In vitro conservation and sustained production of breadfruit (Artocarpus altilis, Moraceae): modern technologies for a traditional tropical crop.
Breadfruit (Artocarpus altilis, Moraceae) is a traditionally cultivated, high-energy, high-yield crop, but widespread use of the plant for food is limited by poor quality and poor storage properties of the fruit. A unique field genebank of breadfruit species and cultivars exists at the National Tropical Botanical Garden in the Hawaiian Islands and is an important global resource for conservation and sustainable use of breadfruit. However, this plant collection could be damaged by a random natural disaster such as a hurricane. We have developed a highly efficient in vitro plant propagation system to maintain, conserve, mass propagate, and distribute elite varieties of this important tree species. Mature axillary shoot buds were collected from three different cultivars of breadfruit and proliferated using a cytokinin-supplemented medium. The multiple shoots were maintained as stock cultures and repeatedly used to develop whole plants after root differentiation on a basal or an auxin-containing medium. The plantlets were successfully grown under greenhouse conditions and were reused to initiate additional shoot cultures for sustained production of plants. Flow cytometry was used to determine the nuclear deoxyribonucleic acid content and the ploidy status of the in vitro grown population. The efficacy of the micropropagation protocols developed in this study represents a significant advancement in the conservation and sustained mass propagation of breadfruit germplasm in a controlled environment free from contamination.


CAR T cells targeting tumor endothelial marker CLEC14A inhibit tumor growth.
Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.


Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes.
Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation.


Autoantibodies, autoimmune disease, and the birth of immune diagnostics.
The appearance of autoantibody to DNA followed sequentially by the disappearance of anti-DNA and appearance of DNA antigen in a patient with systemic lupus erythematosus demonstrated that autoantibodies participate in immune complex-mediated pathogenesis. Continuing studies showed that autoantibodies are also useful biomarkers in clinical diagnosis and important reagents for elucidating the structure and function of intracellular proteins in cell biology. Recently, autoantibodies to tumor-associated antigens have been identified in cancer, and these findings have expanded the field of cancer immunodiagnostics.


Relations between infants' emerging reach-grasp competence and event-related desynchronization in EEG.
Recent reports of similar patterns of brain electrical activity (electroencephalogram: EEG) during action execution and observation, recorded from scalp locations over motor-related regions in infants and adults, have raised the possibility that two foundational abilities--controlling one's own intentional actions and perceiving others' actions--may be integrally related during ontogeny. However, to our knowledge, there are no published reports of the relations between developments in motor skill (i.e. recording actual motor skill performance) and EEG during both action execution and action observation. In the present study we collected EEG from 21 9-month-olds who were given opportunities to reach for toys and who also observed an experimenter reach for toys. Event-related desynchronization (ERD) was computed from the EEG during the reaching events. We assessed infants' reaching-grasping competence, including reach latency, errors, preshaping of the hand, and bimanual reaches, and found that desynchronization recorded in scalp electrodes over motor-related regions during action observation was associated with action competence during execution. Infants who were more competent reachers, compared to less competent reachers, exhibited greater ERD while observing reaching-grasping. These results provide initial evidence for an early emerging neural system integrating one's own actions with the perception of others' actions.


MicroRNAs in the skin: role in development, homoeostasis and regeneration.
The skin is the largest organ of the integumentary system and possesses a vast number of functions. Due to the distinct layers of the skin and the variety of cells which populate each, a tightly regulated network of molecular signals control development and regeneration, whether due to programmed cell termination or injury. MicroRNAs (miRs) are a relatively recent discovery; they are a class of small non-coding RNAs which possess a multitude of biological functions due to their ability to regulate gene expression via post-transcriptional gene silencing. Of interest, is that a plethora of data demonstrates that a number of miRs are highly expressed within the skin, and are evidently key regulators of numerous vital processes to maintain non-aberrant functioning. Recently, miRs have been targeted as therapeutic interventions due to the ability of synthetic 'antagomiRs' to down-regulate abnormal miR expression, thereby potentiating wound healing and attenuating fibrotic processes which can contribute to disease such as systemic sclerosis (SSc). This review will provide an introduction to the structure and function of the skin and miR biogenesis, before summarizing the literature pertaining to the role of miRs. Finally, miR therapies will also be discussed, highlighting important future areas of research.


The role of nitric oxide and reactive oxygen species in the positive inotropic response to mechanical stretch in the mammalian myocardium.
The endothelial nitric oxide synthase (eNOS) has been implicated in the rapid (Frank-Starling) and slow (Anrep) cardiac response to stretch. Our work and that of others have demonstrated that a neuronal nitric oxide synthase (nNOS) localized to the myocardium plays an important role in the regulation of cardiac function and calcium handling. However, the effect of nNOS on the myocardial response to stretch has yet to be investigated. Recent evidence suggests that the stretch-induced release of angiotensin II (Ang II) and endothelin 1 (ET-1) stimulates myocardial superoxide production from NADPH oxidases which, in turn, contributes to the Anrep effect. nNOS has also been shown to regulate the production of myocardial superoxide, suggesting that this isoform may influence the cardiac response to stretch or ET-1 by altering the NO-redox balance in the myocardium. Here we show that the increase in left ventricular (LV) myocyte shortening in response to the application of ET-1 (10 nM, 5 min) did not differ between nNOS(-/-) mice and their wild type littermates (nNOS(+/+)). Pre-incubating LV myocytes with the NADPH oxidase inhibitor, apocynin (100 microM, 30 min), reduced cell shortening in nNOS(-/-) myocytes only but prevented the positive inotropic effects of ET-1 in both groups. Superoxide production (O(2)(-)) was enhanced in nNOS(-/-) myocytes compared to nNOS(+/+); however, this difference was abolished by pre-incubation with apocynin. There was no detectable increase in O(2)(-) production in ET-1 pre-treated LV myocytes. Inhibition of protein kinase C (chelerythrine, 1 microM) did not affect cell shortening in either group, however, protein kinase A inhibitor, PKI (2 microM), significantly reduced the positive inotropic effects of ET-1 in both nNOS(+/+) and nNOS(-/-) myocytes. Taken together, our findings show that the positive inotropic effect of ET-1 in murine LV myocytes is independent of nNOS but requires NADPH oxidases and protein kinase A (PKA)-dependent signaling. These results may further our understanding of the signaling pathways involved in the myocardial inotropic response to stretch.


Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells.
Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the alpha1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le(x)). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-alpha expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.


Effects of oral contraceptive pills on drug metabolizing enzymes and acid soluble sulfhydryl level in mouse liver.
The present paper reports the modulatory influence of two widely used combined oral contraceptive pills "OVRAL" and "NORACYCLINE" on hepatic phase I and II drug metabolizing enzymes and acid soluble sulfhydryl group of the mouse. Three different doses of the pills were used in this study i.e. D1 (1/2000th of a pill), D2 (1/200th of a pill) and D3 (1/20th of a pill). The sulfhydryl group increased significantly with the D1 and D2 dose of Ovral and the D2 dose of Noracycline. Dose D2 of both pills decreased cyt.P450 and cyt.b5 contents. D3 of Noracycline, however increased both the cytochrome levels. Dose D3 of Ovral and all three doses of Noracycline reduced the glutathione S-transferase activity.


Cracking under Internal Pressure: Photodynamic Behavior of Vinyl Azide Crystals through N2 Release.
When exposed to UV light, single crystals of the vinyl azides 3-azido-1-phenylpropenone (1a), 3-azido-1-(4-methoxyphenyl)propenone (1b), and 3-azido-1-(4-chlorophenyl)propenone (1c) exhibit dramatic mechanical effects by cracking or bending with the release of N2. Mechanistic studies using laser flash photolysis, supported by quantum mechanical calculations, show that each of the vinyl azides degrades through a vinylnitrene intermediate. However, despite having very similar crystal packing motifs, the three compounds exhibit distinct photomechanical responses in bulk crystals. While the crystals of 1a delaminate and release gaseous N2 indiscriminately under paraffin oil, the crystals of 1b and 1c visibly expand, bend, and fracture, mainly along specific crystallographic faces, before releasing N2. The photochemical analysis suggests that the observed expansion is due to internal pressure exerted by the gaseous product in the crystal lattices of these materials. Lattice energy calculations, supported by nanoindentation experiments, show significant differences in the respective lattice energies. The calculations identify critical features in the crystal structures of 1b and 1c where elastic energy accumulates during gas release, which correspond to the direction of the observed cracks. This study highlights the hitherto untapped potential of photochemical gas release to elicit a photomechanical response and motility of photoreactive molecular crystals.


γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type.
Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1-3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes.


Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor.
A family of peptides with broad-spectrum antimicrobial activity has been isolated from the skin of the African clawed frog Xenopus laevis. It consists of two closely related peptides that are each 23 amino acids and differ by two substitutions. These peptides are water soluble, nonhemolytic at their effective antimicrobial concentrations, and potentially amphiphilic. At low concentrations they inhibit growth of numerous species of bacteria and fungi and induce osmotic lysis of protozoa. The sequence of a partial cDNA of the precursor reveals that both peptides derive from a common larger protein. These peptides appear to represent a previously unrecognized class of vertebrate antimicrobial activities.


Intraoperative long range optical coherence tomography as a novel method of imaging the pediatric upper airway before and after adenotonsillectomy.
While upper airway obstruction is a common problem in the pediatric population, the first-line treatment, adenotonsillectomy, fails in up to 20% of patients. The decision to proceed to surgery is often made without quantitative anatomic guidance. We evaluated the use of a novel technique, long-range optical coherence tomography (LR-OCT), to image the upper airway of children under general anesthesia immediately before and after tonsillectomy and/or adenoidectomy. We investigated the feasibility of LR-OCT to identify both normal anatomy and sites of airway narrowing and to quantitatively compare airway lumen size in the oropharyngeal and nasopharyngeal regions pre- and post-operatively. 46 children were imaged intraoperatively with a custom-designed LR-OCT system, both before and after adenotonsillectomy. These axial LR-OCT images were both rendered into 3D airway models for qualitative analysis and manually segmented for quantitative comparison of cross-sectional area. LR-OCT images demonstrated normal anatomic structures (base of tongue, epiglottis) as well as regions of airway narrowing. Volumetric rendering of pre- and post-operative images clearly showed regions of airway collapse and post-surgical improvement in airway patency. Quantitative analysis of cross-sectional images showed an average change of 70.52mm(2) (standard deviation 47.87mm(2)) in the oropharynx after tonsillectomy and 105.58mm(2) (standard deviation 60.62mm(2)) in the nasopharynx after adenoidectomy. LR-OCT is an emerging technology that rapidly generates 3D images of the pediatric upper airway in a feasible manner. This is the first step toward development of an office-based system to image awake pediatric subjects and thus better identify loci of airway obstruction prior to surgery.


Alendronate therapy in cyclosporine-induced alveolar bone loss in rats.
Cyclosporine A is an immunosuppressive drug that is widely used in organ transplant patients as well as to treat a number of autoimmune conditions. Bone loss is reported as a significant side-effect of cyclosporine A use because this can result in serious morbidity of the patients. As we have shown that cyclosporine A-associated bone loss can also affect the alveolar bone, the purpose of this study was to evaluate the effect of the concomitant administration of alendronate on alveolar bone loss in a rat model. Forty Wistar rats (10 per group) were given cyclosporine A (10 mg/kg, daily), alendronate (0.3 mg/kg, weekly), or both cyclosporine A and alendronate, for 60 d. The control group received daily injections of sterile saline. The expression of proteins associated with bone turnover, including osteocalcin, alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP), and also the calcium levels, were evaluated in the serum. Analysis of the bone volume, alveolar bone surface, the number of osteoblasts per bone surface and the number of osteoclasts per bone surface around the lower first molars was also performed. The results indicate that cyclosporine A treatment was associated with bone resorption, represented by a decrease in the bone volume, alveolar bone surface and the number of osteoblasts per bone surface and by an increase in the number of osteoclasts per bone surface and TRAP-5b. These effects were effectively counteracted by concomitant alendronate administration. It is concluded that concomitant administration of alendronate can prevent cyclosporine A-associated alveolar bone loss.


Fluoride in Human Health and Nutrition.
Fluorine is in the form of fluorides ubiquitous in nature and as such an inevitable part of our environment. In small amounts, it is known to have beneficial effects on dental health. On the other hand, excessive chronic intakes can result in adverse effects including the development of dental fluorosis in children and/or skeletal fluorosis in both children and adults. The adequate intake has been set, based on empirical observations, at 0.05 mg/day/kg body weight however, the threshold between beneficial and harmful effects is narrow. Despite numerous studies, knowledge on the fluoride toxicity is still relatively poor. In this review, the role and the effects of fluoride on human health are enlightened. Some of the fluoride controversies are discussed and future research directions suggested.


Creating a model to detect dairy cattle farms with poor welfare using a national database.
The objective of this study was to determine whether dairy farms with poor cow welfare could be identified using a national database for bovine identification and registration that monitors cattle deaths and movements. The welfare of dairy cattle was assessed using the Welfare Quality(®) protocol (WQ) on 24 Portuguese dairy farms and on 1930 animals. Five farms were classified as having poor welfare and the other 19 were classified as having good welfare. Fourteen million records from the national cattle database were analysed to identify potential welfare indicators for dairy farms. Fifteen potential national welfare indicators were calculated based on that database, and the link between the results on the WQ evaluation and the national cattle database was made using the identification code of each farm. Within the potential national welfare indicators, only two were significantly different between farms with good welfare and poor welfare, 'proportion of on-farm deaths' (p<0.01) and 'female/male birth ratio' (p<0.05). To determine whether the database welfare indicators could be used to distinguish farms with good welfare from farms with poor welfare, we created a model using the classifier J48 of Waikato Environment for Knowledge Analysis. The model was a decision tree based on two variables, 'proportion of on-farm deaths' and 'calving-to-calving interval', and it was able to correctly identify 70% and 79% of the farms classified as having poor and good welfare, respectively. The national cattle database analysis could be useful in helping official veterinary services in detecting farms that have poor welfare and also in determining which welfare indicators are poor on each particular farm.


Dynamical stability of water distribution networks.
Water distribution networks are hydraulic infrastructures that aim to meet water demands at their various nodes. Water flows through pipes in the network create nonlinear dynamics on networks. A desirable feature of water distribution networks is high resistance to failures and other shocks to the system. Such threats would at least transiently change the flow rate in various pipes, potentially undermining the functionality of the whole water distribution system. Here we carry out a linear stability analysis for a nonlinear dynamical system representing the flow rate through pipes that are interconnected through an arbitrary pipe network with reservoirs and consumer nodes. We show that the steady state is always locally stable and develop a method to calculate the eigenvalue that corresponds to the mode that decays the most slowly towards the equilibrium, which we use as an index for resilience of the system. We show that the proposed index is positively correlated with the recovery rate of the pipe network, which was derived from a realistic and industrially popular simulator. The present analytical framework is expected to be useful for deploying tools from nonlinear dynamics and network analysis in the design, resilience management and scenario testing of water distribution networks.


Final analyses of OPTiM: a randomized phase III trial of talimogene laherparepvec versus granulocyte-macrophage colony-stimulating factor in unresectable stage III-IV melanoma.
Talimogene laherparepvec is an oncolytic immunotherapy approved in the US, Europe, Australia and Switzerland. We report the final planned analysis of OPTiM, a randomized open-label phase III trial in patients with unresectable stage IIIB-IVM1c melanoma. Patients were randomized 2:1 to receive intratumoral talimogene laherparepvec or subcutaneous recombinant GM-CSF. In addition to overall survival (OS), durable response rate (DRR), objective response rate (ORR), complete responses (CR), and safety are also reported. All final analyses are considered to be descriptive and treatment responses were assessed by the investigators. Of 436 patients in the intent-to-treat population, 295 were allocated to talimogene laherparepvec and 141 to GM-CSF. Median follow-up in the final OS analysis was 49 months. Median OS was 23.3 months (95% confidence interval [CI], 19.5-29.6) and 18.9 months (95% CI, 16.0-23.7) in the talimogene laherparepvec and GM-CSF arms, respectively (unstratified hazard ratio, 0.79; 95% CI, 0.62-1.00; p = 0.0494 [descriptive]). DRR was 19.0 and 1.4% (unadjusted odds ratio, 16.6; 95% CI, 4.0-69.2; p < 0.0001); ORR was 31.5 and 6.4%. Fifty (16.9%) and 1 (0.7%) patient in the talimogene laherparepvec and GM-CSF arms, respectively, achieved CR. In talimogene laherparepvec-treated patients, median time to CR was 8.6 months; median CR duration was not reached. Among patients with a CR, 88.5% were estimated to survive at a 5-year landmark analysis. Talimogene laherparepvec efficacy was more pronounced in stage IIIB-IVM1a melanoma as already described in the primary analysis. The safety reporting was consistent with the primary OPTiM analysis. In this final planned OPTiM analysis, talimogene laherparepvec continued to result in improved longer-term efficacy versus GM-CSF and remained well tolerated. The final analysis also confirms that talimogene laherparepvec was associated with durable CRs that were associated with prolonged survival. ClinicalTrials.gov identifier: NCT00769704 .


