Activation and reconfiguration of fictive feeding by the octopamine-containing modulatory OC interneurons in the snail Lymnaea.
We describe the role of the octopamine-containing OC interneurons in the buccal feeding system of Lymnaea stagnalis. OC neurons are swallowing phase interneurons receiving inhibitory inputs in the N1 and N2 phases, and excitatory inputs in the N3 phase of fictive feeding. Although the OC neurons do not always fire during feeding, the feeding rate is significantly (P < 0.001) higher when both SO and OC fire in each cycle than when only the SO fires. In 28% of silent preparations, a single stimulation of an OC interneuron evokes the feeding pattern. Repetitive stimulation of the OC interneuron increases the proportion of responsive preparations to 41%. The OC interneuron not only changes both the feeding rate and reconfigures the pattern. Depolarization of the OC interneurons increases the feeding rate and removes the B3 motor neuron from the firing sequence. Hyperpolarization slows it down (increasing the duration of N1 and N3 phases) and recruits the B3 motor neuron. OC interneurons form synaptic connections onto buccal motor neurons and interneurons but not onto the cerebral (cerebral giant cell) modulatory neurons. OC interneurons are electrically coupled to all N3 phase (B4, B4Cl, B8) feeding motor neurons. They form symmetrical connections with the N3p interneurons having dual electrical (excitatory) and chemical (inhibitory) components. OC interneurons evoke biphasic synaptic inputs on the protraction phase interneurons (SO, N1L, N1M), with a short inhibition followed by a longer lasting depolarization. N2d interneurons are hyperpolarized, while N2v interneurons are slowly depolarized and often fire a burst after OC stimulation. Most motor neurons also receive synaptic responses from the OC interneurons. Although OC and N3p interneurons are both swallowing phase interneurons, their synaptic contacts onto follower neurons are usually different (e.g., the B3 motor neurons are inhibited by OC, but excited by N3p interneurons). Repetitive stimulation of OC interneuron facilitates the excitatory component of the biphasic responses evoked on the SO, N1L, and N1M interneurons, but neither the N2 nor the N3 phase interneurons display a similar longer-lasting excitatory effect. OC interneurons are inhibited by all the buccal feeding interneurons, but excited by the serotonergic modulatory CGC neurons. We conclude that OC interneurons are a new kind of swallowing phase interneurons. Their connections with the buccal feeding interneurons can account for their modulatory effects on the feeding rhythm. As they contain octopamine, this is the first example in Lymnaea that monoaminergic modulation and reconfiguration are provided by an intrinsic member of the buccal feeding network.


Daily 300 mg dose of linezolid for the treatment of intractable multidrug-resistant and extensively drug-resistant tuberculosis.
Although previous studies have suggested that linezolid may be effective for treating multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), the optimal dose of linezolid for intractable MDR/XDR-TB is not clear. Twenty-four patients with intractable MDR/XDR-TB were treated with a daily 300 mg dose of linezolid as part of their anti-TB drug regimen. The patients were treated with linezolid for a median duration of 359 days [interquartile range (IQR) 268-443 days]. Seventeen (71%) patients received 300 mg of linezolid once daily from the beginning of treatment for a median duration of 289 days (IQR 233-405 days). Of these patients, four developed peripheral neuropathy, one of whom discontinued linezolid. In seven (29%) patients, 600 mg/day linezolid was administered initially for a median duration of 104 days (IQR 26-145 days) followed by 300 mg/day linezolid for a median duration of 348 days (IQR 298-427 days). In five of these seven patients, the reason for changing from 600 to 300 mg/day was due to side effects of 600 mg/day linezolid (peripheral neuropathy in four patients and leucopenia in one patient). After reducing the dose to 300 mg/day, linezolid could be continued in six of the seven patients. Negative sputum conversion was achieved in 22 (92%) patients after a median of 89 days from the start of linezolid treatment (IQR 48-160 days). A daily 300 mg dose of linezolid may be useful for increasing the chances of culture conversion in the treatment of patients with intractable MDR/XDR-TB and might have fewer side effects, especially neurotoxicity, compared with a daily 600 mg dose of linezolid therapy. The present results encourage further research into the use of a 300 mg dose of linezolid for MDR/XDR-TB patients.


Metamaterials-based broadband generation of orbital angular momentum carrying vector beams.
We propose a simple approach to broadband generation of orbital angular momentum (OAM) carrying vector beams based on compact metamaterials. It consists of two concentric rings in a gold film, where each ring is composed of subwavelength rectangular apertures with gradually varied orientation. The subwavelength rectangular aperture serves as a localized spatial polarizer. We show the generation of different OAM-carrying vector beams with OAM charge number and polarization order varying from -3 to +3 using a 11.2×11.2 μm device. The extinction ratio can exceed 20 dB, and the operation bandwidth (1500 nm) can cover from 1000 to 2500 nm (from near-infrared to mid-infrared). The device provides three degrees of freedom (polarization order l, polarization of input beam σ, and initial orientation angle α(0)) to flexibly generate different OAM-carrying vector beams. We can use a single device to generate two OAM-carrying vector beams with opposite charge sign of OAM by simply controlling the polarization of the input beam. We further study the performance dependence of the designed metamaterials on the offset of the initial orientation angle, length, and width of the rectangular apertures. The obtained results indicate favorable fabrication tolerance.


The Chinatown Patient Navigation Program: Adaptation and Implementation of Breast and Cervical Cancer Patient Navigation in Chicago's Chinatown.
As health care reform continues within the United States, navigators may play increasingly diverse and vital roles across the health care continuum. The growing interest in patient navigation programs for underserved populations calls for detailed descriptions of intervention components to facilitate implementation and dissemination efforts. In Chicago's Chinatown, Chinese immigrant women face language, cultural, and access barriers in obtaining breast and cervical cancer screening and follow-up. These barriers spurred the research partnership between Northwestern University, the Chinese American Service League, Mercy Hospital & Medical Center, and Rush University Medical Center to formalize the Chinatown Patient Navigation Collaborative for adapting and implementing previously developed patient navigation models. In this report, we describe the adaptation of patient navigation to build the Chinatown Patient Navigation Collaborative's community-based patient navigation program for breast and cervical cancer. We offer insights into the roles of community patient navigators in safety net hospital and underserved Chinese immigrant communities, and describe implications for patient navigation initiatives to maximize community benefits by improving access to health care for vulnerable populations. Our adaptation and implementation of a patient navigation intervention in Chicago's Chinatown illustrates promising approaches for future navigator research.


Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.
Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.


Temporal influences on the prediction of postinfarction mortality by heart rate variability: a comparison with the left ventricular ejection fraction.
To examine the influence of the duration of follow up on the values of heart rate variability (HRV) and the left ventricular ejection fraction (LVEF) for predicting mortality after infarction. HRV is an index of autonomic balance that identifies patients at a high risk of arrhythmic events. The index is most depressed during the first few weeks after myocardial infarction whereas left ventricular function tends to deteriorate with time. The value of depressed HRV measured before discharge from hospital for predicting mortality after infarction should decline with time. The HRV and the LVEF were assessed in 433 survivors of a first acute myocardial infarction: HRV < 20 units and LVEF < 40% were taken as cut off points. Kaplan-Meier survival functions for total cardiac mortality and sudden cardiac death were calculated for the whole five year follow up period and for different intervening periods. During follow up of four weeks to five years there were 46 (10.6%) deaths and 15 (3.5%) patients died suddenly. Within the whole follow up period, HRV < 20 units and LVEF < 40% were both strongly associated with total cardiac mortality (p < 0.0001), but HRV was an independent predictor of total cardiac mortality only during the first six months of follow up. There were no deaths predicted by HRV < 20 units after the first year of follow up whereas LVEF < 40% had a sensitivity of 43% and a positive predictive accuracy of 9% for predicting death during this period. HRV < 20 units was better than LVEF < 40% in predicting sudden deaths during the first year of follow up but was an independent predictor only of those sudden deaths occurring within six months of infarction. The duration of follow up affects the prediction of sudden death and total cardiac mortality from HRV. Reduced HRV as measured before discharge from hospital does not seem to retain independent prognostic value after six months of follow up. These findings have potential implications for the serial evaluation of HRV and for the prevention of sudden death after myocardial infarction.


Creative motivation: creative achievement predicts cardiac autonomic markers of effort during divergent thinking.
Executive approaches to creativity emphasize that generating creative ideas can be hard and requires mental effort. Few studies, however, have examined effort-related physiological activity during creativity tasks. Using motivational intensity theory as a framework, we examined predictors of effort-related cardiac activity during a creative challenge. A sample of 111 adults completed a divergent thinking task. Sympathetic (PEP and RZ) and parasympathetic (RSA and RMSSD) outcomes were assessed using impedance cardiography. As predicted, people with high creative achievement (measured with the Creative Achievement Questionnaire) showed significantly greater increases in sympathetic activity from baseline to task, reflecting higher effort. People with more creative achievements generated ideas that were significantly more creative, and creative performance correlated marginally with PEP and RZ. The results support the view that creative thought can be a mental challenge.


