy to collect items, the probability of mortality within six months was accurately predicted within the MDS database. The items are: admission to the care home within three months; lost weight unintentionally in past three months; renal failure; chronic heart failure; poor appetite; male; dehydrated; short of breath; active cancer diagnosis; age; deteriorated cognitive skills in past three months; activities of daily living score. A lack of recognition of the proximity of death is often blamed for inappropriate admission to hospital at the end of an older person's life. An accurate prognosis for older adults living in a residential or nursing home can facilitate end of life decision making and planning for preferred place of care at the end of life. The original MMRI was derived and validated from a large database of long term care residents in the USA. However, this simplification of the revised index (MMRI-R) may provide a means for facilitating prognostication and end of life discussions for application outside the USA where the MDS is not in use. Prospective testing is needed to further test the accuracy of the MMRI-R and its application in the UK and other non-MDS settings.


Novel synthetic benzimidazole-derived oligosaccharide, M3BIM, prevents ex vivo platelet aggregation and in vivo thromboembolism.
Thrombus formation, a phenomenon primarily related to increased platelet activation, plays a key role in cardiovascular and cerebrovascular diseases. Although the established antiplatelet agents, such as aspirin and clopidogrel, have been shown to be beneficial in treating thromboembolic diseases, they have considerable limitations. Hence, the development of more effective and safe antithrombotic agents is necessary to satisfy a substantial unmet clinical need. In recent years, the favorable properties of imidazole-related drugs have prompted medicinal chemists to synthesize numerous novel therapeutic agents. The chemical structure of the benzimidazole backbone has proven antiplatelet properties. Moreover, synthetic oligosaccharides have exhibited antiplatelet properties. Therefore, we developed a new aldo-benzimidazole-derived oligosaccharide compound, M3BIM, for achieving a stronger antiplatelet effect than the drugs which are being used in clinical aspects. We investigated the effects of M3BIM on platelet activation ex vivo and its antithrombotic activity in vivo. M3BIM (10-50 μM) exhibited a more potent activity in inhibiting platelet aggregation stimulated by collagen than it did in inhibiting that stimulated by thrombin in washed human platelets. The M3BIM treatment revealed no cytotoxicity in zebrafish embryos, even at the highest concentration of 100 μM. In addition, M3BIM inhibited the phosphorylation of phospholipase Cγ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 2 and c-Jun N-terminal kinase 1), and markedly reduced the ATP-release reaction and intracellular calcium mobilization in collagen-activated platelets. By contrast, M3BIM showed no effects on either collagen-induced p38 MAPK and Akt phosphorylation or phorbol 12, 13-dibutyrate-induced PKC activation and platelet aggregation. Moreover, the M3BIM treatment substantially prolonged the closure time in human whole blood, and increased the occlusion time in mesenteric microvessels and attenuated cerebral infarction in mice. For the study of anticoagulant activities, M3BIM showed no significant effects in the prolongation of activated partial thromboplastin time and prothrombin time in mice. The findings of our study suggest that M3BIM is a potential therapeutic agent for preventing or treating thromboembolic disorders.


The nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes.
The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha 1, alpha 2, and alpha 3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZR alpha and RZR beta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZR beta is expressed, but also in peripheral tissues, where RZR alpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZR alpha and ROR alpha 1, but not ROR alpha 2 and alpha 3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZR alpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZR alpha. This indicates that 5-lipoxygenase is the first natural RZR alpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.


Living arrangements, knowledge of health risks, and stress as determinants of health-risk behavior among college students.
The association of knowledge of health risks, living arrangements, and perceived stress with health-risk behaviors was examined in a sample of college students included in the Health Promotion and Disease Prevention Supplement of the National Health Interview Survey. Regressions of each health-risk behavior (dependent variable) were performed on the predicted correlates. Although knowledge was not associated with participation in physical activity or smoking, the study found that students who knew more about the harmful effects of alcohol drank less, and those with greater knowledge of health risks practiced fewer risky behaviors. Students living independently were more likely to smoke, and those living in residence halls were less like to do so. Drinking, however, was more common among students living in residence halls or independently than among those living at home. Hall residents engaged in more group physical activity than other students did, but their physical activity was unrelated to health-risk behaviors. Stress was associated with smoking but not with other health practices. The findings suggest that smoking may be less influenced by health knowledge and more associated than drinking is with a response to stress. Drinking appears to be a social activity associated with living among peers and is potentially modifiable by increased knowledge about the effects of alcohol on health.


Can They Stay or Will They Go? A Cross Sectional Study of Managers' Attitudes towards Their Senior Employees.
A larger amount of older people need to participate in working life due to the global demographic change. It is the employer, through the manager, who enables employees to have access to measures in the workplace that facilitate and enable a sustainable extended working life. The aim of this study was to evaluate work life factors associated with managers believing their employees can work versus wanting to work until age 65 or older. This cross-sectional study included 249 managers in the Swedish municipality sector. Logistic regression analysis was used to investigate associations between different univariate estimates and in data modelling using the SwAge-model. The result stated that 79% of managers believed their employees 'can' work and 58% of managers believed their employees 'want to' work until age 65 or older. Health, physical work environment, skills and competence are associated the strongest to managers believing employees 'can' work until age 65 or older. Insufficient social support at work and lacking possibilities for relocations associated the strongest to managers believing employees would not 'want to' work until age 65 or older. Though, several countries (especially in Europe) have included in their social policy measures that retirement age be increased after 65, proposing ages approaching 70. When these proposals become laws, through obligation, people will have no choice (if they want to or if they can continue working). However, people's attitudes to work may be different (especially after the COVID-19 pandemic), and this analysis of the participating managers' attitudes showed there is a difference between why employees 'can' versus 'want' to work respectively. Therefore, different strategies may be needed to contribute to employees both being able to and willing to participate in working life until an older age. These findings on managers' perspectives, regarding whether they believe employees would be able to versus would want to work and the SwAge-model, will hopefully contribute to an increased understanding of organisational actions and measures in the process of creating a sustainable extended working life and to increase senior employees' employability.


A trial of prophylactic replacement therapy in haemophilia and Christmas disease.
A trial of prophylactic replacement therapy in low dosage once a week is described in two patients with classical haemophilia and one patient with Christmas disease, using concentrates of factor VIII and factor IX respectively. The clinical effectiveness and complications of the therapy are assessed and discussed. It was concluded that the patient suffering from Christmas disease showed both objective and subjective improvement while on prophylaxis and merited further study. Neither of the patients with haemophilia showed objective evidence of improvement, but both stated that they felt fitter and more able to lead a normal life while on the trial.


Telemedicine from research to practice during the pandemic. "Instant paper from the field" on rehabilitation answers to the COVID-19 emergency.
COVID-19 pandemic is creating collateral damage to outpatients, whose rehabilitation services have been disrupted in most of the European countries. Telemedicine has been advocated as a possible solution. This paper reports the contents of the third Italian Society of Physical and Rehabilitation Medicine (SIMFER) webinar on "experiences from the field" COVID-19 impact on rehabilitation ("Covinars"). It provides readily available, first-hand information about the application of telemedicine in rehabilitation. The experiences reported were very different for population (number and health conditions), interventions, professionals, service payment, and technologies used. Commonalities included the pushing need due to the emergency, previous experiences, and a dynamic research and innovation environment. Lights included feasibility, results, reduction of isolation, cost decrease, stimulation to innovation, satisfaction of patients, families, and professionals beyond the starting diffidence. Shadows included that telemedicine can integrate but will never substitute face-to-face rehabilitation base on the encounter among human beings; age, and technology barriers (devices absence, bad connection and human diffidence) have also been reported. Possible issues included privacy and informed consent, payments, cultural difficulties in understanding that telemedicine is a real rehabilitation intervention. There was a final agreement that this experience will be incorporated by participants in their future services: technology is ready, but the real challenge is to change PRM physicians' and patients' habits, while better specific regulation is warranted.


Comparison of metal ion levels in patients with hip resurfacing versus total hip arthroplasty.
Mechanical wear at the articular surface and corrosive processes at modular junctions, such as the trunnion, are responsible for metal ions production. We retrospectively reviewed 64 patients who underwent THA with a metal on metal bearing surface and 34 patients with hip Resurfacing. Metal ion measurements, six-week post-op radiographs, and functional scores were included in the analysis. Cobalt ion levels were significantly elevated in THA patients, 2.95 μg/L as compared to resurfacing patients, (2.95 versus 1.30 μg/L, p < 0.0005). Chromium levels were not significantly different between THA patients and resurfacing patients (1.05 versus 1.00 μg/L, p = 0.529).


