Mitsugumin 53 regulates extracellular Ca2+ entry and intracellular Ca2+ release via Orai1 and RyR1 in skeletal muscle.
Mitsugumin 53 (MG53) participates in the membrane repair of various cells, and skeletal muscle is the major tissue that expresses MG53. Except for the regulatory effects of MG53 on SERCA1a, the role(s) of MG53 in the unique functions of skeletal muscle such as muscle contraction have not been well examined. Here, a new MG53-interacting protein, Orai1, is identified in skeletal muscle. To examine the functional relevance of the MG53-Orai1 interaction, MG53 was over-expressed in mouse primary or C2C12 skeletal myotubes and the functional properties of the myotubes were examined using cell physiological and biochemical approaches. The PRY-SPRY region of MG53 binds to Orai1, and MG53 and Orai1 are co-localized in the plasma membrane of skeletal myotubes. MG53-Orai1 interaction enhances extracellular Ca2+ entry via a store-operated Ca2+ entry (SOCE) mechanism in skeletal myotubes. Interestingly, skeletal myotubes over-expressing MG53 or PRY-SPRY display a reduced intracellular Ca2+ release in response to K+-membrane depolarization or caffeine stimulation, suggesting a reduction in RyR1 channel activity. Expressions of TRPC3, TRPC4, and calmodulin 1 are increased in the myotubes, and MG53 directly binds to TRPC3, which suggests a possibility that TRPC3 also participates in the enhanced extracellular Ca2+ entry. Thus, MG53 could participate in regulating extracellular Ca2+ entry via Orai1 during SOCE and also intracellular Ca2+ release via RyR1 during skeletal muscle contraction.


Cryptic terminal rearrangement of chromosome 22q13.32 detected by FISH in two unrelated patients.
Two unrelated patients with cryptic subtelomeric deletions of 22q13.3 were identified using FISH with the commercially available Oncor probe, D22S39. Proband 1 was found to have a derivative chromosome 22 resulting from the unbalanced segregation of a t(1;22)(q44;q13.32) in her mother. Additional FISH analysis of proband 1 and her mother placed the breakpoint on chromosome 22 in this family proximal to D22S55 and D22S39 and distal to D22S45. We have mapped D22S39 to within 170 kb of D22S21 using pulsed field gel electrophoresis. D22S21 is genetically mapped between D22S55 and D22S45. These data indicate that the deletion in proband 1 is smaller than in eight of nine reported del(22)(q13.3) patients. Probands 1 and 2 share features of hypotonia, developmental delay, and expressive language delay, also seen in previously reported del(22)(q13.3) patients, although proband 1 appears to be more mildly affected. Proband 1 is also trisomic for the region 1q44-->qter. This very small duplication has been previously reported only once and the patient had idiopathic mental retardation. This is the first report where 22q13.3 terminal deletion patients have been identified through the use of FISH, and the first report of a deletion of this region occurring because of missegregation of a parental balanced cryptic translocation. We feel that investigation of the frequency of del(22)(q13.3) in the idiopathic mentally retarded population is warranted and may be aided by the ability to use a commercially available probe (D22S39), which is already currently in use in a large number of cytogenetic laboratories.


SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation.
Simple, efficient reactions for connecting biological building-blocks open up many new possibilities. Here we have designed SnoopLigase, a protein that catalyzes site-specific transamidation, forming an isopeptide bond with more than 95% efficiency between two peptide tags, SnoopTagJr and DogTag. We initially developed these components by three-part splitting of the Streptococcus pneumoniae adhesin RrgA. The units were then engineered, guided by structure, bioinformatic analysis of sequence homology, and computational prediction of stability. After engineering, SnoopLigase demonstrated high-yield coupling under a wide range of buffers and temperatures. SnoopTagJr and DogTag were functional at the N- or C-terminus, while DogTag was also functional at internal sites in proteins. Having directed reaction of SnoopTagJr and DogTag, SnoopLigase remained stably bound to the ligated product, thus reconstituting the parent domain. Separating products from unreacted starting material and catalyst is often as challenging as reactions themselves. However, solid-phase immobilization of SnoopLigase enabled the ligated SnoopTagJr-DogTag product to be eluted with high purity, free from SnoopLigase or unligated substrates. The solid-phase catalyst could then be reused multiple times. In search of a generic route to improve the resilience of enzymes, we fused SnoopTagJr to the N-terminus and DogTag to the C-terminus of model enzymes, allowing cyclization via SnoopLigase. While wild-type phytase and β-lactamase irreversibly aggregated upon heating, cyclization using SnoopLigase conferred exceptional thermoresilience, with both enzymes retaining solubility and activity following heat treatment up to 100 °C. SnoopLigase should create new opportunities for conjugation and nanoassembly, while illustrating how to harness product inhibition and extend catalyst utility.


Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry.
Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.


Analysis of the barley bract suppression gene Trd1.
A typical barley (Hordeum vulgare) floret consists of reproductive organs three stamens and a pistil, and non-reproductive organs-lodicules and two floral bracts, abaxial called 'lemma' and adaxial 'palea'. The floret is subtended by two additional bracts called outer or empty glumes. Together these organs form the basic structural unit of the grass inflorescence, a spikelet. There are commonly three spikelets at each rachis (floral stem of the barley spike) node, one central and two lateral spikelets. Rare naturally occurring or induced phenotypic variants that contain a third bract subtending the central spikelets have been described in barley. The gene responsible for this phenotype was called the THIRD OUTER GLUME1 (Trd1). The Trd1 mutants fail to suppress bract growth and as a result produce leaf-like structures that subtend each rachis node in the basal portion of the spike. Also, floral development at the collar is not always suppressed. In rice and maize, recessive mutations in NECK LEAF1 (Nl1) and TASSEL SHEATH1 (Tsh1) genes, respectively, have been shown to be responsible for orthologous phenotypes. Fine mapping of the trd1 phenotype in an F(3) recombinant population enabled us to position Trd1 on the long arm of chromosome 1H to a 10 cM region. We anchored this to a conserved syntenic region on rice chromosome Os05 and selected a set of candidate genes for validation by resequencing PCR amplicons from a series of independent mutant alleles. This analysis revealed that a GATA transcription factor, recently proposed to be Trd1, contained mutations in 10 out of 14 independent trd1 mutant alleles that would generate non-functional TRD1 proteins. Together with genetic linkage data, we confirm the identity of Trd1 as the GATA transcription factor ortholog of rice Nl1 and maize Tsh1 genes.


Phylogenetic and population genetic analyses of diploid Leucaena (Leguminosae; Mimosoideae) reveal cryptic species diversity and patterns of divergent allopatric speciation.
Leucaena comprises 17 diploid species, five tetraploid species, and a complex series of hybrids whose evolutionary histories have been influenced by human seed translocation, cultivation, and subsequent spontaneous hybridization. Here we investigated patterns of evolutionary divergence among diploid Leucaena through comprehensively sampled multilocus phylogenetic and population genetic approaches to address species delimitation, interspecific relationships, hybridization, and the predominant mode of speciation among diploids. Parsimony- and maximum-likelihood-based phylogenetic approaches were applied to 59 accessions sequenced for six SCAR-based nuclear loci, nrDNA ITS, and four cpDNA regions. Population genetic comparisons included 1215 AFLP loci representing 42 populations and 424 individuals. Phylogenetic results provided a well-resolved hypothesis of divergent species relationships, recovering previously recognized clades of diploids as well as newly resolved relationships. Phylogenetic and population genetic assessments identified two cryptic species that are consistent with geography and morphology. Findings from this study highlight the importance and utility of multilocus data in the recovery of complex evolutionary histories. The results are consistent with allopatric divergence representing the predominant mode of speciation among diploid Leucaena. These findings contrast with the potential hybrid origin of several tetraploid species and highlight the importance of human translocation of seed to the origin of these tetraploids. The recognition of one previously unrecognized species (L. cruziana) and the elevation of another taxon (L. collinsii subsp. zacapana) to specific status (L. zacapana) is consistent with a growing number of newly diagnosed species from neotropical seasonally dry forests, suggesting these communities harbor greater species diversity than previously recognized.


Daily stressors in patients with acute whiplash associated disorders.
Stressors in acute whiplash associated disorders (WAD), as reported on a daily basis, have hitherto been neglected in research. The primary aim of this study was to describe the most stressful daily situation or event reported by individuals with acute WAD within a month of a whiplash trauma. Another aim was to describe the meaning and significance of these daily stressors, i.e. primary appraisal. A descriptive design with a content analysis approach was used. 260 WAD-daily coping assessments (WAD-DCA) generated during 1 week by 51 participants with acute WAD were included in the study. Stressors were analysed using qualitative content analysis. The reported stressors generated 13 categories covering a wide range of stressful situations in daily life related to (i) work, (ii) physical symptoms, (iii) feelings and cognitions, (iv) family and home responsibilities and (v) recreation. The majority of the stressors were appraised as "expected" as well as "disabling". Most threatening stressors were related to work, driving and feelings/cognitions. The wide variety of stressors indicates that it is not only pain itself that influences daily life in acute WAD. Early identification of individual and situation-specific stressors gives new data regarding what bothers individuals suffering from WAD after a collision and may be helpful in understanding the coping process in relation to specific stressors and stressor appraisals.


Benign cervical multi-nodular goiter presenting with acute airway obstruction: a case report.
Benign cervical goiters rarely cause acute airway obstruction. We report the case of a 64-year-old woman of African descent who presented with acute shortness of breath. She required immediate intubation and later a total thyroidectomy for a benign cervical multi-nodular goiter with no retrosternal tracheal compression. Benign multi-nodular goiters are commonly left untreated once euthyroid. Peak inspiratory flow rates should be measured via spirometry in all goiters to assess the degree of tracheal compression. Once tracheal compression is identified, an elective total thyroidectomy should be performed to prevent morbidity and mortality from acute airway obstruction.