Relationship of smartphone use severity with sleep quality, depression, and anxiety in university students.
The usage of smartphones has increased rapidly in recent years, and this has brought about addiction. The aim of the current study was to investigate the relationship between smartphone use severity and sleep quality, depression, and anxiety in university students. In total, 319 university students (203 females and 116 males; mean age = 20.5 ± 2.45) were included in the study. Participants were divided into the following three groups: a smartphone non-user group (n = 71, 22.3%), a low smartphone use group (n = 121, 37.9%), and a high smartphone use group (n = 127, 39.8%). All participants were evaluated using the Pittsburgh Sleep Quality Index, Beck Depression Inventory, Beck Anxiety Inventory; moreover, participants other than those in the smartphone non-user group were also assessed with the Smartphone Addiction Scale. The findings revealed that the Smartphone Addiction Scale scores of females were significantly higher than those of males. Depression, anxiety, and daytime dysfunction scores were higher in the high smartphone use group than in the low smartphone use group. Positive correlations were found between the Smartphone Addiction Scale scores and depression levels, anxiety levels, and some sleep quality scores. The results indicate that depression, anxiety, and sleep quality may be associated with smartphone overuse. Such overuse may lead to depression and/or anxiety, which can in turn result in sleep problems. University students with high depression and anxiety scores should be carefully monitored for smartphone addiction.


Use of several second generation serological assays to determine the true prevalence of hepatitis C virus infection in haemophiliacs treated with non-virus inactivated factor VIII and IX concentrates.
To investigate the prevalence of hepatitis C virus infection in two risk groups, stored serum samples from treated haemophiliacs and intravenous drug users were tested for anti-HCV by both anti-C-100 based and second generation ELISAs (Abbott and Ortho) followed by testing in two confirmatory immunoblot assays that incorporate core as well as other non-structural antigens (Innogenetics LIA and Chiron RIBA-HCV test). Clear evidence of HCV infection was found in all but one of 78 haemophiliacs treated with non-virus inactivated clotting factor concentrates, but in none exposed only to super dry heat-treated concentrates. Only four samples gave rise to conflicting serological results between the four tests, two of these occurred in patients with advanced HIV related disease and almost certainly reflected loss of humoral immunity associated with disease progression, and the others occurred in the only two patients tested who were chronic carriers of hepatitis B infection and may reflect an interaction between the two viruses. Comparison of anti-C-100 versus second generation tests in immunocompetent drug users revealed a false negative rate of 20% using C-100 alone, indicating the advantage of using second generation assays for detection of past or current HCV infection. Of all of the antigens used in the confirmatory assay, positive sera showed strongest and most frequent reactivity with the C22 and C33c proteins (Ortho RIBA).


Exploring Bayesian Approaches to eQTL Mapping Through Probabilistic Programming.
The discovery of genomic polymorphisms influencing gene expression (also known as expression quantitative trait loci or eQTLs) can be formulated as a sparse Bayesian multivariate/multiple regression problem. An important aspect in the development of such models is the implementation of bespoke inference methodologies, a process which can become quite laborious, when multiple candidate models are being considered. We describe automatic, black-box inference in such models using Stan, a popular probabilistic programming language. The utilization of systems like Stan can facilitate model prototyping and testing, thus accelerating the data modeling process. The code described in this chapter can be found at https://github.com/dvav/eQTLBookChapter .


Model of the mechanism of Ca loss by bones under microgravity and earth conditions.
A comparative characterization of crystal structure, morphology, sizes, and orientation in Ca phosphate precipitation from aqueous solutions, the mineral phase in bones, and mineral deposits on cardiac valves has been performed by high-resolution transmission electron microscopy to model possible mechanisms of Ca loss by bones. Physiological changes occurring in organisms can lead to deep perturbations of the natural calcium phosphate supersaturation and its local distribution, which in turn influences the phase composition, morphology, and organization of the mineral phase. Formation of crystals with larger size or of two distinct phases instead of the single hydroxyapatite one can result in the deterioration of the Ca balance in bone and tissue destruction as well as the possible misorientation (or spread of orientation) between HAP crystals newly formed in the bone.


Phytochip: development of a DNA-microarray for rapid and accurate identification of Pseudo-nitzschia spp and other harmful algal species.
Detection of harmful algal blooms has become a challenging concern because of the direct impacts on public health and economy. The identification of toxic dinoflagellates and diatoms in monitoring programs requires an extensive taxonomic expertise and is time consuming. Advances in molecular biology have allowed the development of new approaches, more rapid, accurate and cost-effective for detecting these microorganisms. In this context, we developed a new DNA microarray (called, Phytochip) for the simultaneous detection of multiple HAB species with a particular emphasis on Pseudo-nitzschia species. Oligonucleotide probes were designed along the rRNA operon. After DNA extraction, the target rDNA genes were amplified and labeled using an asymmetric PCR; then, the amplicons were hybridized to the oligonucleotide probes present on the chips. The total assay from seawater sampling to data acquisition can be performed within a working day. Specificity and sensitivity were assessed by using monoclonal cultures, mixtures of species and field samples spiked with a known amount of cultured cells. The Phytochip with its 81 validated oligonucleotide probes was able to detect 12 species of Pseudo-nitzschia and 11 species of dinoflagellates among which were 3 species of Karenia and 3 species of Alexandrium. The Phytochip was applied to environmental samples already characterized by light microscopy and cloned into DNA libraries. The hybridizations on the Phytochip were in good agreement with the sequences retrieved from the clone libraries and the microscopic observations. The Phytochip enables a reliable multiplex detection of phytoplankton and can assist a water quality monitoring program as well as more general ecological research.


Disentangling the contribution of the transboundary out-flow from the Asian continent to Tokyo, Japan.
We assessed the contribution of transboundary air pollutants (TAPs) transported from China to Tokyo using the Pb(0.5<Dp < 1)/Cu(2.5<Dp < 10) index. We conducted intensive observations over four seasons during 2014-2015 and analyzed ionic components and thirteen elements in size-segregated aerosols collected at the top of a 30-m-high tower in a hilly forested area of the Field Museum Tamakyuryo (FM Tama), in a western suburb of Tokyo. In order to evaluate if the Pb(0.5<Dp < 1)/Cu(2.5<Dp < 10) ratio can be applied in Tokyo as the megacity, the annual average concentration of Pb at Cape Hedo, Kumamoto city, on the west side of Japan, was higher than that of Pb at Tokyo, on the east side; this result indicates that TAPs affect western Japan more than eastern Japan. We inferred the main source of Cu to be braking abrasion from cars at local scale on the basis of its enrichment factor and size distribution. From the Pb(0.5<Dp < 1)/Cu(2.5<Dp < 10) ratio and sulfate concentration, we inferred that the highest contribution of TAPs to the total mass concentration in Tokyo occurred in spring, when TAPs accounted for 55%, 72%, and 53% of the total mass concentration in the 0.1 < Dp < 0.5, 0.5 < Dp < 1, and 1 < Dp < 2.5 fractions, respectively. These results can contribute to assessment of TAPs in Tokyo.


Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform. In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples. Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.


Response of macrophyte communities to flow regulation in mountain streams.
River macrophytes are widely used in freshwater ecosystem assessment because of their sensitivity to anthropogenic pressures, even if there are only a few studies that investigated how macrophytes respond to water regime alterations. In this study, we analyzed the effects of dams on river macrophyte communities through a comparison between upstream and downstream sides from 18 dams located in Alps and Apennines. A co-inertia analysis and a Mantel test were applied to assess if the analysis of environmental parameters could be effective in predicting macrophyte community structure. We analyzed morphological and physicochemical inter-site differences and tested the influence of dams on various aspects of community structure (composition, richness, diversity, dominance, coverage) using multivariate randomized block permutation procedure. Plant similarity between sites was evaluated at the level of phylum, and indicator species analysis was performed to identify the taxa most sensitive or tolerant to water regulation. We found that the overall environmental setting overwhelms the dam impact and that the influence of hydrological alteration became apparent when comparing upstream and downstream assemblages at the same dam. In particular, we found that most of taxa had a higher affinity with the downstream side and that in general, stream regulation increases plant richness and coverage, but reduces community evenness. Analyses based on higher taxonomic groups (phyla) demonstrated that this community can be effectively used in bioassessment even at phylum level analysis. In particular, we found that bryophytes, strictly linked with changes in substrate stability, show particular sensitivity to water regulation in mountain streams.