Dot trajectories in the superposition of random screens: analysis and synthesis.
Moiré effects that occur in the superposition of aperiodic layers such as correlated random dot screens are known as Glass patterns. One of the most interesting properties of such moiré effects, which clearly distinguish them from their periodic counterparts, is undoubtedly the appearence in the superposition of intriguing microstructure dot alignments, also known as dot trajectories. These dot trajectories may have different geometric shapes, depending on the transformations undergone by the superposed layers. In the case of simple linear transformations such as layer rotations or layer scalings, the resulting dot trajectories are rather simple (circular, radial, spiral, elliptic, hyperbolic, linear, etc.); but in more complex layer transformations the dot trajectories can have much more interesting and surprising shapes. A full mathematical analysis of the dot trajectories, their morphology, and their various properties is provided. Furthermore, it is shown how the approach also allows us to synthesize correlated random screens that give in their superposition dot trajectories having any desired geometric shapes. Finally, it is also explained why such dot trajectories are visible only in superpositions of aperiodic screens but not in superpositions of periodic screens.


Brain scans from 21,297 individuals reveal the genetic architecture of hippocampal subfield volumes.
The hippocampus is a heterogeneous structure, comprising histologically distinguishable subfields. These subfields are differentially involved in memory consolidation, spatial navigation and pattern separation, complex functions often impaired in individuals with brain disorders characterized by reduced hippocampal volume, including Alzheimer's disease (AD) and schizophrenia. Given the structural and functional heterogeneity of the hippocampal formation, we sought to characterize the subfields' genetic architecture. T1-weighted brain scans (n = 21,297, 16 cohorts) were processed with the hippocampal subfields algorithm in FreeSurfer v6.0. We ran a genome-wide association analysis on each subfield, co-varying for whole hippocampal volume. We further calculated the single-nucleotide polymorphism (SNP)-based heritability of 12 subfields, as well as their genetic correlation with each other, with other structural brain features and with AD and schizophrenia. All outcome measures were corrected for age, sex and intracranial volume. We found 15 unique genome-wide significant loci across six subfields, of which eight had not been previously linked to the hippocampus. Top SNPs were mapped to genes associated with neuronal differentiation, locomotor behaviour, schizophrenia and AD. The volumes of all the subfields were estimated to be heritable (h2 from 0.14 to 0.27, all p < 1 × 10-16) and clustered together based on their genetic correlations compared with other structural brain features. There was also evidence of genetic overlap of subicular subfield volumes with schizophrenia. We conclude that hippocampal subfields have partly distinct genetic determinants associated with specific biological processes and traits. Taking into account this specificity may increase our understanding of hippocampal neurobiology and associated pathologies.


Scaling up: bringing the transitional care model into the mainstream.
Elderly, chronically ill people experience frequent changes in health status that require transitions among health care providers and settings. This issue brief describes two projects that identified the essential elements of effective care management interventions for this population and the facilitators of translating one such intervention, the Transitional Care Model (TCM), into mainstream practice. Together these projects demonstrate that successful translation of the TCM, which incorporates both in-person contact and a nurse-led, interdisciplinary team approach, can effectively interrupt patterns of frequent rehospitalizations, reduce costs, and improve patient health status. Findings from these projects inform challenges that must be overcome to facilitate the translation of effective care management innovations into mainstream practice.


Saturation of 2',2'-difluorodeoxycytidine 5'-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine.
The plasma and cellular pharmacology of 2',2'-difluorodeoxycytidine (dFdC, Gemcitabine) was studied during a phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC dose, which ranged between 53 and 1,000 mg/m2 per 30 min. The cellular pharmacokinetics of an active metabolite, dFdC 5'-triphosphate (dFdCTP), were determined in mononuclear cells of 22 patients by anion-exchange high-pressure liquid chromatography. The rate of dFdCTP accumulation and the peak cellular concentration were highest at a dose rate of 350 mg/m2 per 30 min, during which steady-state dFdC levels of 15-20 microM were achieved in plasma. A comparison of patients infused with 800 mg/m2 over 60 min with those receiving the same dose over 30 min demonstrated that the dFdC steady-state concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by mononuclear cells incubated in vitro was maximal at 10-15 microM dFdC. These studies suggest that the ability of mononuclear cells to use dFdC for triphosphate formation is saturable. In the design of future protocols, a dose rate should be considered that produces maximal nucleotide analogue formation, with increased intensity being achieved by prolonging the duration of infusion.


Baseline gene expression predicts sensitivity to gefitinib in non-small cell lung cancer cell lines.
Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.


Reduction of ochratoxin a levels in red wine by bentonite, modified bentonites, and chitosan.
Adsorption method may play an important role to remove ochratoxin A (OTA) from wine by bentonite (B), nonylammonium bentonite (NB), dodecylammonium bentonite (DB), KSF-montmorillonite (KSF), and chitosan bead (CB). The optimum conditions of OTA adsorption from synthetic solutions were revealed at room temperature and pH 3.5. The adsorption equilibria of B and NB were almost established within 120 and 240 min, respectively. DB, KSF, and CB had about 90 min of equilibration time. The adsorption efficiency carried out in the synthetic OTA solution did not change remarkably when the amounts of adsorbents were 25 mg for bentonite, DB, and KSF and 100 mg for NB and CB. Furthermore, 25 mg of adsorbents was used at all adsorption studies in synthetic solution. The adsorption isotherm was fitted with mostly a Freundlich equation with respect to the correlation coefficients. The adsorption data were evaluated using Langmuir and Freundlich equations having Kf values ranging from 0.011 to 9.5 with respect to correlation coefficients (R2 = 0.900-0.977). DB and KSF have the highest adsorption capacity for OTA in synthetic solutions. In wine, the removal of OTA was succeeded at a percentage of 60-100 by KSF and CB. Furthermore, the highest adsorption capacity of OTA for red wine was obtained by using 250 mg of KSF, which caused less damage to the nature of wine and also low adsorption of polyphenols and anthcyans.


Evidence for a common mechanism of SIRT1 regulation by allosteric activators.
A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.


Important roles of dietary taurine, creatine, carnosine, anserine and 4-hydroxyproline in human nutrition and health.
Taurine (a sulfur-containing β-amino acid), creatine (a metabolite of arginine, glycine and methionine), carnosine (a dipeptide; β-alanyl-L-histidine), and 4-hydroxyproline (an imino acid; also often referred to as an amino acid) were discovered in cattle, and the discovery of anserine (a methylated product of carnosine; β-alanyl-1-methyl-L-histidine) also originated with cattle. These five nutrients are highly abundant in beef, and have important physiological roles in anti-oxidative and anti-inflammatory reactions, as well as neurological, muscular, retinal, immunological and cardiovascular function. Of particular note, taurine, carnosine, anserine, and creatine are absent from plants, and hydroxyproline is negligible in many plant-source foods. Consumption of 30 g dry beef can fully meet daily physiological needs of the healthy 70-kg adult human for taurine and carnosine, and can also provide large amounts of creatine, anserine and 4-hydroxyproline to improve human nutrition and health, including metabolic, retinal, immunological, muscular, cartilage, neurological, and cardiovascular health. The present review provides the public with the much-needed knowledge of nutritionally and physiologically significant amino acids, dipeptides and creatine in animal-source foods (including beef). Dietary taurine, creatine, carnosine, anserine and 4-hydroxyproline are beneficial for preventing and treating obesity, cardiovascular dysfunction, and ageing-related disorders, as well as inhibiting tumorigenesis, improving skin and bone health, ameliorating neurological abnormalities, and promoting well being in infants, children and adults. Furthermore, these nutrients may promote the immunological defense of humans against infections by bacteria, fungi, parasites, and viruses (including coronavirus) through enhancing the metabolism and functions of monocytes, macrophages, and other cells of the immune system. Red meat (including beef) is a functional food for optimizing human growth, development and health.


The socio-legal acceptance of new technologies: a close look at artificial insemination.
Heated debates often surround the introduction of an important new technology into society, as exemplified by current controversies surrounding human cloning and privacy protection on the Internet. Underlying these controversies are disruptions to central socio-legal values caused by these new technologies. Whether new technologies will eventually be accepted by society is often contingent on the reaction of the legal system. This mandates the formulation of a conceptual framework for understanding and structuring the way the law should react in cases surrounding the adoption of new technologies. By using the case study of artificial insemination this Article develops the tools for structuring the legal role in the acceptance process of new technologies. The three-century controversy surrounding the innovation of artificial insemination results from the innovations' disruption of the socio-legal value of the family. Artificial Insemination--although invented in the eighteenth-century--was rarely used until the 1930s, and only legalized in the 1960s. Its application to surrogacy and its use by unmarried women extends the controversy into the twenty-first century. The case study demonstrates the nature of the relationship among the technological, social and legal acceptance processes of new technologies, and analyzes the legal acceptance debate. The conceptual framework produced is useful in understanding and structuring the legal role in current debates surrounding the introduction and acceptance of new technologies.


