Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage.
M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.


Soy and its isoflavones: the truth behind the science in breast cancer.
Epidemiological and migratory evidence suggests that dietary soy consumption can lower the risk for breast cancer. The role of soy isoflavones in cancer prevention and promotion is somewhat unclear. There are two views in terms of soy isoflavones and breast cancer. One line of evidence suggests that soy and its isoflavones have exhibited cancer-preventive properties including lengthening the menstrual cycle, altering estrogen metabolism away from cancerous compounds, and demonstrating anti-proliferative properties in vivo. On the contrary, isoflavones found in soy products are suggested to behave as weak estrogens and as such, much speculation surrounds the influence of soy and/or its isoflavones on hormone-receptor-positive cancers. The objective of this review is to present the latest knowledge regarding the role of soy and its isoflavones with the development and advancement of breast cancer, the safety of soy isoflavones for breast cancer survivors, and a comparison of the carcinogenic effects in animal models following soy isoflavone and estrogen administration. This review compares and contrasts literature in terms of the anti-cancer and cancer-promoting effects of soy isoflavones and estrogen in humans and animal models. In conclusion, current human and animal data provide evidence for several anticancer properties of soy and/or its isoflavones. Although the specific quantities and constituents responsible for the observed anti-cancer effects have not been elucidated, it appears that soy isoflavones do not function as an estrogen, but rather exhibit anti-estrogenic properties. However, their metabolism differs between humans and animals and therefore the outcomes of animal studies may not be applicable to humans. The majority of breast cancer cases are hormone-receptor-positive; therefore, soy isoflavones should be considered a potential anti-cancer therapeutic agent and warrant further investigation.


Host genetic polymorphisms and serological response against malaria in a selected population in Sri Lanka.
Antibodies against the merozoite surface protein 1-19 (MSP1-19) and the apical membrane antigen 1 (AMA1) of the malaria parasite (Plasmodium vivax) are proven to be important in protection against clinical disease. Differences in the production/maintenance of antibodies may be due to many factors including host genetics. This paper discusses the association of 4 anti-malarial antibodies with selected host genetic markers. Blood was collected from individuals (n = 242) with a history of malaria within past 15 years for DNA and serum. ELISA was carried out for serum to determine the concentration of anti-malarial antibodies MSP1-19 and AMA1 for both vivax and falciparum malaria. 170 SNPs related to malaria were genotyped. Associations between seropositivity, antibody levels and genetic, non-genetic factors were determined. Age ranged 13-74 years (mean age = 40.21 years). Majority were females. Over 90% individuals possessed either one or more type(s) of anti-malarial antibodies. Five SNPs were significantly associated with seropositivity. One SNP was associated with MSP1-19_Pv(rs739718); 4 SNPs with MSP1-19_Pf (rs6874639, rs2706379, rs2706381 and rs2075820) and1 with AMA1_Pv (rs2075820). Eleven and 7 genotypes (out of 15) were significantly associated with either presence or absence of antibodies. Three SNPs were found to be significantly associated with the antibody levels viz. rs17411697 with MSP1-19_Pv, rs2227491 with AMA1_Pv and rs229587 with AMA1_Pf. Linkage of the markers in the two groups was similar, but lower LOD scores were observed in seropositives compared to seronegatives. The study suggests that several SNPs in the human genome that exist in Sri Lankan populations are significantly associated with anti-malarial antibodies, either with generation and/or maintenance of antibodies for longer periods, which can be due to either individual polymorphisms or most probably a combined effect of the markers.