The role of COX-2 in intestinal cancer.
Cyclooxygenase (COX), the key regulatory enzyme for prostaglandin synthesis is transcribed from two distinct genes. COX-1 is expressed constitutively in most tissues, and COX-2 is induced by a wide variety of stimuli and was initially identified as an immediate-early growth response gene. In addition, COX-2 expression is markedly increased in 85-90% of human colorectal adenocarcinomas, whereas COX-1 levels remain unchanged. Several epidemiological studies have reported a 40-50% reduction in the risk of developing colorectal cancer in persons who chronically take such nonsteroidal anti-inflammatory drugs (NSAIDs) as aspirin, which are classic inhibitors of cyclooxygenase. Genetic evidence also supports a role for COX-2, since mice null for COX-2 have an 86% reduction in tumor multiplicity in a background containing a mutated APC allele. These results strongly suggest that COX-2 contributes to the development of intestinal tumors and that inhibition of COX is chemo-preventative.


Plasmid-based lacZalpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase.
The preparation of a gapped pUC18 derivative, containing the lacZalpha reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZalpha gene with sites for two related nicking endonucleases, enabling the excision of either the coding or non-coding strand. However, the excised strand remains annealed to the plasmid through non-covalent Watson-Crick base-pairing; its removal, therefore, requires a heat-cool cycle in the presence of an exactly complementary competitor DNA. The gapped plasmids can be used to assess DNA polymerase fidelity using in vitro replication, followed by transformation into Escherichia coli and scoring the blue/white colony ratio. Results found with plasmids are similar to the well established method based on gapped M13, in terms of background ( approximately 0.08% in both cases) and the mutation frequencies observed with a number of DNA polymerases, providing validation for this straightforward and technically uncomplicated approach. Several error prone variants of the archaeal family-B DNA polymerase from Pyrococcus furiosus have been investigated, illuminating the potential of the method.


Quantitative chromatin proteomics reveals a dynamic histone post-translational modification landscape that defines asexual and sexual Plasmodium falciparum parasites.
Gene expression in Plasmodia integrates post-transcriptional regulation with epigenetic marking of active genomic regions through histone post-translational modifications (PTMs). To generate insights into the importance of histone PTMs to the entire asexual and sexual developmental cycles of the parasite, we used complementary and comparative quantitative chromatin proteomics to identify and functionally characterise histone PTMs in 8 distinct life cycle stages of P. falciparum parasites. ~500 individual histone PTMs were identified of which 106 could be stringently validated. 46 individual histone PTMs and 30 co-existing PTMs were fully quantified with high confidence. Importantly, 15 of these histone PTMs are novel for Plasmodia (e.g. H3K122ac, H3K27me3, H3K56me3). The comparative nature of the data revealed a highly dynamic histone PTM landscape during life cycle development, with a set of histone PTMs (H3K4ac, H3K9me1 and H3K36me2) displaying a unique and conserved abundance profile exclusively during gametocytogenesis (P < 0.001). Euchromatic histone PTMs are abundant during schizogony and late gametocytes; heterochromatic PTMs mark early gametocytes. Collectively, this data provides the most accurate, complete and comparative chromatin proteomic analyses of the entire life cycle development of malaria parasites. A substantial association between histone PTMs and stage-specific transition provides insights into the intricacies characterising Plasmodial developmental biology.


Human coronary plaque wall thickness correlated positively with flow shear stress and negatively with plaque wall stress: an IVUS-based fluid-structure interaction multi-patient study.
Atherosclerotic plaque progression and rupture are believed to be associated with mechanical stress conditions. In this paper, patient-specific in vivo intravascular ultrasound (IVUS) coronary plaque image data were used to construct computational models with fluid-structure interaction (FSI) and cyclic bending to investigate correlations between plaque wall thickness and both flow shear stress and plaque wall stress conditions. IVUS data were acquired from 10 patients after voluntary informed consent. The X-ray angiogram was obtained prior to the pullback of the IVUS catheter to determine the location of the coronary artery stenosis, vessel curvature and cardiac motion. Cyclic bending was specified in the model representing the effect by heart contraction. 3D anisotropic FSI models were constructed and solved to obtain flow shear stress (FSS) and plaque wall stress (PWS) values. FSS and PWS values were obtained for statistical analysis. Correlations with p < 0.05 were deemed significant. Nine out of the 10 patients showed positive correlation between wall thickness and flow shear stress. The mean Pearson correlation r-value was 0.278 ± 0.181. Similarly, 9 out of the 10 patients showed negative correlation between wall thickness and plaque wall stress. The mean Pearson correlation r-value was -0.530 ± 0.210. Our results showed that plaque vessel wall thickness correlated positively with FSS and negatively with PWS. The patient-specific IVUS-based modeling approach has the potential to be used to investigate and identify possible mechanisms governing plaque progression and rupture and assist in diagnosis and intervention procedures. This represents a new direction of research. Further investigations using more patient follow-up data are warranted.


Nonmyeloablative HLA-matched sibling allogeneic hematopoietic stem cell transplantation for severe sickle cell phenotype.
Myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is curative for children with severe sickle cell disease, but toxicity may be prohibitive for adults. Nonmyeloablative transplantation has been attempted with degrees of preparative regimen intensity, but graft rejection and graft-vs-host disease remain significant. To determine the efficacy, safety, and outcome on end-organ function with this low-intensity regimen for sickle cell phenotype with or without thalassemia. From July 16, 2004, to October 25, 2013, 30 patients aged 16-65 years with severe disease enrolled in this nonmyeloablative transplant study, consisting of alemtuzumab (1 mg/kg in divided doses), total-body irradiation (300 cGy), sirolimus, and infusion of unmanipulated filgrastim mobilized peripheral blood stem cells (5.5-31.7 × 10(6) cells/kg) from human leukocyte antigen-matched siblings. The primary end point was treatment success at 1 year after the transplant, defined as a full donor-type hemoglobin for patients with sickle cell disease and transfusion independence for patients with thalassemia. The secondary end points were the level of donor leukocyte chimerism; incidence of acute and chronic graft-vs-host disease; and sickle cell-thalassemia disease-free survival, immunologic recovery, and changes in organ function, assessed by annual brain imaging, pulmonary function, echocardiographic image, and laboratory testing. Twenty-nine patients survived a median 3.4 years (range, 1-8.6), with no nonrelapse mortality. One patient died from intracranial bleeding after relapse. As of October 25, 2013, 26 patients (87%) had long-term stable donor engraftment without acute or chronic graft-vs-host disease. The mean donor T-cell level was 48% (95% CI, 34%-62%); the myeloid chimerism levels, 86% (95% CI, 70%-100%). Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no graft-vs-host disease. The normalized hemoglobin and resolution of hemolysis among engrafted patients were accompanied by stabilization in brain imaging, a reduction of echocardiographic estimates of pulmonary pressure, and allowed for phlebotomy to reduce hepatic iron. The mean annual hospitalization rate was 3.23 (95% CI, 1.83-4.63) the year before, 0.63 (95% CI, 0.26-1.01) the first year after, 0.19 (95% CI, 0-0.45) the second year after, and 0.11 (95% CI, 0.04-0.19) the third year after transplant. For patients taking long-term narcotics, the mean use per week was 639 mg (95% CI, 220-1058) of intravenous morphine-equivalent dose the week of their transplants and 140 mg (95% CI, 56-225) 6 months after transplant. There were 38 serious adverse events: pain and related management, infections, abdominal events, and sirolimus related toxic effects. Among 30 patients with sickle cell phenotype with or without thalassemia who underwent nonmyeloablative allogeneic HSCT, the rate of stable mixed-donor chimerism was high and allowed for complete replacement with circulating donor red blood cells among engrafted participants. Further accrual and follow-up are required to assess longer-term clinical outcomes, adverse events, and transplant tolerance. clinicaltrials.gov Identifier: NCT00061568.


Effects of seaweed supplementation on blood glucose concentration, lipid profile, and antioxidant enzyme activities in patients with type 2 diabetes mellitus.
The present study was carried out to evaluate the physiological effects of seaweed supplementation on blood glucose levels, lipid profile, and antioxidant enzyme activities in subjects with type 2 diabetes mellitus. Subjects were randomized into either a control group or a seaweed supplementation group. Pills with equal parts of dry powdered sea tangle and sea mustard were provided to the seaweed supplementation group three times a day for 4 weeks. Total daily consumption of seaweed was 48 g. We found that total dietary fiber intake was 2.5 times higher in subjects receiving seaweed supplementation than in the control group. Accordingly, fasting blood glucose levels (p<0.01) and 2-hour postprandial blood glucose measurements (p<0.05) were decreased significantly in those ingesting seaweed. Furthermore, the serum concentrations of triglycerides were decreased and high-density lipoprotein cholesterol was increased significantly in seaweed supplement group (p<0.05). However, the concentrations of total cholesterol and low-density lipoprotein cholesterol were not affected by seaweed supplementation. The level of thiobarbituric acid reactive substances in erythrocytes was significantly lower with seaweed supplementation compared to controls (p<0.05). Catalase and glutathione peroxidase activities with seaweed supplementation were higher than the controls (p<0.05), but superoxide dismutase activity was not affected. We, therefore, conclude that ingestion of seaweed influences glycemic control, lowers blood lipids, and increases antioxidant enzyme activities.


Detection of cystic fibrosis mutations by peptide mass signature genotyping.
The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis. We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149-54) were assessed using the PMSG test for the CFTR gene. Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.