Pathogenesis of malignant pleural mesothelioma.
Malignant pleural mesothelioma (MPM) results from neoplastic transformation of mesothelial cells. Past asbestos exposure represents the major risk factor for MPM, as the link between asbestos fibres and MPM has been largely proved by epidemiological and experimental studies. Asbestos fibres induce DNA and chromosome damage linked to oxidative stress following phagocytosis. Recently, simian virus 40 (SV40) has been implicated in the aetiology of MPM. The origin of human infection has been associated with SV40-contaminated polio vaccines, although to date, no epidemiological data supports this hypothesis. SV40 may act as a coactivator of asbestos in mesothelial oncogenesis. The transforming potency of SV40 results from the activity of two viral proteins, large T and small t antigens. SV40 infection stimulates production of growth factors elsewhere implicated in autocrine growth of mesothelioma cells and inactivates RASSF1, a gene silenced in MPM. Roles for ionising radiation, chemicals or genetic factors have also been suggested from the observation of sporadic MPM cases or animal studies. Genetic alterations in the tumour suppressor genes, P16/CDKN2A and neurofibromatosis 2 (NF2), are found both in human MPM and in asbestos-exposed Nf2-deficient mice. MPM is still of great international concern. Despite a ban on asbestos use in Western countries, the incidence of MPM is increasing, due to the long delay between asbestos exposure and diagnosis. Moreover, asbestos is still used in developing countries. The implication of other risk factors, especially SV40, supports a need for further research into MPM.


Pyocyanin Restricts Social Cheating in Pseudomonas aeruginosa.
Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, such as exoproteases and siderophores, that are public goods utilized by the whole population of bacteria, regardless of whether they invested or not in their production. These public goods can be used by QS defective mutants for growth, and since these mutants do not contribute to public goods production, they are considered social cheaters. Pyocyanin is a phenazine that is a toxic, QS-controlled metabolite produced by P. aeruginosa. It is a redox-active compound and promotes the generation of reactive oxygen species; it also possesses antibacterial properties and increases fitness in competition with other bacterial species. Since QS-deficient individuals are less able to tolerate oxidative stress, we hypothesized that the pyocyanin produced by the wild-type population could promote selection of functional QS systems in this bacterium. Here, we demonstrate, using competition experiments and mathematical models, that, indeed, pyocyanin increases the fitness of the cooperative QS-proficient individuals and restricts the appearance of social cheaters. In addition, we also show that pyocyanin is able to select QS in other bacteria such as Acinetobacter baumannii.


Effect of chronic renal failure on Na,K-ATPase alpha 1 and alpha 2 mRNA transcription in rat skeletal muscle.
Previous studies have suggested that an alteration in the expression of the Na,K-ATPase of muscle may be an important determinant of enhanced insulin sensitivity in chronic renal failure. Therefore, in the present studies we have examined the effect of uremia on the Na,K-ATPase alpha isoforms in skeletal muscle, at the level of mRNA expression and enzymatic activity. The activity of the sodium pump, as measured ouabain-sensitive 86Rb/K uptake in soleus muscle, revealed a reduction in the activity in uremia, related to the increment in plasma creatinine values. The decrement in 86Rb uptake by the rat soleus muscle of experimental animals was associated with changes on Na,K-ATPase gene product. Northern analysis of mRNA revealed isoform-specific regulation of Na,K-ATPase by uremia in skeletal muscle: a decrease of approximately 50% in alpha 1 subunit Na,K-ATPase mRNA, as compared to controls. The decrement in alpha 1 mRNA correlates with the decreased activity of the Na,K-ATPase in uremia, under basal conditions and with the almost complete inhibition of the Na,K-ATPase, of uremic tissue by a concentration of 10(-5) M ouabain. Although the activity of the alpha 2 isoform pump was not modified by uremia, the 3.4-kb message for this enzyme was increased 2.2-fold; this discrepancy is discussed. Altogether these findings demonstrate that the defective extrarenal potassium handling in uremia is at least dependent in the expression of alpha 1 subunit of the Na,K-ATPase.


Toward a cytological characterization of the rice genome.
Rice (Oryza sativa L.) will be the first major crop, as well as the first monocot plant species, to be completely sequenced. Integration of DNA sequence-based maps with cytological maps will be essential to fully characterize the rice genome. We have isolated a set of 24 chromosomal arm-specific bacterial artificial chromosomes to facilitate rice chromosome identification. A standardized rice karyotype was constructed using meiotic pachytene chromosomes of O. sativa spp. japonica rice var. Nipponbare. This karyotype is anchored by centromere-specific and chromosomal arm-specific cytological landmarks and is fully integrated with the most saturated rice genetic linkage maps in which Nipponbare was used as one of the mapping parents. An ideogram depicting the distribution of heterochromatin in the rice genome was developed based on the patterns of 4',6-diamidino-2-phenylindole staining of the Nipponbare pachytene chromosomes. The majority of the heterochromatin is distributed in the pericentric regions with some rice chromosomes containing a significantly higher proportion of heterochromatin than other chromosomes. We showed that pachytene chromosome-based fluorescence in situ hybridization analysis is the most effective approach to integrate DNA sequences with euchromatic and heterochromatic features.


Who Goes Where? Exploring Factors Related to Placement Among Group Homes.
Despite their widespread use as a placement option for youth with mental health problems, there is relatively little research on group homes for youth. Available data highlight concerns with practices and treatment within group homes and mixed results on youth-level outcomes. However, existing research appears to collapse a wide range of group residential settings into a single amorphous category. This article explores potential variations among group homes to examine whether different programs are systematically serving different types of youth. It examines, in particular, placement in homes using the teaching family model (TFM) versus homes that do not. Findings suggest that demographics are not significantly associated with TFM placement. However, custody status, types of mental health problems, and use of psychotropic medications are. Homes appear to be serving distinct niches within a geographic area. Implications for future research and policy/practice are discussed.


An Unusual Cause of Cauda Equina Syndrome: Lumbar Epidural Venous Engorgement.
BACKGROUND Epidural venous plexus (EVP) engorgement occurs due to many conditions, so it can be easily misdiagnosed. This becomes problematic when the diagnosis requires prompt treatment for a good outcome, especially when it results in cauda equina syndrome (CES). We report a case of extensive iliocaval thrombosis leading to epidural venous plexus and ascending lumbar vein engorgement as an outcome of deep venous thrombosis (DVT) due to probable adverse effects of oral combined contraceptive pills (OCCP). CASE REPORT A 42-year-old woman presented to a rural medical facility with bilateral lower-limb swelling and skin darkening for 2 days. She was transferred to a tertiary medical facility where her condition deteriorated to severe CES. A lower-limbs ultrasonography confirmed the presence of extensive DVT extending to the lower segment of the inferior vena cava. Spine magnetic resonance imaging showed abnormal enhancement of the conus medullaris with thick enhanced cauda equina nerve roots, consistent with a possible case of Guillain-Barré syndrome. However, there was engorgement of the EVP extending to the ascending lumbar, azygos, and hemiazygos veins, which was misdiagnosed. The patient was managed immediately with low-molecular-weight heparin and steroids. She died 4 weeks after admission due to hospital-acquired pneumonia and acute respiratory distress syndrome, probably due to the high dose of steroids. CONCLUSIONS Acute CES has a wide differential diagnosis. This report describes an unusual cause of CES and emphasizes the importance of early recognition to avoid misdiagnosis and management delay. Early identification of this clinical entity markedly decreases morbidity and mortality and thus improves the prognosis. Likewise, underlying causing factors such as venous congestion due to OCCP-related DVT should be considered in the diagnosis.


Scaleable production of microbubbles using an ultrasound-modulated microfluidic device.
Surfactant-coated gas microbubbles are widely used as contrast agents in ultrasound imaging and increasingly in therapeutic applications. The response of microbubbles to ultrasound can be strongly influenced by their size and coating properties, and hence the production method. Ultrasonic emulsification (sonication) is the most commonly employed method and can generate high concentrations of microbubbles rapidly, but with a broad size distribution, and there is a risk of contamination and/or degradation of sensitive components. Microfluidic devices provide excellent control over microbubble size, but are often challenging or costly to manufacture, offer low production rates (<106s-1), and are prone to clogging. In this study, a hybrid sonication-microfluidic or "sonofluidic" device was developed. Bubbles of ∼180 μm diameter were produced rapidly in a T-junction and subsequently exposed to ultrasound (71-73 kHz) within a microchannel, generating microbubbles (mean diameter: 1-2 μm) at a rate of >108s-1 using a single device. Microbubbles were prepared using either the sonofluidic device or conventional sonication, and their size, concentration, and stability were comparable. The mean diameter, concentration, and stability were found to be comparable between techniques, but the microbubbles produced by the sonofluidic device were all <5 μm in diameter and thus did not require any post-production fractionation.


LEAP: biomarker inference through learning and evaluating association patterns.
Single nucleotide polymorphism (SNP) high-dimensional datasets are available from Genome Wide Association Studies (GWAS). Such data provide researchers opportunities to investigate the complex genetic basis of diseases. Much of genetic risk might be due to undiscovered epistatic interactions, which are interactions in which combination of several genes affect disease. Research aimed at discovering interacting SNPs from GWAS datasets proceeded in two directions. First, tools were developed to evaluate candidate interactions. Second, algorithms were developed to search over the space of candidate interactions. Another problem when learning interacting SNPs, which has not received much attention, is evaluating how likely it is that the learned SNPs are associated with the disease. A complete system should provide this information as well. We develop such a system. Our system, called LEAP, includes a new heuristic search algorithm for learning interacting SNPs, and a Bayesian network based algorithm for computing the probability of their association. We evaluated the performance of LEAP using 100 1,000-SNP simulated datasets, each of which contains 15 SNPs involved in interactions. When learning interacting SNPs from these datasets, LEAP outperformed seven others methods. Furthermore, only SNPs involved in interactions were found to be probable. We also used LEAP to analyze real Alzheimer's disease and breast cancer GWAS datasets. We obtained interesting and new results from the Alzheimer's dataset, but limited results from the breast cancer dataset. We conclude that our results support that LEAP is a useful tool for extracting candidate interacting SNPs from high-dimensional datasets and determining their probability.