An integrated approach to historical population assessment of the great whales: case of the New Zealand southern right whale.
Accurate estimation of historical abundance provides an essential baseline for judging the recovery of the great whales. This is particularly challenging for whales hunted prior to twentieth century modern whaling, as population-level catch records are often incomplete. Assessments of whale recovery using pre-modern exploitation indices are therefore rare, despite the intensive, global nature of nineteenth century whaling. Right whales (Eubalaena spp.) were particularly exploited: slow swimmers with strong fidelity to sheltered calving bays, the species made predictable and easy targets. Here, we present the first integrated population-level assessment of the whaling impact and pre-exploitation abundance of a right whale, the New Zealand southern right whale (E. australis). In this assessment, we use a Bayesian population dynamics model integrating multiple data sources: nineteenth century catches, genetic constraints on bottleneck size and individual sightings histories informing abundance and trend. Different catch allocation scenarios are explored to account for uncertainty in the population's offshore distribution. From a pre-exploitation abundance of 28 800-47 100 whales, nineteenth century hunting reduced the population to approximately 30-40 mature females between 1914 and 1926. Today, it stands at less than 12% of pre-exploitation abundance. Despite the challenges of reconstructing historical catches and population boundaries, conservation efforts of historically exploited species benefit from targets for ecological restoration.


Exposure science in an age of rapidly changing climate: challenges and opportunities.
Climate change is anticipated to alter the production, use, release, and fate of environmental chemicals, likely leading to increased uncertainty in exposure and human health risk predictions. Exposure science provides a key connection between changes in climate and associated health outcomes. The theme of the 2015 Annual Meeting of the International Society of Exposure Science-Exposures in an Evolving Environment-brought this issue to the fore. By directing attention to questions that may affect society in profound ways, exposure scientists have an opportunity to conduct "consequential science"-doing science that matters, using our tools for the greater good and to answer key policy questions, and identifying causes leading to implementation of solutions. Understanding the implications of changing exposures on public health may be one of the most consequential areas of study in which exposure scientists could currently be engaged. In this paper, we use a series of case studies to identify exposure data gaps and research paths that will enable us to capture the information necessary for understanding climate change-related human exposures and consequent health impacts. We hope that paper will focus attention on under-developed areas of exposure science that will likely have broad implications for public health.


Case-control study of paternal occupational exposures and childhood lymphoma in Great Britain, 1962-2010.
This nationwide study investigates associations between paternal occupational exposure and childhood lymphoma. The UK National Registry of Childhood Tumours provided cases of childhood lymphoma born and diagnosed in Great Britain 1962-2010. Control births, unaffected by childhood cancer, were matched on sex, birth period and birth registration sub-district. Fathers' occupations were assigned to one or more of 33 exposure groups and also coded for occupational social class. We analysed 5033 childhood lymphoma cases and 4990 controls. Total lymphoma and the subgroups Hodgkin, Burkitt and non-Hodgkin lymphoma were considered separately. No one exposure was significantly associated with increased risk within all subgroups and for total lymphoma. However, exposure to "ceramics and glass" was significantly associated with increased risk of total lymphoma, Hodgkin and non-Hodgkin lymphoma. Paternal lead exposure was associated with Burkitt lymphoma and exposure to metal fumes was associated with Hodgkin lymphoma. This study provides no support for previous suggestions of an association between childhood lymphoma and paternal occupational exposure to pesticides, solvents/hydrocarbons or infections potentially transmitted by father's social contacts. An association with exposure to "ceramics and glass" was noted for the two major lymphoma subtypes together comprising 80% of total lymphoma.


Effect of polaprezinc on impaired healing of chronic gastric ulcers in adjuvant-induced arthritic rats--role of insulin-like growth factors (IGF)-1.
Polaprezinc, N-(3-aminopropionyl)-L-histidinatozinc, has been shown to stimulate the production of insulin-like growth factor-1 (IGF-1) in mesenchymal cells, the polypeptide playing a role in the gastric epithelial wound repair. The present study was performed to examine the effect of polaprezinc on the impaired healing of chronic gastric ulcers in adjuvant-induced arthritic rats, in relation to IGF-1. Arthritis was induced in male Dark Agouti (DA) rats by a single injection of Freund's complete adjuvant (FCA), and the gastric ulcers were induced by thermal cauterization (70 degrees C for 30 sec) 7 days after FCA injection. Omeprazole (30 mg/kg) was administered p.o. once daily, while recombinant human IGF-1 (rhIGF-1) (30 micrograms/kg, s.c.) or polaprezinc (3-10 mg/kg, p.o.) was administered twice daily, starting from 3 days after ulceration for 14 days. The healing of gastric ulcers was significantly delayed in arthritic rats as compared to normal rats on day 10 and 17 following ulceration. The expression of IGF-1 mRNA was markedly increased in the ulcerated mucosa, but this response was apparently attenuated in arthritic rats. Repeated administration of polaprezinc accelerated the healing of gastric ulcers in both normal and arthritic rats, in a dose-dependent manner, and this effect was more pronounced in arthritic rats. Likewise, treatment with omeprazole also significantly promoted the healing of gastric ulcers in both normal and arthritic rats. On the other hand, rhIGF-1 significantly promoted the gastric ulcer healing in arthritic rats without any effect on that in normal rats. These results suggest that the impaired healing of chronic gastric ulcers in arthritic rats is, at least partly, accounted for by less expression of IGF-1, and the polaprezinc improves the delayed healing of gastric ulcers in arthritic rats, probably through an increase in IGF-1 production.


Lipid and DHA-production in Aurantiochytrium sp. - Responses to nitrogen starvation and oxygen limitation revealed by analyses of production kinetics and global transcriptomes.
Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine n3 fatty acid docosahexaenoic acid (DHA). DHA production in Aurantiochytrium sp T66 was studied with the aim to provide more knowledge about factors that affect the DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis in thraustochytrids, fatty acid synthetase (FAS) and PUFA-synthase. Fermentations with nitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared to fermentations with nitrogen in excess, obtained by oxygen limitation. The specific productivities of fatty acids originating from FAS were considerably higher under nitrogen starvation than with nitrogen in excess, while the specific productivities of DHA were the same at both conditions. Global transcriptome analysis showed significant up-regulation of FAS under N-deficient conditions, while the PUFA-synthase genes were only marginally upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that N-starvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.


Selection maintains MHC diversity through a natural population bottleneck.
A perceived consequence of a population bottleneck is the erosion of genetic diversity and concomitant reduction in individual fitness and evolutionary potential. Although reduced genetic variation associated with demographic perturbation has been amply demonstrated for neutral molecular markers, the effective management of genetic resources in natural populations is hindered by a lack of understanding of how adaptive genetic variation will respond to population fluctuations, given these are affected by selection as well as drift. Here, we demonstrate that selection counters drift to maintain polymorphism at a major histocompatibility complex (MHC) locus through a population bottleneck in an inbred island population of water voles. Before and after the bottleneck, MHC allele frequencies were close to balancing selection equilibrium but became skewed by drift when the population size was critically low. MHC heterozygosity generally conformed to Hardy-Weinberg expectations except in one generation during the population recovery where there was a significant excess of heterozygous genotypes, which simulations ascribed to strong differential MHC-dependent survival. Low allelic diversity and highly skewed frequency distributions at microsatellite loci indicated potent genetic drift due to a strong founder affect and/or previous population bottlenecks. This study is a real-time examination of the predictions of fundamental evolutionary theory in low genetic diversity situations. The findings highlight that conservation efforts to maintain the genetic health and evolutionary potential of natural populations should consider the genetic basis for fitness-related traits, and how such adaptive genetic diversity will vary in response to both the demographic fluctuations and the effects of selection.


Reversibility of retinal ischemia due to central retinal artery occlusion by hyperbaric oxygen.
Ischemic retinal damage can be reversed by hyperbaric oxygen therapy (HBOT) as long as irreversible infarction damage has not developed. However, the time window till irreversible damage develops is still unknown. The study aim was to evaluate the effect of HBOT and determine possible markers for irreversible retinal damage. Retrospective analysis of 225 patients treated with HBOT for central retinal artery occlusion (CRAO) in 1999-2015. One hundred and twenty-eight patients fulfilled inclusion/exclusion criteria: age >18 years, symptoms <20 hours, and best-corrected visual acuity (BCVA) <0.5 logMAR. Time delay from symptoms to treatment was 7.8±3.8 hours. The BCVA was significantly improved after HBOT, from 2.14±0.50 to 1.61±0.78 (P<0.0001). The proportion of patients with clinically meaningful visual improvement was significantly higher in patients without cherry-red spot (CRS) compared to patients with CRS at presentation (86.0% vs 57.6%, P<0.0001). The percentage of patients with final BCVA better than 1.0 was also significantly higher in patients without CRS vs patients with CRS at presentation (61.0% vs 7.1%, P<0.0001). There was no correlation between CRS and the time from symptoms. HBOT was found to be safe, and only 5.5% of patients had minor, reversible, adverse events. HBOT is an effective treatment for non-arteritic CRAO as long as CRS has not formed. The fundus findings, rather than the time delay, should be used as a marker for irreversible damage.