Inflammatory arthritis disrupts gut resolution mechanisms, promoting barrier breakdown by Porphyromonas gingivalis.
Rheumatoid arthritis is linked with altered host immune responses and severe joint destruction. Recent evidence suggests that loss of gut homeostasis and barrier breach by pathobionts, including Porphyromonas gingivalis, may influence disease severity. The mechanism(s) leading to altered gut homeostasis and barrier breakdown in inflammatory arthritis are poorly understood. In the present study, we found a significant reduction in intestinal concentrations of several proresolving mediators during inflammatory arthritis, including downregulation of the gut-protective mediator resolvin D5n-3 DPA (RvD5n-3 DPA). This was linked with increased metabolism of RvD5n-3 DPA to its inactive 17-oxo metabolite. We also found downregulation of IL-10 expression in the gut of arthritic mice that was coupled with a reduction in IL-10 and IL-10 receptor (IL-10R) in lamina propria macrophages. These changes were linked with a decrease in the number of mucus-producing goblet cells and tight junction molecule expression in the intestinal epithelium of arthritic mice when compared with naive mice. P. gingivalis inoculation further downregulated intestinal RvD5n-3 DPA and Il-10 levels and the expression of gut tight junction proteins. RvD5n-3 DPA, but not its metabolite 17-oxo-RvD5n-3 DPA, increased the expression of both IL-10 and IL-10R in macrophages via the upregulation of the aryl hydrocarbon receptor agonist l-kynurenine. Administration of RvD5n-3 DPA to arthritic P. gingivalis-inoculated mice increased intestinal Il-10 expression, restored gut barrier function, and reduced joint inflammation. Together, these findings uncover mechanisms in the pathogenesis of rheumatoid arthritis, where disruption of the gut RvD5n-3 DPA-IL-10 axis weakens the gut barrier, which becomes permissive to the pathogenic actions of the pathobiont P. gingivalis.


Cell-mediated immunity in acute and chronic hepatitis.
Peripheral lymphocytes from patients with hepatitis-B surface antigen (HBsAg)-positive and -negative acute hepatitis (AH), chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and normal controls were tested for in vitro cytotoxicity and blast transformation. Cytotoxicity was measured by chrominum (21Cr) release into the medium from 51Cr-labeled Chang liver cells after incubation for 6 h with peripheral lymphocytes at a lymphocyte target cell ratio of 200:1. Concomitant 72-h incubation studies were performed to assess thymus cell-dependent (T) lymphocyte function as measured by conccanavalin A (Con A)- stimulated incorporation of tritiated thymidine (blast transformation) and by cytotoxicity. It was found that (a) lymphocytes from patients with AH are cytotoxic to Chang liver cells compared to controls (P less than 0.001); (b) lymphocytes from patients with acute and chronic hepatitis are less cytotoxic when incubated with autologous and homologous HB2Ag-positive and -negative AH, CAH, and CPH are as cytotoxic as normal controls when stimulated with a nonspecific mitogen such as Con A; and (d) lymphocytes from patients with CAH while on prednisone therapy showed marked depression of cytotoxicity when stimulated with Con A. Thus these studies show that patients with AH have circulating T lymphocytes which are capable of causing the destruction of Chang liver cells. There is no defect in T-cell function as measured by Con A-stimulated cytotoxicity. There is a serum factor (s) in patients with acute and chronic hepatitis which inhibits spontaneous and induced lymphocyte cytotoxicity and blast transformation. Finally, prednisone treatment appears to inhibit lymphocyte cytotoxicity in patients with CAH.


To eat or not to eat: ontogeny of hypothalamic feeding controls and a role for leptin in modulating life-history transition in amphibian tadpoles.
Many animal life histories entail changing feeding ecology, but the molecular bases for these transitions are poorly understood. The amphibian tadpole is typically a growth and dispersal life-history stage. Tadpoles are primarily herbivorous, and they capitalize on growth opportunities to reach a minimum body size to initiate metamorphosis. During metamorphic climax, feeding declines, at which time the gastrointestinal (GI) tract remodels to accommodate the carnivorous diet of the adult frog. Here we show that anorexigenic hypothalamic feeding controls are absent in the tadpole, but develop during metamorphosis concurrent with the production of the satiety signal leptin. Before metamorphosis there is a large increase in leptin mRNA in fat tissue. Leptin receptor mRNA increased during metamorphosis in the preoptic area/hypothalamus, the key brain region involved with the control of food intake and metabolism. This corresponded with an increase in functional leptin receptor, as evidenced by induction of socs3 mRNA and phosphorylated STAT3 immunoreactivity, and suppression of feeding behaviour after injection of recombinant frog leptin. Furthermore, we found that immunoneutralization of leptin in tadpoles at metamorphic climax caused them to resume feeding. The absence of negative regulation of food intake in the tadpole allows the animal to maximize growth prior to metamorphosis. Maturation of leptin-responsive neural circuits suppresses feeding during metamorphosis to facilitate remodelling of the GI tract.


Rotational spectrum and internal dynamics of tetrahydrofuran-krypton.
The rotational spectrum of the tetrahydrofuran-krypton van der Waals complex has been investigated by pulsed-jet Fourier transform microwave spectroscopy. The spectra of the (84)Kr and (86)Kr isotopologues have been assigned and the krypton atom is located nearly over the oxygen atom, almost perpendicular to the COC plane. Each rotational transition is split into two component lines due to, according to the observed Coriolis coupling term between the tunneling states, the residual pseudorotational effects of the ring in the complex. The splitting between the two vibrational sublevels is 87.462(2) and 87.062(2) MHz for the (84)Kr and (86)Kr isotopologues, respectively. These splittings have been used to determine the barrier to inversion, B(2) = 67 cm(-1). The dissociation energy has been estimated to be 3.7 kJ mol(-1) from centrifugal distortion effects.


In vitro precision of fit of computer-aided designed and computer-aided manufactured titanium screw-retained fixed dental prostheses before and after ceramic veneering.
To compare the precision of fit of full-arch implant-supported screw-retained computer-aided designed and computer-aided manufactured (CAD/CAM) titanium-fixed dental prostheses (FDP) before and after veneering. The null-hypothesis was that there is no difference in vertical microgap values between pure titanium frameworks and FDPs after porcelain firing. Five CAD/CAM titanium grade IV frameworks for a screw-retained 10-unit implant-supported reconstruction on six implants (FDI tooth positions 15, 13, 11, 21, 23, 25) were fabricated after digitizing the implant platforms and the cuspid-supporting framework resin pattern with a laser scanner (CARES(®) Scan CS2; Institut Straumann AG, Basel, Switzerland). A bonder, an opaquer, three layers of porcelain, and one layer of glaze were applied (Vita Titankeramik) and fired according to the manufacturer's preheating and fire cycle instructions at 400-800 °C. The one-screw test (implant 25 screw-retained) was applied before and after veneering of the FDPs to assess the vertical microgap between implant and framework platform with a scanning electron microscope. The mean microgap was calculated from interproximal and buccal values. Statistical comparison was performed with non-parametric tests. All vertical microgaps were clinically acceptable with values <90 μm. No statistically significant pairwise difference (P = 0.98) was observed between the relative effects of vertical microgap of unveneered (median 19 μm; 95% CI 13-35 μm) and veneered FDPs (20 μm; 13-31 μm), providing support for the null-hypothesis. Analysis within the groups showed significantly different values between the five implants of the FDPs before (P = 0.044) and after veneering (P = 0.020), while a monotonous trend of increasing values from implant 23 (closest position to screw-retained implant 25) to 15 (most distant implant) could not be observed (P = 0.169, P = 0.270). Full-arch CAD/CAM titanium screw-retained frameworks have a high accuracy. Porcelain firing procedure had no impact on the precision of fit of the final FDPs. All implant microgap measurements of each FDP showed clinically acceptable vertical misfit values before and after veneering. Thus, the results do not only show accurate performance of the milling and firing but show also a reproducible scanning and designing process.


Characterization of a novel barley protein, HCP1, that interacts with the Brome mosaic virus coat protein.
Brome mosaic virus (BMV) requires the coat protein (CP) not only for encapsidation but also for viral cell-to-cell and long-distance movement in barley plants. This suggests that BMV infection is controlled by interactions of CP with putative host factors as well as with viral components. To identify the host factors that interact with BMV CP, we screened a barley cDNA library containing 2.4 x 10(6) independent clones, using a yeast two-hybrid system. Using full-length and truncated BMV CPs as baits, four candidate cDNA clones were isolated. One of the candidate cDNAs encodes a unique oxidoreductase enzyme, designated HCP1. HCP1 was found predominantly in the soluble fractions after differential centrifugation of BMV-infected and mock-inoculated barley tissues. A two-hybrid binding assay using a series of truncated BMV CPs demonstrated that a C-terminal portion of CP is essential for its interaction with HCP1. Interestingly, experiments with CP mutants bearing single amino acid substitutions at the C-terminus revealed that the capacity for mutant CP-HCP1 binding correlates well with the infectivity of the corresponding mutant viruses in barley. These results indicate that CP-HCP1 binding controls BMV infection of barley, interacting directly with CP, probably in the cell cytoplasm.