Regionalized calcium signaling in zebrafish fertilization.
Fertilization involves an initial, highly localized signal delivered by the sperm, which becomes amplified by a signal transduction cascade to impact the entire oocyte cytoplasm. The zebrafish oocyte presents a unique opportunity to study this process since fertilization always occurs at the micropyle, allowing the investigator to image the earliest steps in the oocyte activation process. The objective of the present study was to characterize the amplification of the sperm-induced calcium transient in the zebrafish oocyte and test the role of Fyn kinase in this process. Confocal fluorescence microscopy revealed that the sperm-induced calcium transient was composed of two elements, one of which was unique to the oocyte cortex and a second, slower transient that occurred in the central cytoplasm of the oocyte. The cortical transient was initiated immediately deep to the micropyle, became amplified at the animal pole, and progressed peripherally through the oocyte cortex. This was followed by a slower transient that occurred in the central cytoplasm of the oocyte. Several lines of evidence indicate that calcium release in these two compartments may be regulated differently. The calcium transient in the oocyte cortex is highly sensitive to inhibition by Fyn-SH2 domain containing fusion proteins, while the central cytoplasmic transient is relatively resistant to this treatment. Oocytes stimulated by injection of a soluble extract prepared from zebrafish sperm respond only with a cortical calcium transient initiated at the micropyle, while oocytes stimulated parthenogenetically by hypotonic shock exhibit a defective cortical transient but a normal transient in the central cytoplasm. Analysis of the subcellular distribution of Fyn kinase and the IP3 receptor reveal that these important signaling components are highly enriched in the oocyte cortex, a factor which may facilitate a faster propagation of the calcium transient in this compartment. In summary, analysis of calcium signaling in the zebrafish oocyte requires attention to morphologically distinct compartments of the oocyte and it is likely that these compartments are controlled by different biochemical events.


Variants of Psychopathic Traits Follow Distinct Trajectories of Clinical Features Among Children with Conduct Problems.
Original definitions of psychopathy suggest the existence of two variants that present with distinct clinical features among antisocial adults, but whether these clinical differences originate early in life or emerge at some point during childhood remains uncertain. We examined if primary and secondary variants follow distinct developmental trajectories of theoretically relevant clinical features among children with conduct problems (CP). Participants were 370 children (40.3% girls) with CP initially aged 8.49 years old in average (s.d. = 0.93). Variants indicators (callous-unemotional [CU] traits and anxiety [ANX]) and clinical features were measured at six yearly assessments. A dual trajectory modelling approach was used to identify groups and group memberships were entered in conditional growth models predicting trajectories of clinical features. Four groups were identified: CP-only, anxious (CP + ANX), primary (CP + CU), and secondary (CP + CU + ANX). Both variants showed higher initial levels of impairment than the CP-only group on most features. Compared to the primary variant, membership to the secondary variant was associated with more stable patterns of CP, oppositional problems, narcissism-grandiosity and impulsivity-irresponsibility traits. Moreover, children from the secondary variant showed higher initial levels of impairment in terms of cognitive abilities, depression, victimization, and dependency to teachers, with non-significant effects on the slope parameters suggesting that these early differences persist across development. In addition to showing distinct clinical features relatively early in childhood, children from the secondary variant of psychopathic traits are at high risk of experiencing an increasing psychopathological burden across childhood. The early identification and treatment of these children therefore appears particularly important.


Electronic fetal monitoring: what's reassuring?
To determine which combinations of fetal heart rate pattern abnormalities are associated with normal outcome in term pregnancies. A cohort of 2200 consecutive deliveries was examined and the fetal heart rate tracings analyzed. Singleton, term patients without chorioamnionitis or serious malformations were used to perform logistic regression analysis to select those FHR patterns associated with increased risk for Apgar<7 and cord pH<7.15, or immediate adverse outcome. Patients having no fetal heart rate abnormalities, mild variable decelerations, decreased variability, mild bradycardia, or accelerations present, constituted 84% of all fetal heart rate tracings. These tracings alone, or in combination, predicted 5 minute Apgar score> or =7 in 99.7%, cord pH> or =7.15 in 96.9% and no adverse neonatal sequelae in 96.2% of cases. Accelerations were reassuring regardless of FHR pattern. When these patterns were not present, non-reassuring tracings, the risk for immediate adverse outcome increased 50%. The non-reassuring tracings were both without accelerations and had tachycardia, prolonged bradycardia, severe variable or late decelerations, or a combination of these patterns. This increased risk was independent of the risk of confounders: i.e. thick meconium (1.8-fold), prolonged second stage of labor (>50 min., 1.5-fold), maternal disease (e.g. kidney, respiratory, 3-fold), or hypertensive disease (1.9-fold). The great majority of fetal heart rate pattern abnormalities can be considered reassuring as they are within normal variations of a healthy fetus. The non-reassuring ones identify infants that truly require further evaluation by fetal scalp, vibroacoustic stimulation, or fetal scalp blood sampling.


Effects of selective serotonin reuptake inhibitor on treating tinnitus in patients stratified for presence of depression or anxiety.
We evaluated the effects of a selective serotonin reuptake inhibitor, paroxetine, on treating tinnitus.Tinnitus patients stratified for the presence of depression and anxiety were studied retrospectively. Fifty-six patients were observed for more than 6 months. They were initially treated with paroxetine only at a dose of 10 mg/day for 2-4 weeks; thereafter, the dose was increased to 20 mg/day. Tinnitus distress was evaluated with the Tinnitus Handicap Inventory (THI) and with visual analog scales (VASs) for tinnitus loudness and annoyance. Depression and anxiety were measured with the Self-Rating Depression Scale (SDS) and the trait section of the State-Trait Anxiety Inventory (STAI). The patients were grouped according to their SDS and STAI scores, and each variable was compared at baseline and the 6-month follow-up. Changes among these variables were also examined to determine whether reduced tinnitus distress was related to the improvement of depression or anxiety. Patients with both depression and anxiety showed better results (decrease in THI, VASs, SDS and STAI scores) than patients with anxiety alone, or patients without depression and anxiety. In patients with depression and anxiety, changes in tinnitus variables and changes in depression and anxiety scores were strongly correlated. In other patients, however, changes in tinnitus variables and changes in depression and anxiety scores were not correlated. These results suggest that paroxetine is effective in treating distressed tinnitus patients with depression and anxiety by reducing their tinnitus severity as well as their depression and anxiety.


Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii.
The role of reactive oxygen species (ROS) in cell communication, control of gene expression, and oxygen sensing is well established. Inappropriate regulation of ROS levels can damage cells, resulting in a diseased state. In Colletotrichum trifolii, a fungal pathogen of alfalfa, the mutationally activated oncogenic fungal Ras (DARas) elevates levels of ROS, causing abnormal fungal growth and development and eventual apoptotic-like cell death but only when grown under nutrient-limiting conditions. Remarkably, restoration to the wild-type phenotype requires only proline. Here, we describe a generally unrecognized function of proline: its ability to function as a potent antioxidant and inhibitor of programmed cell death. Addition of proline to DARas mutant cells effectively quenched ROS levels and prevented cell death. Treating cells with inhibitors of ROS production yielded similar results. In addition, proline protected wild-type C. trifolii cells against various lethal stresses, including UV light, salt, heat, and hydrogen peroxide. These observations appear to be general because proline also protected yeast cells from lethal levels of the ROS-generating herbicide methyl viologen (paraquat), suggesting a common protective role for proline in response to oxidative stress. The ability of proline to scavenge intracellular ROS and inhibit ROS-mediated apoptosis may be an important and broad-based function of this amino acid in responding to cellular stress, in addition to its well established role as an osmolyte.


Lyme disease enolpyruvyl-UDP-GlcNAc synthase: fosfomycin-resistant MurA from Borrelia burgdorferi, a fosfomycin-sensitive mutant, and the catalytic role of the active site Asp.
MurAs (enolpyruvyl-UDP-GlcNAc synthases) from pathogenic bacteria such as Borrelia burgdorferi (Lyme disease) and tuberculosis are fosfomycin resistant because an Asp-for-Cys substitution prevents them from being alkylated by this epoxide antibiotic. Previous attempts to characterize naturally Asp-containing MurAs have resulted in no protein or no activity. We have expressed and characterized His-tagged Lyme disease MurA (Bb_MurA(H6)). The protein was most soluble at high salt concentrations but maximally active around physiological ionic strength. The steady-state kinetic parameters at pH 7 were k(cat) = 1.07 ± 0.03 s(-1), K(M,PEP) = 89 ± 12 μM, and K(M,UDP-GlcNAc) = 45 ± 7 μM. Mutating the active site Asp to Cys, D116C, caused a 21-fold decrease in k(cat) and rendered the enzyme fosfomycin sensitive. The pH profile of k(cat) was bell-shaped and centered around pH 5.3 for Bb_MurA(H6), with pK(a1) = 3.8 ± 0.2 and pK(a2) = 7.4 ± 0.2. There was little change in pK(a1) with the D116C mutant, 3.5 ± 0.3, but pK(a2) shifted to >11. This demonstrated that the pK(a2) of 7.4 was due to D116, almost 3 pH units above an unperturbed carboxylate, and that it must be protonated for activity. This supports D116's proposed role as a general acid/base catalyst. As fosfomycin does not react with simple thiols, nor most protein thiols, the reactivity of D116C with fosfomycin, combined with the strongly perturbed pK(a2) for D116, strongly implies an unusual active site environment and a chemical role in catalysis for Asp/Cys. There is also good evidence for C115 having a role in product release. Both roles may be operative for both Asp- and Cys-containing MurAs.


A novel metabotropic glutamate receptor 5 positive allosteric modulator acts at a unique site and confers stimulus bias to mGlu5 signaling.
Metabotropic glutamate receptor 5 (mGlu5) is a target for the treatment of central nervous system (CNS) disorders, such as schizophrenia and Alzheimer's disease. Furthermore, mGlu5 has been shown to play an important role in hippocampal synaptic plasticity, specifically in long-term depression (LTD) and long-term potentiation (LTP), which is thought to be involved in cognition. Multiple mGlu5-positive allosteric modulators (PAMs) have been developed from a variety of different scaffolds. Previous work has extensively characterized a common allosteric site on mGlu5, termed the MPEP (2-Methyl-6-(phenylethynyl)pyridine) binding site. However, one mGlu5 PAM, CPPHA (N-(4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl)-2-hydroxybenzamide), interacts with a separate allosteric site on mGlu5. Using cell-based assays and brain slice preparations, we characterized the interaction of a potent and efficacious mGlu5 PAM from the CPPHA series termed NCFP (N-(4-chloro-2-((4-fluoro-1,3-dioxoisoindolin-2-yl)methyl)phenyl)picolinamide). NCFP binds to the CPPHA site on mGlu5 and potentiates mGlu5-mediated responses in both recombinant and native systems. However, NCFP provides greater mGlu5 subtype selectivity than does CPPHA, making it more suitable for studies of effects on mGlu5 in CNS preparations. Of interest, NCFP does not potentiate responses involved in hippocampal synaptic plasticity (LTD/LTP), setting it apart from other previously characterized MPEP site PAMs. This suggests that although mGlu5 PAMs may have similar responses in some systems, they can induce differential effects on mGlu5-mediated physiologic responses in the CNS. Such stimulus bias by mGlu5 PAMs may complicate drug discovery efforts but would also allow for specifically tailored therapies, if pharmacological biases can be attributed to different therapeutic outcomes.