Therapeutic electrical stimulation to improve motor control and functional abilities of the upper extremity after stroke: a systematic review.
Therapeutic electrical stimulation (TES) is a therapeutic strategy aimed at improving impairments of the upper extremity in stroke. Assessment of the available evidence on the effect of TES of the affected upper extremity in improving motor control and functional abilities after stroke. A systematic literature search was performed to identify randomized controlled trials (RCTs) that have studied the effect of TES on motor control and functional abilities. The methodological quality of the studies was assessed systematically by two raters. The reported outcomes were examined to evaluate the effect of TES and to identify a possible relationship with patient characteristics, method of stimulation and methodological quality. When possible, effect sizes were calculated (Hedges' g). Six RCTs were included. The methodological scores ranged from 7 to 16 (maximum 19). All studies assessed the effect on motor control, and four reported a positive effect. Effect sizes calculated in three studies ranged from 0.55 to 1.46. Only two studies assessed the effect on functional ability, one reported a positive effect. Subgroup analyses in two studies suggest a better response to stimulation in less severely affected patients. Apart from this, no relationship between effect and patient characteristics, method of stimulation or methodological quality could be detected. The present review suggests a positive effect of electrical stimulation on motor control. No conclusions can be drawn with regard to the effect on functional abilities.


Management of respiration in MND/ALS patients: an evidence based review.
This systematic review comprises an objective appraisal of the evidence in regard to the management of respiration in patients with motor neuron disease (MND/ALS). Studies were identified through computerised searches of 32 databases. Internet searches of websites of drug companies and MND/ALS research web sites, 'snow balling' and hand searches were also employed to locate any unpublished study or other 'grey literature' on respiration and MND/ALS. Since management of MND/ALS involves a number of health professionals and care workers, searches were made across multiple disciplines. No time frame was imposed on the search in order to increase the probability of identifying all relevant studies, although there was a final limit of March 2005. Recommendations for patient and carer-based guidelines for the clinical management of respiration for MND/ALS patients are suggested on the basis of qualitative analyses of the available evidence. However, these recommendations are based on current evidence of best practice, which largely comprises observational research and clinical opinion. There is a clear need for further evidence, in particular randomised and non-randomised controlled trials on the effects of non-invasive ventilation and additional larger scale cohort studies on the issues of initial assessment of respiratory symptoms, and management and timing of interventions.


High-speed and widefield handheld swept-source OCT angiography with a VCSEL light source.
Optical coherence tomography (OCT) and OCT angiography (OCTA) enable noninvasive structural and angiographic imaging of the eye. Portable handheld OCT/OCTA systems are required for imaging patients in the supine position. Examples include infants in the neonatal intensive care unit (NICU) and operating room (OR). The speed of image acquisition plays a pivotal role in acquiring high-quality OCT/OCTA images, particularly with the handheld system, since both the operator hand tremor and subject motion can cause significant motion artifacts. In addition, having a large field of view and the ability of real-time data visualization are critical elements in rapid disease screening, reducing imaging time, and detecting peripheral retinal pathologies. The arrangement of optical components is less flexible in the handheld system due to the limitation of size and weight. In this paper, we introduce a 400-kHz, 55-degree field of view handheld OCT/OCTA system that has overcome many technical challenges as a portable OCT system as well as a high-speed OCTA system. We demonstrate imaging premature infants with retinopathy of prematurity (ROP) in the NICU, a patient with incontinentia pigmenti (IP), and a patient with X-linked retinoschisis (XLRS) in the OR using our handheld OCT system. Our design may have the potential for improving the diagnosis of retinal diseases and help provide a practical guideline for designing a flexible and portable OCT system.


Refined similarity hypothesis using three-dimensional local averages.
The refined similarity hypotheses of Kolmogorov, regarded as an important ingredient of intermittent turbulence, has been tested in the past using one-dimensional data and plausible surrogates of energy dissipation. We employ data from direct numerical simulations, at the microscale Reynolds number R(λ)∼650, on a periodic box of 4096(3) grid points to test the hypotheses using three-dimensional averages. In particular, we study the small-scale properties of the stochastic variable V=Δu(r)/(rε(r))(1/3), where Δu(r) is the longitudinal velocity increment and ε(r) is the dissipation rate averaged over a three-dimensional volume of linear size r. We show that V is universal in the inertial subrange. In the dissipation range, the statistics of V are shown to depend solely on a local Reynolds number.