Mucilaginibacter xinganensis sp. nov., a phenanthrene-degrading bacterium isolated from wetland soil.
An aerobic, Gram-stain negative, rod-shaped and non-motile strain, BJC16-A31T, was isolated from the wetland soil sample taken from Daxing'anling, Heilongjiang, People's Republic of China. Strain BJC16-A31T was found to be oxidase- and catalase-positive, and produced light orange colonies on modified R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BJC16-A31T is closely related to Mucilaginibacter gotjawali SA3-7T with 96.54% sequence similarity and it formed a separate lineage in the genus Mucilaginibacter. Strain BJC16-A31T contained menaquinone-7 (MK-7) as the predominant isoprenoid quinine. Anteiso-C15:0, C16:0 and anteiso-C15:0 were the major fatty acids. The major polar lipids were phosphatidylethanolamine, six unidentified polar lipid, two unidentified aminophospholipids and one unidentified aminolipid. The genome is composed of a circular 5,301,339 bp chromosome with average G + C percentage of 42.25%. The Average Nucleotide Identity (ANI) between strain BJC16-A31T and M. gotjawali SA3-7T was 77.51%. Combined phenotypic, chemotaxonomic, phylogenetic and genomic characteristics support the conclusion that strain BJC16-A31T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter xinganensis sp. nov. is proposed. The type strain is BJC16-A31T (= CGMCC 1.12728T = NBRC 110384T).


Results of patient specific quality assurance for patients undergoing stereotactic ablative radiotherapy for lung lesions.
Hypofractionated image guided radiotherapy of extracranial targets has become increasingly popular as a treatment modality for inoperable patients with one or more small lesions, often referred to as stereotactic ablative body radiotherapy (SABR). This report details the results of the physical quality assurance (QA) program used for the first 33 lung cancer SABR radiotherapy 3D conformal treatment plans in our centre. SABR involves one or few fractions of high radiation dose delivered in many small fields or arcs with tight margins to mobile targets often delivered through heterogeneous media with non-coplanar beams. We have conducted patient-specific QA similar to the more common intensity modulated radiotherapy QA with particular reference to motion management. Individual patient QA was performed in a Perspex phantom using point dose verification with an ionisation chamber and radiochromic film for verification of the dose distribution both with static and moving detectors to verify motion management strategies. While individual beams could vary by up to 7%, the total dose in the target was found to be within ±2% of the prescribed dose for all 33 plans. Film measurements showed qualitative and quantitative agreement between planned and measured isodose line shapes and dimensions. The QA process highlighted the need to account for couch transmission and demonstrated that the ITV construction was appropriate for the treatment technique used. QA is essential for complex radiotherapy deliveries such as SABR. We found individual patient QA helpful in setting up the technique and understanding potential weaknesses in SABR workflow, thus providing confidence in SABR delivery.


Association of tumor size, location, R.E.N.A.L., PADUA and centrality index score with perioperative outcomes and postoperative renal function.
Multiple scoring systems have been proposed to standardize the description of anatomical features of renal tumors. However, it remains unclear which of these systems, if any, is most useful, or whether any performs better than simply reporting tumor size or location in patients undergoing partial nephrectomy. To clarify these issues we evaluated the association of tumor size, location, R.E.N.A.L. (Radius/Exophytic/Nearness to collecting system/Anterior/Location), PADUA (Preoperative Aspects and Dimensions Used for an Anatomical classification) and centrality index scores with perioperative outcomes. Patients undergoing partial nephrectomy with available preoperative imaging were identified from 2005 to 2011. R.E.N.A.L., PADUA and centrality index scores were assigned according to the described protocols for those systems. Associations between each variable and ischemia time, estimated blood loss, total operative time and change in estimated glomerular filtration rate were examined. A total of 162 patients were identified with a median tumor size of 3.1 cm (IQR 2.2 to 4.6). Median estimated blood loss, ischemia time and total operative time were 200 ml (IQR 100 to 300), 24 minutes (IQR 20 to 30) and 211 minutes (IQR 179 to 249), respectively. Each scoring system was found to have a statistically significant (p <0.001) correlation with ischemia time, with the centrality index system showing the strongest correlation. Furthermore, each of the scoring systems showed a stronger correlation with ischemia time than tumor size or tumor location. Each scoring system outperformed tumor size and location, and may be useful when describing the surgical complexity of renal tumors treated with partial nephrectomy.


Low sensitivity of T-cell based detection of tuberculosis among HIV co-infected Tanzanian in-patients.
To evaluate the performance of QuantiFERON-TB GOLD (QFTG) in a resource-poor setting among patients with and without HIV infection. Cross-sectional study. Two hospitals in Northern Tanzania. Eighty three adult male and female inpatients. All patients were screened for HIV infection and underwent tuberculin skin test (TST) and QFTG. Eighty-three subjects were enrolled, and 29 (35%) of 83 were HIV-infected. QFTG yielded indeterminate results in 12 (22%; 95% CI 12%-34%) of 54 HIV-uninfected and 13 (45%; 95% CI 26%-64%) of 29 HIV-infected subjects (p = 0.0323). Among those with smear-positive pulmonary tuberculosis, TST was positive in 40 (100%; 95% CI 91%-100%) of 40 HIV-uninfected subjects compared with seven (54%; 95% CI 25%-81%) of 13 HIV-infected subjects (p < 0.0001), and QFTG was positive in 28 (70%; 95% CI 53%-83%) of 40 HIV-uninfected subjects compared with three (23%; 95% CI 5%-54%) of 13 HIV-infected subjects (p = 0.0029). Among medical inpatients at risk for latent tuberculosis infection, TST was positive in seven (50%) of 14 HIV-uninfected patients and three (19%) of 16 HIV-infected patients (p = 0.0701) and QFTG was positive among two (14%) of 14 HIV-uninfected patients and three (19%) of 16 HIV-infected patients (p = 0.7437). The presence of HIV co-infection was associated with a significant reduction in sensitivity of both the TST (p < 0.0001) and QFTG (p = 0.0029) for the diagnosis of active M. tuberculosis infection. The high proportion of indeterminate QFTG and lack of sensitivity, particularly among HIV-infected patients, may limit its applicability in settings like Tanzania. Larger studies in resource-poor settings are required.


Optimum phosphor blends for fluorescent lamps.
A theoretical study has been made of the optical properties of two-component phosphor blends for fluorescent lamps. The component spectral energy distributions were taken to be gaussian when the number of emitted photons is plotted vs energy. Mercury lines were added. The widths, positions, and proportions of the components were systematically varied in such a way as to keep the blend chromaticity coordinates constant. Luminosity and color-rendering indices were computed. Optimum blends for daylight, cool white, white, and warm white lamps were determined in terms of a quality index, which measures the combination of brightness and color rendition.


[Analysis of treatments used in infections caused by gram-positive multiresistant cocci in critically ill patients admitted to the ICU].
The appearance of new antimicrobials with activity against Gram-positive multiresistant cocci and knowledge of the limitations of glycopeptides has represented an important change in the use of these antibiotics. To analyze at the national level changes in the use of antibiotics with specific activity against Gram-positive multiresistant cocci in critically ill patients admitted to the ICU as well as the characteristics of patients treated with these agents and the forms of administration. Retrospective cohort study of patients admitted to the ICU for more than 24 hours between 2008 and 2010 in the ENVIN-HELICS national registry. Cases were defined as patients who had received one or more of the following antibiotics: vancomycin, teicoplanin, linezolid or daptomycin. The characteristics of patients who used one or more of these agents were compared with those treated with other antibiotics. Indications and forms of use of each antibiotic were assessed. Descriptive results are presented. A total of 45,757 patients, 27,982 (61.2%) of whom received 63,823 antimicrobials were included in the study. In 6,368 (13.9%) patients, one or more antibiotics specifically active against Gram-positive multiresistant cocci were given. There was a predominance of the use of vancomycin and linezolid and an important increase in the prescription of daptomycin (+320%) and linezolid (+22.4%). In more than 95% of cases, linezolid and daptomycin were prescribed for the treatment of infections, whereas vancomycin and teicoplanin were used for prophylaxis in 20-25% of cases. Between 75% and 80% of indications for treating infections, antibiotics were used empirically except for daptomycin which was used as a directed treatment in 43% of the cases. Only in one third of the indications for empirical treatment, susceptible microorganisms were identified (appropriate treatment). The use of antibiotics with activity against Gram-positive multiresistant cocci remained stable around 14% of all indications. The use of vancomycin and linezolid predominated and there was a clear trend towards an increase in the use of daptomycin and linezolid and a decrease in the use of glycopeptides. Empirical treatments were considered appropriate in only one third of cases.