Oxide versus Nonoxide Cathode Materials for Aqueous Zn Batteries: An Insight into the Charge Storage Mechanism and Consequences Thereof.
Aqueous Zn-ion batteries, which are being proposed as large-scale energy storage solutions because of their unparalleled safety and cost advantage, are composed of a positive host (cathode) material, a metallic zinc anode, and a mildly acidic aqueous electrolyte (pH ≈ 3-7). Typically, the charge storage mechanism is believed to be reversible Zn2+ (de)intercalation in the cathode host, with the exception of α-MnO2, for which multiple vastly different and contradicting mechanisms have been proposed. However, our present study, combining electrochemical, operando X-ray diffraction, electron microscopy in conjunction with energy-dispersive X-ray spectroscopy, and in situ pH evolution analyses on two oxide hosts-tunneled α-MnO2 and layered V3O7·H2O vis-à-vis two nonoxide hosts-layered VS2 and tunneled Zn3[Fe(CN)6]2, suggests that oxides and nonoxides follow two dissimilar charge storage mechanisms. While the oxides behave as dominant proton intercalation materials, the nonoxides undergo exclusive zinc intercalation. Stabilization of H+ on the hydroxyl-terminated oxide surface is revealed to facilitate the proton intercalation by a preliminary molecular dynamics simulation study. Proton intercalation for both oxides leads to the precipitation of layered double hydroxide (LDH)-Zn4SO4(OH)6·5H2O with a ZnSO4/H2O electrolyte and a triflate anion (CF3SO3-)-based LDH with a Zn(SO3CF3)2/H2O electrolyte-on the electrode surface. The LDH precipitation buffers the pH of the electrolytes to a mildly acidic value, sustaining the proton intercalation to deliver large specific capacities for the oxides. Moreover, we also show that the stability of the LDH precipitate is crucial for the rechargeability of the oxide cathodes, revealing a critical link between the charge storage mechanism and the performance of the oxide hosts in aqueous zinc batteries.


Human Islet Response to Selected Type 1 Diabetes-Associated Bacteria: A Transcriptome-Based Study.
Type 1 diabetes (T1D) is a chronic autoimmune disease that results from destruction of pancreatic β-cells. T1D subjects were recently shown to harbor distinct intestinal microbiome profiles. Based on these findings, the role of gut bacteria in T1D is being intensively investigated. The mechanism connecting intestinal microbial homeostasis with the development of T1D is unknown. Specific gut bacteria such as Bacteroides dorei (BD) and Ruminococcus gnavus (RG) show markedly increased abundance prior to the development of autoimmunity. One hypothesis is that these bacteria might traverse the damaged gut barrier, and their constituents elicit a response from human islets that causes metabolic abnormalities and inflammation. We have tested this hypothesis by exposing human islets to BD and RG in vitro, after which RNA-Seq analysis was performed. The bacteria altered expression of many islet genes. The commonly upregulated genes by these bacteria were cytokines, chemokines and enzymes, suggesting a significant effect of gut bacteria on islet antimicrobial and biosynthetic pathways. Additionally, each bacteria displayed a unique set of differentially expressed genes (DEGs). Ingenuity pathway analysis of DEGs revealed that top activated pathways and diseases included TREM1 signaling and inflammatory response, illustrating the ability of bacteria to induce islet inflammation. The increased levels of selected factors were confirmed using immunoblotting and ELISA methods. Our data demonstrate that islets produce a complex anti-bacterial response. The response includes both symbiotic and pathogenic aspects. Both oxidative damage and leukocyte recruitment factors were prominent, which could induce beta cell damage and subsequent autoimmunity.


Prevention of relapse/recurrence in major depressive disorder with either mindfulness-based cognitive therapy or cognitive therapy.
Both Mindfulness Based Cognitive Therapy (MBCT) and Cognitive Therapy (CT) enhance self-management of prodromal symptoms associated with depressive relapse, albeit through divergent therapeutic procedures. We evaluated rates of relapse in remitted depressed patients receiving MBCT and CT. Decentering and dysfunctional attitudes were assessed as treatment-specific process markers. Participants in remission from Major Depressive Disorder (MDD; N = 166) were randomized to 8 weeks of either MBCT (N = 82) or CT (N = 84) and were followed for 24 months, with process markers measured every 3 months. Attendance in both treatments was high (6.3/8 session) and treatment fidelity and competence were evaluated. Relapse was defined as a return of symptoms meeting the criteria for major depression on Module A of the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (SCID). Intention-to-treat analyses indicated no differences between MBCT and CT in either rates of relapse to MDD or time to relapse across 24 months of follow up. Both groups experienced significant increases in decentering and participants in CT reported greater reductions in dysfunctional attitudes. Within both treatments, participants who relapsed evidenced lower decentering scores than those who stayed well over the follow up. This is the first study to directly compare relapse prophylaxis following MBCT and CT directly. The lack of group differences in time to relapse supports the view that both interventions are equally effective and that increases in decentering achieved via either treatment are associated with greater protection. These findings lend credence to Teasdale et al.'s (2002) contention that, even though they may be taught through dissimilar methods, CT and MBCT help participants develop similar metacognitive skills for the regulation of distressing thoughts and emotions. (PsycINFO Database Record


Photocarcinogenesis--molecular mechanisms.
The carcinogenicity (photocarcinogenicity) of sunlight to human skin has been recognized more than a century ago. Last decades numerous experimental studies show that UV rays damage DNA, cause gene mutations leading to the development of malignant tumors such basal cell carcinomas, squamous cell carcinomas and melanomas. The tumors occur most frequently in fair skinned people, and the mutations typically are found at dipyrimidine sites with C-T or / and CC-TT tandem double mutations. The authors briefly summarize their investigation of the p53 suppressor gene, and expose their hypothesis of hTERT involvement in cancerogenesis. Also their underline the importance of UV induced immunosuppression in photocarcinogenesis. Psoriatic patients are exposed to numerous cancerogens in their treatment. A better understanding of the mechanisms of photocarcinogenesis could provide new ways in the treatment of skin tumors.


Neuroprotective effect of single-wall carbon nanotubes with built-in peroxidase-like activity against β-amyloid-induced neurotoxicity.
Carbon nanotubes (CNTs) have emerged as a leading nanomaterial for biomedical applications because of their extraordinary properties, which make them useful as delivery vehicles for drugs, proteins, and DNA into cells. However, the numerous applications of carbon nanotubes inevitably increase the potential risk of this nanomaterial. To address this issue, it is necessary to develop protocols for the effective and safe degradation of CNTs. In this study, we demonstrate a self-degradation route for single-wall carbon nanotubes mediated by the built-in peroxidase-like activity of bacterial magnetic nanoparticles (BMPs). Biocompatible BMPs which originated from Magnetospirillum sp. AMB-1 were directly conjugated through covalent bonding to functionalized SWNTs (f-SWNTs) without any additional functionalization processes. This SWNT-BMP hybrid was proven to exhibit highly synergetic peroxidase-like activity, and BMPs act as a highly effective intrinsic peroxidase for the self-degradation of BMP-decorated SWNTs. Moreover, it was shown to be an inhibitor that reduces the formation of β-amyloid (Aβ) fibrils, which are considered a key element in Alzheimer's disease. Thereby the SWNT-BMP hybrid exerts neuroprotective effects against β-amyloid (Aβ) fibrillation-induced neurotoxicity in SH-SY5Y human neuroblastoma cells. These results suggest that the SWNT-BMP hybrid could offer a new approach for treating or preventing neurodegenerative diseases.


The other side of the leaf: Seizures associated with synthetic cannabinoid use.
There has recently been a marked rise in the medicinal use of cannabis for epilepsy and multiple other conditions. While seizures have been reported in association with synthetic cannabinoids, the clinical features and prognosis have not been studied. Thirty patients with a history of seizures occurring within 24 h of synthetic cannabinoid use were identified from a first seizure clinic database in Perth, Western Australia between 2011 and 2016. Eight had a prior history of seizures, three related to synthetic cannabinoid use, with an additional three patients having risk factors for seizures. The presenting event was a tonic-clonic seizure in 27 patients (90%). "Kronic" was the synthetic cannabinoid used by 16 patients. Absorption was via smoking in all cases, with seizures occurring within 30 min of inhalation in 14 patients (46%). Electroencephalography (EEG) showed epileptiform abnormalities in 11%, and neuroimaging revealed epileptogenic lesions in 12%. Nine of 24 patients with follow-up had subsequent seizures, occurring in the setting of further synthetic cannabinoid use in two patients. This seizure recurrence rate is similar to seizures provoked by other acute systemic insults. In conclusion, smoking of some synthetic cannabinoids is associated with seizures, and this may relate to an intrinsic proconvulsant effect.


Calcium, magnesium, and growth control in the WI-38 human fibroblast cell.
WI-38 and SV40WI-38 cells have been synchronized using centrifugal elutriation. This technique allows for the rapid harvesting of early G1 phase cells from exponentially growing populations of both the normal and transformed cell. Using these cells, as well as WI-38 cells synchronized by serum deprivation, we have examined the effects of extracellular Ca and Mg levels on the progression of cells through G1 phase. A differential sensitivity to both Ca and Mg deprivation is observed between normal and transformed cells. The WI-38 cell requires higher levels of both ions for traversal of G1 phase and for continued proliferation as compared to the transformed cell. The temporal nature of the Ca and Mg requirements for the WI-38 cell has been examined during G1 phase. Ca is strictly required during early and late G1 phase, but not necessarily throughout mid-G1. An early as well as a late G1 Ca requirement is also found in serum-stimulated WI-38 cells. In contrast, the Mg requirement of WI-38 cells does not appear to be temporally well-defined. Mg appears to be a permissive factor, required throughout G1 phase rather than at certain prescribed intervals. On the basis of these data, it seems unlikely that these two cations exert their effects on cell growth entirely through a common competitive mechanism. Ca would appear to be involved in early serum or growth factor-mediated G1 events and later pre-S-phase events, as suggested in previous studies on other cell lines.