In patients stratified by preoperative risk, endovascular repair of ruptured abdominal aortic aneurysms has a lower in-hospital mortality and morbidity than open repair.
Previous studies have reported that endovascular repair (EVAR) of ruptured abdominal aortic aneurysms (RAAAs) has lower postoperative mortality than open repair (OR). However, comparisons involved heterogeneous populations that lacked adjustment for preoperative risk. We hypothesize that for RAAA patients stratified by a validated measure of preoperative mortality risk, EVAR has a lower in-hospital mortality and morbidity than does OR. In-hospital mortality and morbidity after EVAR and OR of RAAA were compared in patients from the Vascular Quality Initiative (2003-2013) stratified by the validated Vascular Study Group of New England RAAA risk score into low-risk (score 0-1), medium-risk (score 2-3), and high-risk (score 4-6) groups. Among 514 patients who underwent EVAR and 651 patients who underwent OR of RAAA, EVAR had lower in-hospital mortality (25% vs 33%, P = .001). In risk-stratified patients, EVAR trended toward a lower mortality in the low-risk group (n = 626; EVAR, 10% vs OR, 15%; P = .07), had a significantly lower mortality in the medium-risk group (n = 457; EVAR, 37% vs OR, 48%; P = .02), and no advantage in the high-risk group (n = 82; EVAR, 95% vs OR, 79%; P = .17). Across all risk groups, cardiac complications (EVAR, 29% vs OR, 38%; P = .001), respiratory complications (EVAR, 28% vs OR, 46%; P < .0001), renal insufficiency (EVAR, 24% vs OR, 38%; P < .0001), lower extremity ischemia (EVAR, 2.7% vs OR, 8.1%; P < .0001), and bowel ischemia (EVAR, 3.9% vs OR, 10%; P < .0001) were significantly lower after EVAR than after OR. Across all risk groups, median (interquartile range) intensive care unit length of stay (EVAR, 2 [1-5] days vs OR, 6 [3-13] days; P < .0001) and hospital length of stay (EVAR, 6 [4-12] days vs OR, 13 [8-22] days; P < .0001) were lower after EVAR. This novel risk-stratified comparison using a national clinical database showed that EVAR of RAAA has a lower mortality and morbidity compared with OR in low-risk and medium-risk patients and that EVAR should be used to treat these patients when anatomically feasible. For RAAA patients at the highest preoperative risk, there is no benefit to using EVAR compared with OR.


On-demand curing of polydimethylsiloxane (PDMS) using the photothermal effect of gold nanoparticles.
The photothermal effect of gold nanoparticles (AuNPs) produces extremely localized heat that can be harnessed to drive large scale chemical reactions by simultaneously generating many individual reactions on the nanoscale. We use the photothermal effect to enhance the curing rate of polydimethylsiloxane (PDMS) by a factor of 4.9 × 109. Photothermal curing occurs via crosslinking reactions between vinyl and Si-H groups of the pre-polymer, and the course of the reaction was followed by monitoring the disappearance of infrared bands associated with these functional groups. Using mass spectroscopy, we verify that the major polymer m/z peaks are identical for both traditionally and photothermally cured polymers, indicating that the photothermal effect of AuNPs is an effective way in which to supply on-demand curing of PDMS.