Simultaneous onset of hemorrhagic and ischemic strokes.
Primary brain hemorrhage and infarction only very rarely occur simultaneously. We report a patient with the simultaneous onset of hemorrhagic and ischemic strokes who had uncontrolled hypertension and atrial fibrillation. Neuroradiologic investigations revealed a large right thalamic hematoma with ventricular extension and an infarct in the territory of the left internal carotid artery. The patient died at the end of the second day because of herniation in spite of anti-edema and antihypertensive medication.


Peri-conception hyperglycaemia and nephropathy are associated with risk of congenital anomaly in women with pre-existing diabetes: a population-based cohort study.
AIMS: The aim of this study was to quantify the risk of major congenital anomaly, and to assess the influence of peri-conception HbA(1c) and other clinical and socio-demographic factors on the risk of congenital anomaly occurrence in offspring of women with type 1 and type 2 diabetes diagnosed before pregnancy. METHODS: This was a population-based cohort study using linked data from registers of congenital anomaly and diabetes in pregnancy. A total of 401,149 singleton pregnancies (1,677 in women with diabetes) between 1996 and 2008 resulting in live birth, fetal death at ≥20 weeks' gestation or termination of pregnancy for fetal anomaly were included. RESULTS: The rate of non-chromosomal major congenital anomaly in women with diabetes was 71.6 per 1,000 pregnancies (95% CI 59.6, 84.9), a relative risk of 3.8 (95% CI 3.2, 4.5) compared with women without diabetes. There was a three- to sixfold increased risk across all common anomaly groups. In a multivariate analysis, peri-conception glycaemic control (adjusted OR [aOR] 1.3 [95% CI 1.2, 1.4] per 1% [11 mmol/mol] linear increase in HbA(1c) above 6.3% [45 mmol/mol]) and pre-existing nephropathy (aOR 2.5 [95% CI 1.1, 5.3]) were significant independent predictors of congenital anomaly. Associations with gestation at booking (aOR 1.1 [95% CI 1.0, 1.1]) and parity (aOR 1.6 [95% CI 1.0, 2. 5]) were not significant. Unadjusted risk was higher for women from deprived areas or who did not take folate. Type and duration of diabetes, ethnicity, age, BMI, preconception care, smoking and fetal sex were not associated with congenital anomaly risk. CONCLUSIONS: Peri-conception glycaemia is the most important modifiable risk factor for congenital anomaly in women with diabetes. The association with nephropathy merits further study.


Planning of catheter interventions for pulmonary artery stenosis: improved measurement agreement with magnetic resonance angiography using identical angulations.
To evaluate the current disagreement between the gold standard X-ray angiography (XRA) and magnetic resonance angiography (MRA) for the measurement of peripheral pulmonary artery stenosis (PPAS). MRA may help planning of catheter interventions for PPAS. However, there are sources of disagreement between XRA and MRA as measures are performed differently. We hypothesized that agreement may improve if identical angulation views are used. In this retrospective study, 17 patients were included. Four independent observers analyzed the pictures in three technique modalities: (1) XRA, (2) cross-sectional-MRA: reformatted to obtain the perpendicular cut-off view of the vessel, and (3) angulated-MRA: reformatted using the same angulations used in XRA. We measured: (a) minimal diameter at stenosis, (b) vessel diameter 1 cm proximal, and (c) 1 cm distal to the stenosis. While in elliptical vessels cross-sectional-MRA provided both the larger and smaller diameter, XRA and angulated-MRA typically provided only the larger diameter due to projections. We found poor agreement between the smaller diameter in cross-sectional-MRA and XRA. XRA underestimate the diameter at the three locations (<15-22%). However, there was a good agreement between the larger cross-sectional-MRA diameter and XRA. This further improved if MRA images were reformatted with the identical angulations used in XRA (angulated-MRA). Conventional cross-sectional-MRA measurements of PPAS only moderately agree with XRA. However, using identical angulations in MRA reduces the bias. Hence, MRA is a valuable tool for planning angulations and measurements of PPAS prior to catheter interventions.


Smoke-free legislation and hospitalizations for acute coronary syndrome.
Previous studies have suggested a reduction in the total number of hospital admissions for acute coronary syndrome after the enactment of legislation banning smoking in public places. However, it is unknown whether the reduction in admissions involved nonsmokers, smokers, or both. Since the end of March 2006, smoking has been prohibited by law in all enclosed public places throughout Scotland. We collected information prospectively on smoking status and exposure to secondhand smoke based on questionnaires and biochemical findings from all patients admitted with acute coronary syndrome to nine Scottish hospitals during the 10-month period preceding the passage of the legislation and during the same period the next year. These hospitals accounted for 64% of admissions for acute coronary syndrome in Scotland, which has a population of 5.1 million. Overall, the number of admissions for acute coronary syndrome decreased from 3235 to 2684--a 17% reduction (95% confidence interval, 16 to 18)--as compared with a 4% reduction in England (which has no such legislation) during the same period and a mean annual decrease of 3% (maximum decrease, 9%) in Scotland during the decade preceding the study. The reduction in the number of admissions was not due to an increase in the number of deaths of patients with acute coronary syndrome who were not admitted to the hospital; this latter number decreased by 6%. There was a 14% reduction in the number of admissions for acute coronary syndrome among smokers, a 19% reduction among former smokers, and a 21% reduction among persons who had never smoked. Persons who had never smoked reported a decrease in the weekly duration of exposure to secondhand smoke (P<0.001 by the chi-square test for trend) that was confirmed by a decrease in their geometric mean concentration of serum cotinine from 0.68 to 0.56 ng per milliliter (P<0.001 by the t-test). The number of admissions for acute coronary syndrome decreased after the implementation of smoke-free legislation. A total of 67% of the decrease involved nonsmokers. However, fewer admissions among smokers also contributed to the overall reduction.


Peripheral blood mononuclear cell fatty acid composition and inflammatory mediator production in adult Crohn's disease.
Crohn's disease (CD) is associated with nutritional deficiencies, altered plasma concentrations of polyunsaturated fatty acids (PUFA) and an anti-inflammatory response to fish oil that contains n-3 PUFA. This suggests that, in CD, immune cells may have altered n-3 PUFA composition with functional consequences. The aim of this study is to investigate n-3 and n-6 PUFA composition and synthetic function of peripheral blood mononuclear cells (PBMC) in the basal state. A case control study of 52 adult CD patients and healthy, age- and sex-matched controls. Composition of PBMC and plasma phospholipids were measured by gas chromatography and production of tumour necrosis factor-alpha, prostaglandin E2 (PGE2) and interferon-gamma (IFN-gamma) by PBMC were measured by ELISA. CD was associated with higher concentrations of eicosapentaenoic acid and other n-3 PUFA, and lower arachidonic acid (AA) (n-6 PUFA) in PBMC. This was not explained by differences in dietary fat intake. Lower rates of production of PGE2 and IFN-gamma by PBMC were noted in quiescent and active CD, respectively, compared to controls. CD is associated with a greater availability, and not a deficiency, of n-3 PUFA in PBMC, but lower concentrations of AA, and lower rates of production of PGE2 and IFN-gamma, compared to healthy controls.


Effects of reactive nitrogen scavengers on NK-cell-mediated killing of K562 cells.
This study explored the effects of reactive nitrogen metabolites (RNMS) on natural-killer- (NK-) cell-mediated killing of K562 cells and the influence of RNM scavengers, such as tiopronin (TIP), glutamylcysteinylglycine (GSH), and histamine dihydrochloride (DHT), on reversing the suppressing effect of RNM. We administered exogenous and endogenous RNM in the NK + K562 culture system and then added RNM scavengers. The concentrations of RNM, TNF-β and IFN-γ, and NK-cell cytotoxicity (NCC) and the percentage of living NK cells were then examined. We found that both exogenous and endogenous RNM caused the KIR to decrease (P < 0.01); however, RNM scavengers such as TIP and GSH rescued this phenomenon dose dependently. In conclusion, our data suggests that RNM scavengers such as TIP and GSH enhance the antineoplasmic activity of NK cells.