Contribution of 20 single nucleotide polymorphisms of 13 genes to dyslipidemia associated with antiretroviral therapy.
HIV-1 infected individuals have an increased cardiovascular risk which is partially mediated by dyslipidemia. Single nucleotide polymorphisms in multiple genes involved in lipid transport and metabolism are presumed to modulate the risk of dyslipidemia in response to antiretroviral therapy. The contribution to dyslipidemia of 20 selected single nucleotide polymorphisms of 13 genes reported in the literature to be associated with plasma lipid levels (ABCA1, ADRB2, APOA5, APOC3, APOE, CETP, LIPC, LIPG, LPL, MDR1, MTP, SCARB1, and TNF) was assessed by longitudinally modeling more than 4400 plasma lipid determinations in 438 antiretroviral therapy-treated participants during a median period of 4.8 years. An exploratory genetic score was tested that takes into account the cumulative contribution of multiple gene variants to plasma lipids. Variants of ABCA1, APOA5, APOC3, APOE, and CETP contributed to plasma triglyceride levels, particularly in the setting of ritonavir-containing antiretroviral therapy. Variants of APOA5 and CETP contributed to high-density lipoprotein-cholesterol levels. Variants of CETP and LIPG contributed to non-high-density lipoprotein-cholesterol levels, a finding not reported previously. Sustained hypertriglyceridemia and low high-density lipoprotein-cholesterol during the study period was significantly associated with the genetic score. Single nucleotide polymorphisms of ABCA1, APOA5, APOC3, APOE, and CETP contribute to plasma triglyceride and high-density lipoprotein-cholesterol levels during antiretroviral therapy exposure. Genetic profiling may contribute to the identification of patients at risk for antiretroviral therapy-related dyslipidemia.


The effects of stimulus type on consistency of responses to conflicting stimuli in Siamese fighting fish.
Individuals must often decide among a range of behavioral responses in a given situation and individuals will likely vary from one another in the choices they make. A rarely explored example of this is when both a male and a female are present simultaneously and a male must decide to fight or court. Consistent individual differences have been found in decision-making in Siamese fighting fish; however, dummies were used to explore this question and it is possible that individuals respond differently in this situation when faced with live stimuli as a result of feedback. Therefore, this study investigated whether males behave similarly to dummy and live stimuli by exposing them to male and female conspecifics both simultaneously and separately. Subjects were tested first with dummies and then in the same situations using live conspecifics. It was hypothesized that males would respond in the same manner to both the dummy and live fish. The results indicated that individuals behaved similarly to the dummy fish and the live fish in corresponding conditions. In addition, repeatability levels suggest that individuals behaved similarly across trials of a given treatment type while differing from one another in their responses. This study demonstrates that males are consistent in their responses even when receiving feedback from conspecifics and supports the continued use of dummy stimuli as an effective tool.


Regional brain volumes changes in adult male FMR1-KO mouse on the FVB strain.
Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disorder (ASD). FMR1-KO mice mimic the etiology and phenotypic manifestations of FXS. Neuroanatomical changes in specific brain regions have been reported in clinical settings and in preclinical models. FMR1-KO mice have been generated in different strains including C57Bl/6 (B6) and FVB. Mice on different genetic backgrounds have stable yet distinct behavioral phenotypes that may lead to unique gene-strain interactions on brain structure. Previous magnetic resonance imaging (MRI) studies have examined FMR1 knockout male mice on a B6 and found few differences compared to wild-type mice. Here, we examine brain volumes in FMR1 knockout male mice on a FVB background using high resolution (multi-channel 7.0Tesla) MRI. We observe multiple differences in the neuroanatomy of male FMR1-/y mice on a FVB background. Significantly larger relative volume (% total brain volume) differences were found in major white matter structures throughout the brain. In addition, there were changes in areas associated with fronto-striatal circuitry and other regions. Functional and structural connectivity differences are often seen in human ASD, and therefore, this increased white matter seen in the FMR1-KO-FVB could be highlighting a structural over-connectivity, which could lead to some of the behavioral abnormalities seen with the FMR1-KO-FVB mice. Furthermore, these results highlight the importance of genetic strain contribution to brain structure.


Phenylketonuria: an inborn error of phenylalanine metabolism.
Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine (Phe) metabolism resulting from deficiency of phenylalanine hydroxylase (PAH). Most forms of PKU and hyperphenylalaninaemia (HPA) are caused by mutations in the PAH gene on chromosome 12q23.2. Untreated PKU is associated with an abnormal phenotype which includes growth failure, poor skin pigmentation, microcephaly, seizures, global developmental delay and severe intellectual impairment. However, since the introduction of newborn screening programs and with early dietary intervention, children born with PKU can now expect to lead relatively normal lives. A better understanding of the biochemistry, genetics and molecular basis of PKU, as well as the need for improved treatment options, has led to the development of new therapeutic strategies.


Exploring the Potential of Starch/Polycaprolactone Aligned Magnetic Responsive Scaffolds for Tendon Regeneration.
The application of magnetic nanoparticles (MNPs) in tissue engineering (TE) approaches opens several new research possibilities in this field, enabling a new generation of multifunctional constructs for tissue regeneration. This study describes the development of sophisticated magnetic polymer scaffolds with aligned structural features aimed at applications in tendon tissue engineering (TTE). Tissue engineering magnetic scaffolds are prepared by incorporating iron oxide MNPs into a 3D structure of aligned SPCL (starch and polycaprolactone) fibers fabricated by rapid prototyping (RP) technology. The 3D architecture, composition, and magnetic properties are characterized. Furthermore, the effect of an externally applied magnetic field is investigated on the tenogenic differentiation of adipose stem cells (ASCs) cultured onto the developed magnetic scaffolds, demonstrating that ASCs undergo tenogenic differentiation synthesizing a Tenascin C and Collagen type I rich matrix under magneto-stimulation conditions. Finally, the developed magnetic scaffolds were implanted in an ectopic rat model, evidencing good biocompatibility and integration within the surrounding tissues. Together, these results suggest that the effect of the magnetic aligned scaffolds structure combined with magnetic stimulation has a significant potential to impact the field of tendon tissue engineering toward the development of more efficient regeneration therapies.


Breastfeeding, Childhood Asthma, and Allergic Disease.
The worldwide prevalence of childhood asthma has been increasing considerably, and the protection afforded by breastfeeding in its development has been the subject of controversy for more than 80 years. Previous systematic reviews have generally found a protective effect of breastfeeding on allergic outcomes, although many studies have methodological limitations. Although breastfeeding is protective against lower respiratory tract infection during infancy, such protection has not been demonstrated for asthma in all studies. Breastfeeding has health benefits for the mother and child. Exclusive breastfeeding for the first 6 months of an infant's life, with continued breastfeeding for up to 2 years or longer, is recognized as the "gold" standard for infant feeding because human milk is uniquely suited to the human infant, and its nutritional content and bioactivity promote a healthy development. There is increasing concern that the practice of delaying complementary foods until 6 months may exacerbate the risk of allergic disease. Breast milk contains immunological components that protect against infections and allergic disease in infancy. The composition of human breast milk is complex, containing factors that interact with the infant immune system and intestinal milieu including allergens, cytokines, immunoglobulins, polyunsaturated fatty acids, and chemokines. Transforming growth factor β is a cytokine in human milk involved in maintaining intestinal homeostasis, inflammation regulation, and oral tolerance development. Modern day society, with increased standards of hygiene, has changed the gut flora of Western infants, potentially impacting the risk of developing immune-mediated diseases including allergic disease and asthma. Microbial diversity is intrinsic to healthy immune maturation and function. Compared to breastfed infants, formula-fed infants had lower bacterial diversity and an altered intestinal microbiota in the first few weeks of life associated with an increased risk of eczema and asthma. Favorable gut colonization through continued breastfeeding may promote tolerance as well as protection when complementary feeding is initiated.


Multiple wavelength heterodyne array interferometry.
Multiple wavelength interferometry is combined with a heterodyne array sensing technique to provide an approach for measuring highly aberrated optical surfaces. Interferometric measurements created with long effective wavelengths are obtained by digitally combining complex exposures collected at different optical wavelengths. The heterodyne array sensing method is straightforward to implement and holds promise for rapid wavefront measurements with high spatial and phase resolution. Measurements of a tilted, flat surface are presented and analyzed.


Parathyroid hormone for the treatment of osteoporosis: a systematic review.
Human parathyroid hormone (hPTH)(1-34) was approved in 2004 for the treatment of severe osteoporosis. Members of the Osteoporosis Canada clinical guidelines committee conducted a systematic review of randomized controlled trials (RCTs) to assess the efficacy and safety of hPTH for fracture prevention in postmenopausal women and men with osteoporosis. We searched MEDLINE, EMBASE, HTA, Current Contents and the Cochrane Controlled Trials Registry for published data from 1966 to February 2005. A systematic literature search for RCTs was conducted using the Cochrane Collaborative approach. We identified 12 trials that randomly assigned patients either to hPTH or placebo or to hPTH or an active comparator and were at least 1 year in duration. Outcomes included change in bone mineral density (BMD), fractures, back pain and adverse events. Two independent reviewers abstracted data on study characteristics and outcomes. hPTH(1-34) significantly increases lumbar spine BMD, with smaller increases at the femoral neck and total hip. hPTH(1-84) significantly increases lumbar spine BMD. The data show a significant reduction in both vertebral and nonvertebral fractures with hPTH(1-34) in postmenopausal women with previous vertebral fractures. There were no data on fractures comparing the approved dose of hPTH(1-34) with active comparators. There is Level I evidence that hPTH(1-34) significantly increases BMD at all skeletal sites except the radius and significantly reduces the risk of new vertebral and nonvertebral fractures in postmenopausal women with prior fractures.


Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes.
E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.