The Lancet Infectious Diseases Commission on antimicrobial resistance: 6 years later.
In 2013, a Lancet Infectious Diseases Commission described the state of antimicrobial resistance worldwide. Since then, greater awareness of the public health ramifications of antimicrobial resistance has led to national actions and global initiatives, including a resolution at the high-level meeting of the UN General Assembly in 2016. Progress in addressing this issue has ranged from a ban on irrational drug combinations in India to commitments to ban colistin as a growth promoter in animals, improve hospital infection control, and implement better antimicrobial stewardship. Funds have been mobilised, and regulatory barriers to new antibiotic development have been relaxed. These efforts have been episodic and uneven across countries, however. Sustained funding for antimicrobial resistance and globally harmonised targets to monitor progress are still urgently needed. Except for in a few leading countries, antimicrobial resistance has not captured the sustained focus of national leaders and country-level actors, including care providers.


DNA polymerase beta from brain neurons is a repair enzyme.
DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all beta-polymerases.


Gluten Psychosis: Confirmation of a New Clinical Entity.
Non-celiac gluten sensitivity (NCGS) is a syndrome diagnosed in patients with symptoms that respond to removal of gluten from the diet, after celiac disease and wheat allergy have been excluded. NCGS has been related to neuro-psychiatric disorders, such as autism, schizophrenia and depression. A singular report of NCGS presenting with hallucinations has been described in an adult patient. We report a pediatric case of a psychotic disorder clearly related to NCGS and investigate the causes by a review of literature. The pathogenesis of neuro-psychiatric manifestations of NCGS is unclear. It has been hypothesized that: (a) a "leaky gut" allows some gluten peptides to cross the intestinal membrane and the blood brain barrier, affecting the endogenous opiate system and neurotransmission; or (b) gluten peptides may set up an innate immune response in the brain similar to that described in the gut mucosa, causing exposure from neuronal cells of a transglutaminase primarily expressed in the brain. The present case-report confirms that psychosis may be a manifestation of NCGS, and may also involve children; the diagnosis is difficult with many cases remaining undiagnosed. Well-designed prospective studies are needed to establish the real role of gluten as a triggering factor in neuro-psychiatric disorders.


No evidence for links between autism, MMR and measles virus.
We examined whether, in the UK, there is an increased risk of autism (AD) following exposures, in early life, to: (1) wild measles; (2) live attenuated measles, alone or in combination as MMR; and (3) the alteration of the mumps strain within MMR. We conducted time trend analyses of 2407 AD subjects born between 1959-93; and for comparison, 4640 Down's syndrome (DS) subjects born between 1966-93. Between 1968-86, we correlated variations in AD and DS births with wild measles incidence. Between 1959-93, we tested for abrupt changes in the long-term AD birth trend for the effects of introducing: (1) monovalent measles vaccines in 1968; (2) MMR immunization in 1988; and (3) the 'overnight switch' from mixed use of Urabe MMR to exclusive use of Jeryl-Lynn MMR in 1992. Incidence rate ratios (IRRs) were used as measures of association. We found no significant association between AD births and exposure (prenatal and postnatal up to 18 months age) to population rates of measles infections, and no 'step-up' increase in AD births associated with the introduction of monovalent measles and MMR vaccines, and changing mumps strain. An unexpected reduction in AD births of 21% (95% CI 6.9-33.3%; P=0.005) among the post-1987 birth cohorts was detected. No increased risk of AD following exposures to wild measles and vaccinations with monovalent measles, and Urabe or Jeryl-Lynn variants of MMR was detected. The precise meaning of the detected AD births reduction is unclear. Our study cannot exclude rare complications of MMR, given its correlational design.


Arsenite malignantly transforms human prostate epithelial cells in vitro by gene amplification of mutated KRAS.
Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5μM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q<0.05 by RNA-seq. Notably, KRAS expression was highly elevated in CAsE-PE cells, with pathway analysis supporting increased cell proliferation, cell motility, survival and cancer pathways. Targeted DNA sequencing of KRAS revealed a mutant specific allelic imbalance, 'MASI', frequently found in primary clinical tumors. We found high expression of a mutated KRAS transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by lower expression of a wild-type allele. Parallel cultures of RWPE-1 cells retained a wild-type KRAS genotype. Copy number analysis and sequencing showed amplification of the mutant KRAS allele. KRAS is expressed as two splice variants, KRAS4a and KRAS4b, where variant 4b is more prevalent in normal cells compared to greater levels of variant 4a seen in tumor cells. 454 Roche sequencing measured KRAS variants in each cell type. We found KRAS4a as the predominant transcript variant in CAsE-PE cells compared to KRAS4b, the variant expressed primarily in RWPE-1 cells and in normal prostate, early passage, primary epithelial cells. Overall, gene expression data were consistent with KRAS-driven proliferation pathways found in spontaneous tumors and malignantly transformed cell lines. Arsenite is recognized as an important environmental carcinogen, but it is not a direct mutagen. Further investigations into this in vitro transformation model will focus on genomic events that cause arsenite-mediated mutation and overexpression of KRAS in CAsE-PE cells.


Gravity modes as a way to distinguish between hydrogen- and helium-burning red giant stars.
Red giants are evolved stars that have exhausted the supply of hydrogen in their cores and instead burn hydrogen in a surrounding shell. Once a red giant is sufficiently evolved, the helium in the core also undergoes fusion. Outstanding issues in our understanding of red giants include uncertainties in the amount of mass lost at the surface before helium ignition and the amount of internal mixing from rotation and other processes. Progress is hampered by our inability to distinguish between red giants burning helium in the core and those still only burning hydrogen in a shell. Asteroseismology offers a way forward, being a powerful tool for probing the internal structures of stars using their natural oscillation frequencies. Here we report observations of gravity-mode period spacings in red giants that permit a distinction between evolutionary stages to be made. We use high-precision photometry obtained by the Kepler spacecraft over more than a year to measure oscillations in several hundred red giants. We find many stars whose dipole modes show sequences with approximately regular period spacings. These stars fall into two clear groups, allowing us to distinguish unambiguously between hydrogen-shell-burning stars (period spacing mostly ∼ 50 seconds) and those that are also burning helium (period spacing ∼ 100 to 300 seconds).


Decay of elevated antibiotic resistance genes in natural river sediments after sedimentation of wastewater particles.
Large-scale wastewater treatment plants (WWTPs) discharge hundreds of tons of total suspended solids (TSS) into surface waters every year. Additionally, a comparable amount is released by sewer overflows during heavy rain events in case of combined sewer systems. Along with sedimentation, particle-attached microorganisms and their antibiotic resistance genes (ARGs) are herewith transported to the riverbed of the receiving water. To better understand the dynamics of this process, a particulate wastewater fraction was added into batch reactors, which were previously filled with natural river sediments and tap water. In parallel, antibiotics (ABs) (erythromycin, tetracycline, ciprofloxacin, roxithromycin, penicillin V, and sulfamethoxazole) were spiked to investigate their capability to select for resistance. The abundance of six ARGs (ermB, tetM, blaTEM, sul1, CTX-M-32, and qnrS) as well as total bacteria (16S rDNA) was monitored in waters and in sediments for a duration of two months using quantitative PCR. Despite a continuous exposure to ABs (5 μg/L each), the abundance of ARGs remained unaffected. Addition of wastewater particles resulted in a sudden and strong increase of ARGs in waters (3-5 log units) and sediments (1-4 log units), however, elevated ARGs underwent a particular and complete decay. Our results indicate that the increased ARG abundances in receiving rivers are the result of a continuous import of ARGs from WWTP discharges or sewer overflow events. They further imply that elevated ARGs do not persist in receiving rivers, if this continuous import is removed. This seems to be the case merely for ARGs introduced by wastewater, given that a stable background concentration of ARGs was observed for the native population.


Developing a community-based genetic nomenclature for anole lizards.
Comparative studies of amniotes have been hindered by a dearth of reptilian molecular sequences. With the genomic assembly of the green anole, Anolis carolinensis available, non-avian reptilian genes can now be compared to mammalian, avian, and amphibian homologs. Furthermore, with more than 350 extant species in the genus Anolis, anoles are an unparalleled example of tetrapod genetic diversity and divergence. As an important ecological, genetic and now genomic reference, it is imperative to develop a standardized Anolis gene nomenclature alongside associated vocabularies and other useful metrics. Here we report the formation of the Anolis Gene Nomenclature Committee (AGNC) and propose a standardized evolutionary characterization code that will help researchers to define gene orthology and paralogy with tetrapod homologs, provide a system for naming novel genes in Anolis and other reptiles, furnish abbreviations to facilitate comparative studies among the Anolis species and related iguanid squamates, and classify the geographical origins of Anolis subpopulations. This report has been generated in close consultation with members of the Anolis and genomic research communities, and using public database resources including NCBI and Ensembl. Updates will continue to be regularly posted to new research community websites such as lizardbase. We anticipate that this standardized gene nomenclature will facilitate the accessibility of reptilian sequences for comparative studies among tetrapods and will further serve as a template for other communities in their sequencing and annotation initiatives.