Same or different? A neural circuit mechanism of similarity-based pattern match decision making.
The ability to judge whether sensory stimuli match an internally represented pattern is central to many brain functions. To elucidate the underlying mechanism, we developed a neural circuit model for match/nonmatch decision making. At the core of this model is a "comparison circuit" consisting of two distinct neural populations: match enhancement cells show higher firing response for a match than a nonmatch to the target pattern, and match suppression cells exhibit the opposite trend. We propose that these two neural pools emerge from inhibition-dominated recurrent dynamics and heterogeneous top-down excitation from a working memory circuit. A downstream system learns, through plastic synapses, to extract the necessary information to make match/nonmatch decisions. The model accounts for key physiological observations from behaving monkeys in delayed match-to-sample experiments, including tasks that require more than simple feature match (e.g., when BB in ABBA sequence must be ignored). A testable prediction is that magnitudes of match enhancement and suppression neural signals are parametrically tuned to the similarity between compared patterns. Furthermore, the same neural signals from the comparison circuit can be used differently in the decision process for different stimulus statistics or tasks; reward-dependent synaptic plasticity enables decision neurons to flexibly adjust the readout scheme to task demands, whereby the most informative neural signals have the highest impact on the decision.


The circadian dynamics of small nucleolar RNA in the mouse liver.
The circadian regulation of gene expression allows plants and animals to anticipate predictable environmental changes. While the influence of the circadian clock has recently been shown to extend to ribosome biogenesis, the dynamics and regulation of the many small nucleolar RNA that are required in pre-ribosomal RNA folding and modification are unknown. Using a novel computational method, we show that 18S and 28S pre-rRNA are subject to circadian regulation in a nuclear RNA sequencing time course. A population of snoRNA with circadian expression is identified that is functionally associated with rRNA modification. More generally, we find the abundance of snoRNA known to modify 18S and 28S to be inversely correlated with the abundance of their target. Cyclic patterns in the expression of a number of snoRNA indicate a coordination with rRNA maturation, potentially through an upregulation in their biogenesis, or their release from mature rRNA at the end of the previous cycle of rRNA maturation, in antiphase with the diurnal peak in pre-rRNA. Few cyclic snoRNA have cyclic host genes, indicating the action of regulatory mechanisms in addition to transcriptional activation of the host gene. For highly expressed independently transcribed snoRNA, we find a characteristic RNA polymerase II and H3K4me3 signature that correlates with mean snoRNA expression over the day.


Why should mesenchymal stem cells (MSCs) cure autoimmune diseases?
The adult stem/progenitor cells from bone marrow and other tissues referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) display a significant therapeutic plasticity as reflected by their ability to enhance tissue repair and influence the immune response both in vitro and in vivo. In this review we will focus on the paradigmatic preclinical experience achieved testing MSCs in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. We will emphasize how the paradigm changed over time from the original prediction that MSCs would enhance tissue repair through their transdifferentiation into somatic cells to the current paradigm that they can produce therapeutic benefits without engraftment into the injured tissues. The data will be reviewed in terms of the potentials of MSCs for therapy of autoimmune diseases.


Strawberry disease lesions in rainbow trout from southern Idaho are associated with DNA from a Rickettsia-like organism.
Strawberry disease (SD) in the USA is a skin disorder of unknown etiology that occurs in rainbow trout Oncorhynchus mykiss and is characterized by bright red inflammatory lesions. To identify a candidate bacterial agent responsible for SD, we constructed 16S rDNA libraries from 7 SD lesion samples and 2 apparently healthy skin samples from SD-affected fish. A 16S rDNA sequence highly similar to members of the order Rickettsiales was present in 3 lesion libraries at 1%, 32% and 54% prevalence, but this sequence was not found in either healthy tissue library. Based on phylogenetic analysis, this Rickettsia-like organism (RLO) sequence is most closely related to 16S rDNA sequences of bacteria that may form a novel lineage within the Rickettsiales. We used nested PCR assays to screen 25 SD-affected fish for RLO or Flavobacterium psychrophilum DNA. Sixteen lesion samples were positive for the RLO sequence and 4 of the matched healthy samples were positive resulting in a significant association between SD lesions and presence of RLO DNA. While F. psychrophilum is reportedly associated with 'cold water strawberry disease' in the UK, we found no significant association between SD lesions and the presence of F. psychrophilum DNA. The statistical association between SD lesions and presence of RLO DNA is not proof of etiology, but these data suggest that RLO may play a role in SD in southern Idaho, USA.


Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases.
Most of our current knowledge about cellular phenotypes in neurodevelopmental and neurodegenerative diseases in humans was gathered from studies in postmortem brain tissues. These samples often represent the end-stage of the disease and therefore are not always a fair representation of how the disease developed. Moreover, under these circumstances, the pathology observed could be a secondary effect rather than the authentic disease cellular phenotype. Likewise, the rodent models available do not always recapitulate the pathology from human diseases. In this review, we will examine recent literature on the use of induced pluripotent stem cells to model neurodegenerative and neurodevelopmental diseases. We highlight the characteristics of diseases like spinal muscular atrophy and familial dysautonomia that allowed partial modeling of the disease phenotype. We review human stem cell literature on common neurodegenerative late-onset diseases such as Parkinson's disease and amyotrophic lateral sclerosis where patients' cells have been successfully reprogrammed but a disease phenotype has not yet been described. So far, the technique is of great interest for early onset monogenetic neurodevelopmental diseases. We speculate about potential further experimental requirements and settings for reprogrammed neurons for in vitro disease modeling and drug discovery.


Watershed 'Chemical Cocktails': Forming Novel Elemental Combinations in Anthropocene Fresh Waters.
In the Anthropocene1, watershed chemical transport is increasingly dominated by novel combinations elements, which are hydrologically linked together as 'chemical cocktails.' Chemical cocktails are novel because human activities greatly enhance elemental concentrations and their probability for biogeochemical interactions and shared transport along hydrologic flowpaths. A new chemical cocktail approach advances our ability to: trace contaminant mixtures in watersheds, develop chemical proxies with high-resolution sensor data, and manage multiple water quality problems. We explore the following questions: (1) Can we classify elemental transport in watersheds as chemical cocktails using a new approach? (2) What is the role of climate and land use in enhancing the formation and transport of chemical cocktails in watersheds? To address these questions, we first analyze trends in concentrations of carbon, nutrients, metals, and salts in fresh waters over 100 years. Next, we explore how climate and land use enhance the probability of formation of chemical cocktails of carbon, nutrients, metals, and salts. Ultimately, we classify transport of chemical cocktails based on solubility, mobility, reactivity, and dominant phases: (1) sieved chemical cocktails (e.g., particulate forms of nutrients, metals and organic matter); (2) filtered chemical cocktails (e.g., dissolved organic matter and associated metal complexes); (3) chromatographic chemical cocktails (e.g., ions eluted from soil exchange sites); and (4) reactive chemical cocktails (e.g., limiting nutrients and redox sensitive elements). Typically, contaminants are regulated and managed one element at a time, even though combinations of elements interact to influence many water-quality problems such as toxicity to life, eutrophication, infrastructure and water treatment. A chemical cocktail approach significantly expands evaluations of water-quality signatures and impacts beyond single elements to mixtures. High-frequency sensor data (pH, specific conductance, turbidity, etc.) can serve as proxies for chemical cocktails and improve real-time analyses of water-quality violations, identify regulatory needs, and track water quality recovery following and extreme climate events. Ultimately, a watershed chemical cocktail approach is necessary for effectively co-managing groups of contaminants and provides a more holistic approach for studying, monitoring, and managing water quality in the Anthropocene.


Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II.
Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.


Diet-induced insulin resistance in mice lacking adiponectin/ACRP30.
Here we investigated the biological functions of adiponectin/ACRP30, a fat-derived hormone, by disrupting the gene that encodes it in mice. Adiponectin/ACRP30-knockout (KO) mice showed delayed clearance of free fatty acid in plasma, low levels of fatty-acid transport protein 1 (FATP-1) mRNA in muscle, high levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in adipose tissue and high plasma TNF-alpha concentrations. The KO mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1 (IRS-1)-associated phosphatidylinositol 3 kinase (PI3-kinase) activity in muscle. Viral mediated adiponectin/ACRP30 expression in KO mice reversed the reduction of FATP-1 mRNA, the increase of adipose TNF-alpha mRNA and the diet-induced insulin resistance. In cultured myocytes, TNF-alpha decreased FATP-1 mRNA, IRS-1-associated PI3-kinase activity and glucose uptake, whereas adiponectin increased these parameters. Our results indicate that adiponectin/ACRP30 deficiency and high TNF-alpha levels in KO mice reduced muscle FATP-1 mRNA and IRS-1-mediated insulin signaling, resulting in severe diet-induced insulin resistance.