FBXW12, a novel F box protein-encoding gene, is deleted or methylated in some cases of epithelial ovarian cancer.
Epithelial ovarian cancer is one of the most lethal of gynecological malignancies. Due to its lack of early symptoms, detection usually occurs when the tumor is no longer confined to the ovary. We previously identified Fbxw15, a gene encoding an F-box protein in the mouse ovary, and showed that its expression is developmentally regulated. Here we report the molecular analysis of its human homologue, FBXW12 in epithelial ovarian tumors. To search for FBXW12 gene mutations, we PCR-amplified and sequenced the coding region of FBXW12, the gene's 5-untranslated region and the proximal promoter in each of 30 EOC tumors. Promoter methylation was determined by DNA bisulfite conversion, followed by methylation specific PCR. FBXW12 intracellular localization was identified by means of immunohistochemistry. A complete deletion of the gene's coding region, the 5'-UTR and the proximal promoter, was observed in 3 EOC samples. Eight of the remaining 27, had a deletion of the 5'-UTR, and the proximal promoter. FBXW12 mRNA was detected in 2 of the 19 samples without deletions. The methylation specific PCR results demonstrated CpGs methylation in the FBXW12 proximal promoter. Immunohistochemistry assay revealed that within the normal ovary, FBXW12 has an oocyte specific expression, whereas in EOC samples it is present in the ovarian surface epithelium. Our results indicate that the FBXW12 gene is deleted in approximately ten percent of the EOC cases studied; such deletions comprised either the FBXW12 promoter or the mRNA-encoding region. Moreover, FBXW12 could be epigenetically silenced by CpGs methylation in some of these EOC cases.


Equilibrium properties of a voltage-dependent junctional conductance.
The conductance of junctions between amphibian blastomeres is strongly voltage dependent. Isolated pairs of blastomeres from embryos of Ambystoma mexicanum, Xenopus laevis, and Rana pipiens were voltage clamped, and junctional current was measured during transjunctional voltage steps. The steady-state junctional conductance decreases as a steep function of transjunctional voltage of either polarity. A voltage-insensitive conductance less than 5% of the maximum remains at large transjunctional voltages. Equal transjunctional voltages of opposite polarities produce equal conductance changes. The conductance is half maximal at a transjunctional voltage of approximately 15 mV. The junctional conductance is insensitive to the potential between the inside and outside of the cells. The changes in steady-state junctional conductance may be accurately modeled for voltages of each polarity as arising from a reversible two-state system in which voltage linearly affects the energy difference between states. The voltage sensitivity can be accounted for by the movement of about six electron charges through the transjunctional voltage. The changes in junctional conductance are not consistent with a current-controlled or ionic accumulation mechanism. We propose that the intramembrane particles that comprise gap junctions in early amphibian embryos are voltage-sensitive channels.


The Type I Interferon Response Determines Differences in Choroid Plexus Susceptibility between Newborns and Adults in Herpes Simplex Virus Encephalitis.
Newborns are significantly more susceptible to severe viral encephalitis than adults, with differences in the host response to infection implicated as a major factor. However, the specific host signaling pathways responsible for differences in susceptibility and neurologic morbidity have remained unknown. In a murine model of HSV encephalitis, we demonstrated that the choroid plexus (CP) is susceptible to herpes simplex virus 1 (HSV-1) early in infection of the newborn but not the adult brain. We confirmed susceptibility of the CP to HSV infection in a human case of newborn HSV encephalitis. We investigated components of the type I interferon (IFN) response in the murine brain that might account for differences in cell susceptibility and found that newborns have a dampened interferon response and significantly lower basal levels of the alpha/beta interferon (IFN-α/β) receptor (IFNAR) than do adults. To test the contribution of IFNAR to restricting infection from the CP, we infected IFNAR knockout (KO) adult mice, which showed restored CP susceptibility to HSV-1 infection in the adult. Furthermore, reduced IFNAR levels did not account for differences we found in the basal levels of several other innate signaling proteins in the wild-type newborn and the adult, including protein kinase R (PKR), that suggested specific regulation of innate immunity in the developing brain. Viral targeting of the CP, a region of the brain that plays a critical role in neurodevelopment, provides a link between newborn susceptibility to HSV and long-term neurologic morbidity among survivors of newborn HSV encephalitis. Compared to adults, newborns are significantly more susceptible to severe disease following HSV infection. Over half of newborn HSV infections result in disseminated disease or encephalitis, with long-term neurologic morbidity in 2/3 of encephalitis survivors. We investigated differences in host cell susceptibility between newborns and adults that contribute to severe central nervous system disease in the newborn. We found that, unlike the adult brain, the newborn choroid plexus (CP) was susceptible early in HSV-1 infection. We demonstrated that IFN-α/β receptor levels are lower in the newborn brain than in the adult brain and that deletion of this receptor restores susceptibility of the CP in the adult brain. The CP serves as a barrier between the blood and the cerebrospinal fluid and plays a role in proper neurodevelopment. Susceptibility of the newborn choroid plexus to HSV-1 has important implications in viral spread to the brain and, also, in the neurologic morbidity following HSV encephalitis.


RcsB plays a central role in H-NS-dependent regulation of motility and acid stress resistance in Escherichia coli.
In Escherichia coli, hns mutants lack flagellar motility and display an increase in acid stress resistance. Spontaneous phenotypic revertants showed reversion of both H-NS-controlled phenotypes. In the present study, suppressor mutations were identified in the rcsB gene. In addition to RcsA, our experiments establish that H-NS indirectly controlled the RcsB regulator via repression of RcsD. We also show that RcsB(D56E), mimicking phosphorylated RcsB, interacts with GadE to form a RcsB-P/GadE complex, a general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways. In addition, we showed that H-NS positively affects motility via the flhDC master operon repression by RcsB. This substantiates the central role of RcsB in H-NS-mediated control of motility and acid stress resistance.


Investigation of Local Conduction Mechanisms in Ca and Ti-Doped BiFeO3 Using Scanning Probe Microscopy Approach.
In this work we demonstrate the role of grain boundaries and domain walls in the local transport properties of n- and p-doped bismuth ferrites, including the influence of these singularities on the space charge imbalance of the energy band structure. This is mainly due to the charge accumulation at domain walls, which is recognized as the main mechanism responsible for the electrical conductivity in polar thin films and single crystals, while there is an obvious gap in the understanding of the precise mechanism of conductivity in ferroelectric ceramics. The conductivity of the Bi0.95Ca0.05Fe1-xTixO3-δ (x = 0, 0.05, 0.1; δ = (0.05 - x)/2) samples was studied using a scanning probe microscopy approach at the nanoscale level as a function of bias voltage and chemical composition. The obtained results reveal a distinct correlation between electrical properties and the type of charged defects when the anion-deficient (x = 0) compound exhibits a three order of magnitude increase in conductivity as compared with the charge-balanced (x = 0.05) and cation-deficient (x = 0.1) samples, which is well described within the band diagram representation. The data provide an approach to control the transport properties of multiferroic bismuth ferrites through aliovalent chemical substitution.


Prognostic impact of circulating Her-2-reactive T-cells producing pro- and/or anti-inflammatory cytokines in elderly breast cancer patients.
Treating elderly breast cancer patients remains a challenge but the increasing availability of immunotherapeutic approaches instills optimism that these tumours may also be susceptible to immune control. Because aging leads to a number of alterations in the immune system ("immunosenescence") reflecting potential exhaustion which could compromise immunomodulatory antibody therapy, here we have assessed the immunocompetence of elderly breast cancer patients compared with a group of younger patients, and related this to the 5-year survival of the former. T-cell responses to Her-2 peptide pools in vitro were assessed by analyzing pro- and anti-inflammatory cytokine production by CD4+ and CD8+ T-cells in 40 elderly and 35 younger breast cancer patients. The proportions of older and younger patients whose peripheral T-cells responded to Her-2 peptides in vitro were found to be similar, although a significantly higher fraction of younger patients possessed IL-2-producing CD4+ Her-2-reactive T-cells than in the elderly (p = 0.03). However, IL-2 production did not impart a survival benefit to the latter. In contrast, there was a survival benefit of possessing Her-2-reactive CD8+ T-cells, but this was abrogated in patients if they also had CD4+ Her-2-responsive T-cells that producedIL-5 and/or IL-17 (p = 0.01). This resulted in a 5-yr survival rate of only 29 % compared to 76 % for patients whose her-2-reactive CD4+ T-cells did not produceIL-5 and/or IL-17. Additionally, patients whose CD8+ T-cells produced TNF had a significantly better survival than those that did not (93 % compared to 52 %, p = 0.01), whereas no survival benefit was attributable to possessing IFN-γ-producing cells. Elderly breast cancer patients appear perfectly immunocompetent to respond to Her-2 peptide pools in vitro, with response patterns very similar to younger patients. The nature of this response is associated with 5-year survival of these elderly patients, suggesting that boosting anti-tumor responses and modulating the nature of the T-cell response is likely to be effective even in potentially immunosenescent elderly breast cancer patients, and might be useful for predicting which patients are most likely to benefit from such treatments.


Loss of heterozygosity at 3p in non-small cell lung cancer and its prognostic implication.
We examined 110 patients with non-small cell lung cancer who underwent consecutive pulmonary resection for loss of heterozygosity (LOH) at the short arm of chromosome 3 (3p). We performed a PCR-based microsatellite polymorphism analysis for detection of LOH. The microsatellite markers used were D3S966 (3p21.3), D3S1007 (3p21. 3-22), and D3S1228 (3p14.1-14.3). Of 98 informative cases, 3p LOH was found in 45 (46%). 3p LOH was more prevalent in squamous cell carcinoma (24/35, 69%) than in adenocarcinoma (18/52, 35%; P = 0.0019). There was no significant association between 3p LOH and sex, disease stage, or grade of differentiation. However, patients with 3p LOH tended to survive for a shorter period of time (P = 0.0631, log rank test). There was no such tendency in squamous cell carcinoma (P = 0.7513), but in adenocarcinoma, the difference of survival was significant (P = 0.0015). Cox's proportional hazards model also predicted that 3p LOH was an independent poor prognostic marker in adenocarcinoma (P = 0.0502) but not in squamous cell carcinoma or in the entire cohort (P = 0.7866 and 0.1371, respectively). LOH at 3p may help to identify non-small cell lung cancer patients with a poor prognosis, who thus need an intensive postoperative follow-up protocol or who are suitable for novel investigational therapeutic approaches. It is also suggested that the putative tumor suppressor gene at 3p may have a different role in squamous cell carcinoma and adenocarcinoma of the lung.