Creating safe spaces to prevent unintentional childhood injuries among the Bedouins in southern Israel: A hybrid model comprising positive deviance, community-based participatory research, and entertainment-education.
Despite several intervention programs, the Bedouin population living in the Southern District of Israel has the highest mortality rate among children and adolescents from unintentional injuries. Our research questions asked: (1) How does increasing the involvement and participation of Bedouin community members influence the issue of unintentional injuries among children? (2) How does reframing of the technical issue of safety into security influence community involvement and cooperation? 1) To identify effective and efficacious positive deviance practices through community-based participatory research with adults, children, and professionals in the Bedouin community. 2) To create wider and deeper connections and cohesion between and among diverse Bedouin communities by seeding and sparking opportunities for social networking and cross-learning. The study used a qualitative multi-method approach to generate a hybrid intervention model for reducing unintentional childhood injuries among the Bedouins. To frame the issue of unintentional injuries from the lived perspective of the Bedouins, we employed the Positive Deviance (PD) and Community Based Participatory Research (CBPR) approach. Drawing upon theatrical traditions, entertainment-education (EE), was employed as a way to narratively engage and persuade the Bedouins. Our research resulted in: (1) the emergence of several PD ideas and practices for preventing and avoiding children's injuries; (2) the actual creation of a safe and secure playroom for children at a neighborhood mosque; and (3) the creation of cascading and cross-learning social networks between and among members of the Bedouin community spread across various locations. This study helped in reframing the technical issue of accidents and safety into the notion of sacredness and security, enhanced the association between emotions and cognition by means of experiential and EE methods, and stimulated creative thinking and the emergence of new culturally and contextually relevant ideas and practices through the PD process. It demonstrated the synergistic power of using a hybrid model that combined the rigor and vigor of different health communication approaches to address a significant disparity in the burden of child accidents faced by the Bedouins. Our study generated solutions that emerged from, and directly benefitted, Bedouin children-those, who face overwhelming risk of injury and death from preventable accidents.


Multi-faceted informatics system for digitising and streamlining the reablement care model.
Reablement is new paradigm to increase independence in the home amongst the ageing population. And it remains a challenge to design an optimal electronic system to streamline and integrate reablement into current healthcare infrastructure. Furthermore, given reablement requires collaboration with a range of organisations (including national healthcare institutions and community/voluntary service providers), such a system needs to be co-created with all stakeholders involved. Thus, the purpose of this study is, (1) to bring together stakeholder groups to elicit a comprehensive set of requirements for a digital reablement system, (2) to utilise emerging technologies to implement a system and a data model based on the requirements gathered and (3) to involve user groups in a usability assessment of the system. In this study we employed a mixed qualitative approach that included a series of stakeholder-involved activities. Collectively, 73 subjects were recruited to participate in an ideation event, a quasi-hackathon and a usability study. The study unveiled stakeholder-led requirements, which resulted in a novel cloud-based system that was created using emerging web technologies. The system is driven by a unique data model and includes interactive features that are necessary for streamlining the reablement care model. In summary, this system allows community based interventions (or services) to be prescribed to occupants whilst also monitoring the occupant's progress of independent living.


Gastro-oesophageal reflux in preterm infants.
Gastro-oesophageal reflux in very low birthweight infants was studied using a new 1 mm monocrystalline antimony oesophageal pH electrode. Gastro-oesophageal reflux was detected in 30 (85%) subjects. The mean (SEM) number of episodes of reflux in 24 hours was 12.1 (2.1), and 3.2 (0.6) lasted over five minutes. The mean reflux index was 4.5 (1.0)%, and the longest episode 17.1 (4.6) 17.1. Reflux was unrelated to postconceptional age or to resting lower oesophageal sphincter pressure. The mean reflux index was low at rest before feeds, being 1.8 (0.6)%, and increased slightly after feeds (3.8 (1.0)%), but was significantly increased after nursing care to 16.4 (3.0)%, and while xanthines were being given (5.9 (1.6)%. A subgroup of seven infants with xanthine resistant apnoea had severe gastro-oesophageal reflux that was not clinically apparent (reflux index 27.4 (3.6)%). Successful treatment of the reflux (reflux index: 3.6 (1.2)%) was associated with cessation of the apnoea. We conclude that gastro-oesophageal reflux is common, and is usually not clinically apparent, even when severe. It is important to consider gastro-oesophageal reflux in the differential diagnosis of xanthine resistant apnoea in preterm infants.


Promoting, building and sustaining a regional laboratory network in a changing environment.
The Queen's University Department of Pathology and its affiliated hospital laboratories (Kingston, Canada) have operated a successful laboratory outreach program for more than a decade in Southeastern Ontario. The outreach program provides high quality reference testing and technical and professional expertise in laboratory medicine to largely rural and small urban community hospitals. As a consequence of dramatic cuts to the publicly funded health-care system in the Province of Ontario, the environment in which laboratory medicine is practiced has altered irrevocably. This article discusses some of the difficult internal and external challenges faced by the outreach program within the region and how they were effectively managed, not only to maintain but to enhance the program's services. The result has been a continued improvement in the quality of laboratory services in the region with significantly increased cost-effectiveness, largely through reengineering and consolidation.


Sacral rhizotomies and electrical bladder stimulation in spinal cord injury. 2. Cost-effectiveness and quality of life analysis. Dutch Study Group on Sacral Anterior Root Stimulation.
To present a cost-effectiveness analysis of sacral rhizotomies and electrical bladder stimulation compared with conventional care of neurogenic bladder dysfunction in patients with spinal cord injury. During a 3-year inclusion period, data on costs and quality of life before the intervention were collected to describe conventional care. Data on the pre-implantation period, the implantation and a follow-up period of 2 years were collected following a strict protocol simultaneous with medical and urodynamic data and were used to calculate the costs and effects on quality of life of the implantation of the stimulator. Between June 1991 and June 1994, 52 patients with complete cervical or thoracic spinal cord lesions underwent sacral posterior rhizotomies and implantation of a Finetech-Brindley sacral anterior root stimulator. Although the initial costs of sacral anterior root stimulation are high, they are earned back in this series in about 8 years after the implantation. General indicators of the quality of life show no significant changes after the implantation. Factors related to psychological well-being and the patients' satisfaction with the emptying of the bladder increased significantly whereas the experienced problems of micturition and incontinence all decreased significantly. Sacral rhizotomies and electrical bladder stimulation make a cost-effective method of treatment of lower urinary tract dysfunction in patients with spinal cord injury. Considerable savings on health care costs are possible in the long run with simultaneous positive effects on aspects of health status.


Intranasal immunization with recombinant Toxoplasma gondii actin depolymerizing factor confers protective efficacy against toxoplasmosis in mice.
Toxoplasma gondii is an opportunistic protozoan closely associated with AIDS and vertical transmission. T. gondii actin depolymerizing factor (TgADF) plays an important role in actin cytoskeleton remodeling, and it is required to invade host cells. TgADF was a promising vaccine candidate. To observe the immunological changes and protective efficacy of recombinant TgADF protein (rTgADF) against T. gondii infection, we optimized the intranasal immunization dose of rTgADF and analyzed the survival rate and tachyzoite loads in mouse tissues after oral challenge with T. gondii tachyzoites. rTgADF was prepared, purified, and combined with mouse anti-His antibody and rabbit anti-T. gondii serum. After intranasal immunization with 10 μg, 20 μg, 30 μg, or 40 μg of rTgADF, the 30-μg group elicited high levels of secretory IgA (sIgA) in nasal, intestinal, and vesical washes, raised IgG titres in the sera, strong proliferation of splenocytes, and increased secretion of IL-2 and IFN-γ when compared with the control group. When the mice were orally challenged with T. gondii, an increase in the survival rate (36.36 %) and a decrease in the tachyzoite loads in the liver (67.77 %) and brain (51.01 %) were observed. Our findings demonstrate that intranasal immunization with rTgADF can simultaneously trigger mucosal and systemic immune responses and protect the mice against T. gondii infection.


'Fractional recovery' analysis of a presynaptic synaptotagmin 1-anchored endocytic protein complex.
The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG), has also been implicated in synaptic vesicle (SV) recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE) from presynaptic nerve terminals and have used a novel fractional recovery (FR) assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. The first step in FR analysis was to immunoprecipitate (IP) the complex with an antibody against one protein component (the IP-protein). The immobilized complex was then exposed to a high salt (1150 mM) stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins). A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot) was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized), and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method) at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. We hypothesize that the SAE serves as a recognition site and also as a seed complex for clathrin-mediated synaptic vesicle recovery. The combination of FR analysis with quantitative immunocytochemistry provides a novel and effective strategy for the identification and characterization of biologically-relevant multi-molecular complexes.


Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis via Bacterial Antigens.
The diagnosis of active tuberculosis (TB) cases primarily relies on methods that detect Mycobacterium tuberculosis (Mtb) bacilli or their DNA in patient samples (e.g., mycobacterial culture and Xpert MTB/RIF assays), but these tests have low clinical sensitivity for patients with paucibacillary TB disease. Our goal was to evaluate the clinical performance of a newly developed assay that can rapidly diagnose active TB cases by direct detection of Mtb-derived antigens in patients' blood samples. Nanoparticle (NanoDisk)-enriched peptides derived from the Mtb virulence factors CFP-10 (10-kDa culture factor protein) and ESAT-6 (6-kDa early secretory antigenic target) were analyzed by high-throughput mass spectrometry (MS). Serum from 294 prospectively enrolled Chinese adults were analyzed with this NanoDisk-MS method to evaluate the performance of direct serum Mtb antigen measurement as a means for rapid diagnosis of active TB cases. NanoDisk-MS diagnosed 174 (88.3%) of the study's TB cases, with 95.8% clinical specificity, and with 91.6% and 85.3% clinical sensitivity for culture-positive and culture-negative TB cases, respectively. NanoDisk-MS also exhibited 88% clinical sensitivity for pulmonary and 90% for extrapulmonary TB, exceeding the diagnostic performance of mycobacterial culture for these cases. Direct detection and quantification of serum Mtb antigens by NanoDisk-MS can rapidly and accurately diagnose active TB in adults, independent of disease site or culture status, and outperform Mycobacterium-based TB diagnostics.


Preferential phosphorylation of the 150,000 molecular weight component of neurofilaments by a cyclic AMP-dependent, microtubule-associated protein kinase.
Highly purified preparations of bovine brain and rabbit nerve root neurofilaments were found to be lacking in protein kinase activity when either histone FIIA or the neurofilaments themselves were used as acceptors. There was no augmentation of activity in the presence of cyclic AMP. Addition of microtubule proteins prepared by cycles of assembly and disassembly resulted in phosphorylation of histone, phosphorylation of tubulin and the microtubule-associated proteins, and phosphorylation of neurofilament subunits. The phosphorylation of neurofilaments was predominantly in the 150,000-dalton species and was completely cyclic AMP dependent.


Dynamic response of an all-fiber ring resonator.
An analysis of the dynamic response of a single-mode all-fiber ring resonator is presented in which a time-varying phase shift is induced in the ring by a phase modulator. In particular, the deviation of the response from that expected from the steady-state analysis is discussed.


Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing.
The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.


Antibiotic prescribing and outcomes following treatment of symptomatic urinary tract infections in older women.
To describe antibiotic prescribing patterns for the treatment of urinary tract infections (UTI) among older women female long-term care facility residents and evaluate factors associated with adverse outcomes Population-based retrospective cohort study. Population and sampling frame: All female residents of Olmsted County, Minnesota, aged 65 years and older, who were treated for urinary tract infections during calendar year 1996 were included in the study. Subjects' complete medical records were reviewed and abstracted. Data collected included age, weight, serum creatinine, antibiotic treatment including dosage and duration, as well as retreatment and adverse drug events. A total of 196 subjects, ranging in age from 65 to 98 years, were diagnosed with UTI during calendar year 1996. Forty-four subjects were nursing home residents, and 152 resided in the community. The odds of prolonged antibiotic therapy (7 days or more) were 83 times higher among nursing home residents (Odds ratio = 82.7, 95% Confidence Interval, 11.1-617.7). The odds of receiving treatment for 10 days or longer were 5 times higher among nursing home residents compared to community subjects. The odds of underlying renal impairment necessitating dosage reduction were 3.6 times higher among nursing facility residents. Nursing facility residents were 7.3 times more likely to be treated with excessive dosages of antibiotics, 9.6 times more likely to experience adverse drug events, and 2.6 times more likely to receive retreatment for persistent or recurrent symptoms within one week of antibiotic discontinuation, compared to community subjects with UTI. Nursing home residents with UTI are treated for longer durations than community elderly. They experience a greater likelihood of receiving antibiotics in excessive dosage, a greater likelihood of adverse drug events, and a greater likelihood of retreatment compared with community subjects with UTI.


Structuring a college alcohol prevention program on the low level of response to alcohol model: a pilot study.
New approaches are needed to bolster the modest effects of campus drinking prevention programs. However, more definitive research on new paradigms is very expensive, and in the current economic climate, progress can be made by evaluating smaller pilot studies. This study describes one such approach. A sample of 18-year-old or older, healthy, drinking freshmen at our university was assigned to 2 groups stratified to be similar on demography, drinking histories, and their level of response (LR) to alcohol. In the spring quarter of the school year, the 32 subjects in each of 2 groups viewed four 45-minute Internet-based videotapes as part of 4 prevention sessions. All 8 modules were based on the same techniques and general content, but the 4 videos for the first group were structured around the validated model of how a low LR affects heavy drinking (the low level of response-based [LRB] Group), with partial mediation by heavier drinking peers, positive alcohol expectancies, and drinking to cope with stress. Videos for the state-of-the-art (SOTA) comparison group did not place the similar prevention messages into the low LR framework. Changes in drinking were evaluated at 3 times: before Module 1, before Module 4, and 1 month after Module 4. Usual and maximum drinks per occasion decreased over time for both high and low LR subjects in both LRB and SOTA groups. As predicted, the low LR students showed greater decreases in the LRB Group, while high LR students showed greater decreases in the more generic SOTA Group. The results support the hypothesis that tailoring prevention efforts to address specific predisposing factors, such as a low LR, may be associated with beneficial effects on drinking quantity. We hope that these data will encourage additional efforts to validate the low LR-based prevention paradigm and test other interventions that are targeted toward predisposing phenotypes such as impulsivity and negative affect.


The effects of limb posture on relationships between in vitro radial hoof strain, load and joint angles.
Radial strain in normal hooves has been found to vary with strain gauge location, limb posture and sample limb but reported magnitudes were considered to be low. More accurate measurement of radial strain may enhance the understanding of hoof function. To explore in vitro radial hoof strain in relation other kinetic and kinematic variables that may be related. Five normal forelimbs were removed at the proximal articular surface of the third metacarpal bone (McIII). The limbs were loaded using a modified Instron test machine. Six calibrated infrared cameras captured movement from markers on the hoof and bone fixed markers on the second and first phalanxes and McIII, whilst radial hoof strain was measured using a calibrated instrumented plug. Change in strain, joint angle and load were found at simulated walking postures and bivariate correlations were used to compare the relationships between them. Radial strain was moderately correlated with proximal interphalangeal joint (PIPJ) rotation (r = -0.519). Large reductions in radial strain were found in loading and midstance with 10 degrees of heel lift postures. PIPJ rotation has previously been linked to the magnitude of deep digital flexor tendon (DDFT) loads and it is therefore suspected that these loads may have the greatest influence on radial strain magnitudes. Further investigation of radial strain is needed to describe the patterns fully during the stance phase in vivo.


The alternative sigma factor, sigmaE, is critically important for the virulence of Salmonella typhimurium.
In Escherichia coli, extracytoplasmic stress is partially controlled by the alternative sigma factor, RpoE (sigmaE). In response to environmental stress or alteration in the protein content of the cell envelope, sigmaE upregulates the expression of a number of genes, including htrA. It has been shown that htrA is required for intramacrophage survival and virulence in Salmonella typhimurium. To investigate whether sigmaE-regulated genes other than htrA are involved in salmonella virulence, we inactivated the rpoE gene of S. typhimurium SL1344 by allelic exchange and compared the phenotype of the mutant (GVB311) in vitro and in vivo with its parent and an isogenic htrA mutant (BRD915). Unlike E. coli, sigmaE is not required for the growth and survival of S. typhimurium at high temperatures. However, GVB311 did display a defect in its ability to utilize carbon sources other than glucose. GVB311 was more sensitive to hydrogen peroxide, superoxide, and antimicrobial peptides than SL1344 and BRD915. Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant was defective in its ability to survive and proliferate in both cell lines. The effect of the rpoE mutation on the intracellular behavior of S. typhimurium was greater than that of the htrA mutation. Both GVB311 and BRD915 were highly attenuated in mice. Neither strain was able to kill mice via the oral route, and the 50% lethal dose (LD50) for both strains via the intravenous (i.v.) route was very high. The i.v. LD50s for SL1344, BRD915, and GVB311 were <10, 5.5 x 10(5), and 1.24 x 10(7) CFU, respectively. Growth in murine tissues after oral and i.v. inoculation was impaired for both the htrA and rpoE mutant, with the latter mutant being more severely affected. Neither mutant was able to translocate successfully from the Peyer's patches to other organs after oral infection or to proliferate in the liver and spleen after i.v. inoculation. However, the htrA mutant efficiently colonized the livers and spleens of mice infected i.v., but the rpoE mutant did not. Previous studies have shown that salmonella htrA mutants are excellent live vaccines. In contrast, oral immunization of mice with GVB311 was unable to protect any of the mice from oral challenge with SL1344. Furthermore, i.v. immunization with a large dose ( approximately 10(6) CFU) of GVB311 protected less than half of the orally challenged mice. Thus, our results indicate that genes in the sigmaE regulon other than htrA play a critical role in the virulence and immunogenicity of S. typhimurium.