Angiotensinogen gene expression is dependent on signal transducer and activator of transcription 3-mediated p300/cAMP response element binding protein-binding protein coactivator recruitment and histone acetyltransferase activity.
Angiotensin II, a potent vasoactive peptide produced by proteolysis of the angiotensinogen (AGT) prohormone, plays a critical role in cardiovascular homeostasis. Recently we showed that IL-6 induces human (h)AGT transcription by activating the signal transducers and activators of transcription (STATs). Here we investigated the role of the coactivator p300/cAMP response element binding protein-binding protein (CBP) in STAT3-mediated hAGT gene expression. Overexpression of adenovirus 12S E1A, which binds and inactivates p300/CBP, strongly inhibited basal and stimulated hAGT transcription, whereas a mutant E1A defective in binding p300/CBP did not. Conversely, ectopic expression of p300 and CBP potentiated inducible hAGT promoter activity. Coimmunoprecipitation assays revealed STAT3-p300 interaction upon IL-6 stimulation. The STAT3-p300 association requires the STAT3 C-terminal transactivation domain, as STAT3 deleted of transactivation functions as a dominant-negative inhibitor and does not associate with p300/CBP. The observation that IL-6 stimulation increases histone H4 acetylation of the endogenous hAGT promoter, and expression of p300 deficient in histone acetyltransferase activity down-regulates hAGT promoter activity both suggest that p300 histone acetyltransferase activity is required for hAGT expression. Finally, treatment of HepG2 cells with a histone deacetylase inhibitor increased the hAGT mRNA abundance by 2- to 3-fold. Taken together, our results indicate that IL-6-inducible expression of the hAGT promoter is mediated by physical association of the COOH terminus of STAT3 with p300/CBP, the recruitment of which targets histone acetylation and results in chromatin remodeling.


Typical electronic health record use in primary care practices and the quality of diabetes care.
Recent efforts to encourage meaningful use of electronic health records (EHRs) assume that widespread adoption will improve the quality of ambulatory care, especially for complex clinical conditions such as diabetes. Cross-sectional studies of typical uses of commercially available ambulatory EHRs provide conflicting evidence for an association between EHR use and improved care, and effects of longer-term EHR use in community-based primary care settings on the quality of care are not well understood. We analyzed data from 16 EHR-using and 26 non-EHR-using practices in 2 northeastern states participating in a group-randomized quality improvement trial. Measures of care were assessed for 798 patients with diabetes. We used hierarchical linear models to examine the relationship between EHR use and adherence to evidence-based diabetes care guidelines, and hierarchical logistic models to compare rates of improvement over 3 years. EHR use was not associated with better adherence to care guidelines or a more rapid improvement in adherence. In fact, patients in practices that did not use an EHR were more likely than those in practices that used an EHR to meet all of 3 intermediate outcomes targets for hemoglobin A(1c), low-density lipoprotein cholesterol, and blood pressure at the 2-year follow-up (odds ratio = 1.67; 95% CI, 1.12-2.51). Although the quality of care improved across all practices, rates of improvement did not differ between the 2 groups. Consistent use of an EHR over 3 years does not ensure successful use for improving the quality of diabetes care. Ongoing efforts to encourage adoption and meaningful use of EHRs in primary care should focus on ensuring that use succeeds in improving care. These efforts will need to include provision of assistance to longer-term EHR users.


A Comparision of the Effect of Sugammadex on the Recovery Period and Postoperative Residual Block in Young Elderly and Middle-Aged Elderly Patients.
The importance of the characteristics of anesthesia and postoperative residual curarization (PORC) in the elderly population should be a growing concern in this century. To investigate the effect of sugammadex on the duration of the recovery from neuromuscular blocking agents and postoperative residual curarization in the young elderly and middle-aged elderly patients who underwent elective laparoscopic cholecystectomy, followed by a train of four (TOF) watch monitorization. Prospective clinical trial study. Sixty patients over the age of 65 with American Society of Anesthesiologists I-III were divided into two groups according to their age (65-74 years old and ≥75 years old). Patients received sugammadex (2.0 mg/kg iv) at the reappearance of the second twitch of the TOF as an agent for reversal of neuromuscular blockage at the end of surgery. Patients were extubated at the time of TOF ≥0.9. The patients' TOF responses were evaluated with regards to PORC in at the 5th minute and were followed up for one hour in the recovery room. Reintubation was applied for those patients who developed PORC and had peripheric oxygen saturation <90% despite being given 6 L oxygen per min with a face mask. The onset time of neuromuscular blocking agent and time from T2 to achieve TOF ratio 90% (the duration of sugammadex effect) or over were found to be longer in the middle-aged elderly group than in the young elderly group. A statistically significant relationship was found between age and the duration of TOF ratio to reach 0.9 in the same direction. The PORC incidence and rate of reintubation were found to be 1.7% in all patients. In our opinion, it is necessary to remember that the duration of sugammadex effect on the recovery period is prolonged for patients who are aged ≥75 years compared to patients aged between 65-74 years. (ClinicalTrials.gov Identifier: ACTRN12615000758505).


Brain response to food cues varying in portion size is associated with individual differences in the portion size effect in children.
Large portions promote intake of energy dense foods (i.e., the portion size effect--PSE), but the neurobiological drivers of this effect are not known. We tested the association between blood oxygen level dependent (BOLD) brain response to food images varied by portion size (PS) and energy density (ED) and children's intake at test-meals of high- and low-ED foods served at varying portions. Children (N = 47; age 7-10 years) participated in a within-subjects, crossover study consisting of 4 meals of increasing PS of high- and low-ED foods and 1 fMRI to evaluate food images at 2 levels of PS (Large, Small) and 2 levels of ED (High, Low). Contrast values between PS conditions (e.g., Large PS - Small PS) were calculated from BOLD signal in brain regions implicated in cognitive control and reward and input as covariates in mixed models to determine if they moderated the PSE curve. Results showed a significant effect of PS on intake. Responses to Large relative to Small PS in brain regions implicated in salience (e.g., ventromedial prefrontal cortex and orbitofrontal cortex) were positively associated with the linear slope (i.e., increase in intake from baseline) of the PSE curve, but negatively associated with the quadratic coefficient for the total meal. Responses to Large PS High ED relative to Small PS High ED cues in regions associated with cognitive control (e.g., dorsolateral prefrontal cortex) were negatively associated with the linear slope of the PSE curve for high-ED foods. Brain responses to PS cues were associated with individual differences in children's susceptibility to overeating from large portions. Responses in food salience regions positively associated with PSE susceptibility while activation in control regions negatively associated with PSE susceptibility.


Expectancy and surprise predict neural and behavioral measures of attention to threatening stimuli.
Attention is preferentially deployed toward those stimuli which are threatening and those which are surprising. The current paper examines the intersection of these phenomena; how do expectations about the threatening nature of stimuli influence the deployment of attention? The predictions tested were that individuals would direct attention toward stimuli which were expected to be threatening (regardless of whether they were or not) and toward stimuli which were surprising. As anxiety has been associated with deficient control of attention to threat, it was additionally predicted that high levels of trait anxiety would be associated with deficits in the use of threat-expectation to guide attention. During fMRI scanning, 29 healthy volunteers completed a simple task in which threat-expectation was manipulated by altering the frequency with which fearful or neutral faces were presented. Individual estimates of threat-expectation and surprise were created using a Bayesian computational model. The degree to which the model derived estimates of threat-expectation and surprise were able to explain both a behavioral measure of attention to the faces and activity in the visual cortex and anterior attentional control areas was then tested. As predicted, increased threat-expectation and surprise were associated with increases in both the behavioral and neuroimaging measures of attention to the faces. Additionally, regions of the orbitofrontal cortex and left amygdala were found to covary with threat-expectation whereas anterior cingulate and lateral prefrontal cortices covaried with surprise. Individuals with higher levels of trait anxiety were less able to modify neuroimaging measures of attention in response to threat-expectation. These results suggest that continuously calculated estimates of the probability of threat may plausibly be used to influence the deployment of visual attention and that use of this information is perturbed in anxious individuals.