Modulation of ANP-dependent effects of endothelin by inhibitors of neutral endopeptidase and cGMP phosphodiesterase.
Endothelin 3 (ET3) infusion increases the concentration of atrial natriuretic peptide (ANP) in plasma, which in turn raises hematocrit (Hct) through a transcapillary shift of plasma fluid and proteins into the interstitium, thereby reducing plasma volume (PV). The level of ANP bioactivity in peripheral target tissues is a function of ANP secretory rate and the turnover rate of ANP and its intracellular effector mechanisms. Neutral endopeptidase (NEP) is a widely distributed enzyme that participates in ANP catabolism, whereas cGMP-phosphodiesterase (PDE) degrades the intracellular second messenger of ANP. Therefore, we examined the consequences of inhibition of NEP and PDE on the ANP-dependent activity described above using the NEP inhibitor SQ 28,603 and the cGMP-PDE inhibitor zaprinast (M&B 22,948). In anesthetized, bilaterally nephrectomized rats, infusion of SQ 28,603 alone reduced mean arterial pressure (MAP) by 2.5 +/- 0.5% and increased Hct by 4.6 +/- 0.3% (P < .01 for both), leading to a calculated decrease in PV of 7.5 +/- 0.6%. These changes were prevented by pretreatment with rabbit anti-rat ANP antiserum. Simultaneous infusion of ET3 (25 ng/kg/min) and SQ 28,603 caused MAP to increase by 12.8 +/- 2.2%, an effect identical with that observed after ET3 alone (12.7 +/- 2.3%), whereas the increase in Hct of 9.4 +/- 0.4% was greater (P < .05) than the 7.5 +/- 0.4% increase seen with ET3 alone. ET3 increased plasma ANP concentration (599 +/- 135 vs. 108 +/- 13 pg/ml in vehicle-infused rats; P < .0001); ET3 and SQ 28,603 infused simultaneously increased plasma ANP even further (810 +/- 166 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of progressive exercise performance of normal subjects and patients with primary pulmonary hypertension.
The extent of exercise limitation and the mechanisms for that limitation in 11 patients with primary pulmonary hypertension (PPH) were studied by progressive, upright cycle ergometry. All patients had a mean pulmonary artery pressure of 30 mm Hg or higher (mean, 56 +/- 15), normal pulmonary function testing, normal pulmonary capillary wedge pressure, and pulmonary angiography consistent with the diagnosis. Rest and exercise data obtained from the patients with PPh were compared with data obtained from 11 matched, sedentary control subjects. Mean maximal oxygen consumption (VO2) was 13 +/- 4 ml/kg/min in the PPH group compared with 28 +/- 7 ml/kg/min in the controls. At maximal VO2 the minute ventilation (VE) was similar; however, the VE at any level of carbon dioxide production (VCO2) during rest and exercise was significantly higher in the PPH group. Maximal heart rate and oxygen pulse (VO2/heart rate) was significantly higher in the control group (148 +/- 18 vs 180 +/- 24, and 6.3 +/- 2.2 vs 9.9 +/- 3.9, respectively). Anaerobic threshold occurred earlier during progressive exercise in the PPH group and correlated positively with the maximal oxygen pulse achieved in patients with PPH. In conclusion, patients with PPH have severe exertional limitation due to cardiovascular factors with an inability to maintain appropriate oxygen delivery to the body during exercise. No respiratory impairment was recognized; however, an exaggerated ventilatory response to exercise at any level of VCO2 was found.


Repair of Adult Mammalian Heart After Damages by Oral Intake of Gu Ben Pei Yuan San.
Adult mammalian heart repair after myocardial damage is highly inefficient due to the post-mitotic nature of cardiomyocytes. Interestingly, in traditional Chinese medicine (TCM), there are reported effective treatments of myocardial infarction (MI) and heart failure in adult humans by oral intake of a TCM concoction named Gu Ben Pei Yuan San (GBPYS), which is composed of Panax ginseng, velvet antler, Gekko gecko Linnaeus tail, human placenta, Trogopterus dung, Panax notoginseng, and amber. We fed mice with GBPYS after myocardial damages through everyday self-feeding. We then examined the effect of everyday oral intake of GBPYS on improving cardiac function and myocardial repair in adult mice after apical resection or MI. We found that long-term oral intake of GBPYS significantly improved cardiac function after myocardial damages in adult mice. BrdU, phospho-histone 3, and AuroraB staining indicated increased cell proliferation at the border zone of MI after TCM feeding. GBPYS feeding reduced organ inflammation, induced angiogenesis, and is non-toxic to mice after long-term oral intake. Further, serum derived from TCM-fed MI rats promoted division of both neonatal rat cardiomyocytes and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes in vitro. Oral intake of GBPYS improved heart repair after myocardial damages in adult mice. Our results suggest that there are substances present in GBPYS that help improve adult mammalian heart repair after MI. Also, it could be a good choice of non-invasive alternative therapy for myocardial damages and heart failure after rigorous clinical study in the future.