Effects of suboptimal adherence of CPAP therapy on symptoms of obstructive sleep apnoea: a randomised, double-blind, controlled trial.
Continuous positive airway pressure (CPAP) is currently the treatment of choice for sleepiness in patients with obstructive sleep apnoea (OSA); however, adherence is often thought to be suboptimal. We investigated the effects of suboptimal CPAP usage on objective and subjective sleepiness parameters in patients with OSA. In this 2-week, parallel, double-blind, randomised controlled trial we enrolled moderate-to-severe OSA patients with excessive pre-treatment daytime sleepiness (Epworth sleepiness scale (ESS) score >10 points) who had suboptimal CPAP adherence over ≥12 months (mean nightly usage time 3-4 h). Patients were allocated through minimisation to either subtherapeutic CPAP ("sham CPAP") or continuation of CPAP ("therapeutic CPAP"). A Bayesian analysis with historical priors calculated the posterior probability of superiority. Between May, 2016 and November, 2018, 57 patients (aged 60±8 years, 79% male, 93% Caucasian) were allocated in total, and 52 who completed the study (50% in each arm) were included in the final analysis. The unadjusted ESS score increase was 2.4 points (95% CI 0.6-4.2, p=0.01) in the sham-CPAP group when compared to continuing therapeutic CPAP. The probability of superiority of therapeutic CPAP over sham CPAP was 90.4% for ESS, 90.1% for systolic blood pressure and 80.3% for diastolic blood pressure. Patients with moderate-to-severe OSA and daytime sleepiness are still getting a substantial benefit from suboptimal CPAP adherence, albeit not as much as they might get if they adhered more. Whether a similar statement can be made for even lower adherence levels remains to be established in future trials.


[Glutathione metabolism during postnatal development of the rat lung].
Significant changes in lung antioxidants occur in preparation for birth. Little is known about physiological regulation of antioxidants in the postnatal period. To study the glutathione system in the lungs during postnatal development. In the lungs of 7, 15, 21, 50 and 70 days old Sprague-Dawley rats we measured total and oxidized glutathione content as well as the activity of the limiting enzyme in glutathione synthesis (y-GCS) and of the enzymes glutathione peroxidase (GPx) and glutathione reducíase (GRd). Between 7 and 15 days the activities of GPx and GRd increase 32% and 26%, respectively (p <0.001). Whereas GPx activity remains high throughout the rest of the study period, GRd activity decreases progressively reaching adulthood values at 7 days. y-GCS activity shows a gradual increase that reaches significance at 50 days when it doubles values observed at 7 days (p <0.05). A significant correlation was found between GPx and GRd activities over the entire period (r =0.62, p <0.0001). Strength of the correlation is age dependent due to the differences in time course of the enzyme changes. Whereas total GSH does not change, oxidized glutathione decreases from 7% at 7 and 15 days to 4% later on (p <0.01). The activity of several enzymes involved in glutathione metabolism increases during postnatal development of the rat lung. Interpretation of lung responses to injurious agents needs to be done taking into consideration the physiological regulation of antioxidants during postnatal development.


Acetate Promotes T Cell Effector Function during Glucose Restriction.
Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer.


Unplanned Hospitalization Among Individuals With Cancer in the Year After Diagnosis.
Reducing acute care use is an important strategy for improving value in cancer care. However, little information is available to describe and compare population-level hospital use across cancer types. Our aim was to estimate unplanned hospitalization rates and to describe the reasons for hospitalization in a population-based cohort recently diagnosed with cancer. California Cancer Registry data linked with administrative inpatient data were used to examine unplanned hospitalization among individuals diagnosed with cancer between 2009 and 2012 (n = 412,850). Hospitalizations for maintenance chemotherapy, radiotherapy, or planned surgery were excluded. Multistate models were used to estimate age-adjusted unplanned hospitalization rates, accounting for survival. Approximately 67% of hospitalizations in the year after diagnosis were unplanned, 35% of newly diagnosed individuals experienced an unplanned hospitalization, and 67% of unplanned hospitalizations originated in the emergency department (ED). Nonmalignancy principal diagnoses most frequently associated with unplanned hospitalization included infection (15.8%) and complications of a medical device or care (6.5%). Unplanned hospitalization rates were highest for individuals with hepatobiliary or pancreatic cancer (2.08 unplanned hospitalizations per person-year at risk), lung cancer (1.58 unplanned hospitalizations), and brain or CNS cancer (1.47 unplanned hospitalizations), and were lowest among individuals with prostate cancer (0.18 unplanned hospitalizations) and melanoma (0.25 unplanned hospitalizations). The population burden of unplanned hospitalization among individuals newly diagnosed with cancer is substantial. Many unplanned hospitalizations originate in the ED and are associated with potentially preventable admission diagnoses. Efforts to reduce unplanned hospitalization might target subgroups at higher risk and focus on the ED as a source of admission.


Sleep impairment, mood symptoms, and psychosocial functioning in adolescent bipolar disorder.
Few empirical studies have investigated the role of sleep impairment in the course of adolescent bipolar spectrum disorders (BSD). The present study examined the longitudinal associations between sleep disruption, mood symptom severity, and psychosocial functioning in a 2-year follow-up of patients with adolescent BSD. Fifty-three adolescents with BSD (mean [S.D.] age: 14.6 [1.6]) participated in a two-site randomized trial of family focused treatment for adolescents (FFT-A) or enhanced care, a briefer psychoeducational treatment; both treatments were administered with pharmacotherapy. Sleep disturbance was assessed with the Adolescent Sleep Habits Questionnaire (ASHQ) filled out by patients every 6 weeks in the first study year and every 3 months in the second year. Main outcomes included clinician-rated measures of mania, depression and psychosocial impairment over 2 years. Sleep impairment was significantly associated with mania and depression severity scores and psychosocial impairment ratings across the 2-year follow-up. Despite its efficaciousness in reducing mood symptoms, FFT-A was not more effective than enhanced care in improving sleep habits. Sleep impairment may play a substantial role in the course of adolescent BSD. Youth with BSDs may benefit from targeted psychosocial interventions that emphasize sleep regularity.


Proresolving Lipid Mediators and Receptors in Stem Cell Biology: Concise Review.
Accumulating evidence indicates that stem cells (SCs) possess immunomodulatory, anti-inflammatory, and prohealing properties. The mechanisms underlying these functions are being investigated with the final goal to set a solid background for the clinical use of SCs and/or their derivatives. Specialized proresolving lipid mediators (SPMs) are small lipids formed by the enzymatic metabolism of polyunsaturated fatty acids. They represent a leading class of molecules that actively and timely regulate the resolution of inflammation and promote tissue/organ repair. SC formation of these mediators as well as expression of their receptors has been recently reported, suggesting that SPMs may be involved in the immunomodulatory, proresolving functions of SCs. In the present review, we summarize the current knowledge on SPMs in SCs, focusing on biosynthetic pathways, receptors, and bioactions, with the intent to provide an integrated view of SPM impact on SC biology. Stem Cells Translational Medicine 2019;8:992-998.


Taste disorders in patients with end-stage chronic kidney disease.
The authors aimed to explore taste distortion in patients with chronic kidney disease (CKD). One hundred and four patients were divided into a control group and a study group. The data was collected through a questionnaire and was statistically analyzed. The results showed that 28.7% of respondents had a loss of taste (96.60% CKD patients). There was a statistically significant correlation between the duration of treatment and taste loss, between patients' age and taste impairment, and between patients' age and the sense of a metallic taste in the mouth. Distortion in the sense of taste is an oral manifestation characteristic of CKD patients.


Isoforms of vitamin E differentially regulate inflammation.
Vitamin E regulation of disease has been extensively studied in humans, animal models and cell systems. Most of these studies focus on the α-tocopherol isoform of vitamin E. These reports indicate contradictory outcomes for anti-inflammatory functions of the α-tocopherol isoform of vitamin E, especially with regards to clinical studies of asthma and atherosclerosis. These seemingly disparate clinical results are consistent with recently reported unrecognized properties of isoforms of vitamin E. Recently, it has been reported that physiological levels of purified natural forms of vitamin E have opposing regulatory functions during inflammation. These opposing regulatory functions by physiological levels of vitamin E isoforms impact interpretations of previous studies on vitamin E. Moreover, additional recent studies also indicate that the effects of vitamin E isoforms on inflammation are only partially reversible using physiological levels of a vitamin E isoform with opposing immunoregulatory function. Thus, this further influences interpretations of previous studies with vitamin E in which there was inflammation and substantial vitamin E isoforms present before the initiation of the study. In summary, this review will discuss regulation of inflammation by vitamin E, including alternative interpretations of previous studies in the literature with regards to vitamin E isoforms.