A chronicle of SARS-CoV-2: Part-I - Epidemiology, diagnosis, prognosis, transmission and treatment.
In order to benefit the public, community workers and scientific community, we hereby present a chronicle of SARS-CoV-2 that leads to the unseen precedent of social distancing and lockdown owing to coronavirus disease (COVID-19). Information on this life-threatening pandemic of COVID-19 is sparse and discrete; and the urgency is such that the dissemination of information is increasing with numerous daily publications on the topic. Therefore, we developed a comprehensive review on various aspects of SARS-CoV-2 and COVID-19. We scientifically compiled published research, news, and reports from various sources to comprehend and summarize the information and findings on Coronaviruses. The review explicitly covers the aspects like genome and pedigree of SARS-CoV-2; epidemiology, prognosis, pathogenesis, symptoms and diagnosis of COVID-19 in order to catalog the right information on transmission route, and influence of environmental factors on virus transmissions, for the robust understanding of right strategical steps for proper COVID-19 management. We have explicitly highlighted several useful information and facts like: i) No established relationship between progression of SARS-CoV-2 with temperature, humidity and/or both, ii) The underlying mechanism of SARS-CoV-2 is not fully understood, iii) Respiratory droplet size determines drop and airborne-based transmission, iv) Prognosis of COVID-19 can be done by its effects on various body organs, v) Infection can be stopped by restricting the binding of S protein and AE2, vi) Hydroxychloroquine is believed to be better than chloroquine for COVID-19, vii) Ivermectin with Vero-hSLAM cells is able to reduce infection by ~5000 time within 2 days, and viii) Nafamostat mesylate can inhibit SARS-CoV-2 S protein-initiated membrane fusion. We have also suggested future research perspectives, challenges and scope.


Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins.
ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.


Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein.
Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.


Mcm10 self-association is mediated by an N-terminal coiled-coil domain.
Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.


Sixty-four children with persistent itching nodules and contact allergy to aluminium after vaccination with aluminium-adsorbed vaccines-prognosis and outcome after booster vaccination.
Persistent itching subcutaneous nodules and aluminium (Al) allergy have been described after vaccination with Al-adsorbed vaccines but are considered rare. Little is known about the prognosis. Sixty-four children with itching nodules following vaccination with diphtheria-tetanus-pertussis (DTP) vaccines currently used in Sweden (Infanrix® and Pentavac®) were spontaneously reported to the authors from 1999 and followed for up to 12 years. The median duration of itching was 5 years in the 44 children who were free or almost free from symptoms at the latest follow-up. Typical findings were a long interval between vaccination and onset of symptoms (months or years) and intensified itching during intercurrent infections. Contact allergy to aluminium was demonstrated in 60/63 children (95 %). Neither the incidence nor differences between the two vaccines can be estimated from this study, but vaccine-induced itching nodules are probably more common than hitherto realised. The median interval between onset of symptoms and diagnosis was 8 months in a region where nurses were educated to recognise the condition compared to 2 years in other regions. Booster vaccination with DTP-polio was postponed or declined by 15/40 families in fear for new problems. Out of 25 children who received a booster dose, only two had new itching nodules. Intensely itching subcutaneous nodules (vaccination granulomas) and contact allergy to aluminium may occur after primary vaccination with the two most commonly used DTP vaccines in Europe. The condition is probably underreported. Symptoms may last for at least 4-5 years but eventually seem to subside.


From a single double helix to paired double helices and back.
The propagation of our genomes during cell proliferation depends on the movement of sister DNA molecules produced by DNA replication to opposite sides of the cell before it divides. This feat is achieved by microtubules in eukaryotic cells but it has long remained a mystery how cells ensure that sister DNAs attach to microtubules with opposite orientations, known as amphitelic attachment. It is currently thought that sister chromatid cohesion has a crucial role. By resisting the forces exerted by microtubules, sister chromatid cohesion gives rise to tension that is thought essential for stabilizing kinetochore-microtubule attachments. Efficient amphitelic attachment is therefore achieved by an error correction mechanism that selectively eliminates connections that do not give rise to tension. Cohesion between sister chromatids is mediated by a multisubunit complex called cohesin which forms a gigantic ring structure. It has been proposed that sister DNAs are held together owing to their becoming entrapped within a single cohesin ring. Cohesion between sister chromatids is destroyed at the metaphase to anaphase transition by proteolytic cleavage of cohesin's Scc1 subunit by a thiol protease called separase, which severs the ring and thereby releases sister DNAs.


Functional manipulation of dendritic cells by photoswitchable generation of intracellular reactive oxygen species.
Reactive oxygen species (ROS) play an important role in cellular signaling as second messengers. However, studying the role of ROS in physiological redox signaling has been hampered by technical difficulties in controlling their generation within cells. Here, we utilize two inert components, a photosensitizer and light, to finely manipulate the generation of intracellular ROS and examine their specific role in activating dendritic cells (DCs). Photoswitchable generation of intracellular ROS rapidly induced cytosolic mobilization of Ca(2+), differential activation of mitogen-activated protein kinases, and nuclear translocation of NF-κB. Moreover, a transient intracellular ROS surge could activate immature DCs to mature and potently enhance migration in vitro and in vivo. Finally, we observed that intracellular ROS-stimulated DCs enhanced antigen specific T-cell responses in vitro and in vivo, which led to delayed tumor growth and prolonged survival of tumor-bearing mice when immunized with a specific tumor antigen. Therefore, a transient intracellular ROS surge alone, if properly manipulated, can cause immature DCs to differentiate into a motile state and mature forms that are sufficient to initiate adaptive T cell responses in vivo.


Improving Endotracheal Cuff Inflation Pressures: An Evidence-Based Project in a Military Medical Center.
Poor management of endotracheal tube cuff pressures occurs in more than 50% of all general anesthetics, leading to tracheal ischemia, tracheal rupture, sore throats, recurrent laryngeal nerve palsy, tracheal stenosis, microaspiration, and/or inadequate ventilation. General endotracheal anesthesia is common practice throughout the world. Endotracheal tube cuffs are filled with a fluid (gas or liquid) to a safe and adequate pressure of 20 to 30 cm H2O to protect the lung parenchyma from aspiration while also ensuring positive pressure can be generated to oxygenate/ventilate patients. An evidence-based project to improve anesthesia providers' management of endotracheal tube cuff pressures was performed at a military medical center in the southwestern United States. The intervention consisted of an education presentation, availability of cuff manometers in all operating rooms, a charting reminder to document cuff pressures, and a visual prompt in the electronic anesthesia record. The intervention resulted in a statistically significant increase in safe cuff pressures (P = .0032; odds ratio = 4.41, 95% CI = 1.71-11.3).


Coronin 1 regulates cognition and behavior through modulation of cAMP/protein kinase A signaling.
Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic-AMP-protein kinase A-dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1-deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes.


Constraints and Affordances of Online Engagement With Scientific Information-A Literature Review.
Many urgent problems that societies currently face-from climate change to a global pandemic-require citizens to engage with scientific information as members of democratic societies as well as to solve problems in their personal lives. Most often, to solve their epistemic aims (aims directed at achieving knowledge and understanding) regarding such socio-scientific issues, individuals search for information online, where there exists a multitude of possibly relevant and highly interconnected sources of different perspectives, sometimes providing conflicting information. The paper provides a review of the literature aimed at identifying (a) constraints and affordances that scientific knowledge and the online information environment entail and (b) individuals' cognitive and motivational processes that have been found to hinder, or conversely, support practices of engagement (such as critical information evaluation or two-sided dialogue). Doing this, a conceptual framework for understanding and fostering what we call online engagement with scientific information is introduced, which is conceived as consisting of individual engagement (engaging on one's own in the search, selection, evaluation, and integration of information) and dialogic engagement (engaging in discourse with others to interpret, articulate and critically examine scientific information). In turn, this paper identifies individual and contextual conditions for individuals' goal-directed and effortful online engagement with scientific information.