Markets, Medicare, and making do: business strategies after national health care reform.
This essay examines the role of business health care purchasers in keeping market solutions at the center of the health system. One might assume that employers would have a clear ideological preference for market solutions, but big business managers are ambivalent about market interventions at both the firm and public policy levels. Although currently enthusiastic about market-oriented managed care, large employers have been periodically disappointed by firm-level market experiments during the past two decades. They viewed with skepticism the Republican proposal to apply private-sector market cures to the public Medicare and Medicaid, fearing that the proposals would accelerate cost-shifting to private business payers. Big business objections have been muted, however, by the organizational weakness so vividly illustrated during the national health reform debate.


Perception and production of mandarin tones in prelingually deaf children with cochlear implants.
Mandarin is a lexical tone language in which four tones are crucial for determining lexical meanings. Acquisition of such a tone system may be challenging to prelingually deaf children with cochlear implants because, as recent studies have shown, cochlear implant devices are ineffective in encoding voice pitch information required for tone recognition. This study aimed to investigate Mandarin tone production and perception skills of children with cochlear implants. Thirty prelingually deaf children with cochlear implants, ages 6;0 (yr;mo) to 12;6, participated. These children received their implants at an average age of 5;8, with a range from 2;3 to 10;3. The average length of their cochlear implant experience was 3;7, with a range from 1;7 to 6;5. Tasks of tone production and tone identification involved a pictorial protocol of 48 words containing the targeted tones in either monosyllabic or disyllabic forms. The average scores for tone production was 53.09% (SD = 15.42), and for tone identification was 72.88% (SD = 19.68; chance level = 50%). Significant differences were found in the percentages across the production or identification of tone types or tone pairs. The children with exceptional performance in tone production tended to also perform well in tone identification. The children's performance levels in tone identification and production were also discussed in relation to the factors of age at implantation and length of cochlear implant experience. The present results suggest that the majority of prelingually deaf children with cochlear implants did not master Mandarin tone production. However, a small group of participants demonstrated nearly perfect skills of Mandarin tone production in addition to tone perception. Thus, it is necessary to consider factors other than the device's limitations to explain these high levels of performance in the perception and production of Mandarin lexical tones.


The architecture of the connective tissue in the musculoskeletal system-an often overlooked functional parameter as to proprioception in the locomotor apparatus.
The architecture of the connective tissue, including structures such as fasciae, sheaths, and membranes, is more important for understanding functional meaning than is more traditional anatomy, whose anatomical dissection method neglects and denies the continuity of the connective tissue as integrating matrix of the body.The connective tissue anatomy and architecture exhibits two functional tendencies that are present in all areas of the body in different ways and relationships. In body cavities, the "disconnecting" quality of shaping space enables mobility; between organs and body parts, the "connecting" dimension enables functional mechanical interactions. In the musculoskeletal system, those two features of the connective tissue are also present. They cannot be found by the usual analytic dissection procedures. An architectural description is necessary.This article uses such a methodologic approach and gives such a description for the lateral elbow region. The result is an alternative architectural view of the anatomic substrate involved in the transmission and conveyance of forces over synovial joints. An architectural description of the muscular and connective tissue organized in series with each other to enable the transmission of forces over these dynamic entities is more appropriate than is the classical concept of "passive" force-guiding structures such as ligaments organized in parallel to actively force-transmitting structures such as muscles with tendons.The discrimination between so-called joint receptors and muscle receptors is an artificial distinction when function is considered. Mechanoreceptors, also the so-called muscle receptors, are arranged in the context of force circumstances-that is, of the architecture of muscle and connective tissue rather than of the classical anatomic structures such as muscle, capsules, and ligaments. In the lateral cubital region of the rat, a spectrum of mechanosensitive substrate occurs at the transitional areas between regular dense connective tissue layers and the muscle fascicles organized in series with them. This substrate exhibits features of type and location of the mechanosensitive nerve terminals that usually are considered characteristic for "joint receptors" as well as for "muscle receptors."The receptors for proprioception are concentrated in those areas where tensile stresses are conveyed over the elbow joint. Structures cannot be divided into either joint receptors or muscle receptors when muscular and collagenous connective tissue structures function in series to maintain joint integrity and stability. In vivo, those connective tissue structures are strained during movements of the skeletal parts, those movements in turn being induced and led by tension in muscular tissue. In principle, because of the architecture, receptors can also be stimulated by changes in muscle tension without skeletal movement, or by skeletal movement without change in muscle tension. A mutual relationship exists between structure (and function) of the mechanoreceptors and the architecture of the muscular and regular dense connective tissue. Both are instrumental in the coding of proprioceptive information to the central nervous system.