Adhesive Hydrogels for Maxillofacial Tissue Regeneration Using Minimally Invasive Procedures.
Minimally invasive surgical procedures aiming to repair damaged maxillofacial tissues are hampered by its small, complex structures and difficult surgical access. Indeed, while arthroscopic procedures that deliver regenerative materials and/or cells are common in articulating joints such as the knee, there are currently no treatments that surgically place cells, regenerative factors or materials into maxillofacial tissues to foster bone, cartilage or muscle repair. Here, hyaluronic acid (HA)-based hydrogels are developed, which are suitable for use in minimally invasive procedures, that can adhere to the surrounding tissue, and deliver cells and potentially drugs. By modifying HA with both methacrylate (MA) and 3,4-dihydroxyphenylalanine (Dopa) groups using a completely aqueous synthesis route, it is shown that MA-HA-Dopa hydrogels can be applied under aqueous conditions, gel quickly using a standard surgical light, and adhere to tissue. Moreover, upon oxidation of the Dopa, human marrow stromal cells attach to hydrogels and survive when encapsulated within them. These observations show that when incorporated into HA-based hydrogels, Dopa moieties can foster cell and tissue interactions, ensuring surgical placement and potentially enabling delivery/recruitment of regenerative cells. The findings suggest that MA-HA-Dopa hydrogels may find use in minimally invasive procedures to foster maxillofacial tissue repair.


Catalytic oxidation and reduction reactions of hydrophilic carbon clusters with NADH and cytochrome C: features of an electron transport nanozyme.
Previously, our group reported on the promising efficacy of poly(ethylene glycol)-hydrophilic carbon clusters (PEG-HCCs) to work as broadly active and high capacity antioxidants in brain ischemia and injury models including stroke and traumatic brain injury coupled with hemorrhagic shock. PEG-HCCs are a carbon nanomaterial derived from harsh oxidation of single wall carbon nanotubes and covalently modified with poly(ethylene glycol). They retain no tubular remnants and are composed of a highly oxidized carbon core functionalized with epoxy, peroxyl, quinone, ketone, carboxylate, and hydroxyl groups. HCCs are the redox active carbon core of PEG-HCCs, which have a broad reduction potential range starting at +200 mV and extending to -2 V. Here we describe a new property of these materials: the ability to catalytically transfer electrons between key surrogates and proteins of the mitochondrial electron transport complex in a catalytic fashion consistent with the concept of a nanozyme. The estimated reduction potential of PEG-HCCs is similar to that of ubiquinone and they enabled the catalytic transfer of electrons from low reduction potential species to higher reduction electron transport complex constituents. PEG-HCCs accelerated the reduction of resazurin (a test indicator of mitochondrial viability) and cytochrome c by NADH and ascorbic acid in solution. Kinetic experiments suggested a transient tertiary complex. Electron paramagnetic resonance demonstrated NADH increased the magnitude of PEG-HCCs' intrinsic radical, which then reduced upon subsequent addition of cytochrome c or resazurin. Deconvolution microscopy identified PEG-HCCs in close proximity to mitochondria after brief incubation with cultured SHSY-5Y human neuroblastoma cells. Compared to methylene blue (MB), considered a prototypical small molecule electron transport shuttle, PEG-HCCs were more protective against toxic effects of hydrogen peroxide in vitro and did not demonstrate impaired cell viability as did MB. PEG-HCCs were protective in vitro when cells were exposed to sodium cyanide, a mitochondrial complex IV poison. Because mitochondria are a major source of free radicals in pathology, we suggest that this newly described nanozyme action helps explain their in vivo efficacy in a range of injury models. These findings may also extend their use to mitochondrial disorders.


Climate change, conflict and health.
Future climate change is predicted to diminish essential natural resource availability in many regions and perhaps globally. The resulting scarcity of water, food and livelihoods could lead to increasingly desperate populations that challenge governments, enhancing the risk of intra- and interstate conflict. Defence establishments and some political scientists view climate change as a potential threat to peace. While the medical literature increasingly recognises climate change as a fundamental health risk, the dimension of climate change-associated conflict has so far received little attention, despite its profound health implications. Many analysts link climate change with a heightened risk of conflict via causal pathways which involve diminishing or changing resource availability. Plausible consequences include: increased frequency of civil conflict in developing countries; terrorism, asymmetric warfare, state failure; and major regional conflicts. The medical understanding of these threats is inadequate, given the scale of health implications. The medical and public health communities have often been reluctant to interpret conflict as a health issue. However, at times, medical workers have proven powerful and effective peace advocates, most notably with regard to nuclear disarmament. The public is more motivated to mitigate climate change when it is framed as a health issue. Improved medical understanding of the association between climate change and conflict could strengthen mitigation efforts and increase cooperation to cope with the climate change that is now inevitable.


Resin bonding to a feldspar ceramic after different ceramic surface conditioning methods: evaluation of contact angle, surface pH, and microtensile bond strength durability.
To evaluate the surface pH, contact angle and microtensile bond strength (MTBS) of a resin-cement to feldspathic ceramic after various surface conditioning methods (SC). For pH measurements, 96 glass-ceramic disks were assigned into 12 groups (n = 8): SC1-pH: No ceramic surface conditioning (control); SC2-pH: hydrofluoric-acid (HF) 9% 1 min; SC3-pH: SC2-pH+wash/dry; SC4-pH: SC3-pH+silane; SC5-pH: HF 4% 1 min; SC6-pH: SC5-pH+wash/dry; SC7-pH: SC6 -pH +silane; SC8-pH: HF 5% 1 min; SC9-pH: SC8-pH+wash/dry; SC10-pH: SC9-pH+silane; SC11-pH: SC9-pH +neutralizer+wash/dry+sonic-cleaning; SC12-pH: SC11-pH+silane. For contact angle analysis, 40 disks were divided into 5 groups (n = 8): SC1-ca: no conditioning; SC2-ca: HF 9%+wash/dry; SC3-ca: HF 4%+wash/dry; SC4 -ca: HF 5%+wash/dry; SC5-ca: HF 5%+neutralizer+wash/dry+ultrasonic-cleaning. To evaluate the MTBS, 40 blocks were distributed into 4 groups SC (N = 10): SC1-bond: HF 9% 1 min+silane; SC2-bond: HF 4% 1 min+silane; SC3-bond: HF 5% 1 min+silane; SC4-bond: HF 5% 1 min+neutralizer+wash/dry+ultrasonic cleaning+silane. The resin cement was applied on the treated surfaces and bar specimens were produced that were submitted to 2 conditions: dry: immediate MTBS; TC: storage for 150 days and thermocycling 5000x. Fifty bar specimens were produced per group (n = 50). Contact angle and pH results were submitted to one-way ANOVA and Tukey's test (α = 0.05). Microtensile data were submitted to two-way ANOVA and Tukey's test (a = 0.05). pH values were significantly higher for SC6-ph (11.5 ± 2.6) when compared to those of SC5-pH (2.7 ± 0.4), SC8-pH (2.7 ± 0.2) and SC2-pH (2.2 ± 0.2) (p < 0.00, ANOVA). SC1-ca had the largest contact angle (48 ± 16 degrees) and SC3-ca the smallest (9.4 ± 7.7 degrees). The results of the MTBS test were as follows: independent of the storage condition, SC2-bond = SC1-bond > SC3-bond = SC4-bond. SC4-bond had the lowest MTBS value after TC (10.6 ± 2.6 MPa). The acid neutralization step appears to be dispensable, since the washing/drying promoted similar pH values. That condition promoted a high contact angle and unstable resin microtensile bond strength to glass ceramic.


First U-Pb zircon ages for late Miocene Ashfall Konservat-Lagerstätte and Grove Lake ashes from eastern Great Plains, USA.
This paper documents the first U-Pb zircon ages for Ashfall Fossil Beds (Nebraska, USA), a terrestrial Konservat-Lagerstätte mass-death assemblage that is arguably the most diverse of its type and age. The Ashfall tephra was correlated with ignimbrites from the Bruneau-Jarbidge volcanic field (12.7-10.5 Ma) in southwest Idaho based on geochemical analysis. The methods and geochemical data supporting the original age assessment of the ash bed, however, were never published, and there has been a persistent misconception that dateable heavy minerals (e.g., zircon) are absent. Notwithstanding, we recovered abundant zircons from Ashfall Fossil Beds, and from an ash bed ~6 km to the southeast at Grove Lake, Nebraska, and analyzed them through LA-ICP-MS. Our new zircon U-Pb age of 11.86 ± 0.13 Ma substantiates correlation of the Ashfall Fossil Beds deposit to tuffs originating from the Bruneau-Jarbidge caldera (~12.7-10.5 Ma). Our U-Pb zircon age of 6.42 ± 0.06 Ma for the Grove Lake ash bed coincides with supervolcanic activity in the Heise volcanic field (6.6-4.3 Ma) in eastern Idaho. These new dates improve age constraints of strata comprising the Ogallala Group and the important paleontological site. Moreover, we find that detrital and airfall zircons are unevenly distributed in the stratified ash beds we describe herein and presumably in similar deposits worldwide. Therefore, a higher-resolution sampling scheme is necessary in such cases.


Bacterial contamination and cleanliness of emergency department ultrasound probes.
As ultrasonography is increasingly used in the emergency department (ED), ultrasound equipment has become a potential threat to infection control. Improperly cleaned ultrasound probes may serve as a vector for pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). The primary objective of this study was to determine the prevalence of MRSA colonization on ultrasound probes used in a busy, urban ED. It was hypothesized that cultures of our ED ultrasound probes would yield a significant number of positive results for MRSA. In this observational study, 11 ED ultrasound probes were randomly sampled on 10 different occasions. Samples were taken using a RODAC plate method and were cultured for MRSA and methicillin-sensitive Staphylococcus aureus (MSSA). On half of the randomly assigned sampling occasions, a visual inspection of each ultrasound probe for general cleanliness was conducted and recorded. Data were stratified by ultrasound location in the ED and analyzed using the Fisher exact test, with p < 0.05 deemed to be statistically significant. Of 110 samples, no isolates of MRSA were cultured. One probe yielded a positive culture for MSSA. Probes in the medicine, trauma, and pediatrics areas were found to be clean 65%, 33%, and 70% of the time, respectively. This variability in probe cleanliness by ED location was found to be statistically significant (p < 0.01). Contrary to our hypothesis, MRSA contamination of ultrasound probes was not found. This finding suggests that the spread of MRSA by ED ultrasound machines in a high-volume urban ED is unlikely. Further research at different centres with larger sample sizes is required before these results can be generalized.