Modular reorganization of the global network of gene regulatory interactions during perinatal human brain development.
During early development of the nervous system, gene expression patterns are known to vary widely depending on the specific developmental trajectories of different structures. Observable changes in gene expression profiles throughout development are determined by an underlying network of precise regulatory interactions between individual genes. Elucidating the organizing principles that shape this gene regulatory network is one of the central goals of developmental biology. Whether the developmental programme is the result of a dynamic driven by a fixed architecture of regulatory interactions, or alternatively, the result of waves of regulatory reorganization is not known. Here we contrast these two alternative models by examining existing expression data derived from the developing human brain in prenatal and postnatal stages. We reveal a sharp change in gene expression profiles at birth across brain areas. This sharp division between foetal and postnatal profiles is not the result of pronounced changes in level of expression of existing gene networks. Instead we demonstrate that the perinatal transition is marked by the widespread regulatory rearrangement within and across existing gene clusters, leading to the emergence of new functional groups. This rearrangement is itself organized into discrete blocks of genes, each targeted by a distinct set of transcriptional regulators and associated to specific biological functions. Our results provide evidence of an acute modular reorganization of the regulatory architecture of the brain transcriptome occurring at birth, reflecting the reassembly of new functional associations required for the normal transition from prenatal to postnatal brain development.


Ageing and the haematological profiles of an Australian community.
The Busselton Health Survey was established in 1966 with the aim of identifying factors that influenced the health of the inhabitants of this coastal town in Western Australia. Further surveys were conducted at 3 yearly intervals from 1966 to 1981, however, in these earlier studies little attention was paid to the topic of ageing. Given the increasing global interest in this subject, it was decided that all persons enrolled in the 1994-1995 resurvey should be examined for age-related changes in their haematology. Samples were obtained from 3830 male and female volunteers aged from 16 to 92 years. The data showed a number of significant, age-dependent shifts in the haematological profiles of those tested. In several instances the observed patterns were expected, for example, a gradual decline in the mean red cell count of males over 60 years of age. Other changes, including a significant increase in red cell distribution width which commenced in females aged 40+ years, have not previously been reported. Findings of this nature indicate the presence of an underlying, age-dependent decline in the haematopoietic system. They also reveal a requirement for age-specific normal ranges, to meet the needs of the many human populations with ever-increasing life expectancies.


Sharing of Genes and Pathways Across Complex Phenotypes: A Multilevel Genome-Wide Analysis.
Evidence from genome-wide association studies (GWAS) suggest that pleiotropic effects on human complex phenotypes are very common. Recently, an atlas of genetic correlations among complex phenotypes has broadened our understanding of human diseases and traits. Here, we examine genetic overlap, from a gene-centric perspective, among the same 24 phenotypes previously investigated for genetic correlations. After adopting the multilevel pipeline (freely available at http://grass.cgs.hku.hk/limx/kgg/), which includes intragenic single nucleotide polymorphisms (SNPs), genes, and gene-sets, to estimate genetic similarities across phenotypes, a large amount of sharing of several biologically related phenotypes was confirmed. In addition, significant genetic overlaps were also found among phenotype pairs that were previously unidentified by SNP-level approaches. All these pairs with new genetic links are supported by earlier epidemiological evidence, although only a few of them have pleiotropic genes in the GWAS Catalog. Hence, our gene and gene-set analyses are able to provide new insights into cross-phenotype connections. The investigation on genetic sharing at three different levels presents a complementary picture of how common DNA sequence variations contribute to disease comorbidities and trait manifestations.


eModel-BDB: a database of comparative structure models of drug-target interactions from the Binding Database.
The structural information on proteins in their ligand-bound conformational state is invaluable for protein function studies and rational drug design. Compared to the number of available sequences, not only is the repertoire of the experimentally determined structures of holo-proteins limited, these structures do not always include pharmacologically relevant compounds at their binding sites. In addition, binding affinity databases provide vast quantities of information on interactions between drug-like molecules and their targets, however, often lacking structural data. On that account, there is a need for computational methods to complement existing repositories by constructing the atomic-level models of drug-protein assemblies that will not be determined experimentally in the near future. We created eModel-BDB, a database of 200,005 comparative models of drug-bound proteins based on 1,391,403 interaction data obtained from the Binding Database and the PDB library of 31 January 2017. Complex models in eModel-BDB were generated with a collection of the state-of-the-art techniques, including protein meta-threading, template-based structure modeling, refinement and binding site detection, and ligand similarity-based docking. In addition to a rigorous quality control maintained during dataset generation, a subset of weakly homologous models was selected for the retrospective validation against experimental structural data recently deposited to the Protein Data Bank. Validation results indicate that eModel-BDB contains models that are accurate not only at the global protein structure level but also with respect to the atomic details of bound ligands. Freely available eModel-BDB can be used to support structure-based drug discovery and repositioning, drug target identification, and protein structure determination.


IPAA-related sepsis significantly increases morbidity of ileoanal pouch excision.
Perineal wound complications after ileoanal pouch excision remain a significant cause of morbidity. The purpose of this work was to describe the incidence, outcomes, and predictors of perineal wound complications after pouch excision. This was a retrospective medical chart review. The study was conducted in a single clinical institution. Patients who underwent pouch excision at our institution from July 1992 through July 2012 were identified. Patient and perioperative variables were reviewed. Multivariate and univariate analyses were undertaken. Perineal wound (including perineal wound infection and persistent perineal sinus [nonhealing by 6 months]) and perineal hernia were measured. A total of 47 patients (mean age, 46 years; 42.6% men) with familial adenomatous polyposis (10.6%), mucosal ulcerative colitis (61.7%), or Crohn's disease (27.7%) underwent pouch excision, including 36.2% for IPAA-related sepsis (presacral abscess; perineal-, sacral-, or pouch-vaginal fistula; and anastomotic defect), 44.7% for pouch dysfunction, 10.6% for refractory pouchitis, and 8.5% for neoplasia. Fourteen (29.8%) developed perineal wound complications, including 100% perineal wound infection, 28.6% persistent perineal sinus, and 7.1% perineal hernia. Perineal wound infection was associated with delayed healing (>6 weeks; 71.4% vs 24.2%; p = 0.002) and IPAA-related sepsis (28.6% vs 0%; p = 0.001). Patients with and without perineal wound complications were similar in age, diagnoses, fecal diversion, immunosuppression, comorbid conditions, nutrition, and surgical variables. Most patients underwent intersphincteric dissection (87.2%) with primary perineal closure (97.0%). Perineal wound complications were significantly associated with IPAA-related sepsis as an indication for pouch excision (57.1% vs 27.2%; p = 0.05), intraoperative pouch perforation (35.7% vs 9.1%, p =0.03), and smoking (21.4% vs 3.0%; p = 0.04). IPAA-related sepsis and a current smoking status (OR, 19.3 [95% CI, 1.8 -488.1]) are significant independent predictors on multivariate logistic regression (OR, 6.4 [95% CI, 1.4-30.2]) of perineal wound complications. All of the patients with persistent perineal sinus achieved successful healing at a median of 734 days (range, 363-2182 days), requiring a median of 1.5 procedures. This was a single-center retrospective review with a small sample size. Preoperative IPAA-related sepsis and current smoking are significant risk factors for perineal wound complications after pouch excision.


Pathophysiological and clinical aspects of the diagnosis and treatment of bezoars.
Bezoars are intraluminal conglomerates of indigestible foreign materials that accumulate in the gastrointestinal (GI) tract. They consist of vegetable or fruit fibers, hairs or other substances; accordingly, bezoars are classified as phytobezoars, trichobezoars, pharmacobezoars, etc. Although sometimes asymptomatic, bezoars may cause serious symptoms, such as abdominal discomfort or pain, dysphagia, hematemesis, or even life-threatening entities (GI bleeding, obstruction or perforation). Current technological applications have contributed to the diagnostic and therapeutic approach to these masses, mainly through endoscopic techniques able to diagnose, fragment and extract bezoars, as well as laparoscopic and other surgical modalities that may be used to treat serious complications. Although bezoars were described centuries ago and the term was officially introduced in the mid nineties by Quain, they are still a demanding pathological entity. Their pathophysiology, accurate and prompt diagnosis, as well as successful and minimally invasive treatment, remain under investigation and see continuous progress. Current advances in these challenging areas are discussed in this review, which attempts to present an in-depth study of bezoars along with the well-established modalities and techniques.


Interplay between growth factor and nutrient signaling: lessons from Drosophila TOR.
During normal development, cellular and organismal growth is coordinately regulated. Each cell and each individual organ integrates information about nutrient availability, hormonal signals, and intrinsic growth programs. Describing the signaling pathways involved in these processes and how they are integrated is important to understand how growth is controlled during development and may also permit the development of means to curb uncontrolled growth in disease. In recent years, the biochemical analysis of cellular growth in cultured cells and the genetic dissection of growth control in model organisms has identified two conserved signaling pathways dedicated to cellular growth. The target of rapamycin (TOR) pathway regulates growth in response to nutrients, and the insulin/IGF pathways are involved in coordinating cellular growth in response to endocrine signals. This review discusses recent advances in the understanding of the interaction between these pathways, with a special focus on the contribution of the genetic analysis of these pathways in Drosophila.