Transfer-free synthesis of multilayer graphene using a single-step process in an evaporator and formation confirmation by laser mode-locking.
Multilayer graphene is synthesized by a simplified process employing an evaporator in which a target substrate is deposited with a Ni catalyst layer before being heated to grow graphene directly. Carbon atoms adsorbed onto the surface of the Ni source as impurities from the atmosphere are incorporated into the catalyst layer during the deposition, and diffuse toward the catalyst/substrate interface, where they crystallize as graphene with a thickness of less than 2 nm. The need for a transfer process and external carbon supply is eliminated. The graphene is characterized by conventional analysis approaches, including nano-scale visualization and Raman spectroscopy, and utilizing photonics, graphene-functionalized passive laser mode-locking is demonstrated to confirm the successful synthesis of the graphene layer, resulting in an operating center wavelength of 1569.4 nm, a pulse duration of 1.35 ps, and a repetition rate of 31.6 MHz.


Intraoperative Radiotherapy in the Era of Intensive Neoadjuvant Chemotherapy and Chemoradiotherapy for Pancreatic Adenocarcinoma.
Improved outcomes with FOLFIRINOX or gemcitabine with nab-paclitaxel in the treatment of metastatic pancreatic adenocarcinoma (PDAC) have prompted incorporation of these regimens into neoadjuvant treatment of locally advanced unresectable PDAC. Whereas some patients remain unresectable on surgical exploration, others are able to undergo resection after intensive neoadjuvant treatment. We evaluated outcomes and toxicity associated with use of intensive neoadjuvant treatment followed by intraoperative radiotherapy (IORT) in combination with resection or exploratory laparotomy. We retrospectively analyzed patients with locally advanced unresectable or borderline-resectable PDAC who received intensive neoadjuvant treatment with induction chemotherapy and chemoradiotherapy followed by exploratory laparotomy in an IORT-equipped operating suite between 2010 and 2015. Surgical outcomes and overall survival (OS) were compared. Of 68 patients, 41 (60.3%) underwent resection, 18 (26.5%) had unresectable disease, and 9 (13.2%) had distant metastases. Of 41 resectable patients, 22 received IORT for close/positive resection margins on intraoperative frozen section. There was no significant difference in operative times or morbidity with addition of IORT to resection. Median OS was 26.6 months for all patients who underwent resection, 35.1 months for patients who underwent resection and IORT, and 24.5 months for patients who underwent resection alone (P=NS). Of 18 patients with unresectable disease, all but 1 received IORT, with median OS of 24.8 months. IORT was associated with increased hospital stay (4 vs. 3.5 d), but no significant difference in operative times or morbidity. IORT in addition to intensive neoadjuvant chemotherapy and chemoradiotherapy was not associated with increased toxicity when used with resection or exploratory laparotomy, and was associated with encouraging survival rates in patients with close/positive margins and patients with unresectable disease.


Proliferation of guinea pig tracheal epithelial cells in coculture with rat dorsal root ganglion neural cells.
Neuropeptides secreted by sensory afferent nerves in airways may modulate growth of airway epithelial cells. To determine whether airway sensory C-fiber nerves secrete neuropeptides that stimulate airway epithelial cell proliferation, we measured S-phase traversal in guinea pig tracheal epithelial (GPTE) cells after coculture with rat dorsal root ganglion (DRG) cells. GPTE cells were grown in subconfluent culture on collagen-coated filters for 2 days. DRG cells were harvested from newborn rat pups and grown in primary culture for 7-10 days in separate wells. GPTE and DRG cells then were cocultured for 48 h, and 10 mM bromodeoxyuridine (BrdU), a thymidine analogue, was added in the final 24 h. Control GPTE cells were grown under similar conditions but without DRG cells. Coculture with DRG cells stimulated GPTE cell traversal of S phase. BrdU labeling in cocultured GPTE cells was 42.8 +/- 5.8 compared with 18.1 +/- 7.2% in control GPTE cells (P < 0.001, n = 6). Coculture in the presence of either the neurokinin (NK)1 receptor antagonists LY-297911 or CP-99,994, the NK2 receptor antagonist SR-48,968, or the calcitonin gene-related peptide (CGRP) receptor antagonist hCGRP-(8-37) (10(-7) M of each) during coculture attenuated proliferation of GPTE cells. Treatment with all three antagonists together during coculture decreased BrdU labeling to 2.4 +/- 0.9% of labeled cells vs. 8.5 +/- 0.5% of labeled cells during coculture without antagonists (n = 4, P < 0.02). DRG cells in coculture secreted substantial concentrations of CGRP [71.0 +/- 11.3 (+/- SE) pmol/ml], substance P (1.26 +/- 0.35 pmol/ml), and neurokinin A (0.45 +/- 0.10 pmol/ml) (n = 19 for each).(ABSTRACT TRUNCATED AT 250 WORDS)