Low Spontaneous Breathing Effort during Extracorporeal Membrane Oxygenation in a Porcine Model of Severe Acute Respiratory Distress Syndrome.
A lung rest strategy is recommended during extracorporeal membrane oxygenation in severe acute respiratory distress syndrome (ARDS). However, spontaneous breathing modes are frequently used in this context. The impact of this approach may depend on the intensity of breathing efforts. The authors aimed to determine whether a low spontaneous breathing effort strategy increases lung injury, compared to a controlled near-apneic ventilation, in a porcine severe ARDS model assisted by extracorporeal membrane oxygenation. Twelve female pigs were subjected to lung injury by repeated lavages, followed by 2-h injurious ventilation. Thereafter, animals were connected to venovenous extracorporeal membrane oxygenation and during the first 3 h, ventilated with near-apneic ventilation (positive end-expiratory pressure, 10 cm H2O; driving pressure, 10 cm H2O; respiratory rate, 5/min). Then, animals were allocated into (1) near-apneic ventilation, which continued with the previous ventilatory settings; and (2) spontaneous breathing: neuromuscular blockers were stopped, sweep gas flow was decreased until regaining spontaneous efforts, and ventilation was switched to pressure support mode (pressure support, 10 cm H2O; positive end-expiratory pressure, 10 cm H2O). In both groups, sweep gas flow was adjusted to keep Paco2 between 30 and 50 mmHg. Respiratory and hemodynamic as well as electric impedance tomography data were collected. After 24 h, animals were euthanized and lungs extracted for histologic tissue analysis. Compared to near-apneic group, the spontaneous breathing group exhibited a higher respiratory rate (52 ± 17 vs. 5 ± 0 breaths/min; mean difference, 47; 95% CI, 34 to 59; P < 0.001), but similar tidal volume (2.3 ± 0.8 vs. 2.8 ± 0.4 ml/kg; mean difference, 0.6; 95% CI, -0.4 to 1.4; P = 0.983). Extracorporeal membrane oxygenation settings and gas exchange were similar between groups. Dorsal ventilation was higher in the spontaneous breathing group. No differences were observed regarding histologic lung injury. In an animal model of severe ARDS supported with extracorporeal membrane oxygenation, spontaneous breathing characterized by low-intensity efforts, high respiratory rates, and very low tidal volumes did not result in increased lung injury compared to controlled near-apneic ventilation.


Lipoteichoic Acid Is Involved in the Ability of the Immunobiotic Strain Lactobacillus plantarum CRL1506 to Modulate the Intestinal Antiviral Innate Immunity Triggered by TLR3 Activation.
Studies have demonstrated that lipoteichoic acid (LTA) is involved in the immunomodulatory properties of some immunobiotic lactobacilli. The aim of this work was to evaluate whether LTA contributes to the capacity of Lactobacillus plantarum CRL1506 in modulating the intestinal innate antiviral immune response. A D-alanyl-lipoteichoic acid biosynthesis protein (dltD) knockout CRL1506 strain (L. plantarumΔdltD) was obtained, and its ability to modulate Toll-like receptor (TLR)-3-mediated immune response was evaluated in vitro in porcine intestinal epithelial (PIE) cells and in vivo in Balb/c mice. Wild-type (WT) CRL1506 (L. plantarum WT) was used as positive control. The challenge of PIE cells with the TLR3 agonist poly(I:C) significantly increased interferon (IFN)-β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 expressions. PIE cells pretreated with L. plantarumΔdltD or L. plantarum WT showed higher levels of IFN-β while only L. plantarum WT significantly reduced the expression of IL-6 and MCP-1 when compared with poly(I:C)-treated control cells. The oral administration of L. plantarum WT to mice prior the intraperitoneal injection of poly(I:C) significantly increased IFN-β and IL-10 and reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6, and IL-15) in the intestinal mucosa. Similar to the WT strain, L. plantarumΔdltD-treated mice showed enhanced levels of IFN-β after poly(I:C) challenge. However, treatment of mice with L. plantarumΔdltD was not able to increase IL-10 or reduce CD3+NK1.1+CD8αα+ cells, TNF-α, IL-6, or IL-15 in the intestine. These results indicate that LTA would be a key molecule in the anti-inflammatory effect induced by the CRL1506 strain in the context of TLR3-mediated inflammation.


A mixed-methods approach to understanding barriers to postpartum retention in care among low-income, HIV-infected women.
Optimal retention in HIV care postpartum is necessary to benefit the health and wellbeing of mothers and their infants. However, postpartum retention in HIV care among low-income women is suboptimal, particularly in the Southern United States. A mixed-methods study was conducted to identify factors associated with postpartum retention in care among HIV-infected women. Participants (n=35) were recruited during pregnancy at two county clinics and completed self-report demographic and psychosocial surveys. Twenty-two women who returned for a postpartum appointment completed a semi-structured interview about lifestyle factors and retention in care. Of the participants enrolled at baseline, 71.4% completed a follow-up with an obstetrician (OB), while 57.1% completed a follow-up with a primary care physician (PCP). High CD4 count at delivery, low viral load at baseline, low levels of depression, high interpersonal social support, and fewer other children were significantly associated with completion of postpartum follow-up. Barriers and facilitators to retention identified during qualitative interviews included competing responsibilities for time, lack of social support outside of immediate family members, limited transportation access, experiences of institutionalized stigma, knowledge about the benefits of adherence, and strong relationships with healthcare providers. OB and PCP follow-up postpartum was suboptimal in this sample. Findings underscore the importance of addressing depressive symptoms, social support, viral suppression, competing responsibilities for time, institutionalized stigma, and transportation issues in order to reduce the barriers that inhibit women from seeking postpartum HIV care.


Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation.
Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.


Diagramming Evolution: The Case of Darwin's Trees.
From his earliest student days through the writing of his last book, Charles Darwin drew diagrams. In developing his evolutionary ideas, his preferred form of diagram was the tree. An examination of several of Darwin's trees-from sketches in a private notebook from the late 1830s through the diagram published in the Origin-opens a window onto the role of diagramming in Darwin's scientific practice. In his diagrams, Darwin simultaneously represented both observable patterns in nature and conjectural narratives of evolutionary history. He then brought these natural patterns and narratives into dialogue, allowing him to explore whether the narratives could explain the patterns. But Darwin's diagrams did not reveal their meaning directly to passive readers; they required readers to engage dynamically with them in order to understand the connections they disclosed between patterns and narratives. Moreover, the narratives Darwin depicted in his diagrams did not represent past sequences of events that he claimed had actually occurred; the narratives were conjectural, schematic, and probabilistic. Instead of depicting actual histories in all their particularity, Darwin depicted narratives in his diagrams in order to make general claims about how nature works. The conjunction of these features of Darwin's diagrams is central to how they do their epistemic work.


SGCE missense mutations that cause myoclonus-dystonia syndrome impair epsilon-sarcoglycan trafficking to the plasma membrane: modulation by ubiquitination and torsinA.
Myoclonus-dystonia syndrome (MDS) is a genetically heterogeneous disorder characterized by myoclonic jerks often seen in combination with dystonia and psychiatric co-morbidities and epilepsy. Mutations in the gene encoding epsilon-sarcoglycan (SGCE) have been found in some patients with MDS. SGCE is a maternally imprinted gene with the disease being inherited in an autosomal dominant pattern with reduced penetrance upon maternal transmission. In the central nervous system, epsilon-sarcoglycan is widely expressed in neurons of the cerebral cortex, basal ganglia, hippocampus, cerebellum and the olfactory bulb. epsilon-Sarcoglycan is located at the plasma membrane in neurons, muscle and transfected cells. To determine the effect of MDS-associated mutations on the function of epsilon-sarcoglycan we examined the biosynthesis and trafficking of wild-type and mutant proteins in cultured cells. In contrast to the wild-type protein, disease-associated epsilon-sarcoglycan missense mutations (H36P, H36R and L172R) produce proteins that are undetectable at the cell surface and are retained intracellularly. These mutant proteins become polyubiquitinated and are rapidly degraded by the proteasome. Furthermore, torsinA, that is mutated in DYT1 dystonia, a rare type of primary dystonia, binds to and promotes the degradation of epsilon-sarcoglycan mutants when both proteins are co-expressed. These data demonstrate that some MDS-associated mutations in SGCE impair trafficking of the mutant protein to the plasma membrane and suggest a role for torsinA and the ubiquitin proteasome system in the recognition and processing of misfolded epsilon-sarcoglycan.