The enteric enhancement of glucose-stimulated insulin release. The role of GIP in aging, obesity, and non-insulin-dependent diabetes mellitus.
The effect of aging, obesity, and non-insulin-dependent diabetes mellitus on glucose-stimulated gastric inhibitory polypeptide (GIP) levels was studied in 55 male subjects, ranging in age from 19 to 84 yr, and in obesity, expressed as body mass index, from 21 to 34. Studies were performed using the hyperglycemic glucose clamp technique, in which the blood glucose was maintained at 125 mg/dl above basal for 2 h. Glucose (40 g/m2 body surface) was ingested at 60 min. Plasma immunoreactive GIP (IR-GIP) did not change during intravenous (i.v.) glucose alone, but began to rise within 10 min after glucose ingestion and reached a peak at 30-40 min. Basal and stimulated IR-GIP levels were markedly elevated in diabetic subjects and modestly elevated in obese subjects, compared with appropriately matched controls. In contrast, age had little effect on plasma IR-GIP levels either in the basal state or after glucose ingestion. When IR-GIP responses to oral glucose were expressed as a relative change from basal levels, IR-GIP rose 86% in diabetic subjects and 243% in obese subjects, compared with 185% and 165% in their respective controls. IR-GIP rose 179% in young subjects and 144% in middle-aged subjects, while, in old subjects, the increase was 265%. Plasma IRI levels were reduced in the diabetic subjects, slightly elevated in obese subjects, and were similar in older and younger subjects. Beta cell sensitivity to endogenous GIP decreases with age, and is unchanged in both obesity and nonmedicated diabetes.


Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.
The aim of this study was to attempt to visualize herpes simplex keratitis in an ex vivo model using currently available ophthalmological equipment and anti-herpes simplex virus 1 (HSV-1) fluorescein isothiocynate-labeled antibody. Sixteen donor human corneas were included in this study. Eight corneas were infected with HSV-1, whereas 8 remained uninfected. Abrasions were made on 2 infected and 2 uninfected corneas to assess a possible nonspecific absorption of antibodies in the sites of corneal epithelial defects. Corneas were examined before and after antibody application using a slit lamp, the fluorescein enhancing filter settings of fundus camera, and Confoscan 3. All corneas were further imaged using multiphoton laser confocal microscopy. Before anti-HSV-1 antibody application, no fluorescence was detected in donor corneas with the blue light of the slit lamp and fundus camera at fluorescein enhancing filter settings. Examination with the fundus camera after antibody application detected increased background fluorescence in all the corneas with more highlighted areas of epithelial defects in abraded infected and uninfected corneas. Confoscan 3 did not show a significant difference between the appearances of HSV-1-infected and control corneas with and without application of the antibody. However, specific staining was confirmed by multiphoton laser confocal microscopy in all infected corneas. Further refinement of currently available ophthalmological tools is required to aid in vivo visualization of herpes simplex keratitis using fluorescein isothiocynate-labeled antibodies.


Effect of metformin on the human T98G glioblastoma multiforme cell line.
Metformin is a guanidine derivative found in Galega officinalis that is commonly used to treat diabetes mellitus. The mechanism of action of metformin involves regulation of the adenosine monophosphate-activated protein kinase/mammalian target of rapamycin signaling pathway, which is implicated in the control of protein synthesis and cell proliferation. This led to the hypothesis that metformin reduces the risk of cancer and slows tumor growth. Thus, in the present study, the effectiveness of metformin as an antiglioma agent was evaluated using the human T98G glioblastoma multiforme cell line. The viability of the T98G cells was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was monitored by measuring caspase-3 levels, as well as by terminal deoxynucleotidyl transferase dUTP nick end labeling and staining with acridine orange and ethidium bromide. The results demonstrate that metformin reduced cell viability and caused apoptotic morphological changes in the T98G cells. Furthermore, the caspase-3 levels in the metformin-treated T98G cells were higher than those in the control cells. Metformin induced apoptosis in the T98G cell line in a concentration-dependent manner. Metformin may provide an important contribution to the treatment of glioblastoma multiforme.


Spectral emission of corona discharges.
Corona-discharge devices are commonly used in a variety of research and development applications. They are known to produce a considerable amount of electromagnetic radiation, mainly in the uv region of the spectrum. The mechanism involved is presumed to be the ionization and radiative recombination of the gases surrounding the high-voltage terminal of the device. A series of high-resolution emission spectra of corona discharge has been measured in atmospheres of nitrogen, helium, and air. (The nitrogen and helium are Air Products 99.995% purity, total hydrocarbons less than 0.5 ppm.) These data are presented here and are compared with published spectroscopic reference data. All the emission lines reported in the spectroscopic reference literature were detected, as well as other lines, which may conceivably be due to impurities.


Do "savanna" chimpanzees consume C4 resources?
Several stable carbon isotopic studies have shown that South African australopiths consumed significant quantities of C(4) resources (tropical grasses, sedges, or animals that eat those foods), but relatively little is known about the consumption of such resources by chimpanzees. Here, we present stable carbon isotopic data for 36 chimpanzee hair samples from Fongoli, one of the driest and most open areas inhabited by chimpanzees. These data suggest that the Fongoli chimpanzees consume little in the way of C(4) vegetation or animals that eat such vegetation, even though these resources are locally abundant and preferred fruits are more widely scattered than at most chimpanzee study sites. The homogeneity of the Fongoli results is especially striking and recalls the narrow isotopic distribution of stenotopic savanna mammals. This is in stark contrast to what has been observed for australopiths, which had highly variable diets and consumed about 35% C(4) vegetation on average. Carbon isotope data for modern and fossil Papio depict a dietarily variable genus with a tendency to consume C(4) vegetation. This trophic flexibility, or willingness to consume C(4) savanna resources, may make Papio a more profitable ecological analog for australopiths than chimpanzees.


Homeodomain protein otp and activity-dependent splicing modulate neuronal adaptation to stress.
Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal's adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing.


Double-barrel stent-assisted coiling of a basilar artery fenestration aneurysm.
Basilar artery fenestration aneurysms are rare aneurysms, posing unique challenges for endovascular treatment. We report a case of successful treatment of a wide-necked basilar artery fenestration aneurysm with a novel double-barrel stent-assisted coiling technique. A 50-year-old woman presented with headaches. Evaluation revealed an unruptured 7-mm broad-based basilar artery fenestration aneurysm that incorporated both limbs of the basilar fenestration and both distal vertebral arteries. A 4.5 mm × 22 mm Enterprise stent (Codman Neurovascular, Raynham, MA, USA) was deployed from the right limb of the basilar fenestration into the right distal vertebral artery. Similarly, a 2.5 mm × 20 mm Neuroform EZ stent (Boston Scientific, Natick, MA, USA) was placed from the left limb of the basilar fenestration into the left distal vertebral artery. Following stent deployment, the aneurysm was treated with coil-embolization through a jailed microcatheter. In the case presented, a double-barrel stent configuration enabled dense coil embolization of the aneurysm as well as preservation of both basilar fenestration limbs and both distal vertebral arteries.


Dual effects of n-alcohols on fluid secretion from guinea pig pancreatic ducts.
Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca(2+) concentration ([Ca(2+)](i)) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca(2+)](i) was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3-10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca(2+)](i) in secretin- or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca(2+)](i) in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3-10 mM concentrations significantly reduced fluid secretion and decreased [Ca(2+)](i) in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca(2+)](i) response to ACh as well as the [Ca(2+)](i) elevation induced by thapsigargin, suggesting that they inhibit Ca(2+) influx. Removal of extracellular Ca(2+) reduced [Ca(2+)](i) in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca(2+)](i) in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca(2+) channel.


Regulation of depth and composition of airway surface liquid.
The depth and composition of human airway surface liquid (ASL) may depend on secretion from airway glands, ion transport across the surface epithelium, goblet cell discharge, transepithelial gradients in hydrostatic pressure, and surface tension. Published values for the frequency of airway glands and for the secretory rates of individual glands suggest that total gland secretion in human trachea can amount to approximately 60 microL x cm(-2) x h(-1). Volume absorption directly measured across cultures of surface epithelium from human trachea is approximately 5 microL x cm(-2) x h(-1). These flows should alter the depth of ASL at +10 and -1 microm x min(-1). We have looked for changes in ASL depth of this magnitude using low-temperature scanning electron microscopy (LT-SEM) of rapidly frozen specimens of bovine trachea. Stimulation of gland secretion with methacholine led to an initial increase in depth of approximately 25 microm x min(-1) followed by a decline at approximately 1.5 microm x min(-1). Whereas the initial increase in depth was probably due to transient gland secretion, the later decline reflected active absorption of liquid across the surface epithelium. Finally, we present preliminary data showing that LT-SEM can be combined with X-ray microanalysis to determine the elemental composition of ASL.