Autoradiographic localisation of muscarinic receptors in guinea-pig intestine: distribution of high and low affinity agonist binding sites.
Muscarinic receptors mediate a variety of intestinal functions including smooth muscle contraction, ganglionic transmission and water and electrolyte secretion. In this study, we have used [3H]quinuclidinyl benzilate ([3H]QNB) in an in vitro autoradiographic method to map the distribution of muscarinic receptors in guinea-pig ileum, colon and caecum. In addition, the relative distribution of low and high affinity agonist binding sites was assessed by the addition of the muscarinic agonist, carbachol, to selectively inhibit the binding of [3H]QNB to the high affinity sites. Although quantitative differences existed, the overall distribution of muscarinic receptors was similar in the 3 regions of intestine examined. Autoradiograph grains were found distributed over the myenteric and sub-mucous plexuses, the longitudinal and circular muscle layers and in the case of the colon, the muscularis mucosa. The inclusion of carbachol demonstrated that a greater proportion of high affinity sites were associated with the musculature than with the enteric plexuses. These findings are discussed in relation to the role of muscarinic mechanisms in intestinal motility and secretion.


Interventions for primary (intrinsic) tracheomalacia in children.
Tracheomalacia, a disorder of the large airways where the trachea is deformed or malformed during respiration, is commonly seen in tertiary paediatric practice. It is associated with a wide spectrum of respiratory symptoms from life-threatening recurrent apnoea to common respiratory symptoms such as chronic cough and wheeze. Current practice following diagnosis of tracheomalacia includes medical approaches aimed at reducing associated symptoms of tracheomalacia, ventilation modalities of continuous positive airway pressure (CPAP) and bi-level positive airway pressure (BiPAP), and surgical approaches aimed at improving the calibre of the airway (airway stenting, aortopexy, tracheopexy). To evaluate the efficacy of medical and surgical therapies for children with intrinsic (primary) tracheomalacia. The Cochrane Airways Group searched the Cochrane Central Register of Controlled Trials (CENTRAL), the Cochrane Airways Group's Specialised Register, MEDLINE and EMBASE databases. The Cochrane Airways Group performed the latest searches in March 2012. All randomised controlled trials (RCTs) of therapies related to symptoms associated with primary or intrinsic tracheomalacia. Two reviewers extracted data from the included study independently and resolved disagreements by consensus. We included one RCT that compared nebulised recombinant human deoxyribonuclease (rhDNase) with placebo in 40 children with airway malacia and a respiratory tract infection. We assessed it to be a RCT with overall low risk of bias. Data analysed in this review showed that there was no significant difference between groups for the primary outcome of proportion cough-free at two weeks (odds ratio (OR) 1.38; 95% confidence interval (CI) 0.37 to 5.14). However, the mean change in night time cough diary scores significantly favoured the placebo group (mean difference (MD) 1.00; 95% CI 0.17 to 1.83, P = 0.02). The mean change in daytime cough diary scores from baseline was also better in the placebo group compared to those on nebulised rhDNase, but the difference between groups was not statistically significant (MD 0.70; 95% CI -0.19 to 1.59). Other outcomes (dyspnoea, and difficulty in expectorating sputum scores, and lung function tests at two weeks also favoured placebo over nebulised rhDNase but did not reach levels of significance. There is currently an absence of evidence to support any of the therapies currently utilised for management of intrinsic tracheomalacia. It remains inconclusive whether the use of nebulised rhDNase in children with airway malacia and a respiratory tract infection worsens recovery. It is unlikely that any RCT on surgically based management will ever be available for children with severe life-threatening illness associated with tracheomalacia. For those with less severe disease, RCTs on interventions such as antibiotics and chest physiotherapy are clearly needed. Outcomes of these RCTs should include measurements of the trachea and physiological outcomes in addition to clinical outcomes.


Lactobacillus reuteri DSM 17938 in infantile colic: a randomized, double-blind, placebo-controlled trial.
To test the efficacy of Lactobacillus reuteri on infantile colic and to evaluate its relationship to the gut microbiota. Fifty exclusively breastfed colicky infants, diagnosed according to modified Wessel's criteria, were randomly assigned to receive either L reuteri DSM 17 938 (10(8) colony-forming units) or placebo daily for 21 days. Parental questionnaires monitored daily crying time and adverse effects. Stool samples were collected for microbiologic analysis. Forty-six infants (L reuteri group: 25; placebo group: 21) completed the trial. Daily crying times in minutes/day (median [interquartile range]) were 370 (120) vs 300 (150) (P=.127) on day 0 and 35.0 (85) vs 90.0 (148) (P=.022) on day 21, in the L reuteri and placebo groups, respectively. Responders (50% reduction in crying time from baseline) were significantly higher in the L reuteri group versus placebo group on days 7 (20 vs 8; P=.006), 14 (24 vs 13; P=.007), and 21 (24 vs 15; P=.036). During the study, there was a significant increase in fecal lactobacilli (P=.002) and a reduction in fecal Escherichia coli and ammonia in the L reuteri group only (P=.001). There were no differences in weight gain, stooling frequency, or incidence of constipation or regurgitation between groups, and no adverse events related to the supplementation were observed. L. reuteri DSM 17 938 at a dose of 10(8) colony-forming units per day in early breastfed infants improved symptoms of infantile colic and was well tolerated and safe. Gut microbiota changes induced by the probiotic could be involved in the observed clinical improvement.


Association of neuroinflammation with episodic memory: a [11C]PBR28 PET study in cognitively discordant twin pairs.
Alzheimer's disease is associated with chronic response of innate immune system, referred as neuroinflammation. PET radioligands binding to the 18 kDa translocator protein are potential biomarkers of neuroinflammation. Translocator protein PET studies in mild cognitive impairment and Alzheimer's disease have indicated controversial results, possibly reflecting interindividual variation and heterogeneity of study populations. We controlled for genetic and environmental effects by studying twin pairs discordant for episodic memory performance. Episodic memory impairment is a well-known cognitive hallmark of early Alzheimer's disease process. Eleven same-sex twin pairs (four monozygotic pairs, six female pairs, age 72-77 years) underwent [11C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine ([11C]PBR28) PET imaging, structural magnetic resonance imaging and neuropsychological testing in 2014-17. Main PET outcome was the volume-weighted average standardized uptake value of cortical regions vulnerable to Alzheimer's disease pathology. Ten pairs were discordant for episodic memory performance. In the eight pairs with identical translocator protein genotype, twins with poorer episodic memory had ∼20% higher cortical [11C]PBR28 binding compared with their better-performing co-twins (mean intra-pair difference 0.21 standardized uptake value, 95% confidence interval 0.05-0.37, P = 0.017). The result remained the same when including all discordant pairs and controlling for translocator protein genotype. Increased translocator protein PET signal suggests that increased microglial activation is associated with poorer episodic memory performance. Twins with worse episodic memory performance compared with their co-twins had on average 20% higher uptake of the neuroinflammatory marker translocator protein PET tracer 11[11C]PBR28. The findings support a negative association between neuroinflammation and episodic memory and the use of translocator protein positron emission tomography as a useful indicator of Alzheimer's disease process.


Producing aglycons of ginsenosides in bakers' yeast.
Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal plant that exhibits diverse pharmacological activities. Protopanaxadiol, protopanaxatriol and oleanolic acid are three basic aglycons of ginsenosides. Producing aglycons of ginsenosides in Saccharomyces cerevisiae was realized in this work and provides an alternative route compared to traditional extraction methods. Synthetic pathways of these three aglycons were constructed in S. cerevisiae by introducing β-amyrin synthase, oleanolic acid synthase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and NADPH-cytochrome P450 reductase from different plants. In addition, a truncated 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthase and 2,3-oxidosqualene synthase genes were overexpressed to increase the precursor supply for improving aglycon production. Strain GY-1 was obtained, which produced 17.2 mg/L protopanaxadiol, 15.9 mg/L protopanaxatriol and 21.4 mg/L oleanolic acid. The yeast strains engineered in this work can serve as the basis for creating an alternative way for producing ginsenosides in place of extractions from plant sources.


Brain is a potential sanctuary for subtype C HIV-1 irrespective of ART treatment outcome.
Subtype C HIV-1 is responsible for the largest proportion of people living with HIV-1 infection. However, there is limited information about the roles of the brain and its cell types as a potential sanctuary for this subtype and how the sanctuary may be affected by the administration of anti-retroviral therapy (ART). To address this issue, we collected postmortem brain tissues from ART treated HIV-1 infected Zambian individuals who experienced complete viral suppression and those who did not. Tissues from various brain compartments were collected from each individual as frozen and formalin-fixed paraffin embedded brain specimens, for detection and quantification of HIV-1 genomes and identification of the infected cell type. Genomic DNA and RNA were extracted from frozen brain tissues. The extracted DNA and RNA were then subjected to droplet digital PCR for HIV-1 quantification. RNA/DNAscope in situ hybridization (ISH) for HIV-1 was performed on formalin-fixed paraffin embedded brain tissues in conjugation with immunohistochemistry to identify the infected cell types. Droplet digital PCR revealed that HIV-1 gag DNA and RNA were detectable in half of the cases studied regardless of ART success or failure. The presence of HIV-1 lacked specific tissue compartmentalization since detection was random among various brain tissues. When combined with immunohistochemistry, RNA/DNAscope ISH demonstrated co-localization of HIV-1 DNA with CD68 expressing cells indicative of microglia or peripheral macrophage. Our study showed that brain is a potential sanctuary for subtype C HIV-1, as HIV-1 can be detected in the brain of infected individuals irrespective of ART treatment outcome and no compartmentalization of HIV-1 to specific brain compartments was evident.