Personalising add-on treatment with inhaled corticosteroids in patients with chronic obstructive pulmonary disease: a benefit-harm modelling study.
Since the benefit-harm balance of adding inhaled corticosteroids to long-acting β2-agonists (LABA) and long-acting muscarinic antagonists (LAMA) for patients with chronic obstructive pulmonary disease is unclear, we evaluated this addition for a range of patient profiles. Analyses considered the effects of low-to-moderate doses of inhaled corticosteroids, LABA, and LAMA compared with LABA and LAMA alone, outcome incidences, and preference weights assigned to averted moderate-to-severe exacerbations (benefit) and severe pneumonia, candidiasis, and dysphonia (harm). Using exponential models, we estimated the preference weight-adjusted 2-year net clinical benefit (ie, benefits outweighing harms) indices. Exacerbation risk thresholds for triggering inhaled corticosteroids, LABA, and LAMA were established when the probability of a 2-year net clinical benefit reached 60%. We estimated the proportion of patients benefiting from added inhaled corticosteroids using an externally validated prediction model for acute exacerbations in primary care. Adding low-to-moderate dose inhaled corticosteroids to LABA and LAMA provided a net clinical benefit in patients with a 2-year baseline exacerbation risk of 54-83%. Low-dose inhaled corticosteroids showed a net clinical benefit if the baseline risk was 40-91%, but not at higher doses. The benefit was modified by blood eosinophil count (BEC) and age. Although no net benefit was associated with a BEC of less than 150 cells per μL, patients with a BEC of 150 cells per μL or more had a net benefit from low-dose inhaled corticosteroids with a 2-year exacerbation risk of 32-95% in those aged 40-79 years and 41-93% in those older than 80 years. A moderate dose of inhaled corticosteroids showed a net benefit in patients younger than 80 years with a BEC of 150 cells per μL or more at 52-86% 2-year exacerbation risk. Depending on the subgroups, the proportion of patients with a net benefit from added inhaled corticosteroids ranged from 0 to 68%. The net clinical benefit of adding different inhaled corticosteroid doses to LABA and LAMA varies greatly with exacerbation risk, BEC, and age. Personalised treatment decisions based on these factors and predicted exacerbation risks might reduce overtreatment and undertreatment with inhaled corticosteroids. None.


JC virus T' proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells.
Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T'(135), T'(136), and T'(165), were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T' proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T' mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T' proteins have on the replication process, a set of T' acceptor site mutants was created in which the unique second acceptor splice site of each T' mRNA was altered to eliminate production of one, two or all three T' mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T'(135) acceptor site resulted in the utilization of cryptic splice sites and the generation of new T' species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T' protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T' proteins and the double mutant that only produces T'(136), exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T'(136) replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T' donor site mutant from an earlier study, was impaired significantly.


The Emerging Role of RHOT1/Miro1 in the Pathogenesis of Parkinson's Disease.
The expected increase in prevalence of Parkinson's disease (PD) as the most common neurodegenerative movement disorder over the next years underscores the need for a better understanding of the underlying molecular pathogenesis. Here, first insights provided by genetics over the last two decades, such as dysfunction of molecular and organellar quality control, are described. The mechanisms involved relate to impaired intracellular calcium homeostasis and mitochondrial dynamics, which are tightly linked to the cross talk between the endoplasmic reticulum (ER) and mitochondria. A number of proteins related to monogenic forms of PD have been mapped to these pathways, i.e., PINK1, Parkin, LRRK2, and α-synuclein. Recently, Miro1 was identified as an important player, as several studies linked Miro1 to mitochondrial quality control by PINK1/Parkin-mediated mitophagy and mitochondrial transport. Moreover, Miro1 is an important regulator of mitochondria-ER contact sites (MERCs), where it acts as a sensor for cytosolic calcium levels. The involvement of Miro1 in the pathogenesis of PD was recently confirmed by genetic evidence based on the first PD patients with heterozygous mutations in RHOT1/Miro1. Patient-based cellular models from RHOT1/Miro1 mutation carriers showed impaired calcium homeostasis, structural alterations of MERCs, and increased mitochondrial clearance. To account for the emerging role of Miro1, we present a comprehensive overview focusing on the role of this protein in PD-related neurodegeneration and highlighting new developments in our understanding of Miro1, which provide new avenues for neuroprotective therapies for PD patients.


EEG reactivity testing for prediction of good outcome in patients after cardiac arrest.
To determine the additional value of EEG reactivity (EEG-R) testing to EEG background pattern for prediction of good outcome in adult patients after cardiac arrest (CA). In this post hoc analysis of a prospective cohort study, EEG-R was tested twice a day, using a strict protocol. Good outcome was defined as a Cerebral Performance Category score of 1-2 within 6 months. The additional value of EEG-R per EEG background pattern was evaluated using the diagnostic odds ratio (DOR). Prognostic value (sensitivity and specificity) of EEG-R was investigated in relation to time after CA, sedative medication, different stimuli, and repeated testing. Between 12 and 24 hours after CA, data of 108 patients were available. Patients with a continuous (n = 64) or discontinuous (n = 19) normal voltage background pattern with reactivity were 3 and 8 times more likely to have a good outcome than without reactivity (continuous: DOR, 3.4; 95% confidence interval [CI], 0.97-12.0; p = 0.06; discontinuous: DOR, 8.0; 95% CI, 1.0-63.97; p = 0.0499). EEG-R was not observed in other background patterns within 24 hours after CA. In 119 patients with a normal voltage EEG background pattern, continuous or discontinuous, any time after CA, prognostic value was highest in sedated patients (sensitivity 81.3%, specificity 59.5%), irrespective of time after CA. EEG-R induced by handclapping and sternal rubbing, especially when combined, had highest prognostic value. Repeated EEG-R testing increased prognostic value. EEG-R has additional value for prediction of good outcome in patients with discontinuous normal voltage EEG background pattern and possibly with continuous normal voltage. The best stimuli were clapping and sternal rubbing.


The liver in autosomal dominant polycystic kidney disease. Implications for pathogenesis.
A histomorphometric and clinico-pathologic analysis of 26 autopsy cases of autosomal dominant polycystic kidney disease (ADPKD) showed that (1) the density of biliary microhamartomas (BMHs) and the stage of polycystic liver disease were strongly correlated, and (2) both were positively correlated with the stage of renal dysfunction and age at autopsy. Using multiple linear regression analysis, only the stage of renal dysfunction was significantly predictive of the density of BMHs, but both variables were simultaneously predictive for the stage of polycystic liver disease. On serial sections, 41.4% of cysts were connected to BMHs and 81.0% of BMHs to portal tracts. Bile-like material was found in 10.7% of BMHs. Flat or polypoid hyperplasia of the epithelium was observed in 2.7% of cysts. These results support the long-maintained view that hepatic cysts in ADPKD result from cystic dilatation of BMHs. They indicate, however, that the number of BMHs increases during life. These observations are consistent with the hypothesis that hepatic and renal cysts in ADPKD have similar pathogeneses, that BMHs and hepatic cysts result from hyperplasia of the bile duct epithelium, and that as they grow, the hepatic cysts become disconnected from the biliary ducts from which they are derived.


Stability, electronic structure, and optical properties of protected gold-doped silver Ag29-xAux (x = 0-5) nanoclusters.
In this work, we used density functional theory (DFT) and linear response time-dependent DFT (LR-TDDFT) to investigate the stability, electronic structure, and optical properties of Au-doped [Ag29-xAux(BDT)12(TPP)4]3- nanoclusters (BDT: 1,3-benzenedithiol; TPP triphenylphosphine) with x = 0-5. The aim of this work is to shed light on the most favorable doped structures by comparing our results with previously published experimental data. The calculated relative energies, ranging between 0.8 and 10 meV per atom, indicate that several doped Ag29-xAux nanoclusters are likely to co-exist at room temperature. However, only the Au-doped [Ag29-xAux(BDT)12(TPP)4]3- nanoclusters that have direct bonding between Au dopants and phosphines display an enhancement in the electronic transitions at ∼450 nm. This agrees with the main spectral absorption features that have been experimentally reported for the mixture of Au-doped silver Ag29-xAux nanoclusters. In addition, the formation of the Au-TPP bond could prevent cluster degradation starting from the detachment of the phosphine molecules, since the Au-TPP bond is stronger by ∼0.4 eV than the analogous Ag-TPP one. Thus, the results presented here show the important role of Au-TPP bonding in determining the stability and optical properties of thiolate/phosphine-protected Ag29-xAux nanoclusters.


Current range characteristics of Swiss stone pine (Pinus cembra L.) along the Carpathians revealed by chloroplast SSR markers.
We investigated the diversity pattern of nine Swiss stone pine (Pinus cembra L.) populations along the Carpathian range including the High Tatras, by using six chloroplast DNA microsatellites (cpSSR). Our aim was to detect genetically distinct regions by clustering of populations, and to tackle possible historical colonization routes. Our analysis referred to an investigated geographical range with the two most distant populations situated at about 500 air km. We found that the most diverse populations are situated at the two edges of the investigated part, in the Retezat Mts. (South Carpathians) and the High Tatras, and diversity decreases towards the populations of the Eastern Carpathians. Hierarchical clustering and NMDS revealed that the populations of the South Carpathians with the Tatras form a distinct cluster, significantly separated from those of the Eastern Carpathians. Moreover, based on the most variable chloroplast microsatellites, the four populations of the two range edges are not significantly different. Our results, supported also by palynological and late glacial macrofossil evidences, indicate refugial territories within the Retezat Mts. that conserved rich haplotype composition. From this refugial territory Pinus cembra might have colonized the Eastern Carpathians, and this was accompanied by a gradual decrease in population diversity. Populations of the High Tatras might have had the same role in the colonizing events of the Carpathians, as positive correlation was detected among populations lying from each other at a distance of 280 km, the maximum distance between neighbouring populations.