Effect of allyl alcohol on hepatic transporter expression: zonal patterns of expression and role of Kupffer cell function.
During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.


Serum Sphingolipid Variations Associate with Hepatic Decompensation and Survival in Patients with Cirrhosis.
Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy. We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry. We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival (standard beta = -0.001, p = 0.022). In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies.


Molecular and descriptive epidemiology of intestinal protozoan parasites of children and their pets in Cauca, Colombia: a cross-sectional study.
Parasitic infections, particularly those caused by protozoa, represent a considerable public health problem in developing countries. Blastocystis, Giardia duodenalis, Cryptosporidium spp. and the Entamoeba complex (Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii) are the most common etiological causes of intestinal parasitic infections. We carried out a descriptive cross-sectional study in school-age children attending a daycare institution in commune eight of Popayán, Cauca (Southwest Colombia). A total of 266 fecal samples were collected (258 from children and eight from pets). Blastocystis, G. duodenalis, Cryptosporidium spp. and the Entamoeba complex were identified by microscopy, quantitative real-time PCR (qPCR) and conventional PCR. The concordance of qPCR and microscopy was assessed using the Kappa index. Molecular characterization was conducted to identify Blastocystis subtypes (18S), G. duodenalis assemblages (tpi and gdh) and Cryptosporidium species/subtypes (18S and GP60). Potential associations between intestinal parasitism and sociodemographic factors were examined using bivariate analyses. A total of 258 fecal samples from children were analyzed by microscopy and 255 samples were analyzed by qPCR. The prevalence of Blastocystis was between 25.19% (microscopy) and 39.22% (qPCR), that of G. duodenalis was between 8.14% (microscopy) and 10.59% (qPCR), that of Cryptosporidium spp. was estimated at 9.8% (qPCR), and that of the Entamoeba complex was between 0.39% (conventional PCR) and 0.78% (microscopy). The concordance between microscopy and qPCR was very low. Blastocystis ST1 (alleles 4, 8, and 80), ST2 (alleles 11, 12, and 15), ST3 (alleles 31, 34, 36, 38,57, and 151), and ST4 (alleles 42 and 91), G. duodenalis assemblages AII, BIII, BIV and D, C. parvum subtype IIa and C. hominis subtype IbA9G3R2 were identified. The only identified member of the Entamoeba complex corresponded to E. histolytica. No statistically significant association was identified between parasitic infection and any sociodemographic variable. This study revealed the usefulness of molecular methods to depict the transmission dynamics of parasitic protozoa in southwest Colombia. The presence of some of these protozoa in domestic animals may be involved in their transmission.


Management of Diabetic Bacterial Foot Infections with Organic Extracts of Liverwort Marchantia debilis from Cameroon.
Diabetic bacterial foot infections (DBFIs) are limb-threatening complications in patients with diabetes mellitus, accounting for 50% of diabetes related lower limb amputations in developing countries, representing approximately 20 percent of all diabetes-related hospital admissions with significant healthcare-related costs involved. The widespread problem of bacterial resistance to most commonly used antibiotics places a huge economic burden on the healthcare system, with both increased morbidity and mortality among diabetic patients with foot infections. In this study, the antibacterial activity of organic extracts of the fresh liverwort Marchantia debilis from the North West Region of Cameroon is reported. An exit pool system, where patients presenting with DBFIs consented to be involved in the use of phytomedicines, after long term treatment of ulcers with antibiotics and not yielding significant long term benefit, presented themselves at the Phytobiotechnology Research clinic (PRF). Continuous culture of swabs from foot and toe wounds from 30 infected patients on nutrient agar and MacConkey agars in triplicate as well as Gram stain microscopy, revealed the presence of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Bacillus species. Light petroleum and methanol extracts of the whole bryophyte plants at 100% concentration were tested. In vitro inhibition of the tested bacterial isolates from the diabetic foot ulcers by M debilis was observed only with the light petroleum extract. No inhibition by the extracts was observed for the Pseudomonas aeruginosa isolate. The light petroleum extract of M debilis was formulated into a petroleum oil based cream named BryoCream™. This was administered to 20 of the patients with 90% cure rate in a three week time period. The main nonpolar components were determined by GCMS as lepidozene and β-barbatene, and by NMR as stigmasterol and β-sitosterol. In conclusion, nonpolar extracts from bryophytes from Cameroon could, potentially, be used to treat diabetic bacterial foot infections.


Development and Evaluation of Two Pecan Scab Prediction Models.
Fusicladium effusum (syn. Cladosporium caryigenum), causal agent of pecan scab, is the most economically important pathogen of pecan (Carya illinoinensis). A weather-based advisory is currently used in Oklahoma to assess the need for fungicide application and requires the accumulation of scab hours. A scab hour is defined as an hour with average temperature ≥21.1°C and relative humidity ≥90%. To assess the validity of the thresholds in the advisory, repeated ratings of disease severity were taken on fruit at five locations during the 1994-96 and 2009-10 growing seasons, resulting in a total of eight site years. Hourly weather variables were also examined, including temperature, relative humidity, dew point, dew point depression, total solar radiation, and total rainfall. Rain and disease severity were converted to binomial variables where a rain event (≥2.5 mm) and disease severity (≥25%) were coded as 1 and all other events as 0. Logistic regression models adjusted for correlated data were developed using generalized estimating equations. Two models were developed: a temperature/relative humidity model and a dew point/dew point depression model. For the temperature/relative humidity model, the best fitting model included all main effects. Using this model, validation exercises assuming no rain and total solar radiation of 22.5 MJ m-2 resulted in a 0.45 probability of pecan scab development when the temperature was 21°C and relative humidity was 90%. Findings of this model were further validated during field studies that evaluated different combinations of temperature and relative humidity thresholds for scheduling fungicide applications. These analyses indicated that the current thresholds of temperature and relative humidity are viable, but a modification of the relative humidity component should be considered. For the dew point/dew point depression model, a reduced model, including dew point, dew point depression, and the binomial rain variable, was considered adequate for explaining scab events, which suggests that future model building to describe pecan scab epidemics should include dew point, dew point depression, rain, and total solar radiation as independent variables. This article originally appeared in the January issue, Volume 96, pages 117-123. It was changed to correct errors in a measurement conversion that appeared throughout.


The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.
The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.


Fit to Burst: Toward Noninvasive Estimation of Achilles Tendon Load Using Burst Vibrations.
Tendons are essential components of the musculoskeletal system and, as with any mechanical structure, can fail under load. Tendon injuries are common and can be debilitating, and research suggests that a better understanding of their loading conditions could help mitigate injury risk and improve rehabilitation. To that end, we present a novel method of noninvasively assessing parameters related to mechanical load in the Achilles tendon using burst vibrations. These vibrations, produced by a small vibration motor on the skin superficial to the tendon, are sensed by a skin-mounted accelerometer, which measures the tendon's response to burst excitation under varying tensile load. In this study, twelve healthy subjects performed a variety of everyday tasks designed to expose the Achilles tendon to a range of loading conditions. To approximate the vibration motor-tendon system and provide an explanation for observed changes in tendon response, a 2-degree-of-freedom mechanical systems model was developed. Reliable, characteristic changes in the burst response profile as a function of Achilles tendon tension were observed during all loading tasks. Using a machine learning-based approach, we developed a regression model capable of accurately estimating net ankle moment-which captures general trends in tendon tension-across a range of walking speeds and across subjects (R2 = 0.85). Simulated results of the mechanical model accurately recreated behaviors observed in vivo. Finally, preliminary, proof-of-concept results from a fully wearable system demonstrated trends similar to those observed in experiments conducted using benchtop equipment. These findings suggest that an untethered, unobtrusive system can effectively assess tendon loading during activities of daily life. Access to such a system would have broad implications for injury recovery and prevention, athletic training, and the study of human movement.


Pregnancy-specific malarial immunity and risk of malaria in pregnancy and adverse birth outcomes: a systematic review.
In endemic areas, pregnant women are highly susceptible to Plasmodium falciparum malaria characterized by the accumulation of parasitized red blood cells (pRBC) in the placenta. In subsequent pregnancies, women develop protective immunity to pregnancy-associated malaria and this has been hypothesized to be due to the acquisition of antibodies to the parasite variant surface antigen VAR2CSA. In this systematic review we provide the first synthesis of the association between antibodies to pregnancy-specific P. falciparum antigens and pregnancy and birth outcomes. We conducted a systematic review and meta-analysis of population-based studies (published up to 07 June 2019) of pregnant women living in P. falciparum endemic areas that examined antibody responses to pregnancy-specific P. falciparum antigens and outcomes including placental malaria, low birthweight, preterm birth, peripheral parasitaemia, maternal anaemia, and severe malaria. We searched 6 databases and identified 33 studies (30 from Africa) that met predetermined inclusion and quality criteria: 16 studies contributed estimates in a format enabling inclusion in meta-analysis and 17 were included in narrative form only. Estimates were mostly from cross-sectional data (10 studies) and were heterogeneous in terms of magnitude and direction of effect. Included studies varied in terms of antigens tested, methodology used to measure antibody responses, and epidemiological setting. Antibody responses to pregnancy-specific pRBC and VAR2CSA antigens, measured at delivery, were associated with placental malaria (9 studies) and may therefore represent markers of infection, rather than correlates of protection. Antibody responses to pregnancy-specific pRBC, but not recombinant VAR2CSA antigens, were associated with trends towards protection from low birthweight (5 studies). Whilst antibody responses to several antigens were positively associated with the presence of placental and peripheral infections, this review did not identify evidence that any specific antibody response is associated with protection from pregnancy-associated malaria across multiple populations. Further prospective cohort studies using standardized laboratory methods to examine responses to a broad range of antigens in different epidemiological settings and throughout the gestational period, will be necessary to identify and prioritize pregnancy-specific P. falciparum antigens to advance the development of vaccines and serosurveillance tools targeting pregnant women.


Image-guided corticosteroid injection of calcific tendonitis of gluteus maximus.
Gluteus maximus tendonitis with its clinical and imaging patterns has been described in radiologic, orthopedic, and rheumatology texts and journals. Image-guided treatment, however, is not a familiar approach and has only been described in one case report in the English literature to the best of our knowledge. We present 4 patients with calcific tendonitis of the gluteus maximus who presented with excruciating pain in the posterior upper thigh with limitation of daily activities. The diagnosis was based on the characteristic radiologic appearances of calcifications in the tendon with cortical bone erosion and muscle edema. Treatment was with image-guided (fluoroscopy or computed tomography) injection of a local anesthetic and corticosteroid. All patients were pain-free on follow up.


Expression, purification, and initial kinetic characterization of the large subunit of the human mitochondrial DNA polymerase.
Faulty replication of the human mitochondrial genome is thought to be the cause of many diseases; moreover, the low selectivity of the mitochondrial DNA polymerase has been implicated as the cause of many side effects observed in the treatment of viral infections such as HIV. To better understand how the mitochondrial genome is replicated, we cloned a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit was recombinantly expressed and purified to near homogeneity. The purified enzyme demonstrated both polymerase and 3'-5' exonuclease activity. The purified protein was examined in single nucleotide incorporation assays, demonstrating that the enzyme had a maximum polymerization rate of 3.5 s-1 and a dissociation rate from the DNA substrate of 0.03 s-1, affording a calculated processivity of 116. The dissociation constants for the enzyme binding to DNA and nucleoside triphosphate were 39 nM and 14 microM, respectively. The 3'-5' exonuclease rate was measured at 0. 18 s-1. Though the slow rate of polymerization suggests that the large subunit of human DNA polymerase gamma may require accessory factors to increase its processivity of polymerization, the kinetic parameters indicate that the large subunit of DNA polymerase gamma could replicate the mitochondrial genome in a physiologically relevant time frame. This study provides the initial characterization of the large subunit of DNA polymerase gamma and establishes the baseline for examination of the effects of accessory proteins such as the putative small subunit.


Respiratory viral infections are underdiagnosed in patients with suspected sepsis.
The study aim was to investigate the prevalence and clinical relevance of viral findings by multiplex PCR from the nasopharynx of clinically septic patients during a winter season. During 11 weeks of the influenza epidemic period in January-March 2012, consecutive adult patients suspected to be septic (n = 432) were analyzed with cultures from blood and nasopharynx plus multiplex PCR for respiratory viruses on the nasopharyngeal specimen. The results were compared with those from microbiology analyses ordered as part of standard care. During the winter season, viral respiratory pathogens, mainly influenza A virus, human metapneumovirus, coronavirus, and respiratory syncytial virus were clinically underdiagnosed in 70% of patients positive by the multiplex PCR assay. During the first four weeks of the influenza epidemic, few tests for influenza were ordered by clinicians, indicating low awareness that the epidemic had started. Nasopharyngeal findings of Streptococcus pneumoniae and Haemophilus influenzae by culture correlated to pneumonia diagnosis, and in those patients laboratory signs of viral co-infections were common but rarely suspected by clinicians. The role of respiratory viral infections in patients presenting with a clinical picture of sepsis is underestimated. Specific antiviral treatment might be beneficial in some cases and may reduce spread in a hospital setting. Diagnosing viral infections may promote reduction of unnecessary antibiotic use. It can also be a tool for decisions concerning patient logistics, in order to minimize exposure of susceptible patients and personnel.


Promoting effects of unleaded gasoline and 2,2,4-trimethylpentane on the development of atypical cell foci and renal tubular cell tumors in rats exposed to N-ethyl-N-hydroxyethylnitrosamine.
Unleaded gasoline (UG), a nongenotoxic kidney carcinogen in male, but not female, F344 rats or either sex of mice, and 2,2,4-trimethylpentane (TMP), a representative nephrotoxic isoparaffinic component of UG, were tested for potential promoting and cocarcinogenic effects in a kidney initiation-promotion model. The promotion study was conducted with 305 male and 305 female F344 rats fed 170 ppm N-ethyl-N-hydroxyethylnitrosamine in the drinking water for 2 weeks and then inhalation exposed to 0, 10, 70, or 300 ppm UG or 50 ppm TMP for 24 or 59 to 61 weeks. In a sequence reversal study, 390 male F344 rats were inhalation exposed to 0, 10, 70, or 300 ppm UG or 50 ppm TMP for 24 weeks, followed by 170 ppm N-ethyl-N-hydroxyethylnitrosamine in the drinking water during weeks 28 to 30, and killed at weeks 65 to 67. Renal neoplastic lesions were classified as atypical cell foci (ACF) and renal cell tumors (RCT). In the hydrocarbon promotion study, dose related increases were observed in the incidence of ACF in male rats promoted with UG or 50 ppm TMP for 24 or 60 weeks. A significant linear trend in the incidence of RCT was observed in male rats promoted with UG for 24 weeks. The incidence of ACF or RCT was not elevated in female rats promoted with UG or TMP. In the sequence reversal study, a slight increase in ACF was demonstrated in male rats exposed to 300 ppm UG, whereas no increase in RCT was observed in any exposure group. It is concluded that UG and TMP are promoters of ACF and RCT in male, but not female, rats under the conditions of this study. Data from related investigations suggest that the tumor promoting potential of UG and TMP results from reversible binding of metabolites to alpha 2u-globulin, which leads to decreased renal catabolism of this protein, chronic lysosomal overload, cell death, and compensatory cell proliferation.


Arteriopathy diagnosis in childhood arterial ischemic stroke: results of the vascular effects of infection in pediatric stroke study.
Although arteriopathies are the most common cause of childhood arterial ischemic stroke, and the strongest predictor of recurrent stroke, they are difficult to diagnose. We studied the role of clinical data and follow-up imaging in diagnosing cerebral and cervical arteriopathy in children with arterial ischemic stroke. Vascular effects of infection in pediatric stroke, an international prospective study, enrolled 355 cases of arterial ischemic stroke (age, 29 days to 18 years) at 39 centers. A neuroradiologist and stroke neurologist independently reviewed vascular imaging of the brain (mandatory for inclusion) and neck to establish a diagnosis of arteriopathy (definite, possible, or absent) in 3 steps: (1) baseline imaging alone; (2) plus clinical data; (3) plus follow-up imaging. A 4-person committee, including a second neuroradiologist and stroke neurologist, adjudicated disagreements. Using the final diagnosis as the gold standard, we calculated the sensitivity and specificity of each step. Cases were aged median 7.6 years (interquartile range, 2.8-14 years); 56% boys. The majority (52%) was previously healthy; 41% had follow-up vascular imaging. Only 56 (16%) required adjudication. The gold standard diagnosis was definite arteriopathy in 127 (36%), possible in 34 (9.6%), and absent in 194 (55%). Sensitivity was 79% at step 1, 90% at step 2, and 94% at step 3; specificity was high throughout (99%, 100%, and 100%), as was agreement between reviewers (κ=0.77, 0.81, and 0.78). Clinical data and follow-up imaging help, yet uncertainty in the diagnosis of childhood arteriopathy remains. This presents a challenge to better understanding the mechanisms underlying these arteriopathies and designing strategies for prevention of childhood arterial ischemic stroke.