Reduction of circulating endothelial cells in peripheral blood of ALS patients.
Amyotrophic Lateral Sclerosis (ALS) treatment is complicated by the various mechanisms underlying motor neuron degeneration. Recent studies showed that the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are compromised in an animal model of ALS due to endothelial cell degeneration. A later study demonstrated a loss of endothelium integrity in the spinal cords of ALS patients. Since circulating endothelial cells (CECs) in the peripheral blood are associated with endothelium damage, being detached dysfunctional endothelial cells, we hypothesized that CEC levels may reflect endothelium condition in ALS patients. CEC levels were estimated in whole blood smears from ALS patients with moderate stage ((M)ALS), severe stage ((S)ALS), and healthy controls by CD146 expression using immunocytochemistry. A significant reduction of CECs was detected in (M)ALS and (S)ALS patients. CECs did not predict endothelium state in ALS patients; however, endothelial damage and/or impaired endothelium repair may occur in ALS leading to BBB/BSCB dysfunction. Reduced CECs in peripheral blood of ALS patients may indicate different mechanisms of endothelial damage and repair, rather than only detachment of dysfunctional endothelial cells. Although a potential mechanism of CEC reduction is discussed, establishing a reliable indicator of endothelial dysfunction/damage is important for evaluation of BBB/BSCB status in ALS patients during disease progression.


Variable dopamine release probability and short-term plasticity between functional domains of the primate striatum.
Release of the neuromodulator dopamine (DA) is critical to the control of locomotion, motivation, and reward. However, the probability of DA release is not well understood. Current understanding of neurotransmitter release probability in the CNS is limited to the conventional synaptic amino acid transmitters (e.g., glutamate and GABA). These fast neurotransmitters are released with a repertoire of probabilities according to synapse type, and these probabilities show activity-dependent plasticity according to synapse use. Synapses for neuromodulators such as DA, however, are designed for signaling that diverges temporally and spatially from that for fast neurotransmitters: DA receptors are exclusively metabotropic and at sites that extend to extrasynaptic locations and neighboring synapses. In this study, the release probability of DA was explored in real time in limbicversus motor-associated functional domains of the striatum of a primate (marmoset; Callithrix jacchus) using fast-scan voltammetry at a carbon-fiber microelectrode. We show that the probability of axonal DA release varies with striatal domain. Furthermore, release probability exhibits a short-term, activity-dependent plasticity that ranges from depression to facilitation in motor-through limbic-associated regions, respectively. Rapid plasticity does not result from metabotropic D2-like DA receptor activation or ionotropic GABA(A) receptor effects but is dependent on Ca2+ availability. These data reveal that rapid dynamics in DA release probability will participate in the transmission of the patterns and frequencies encoded by DA neuron action potential discharge. Furthermore, the regional variation in these features indicates that limbic-versus motor-associated DA neurons are permitted to generate diverse DA signals in response to a given firing pattern.


A baseline metabolomic signature is associated with immunological CD4+ T-cell recovery after 36 months of antiretroviral therapy in HIV-infected patients.
Poor immunological recovery in treated HIV-infected patients is associated with greater morbidity and mortality. To date, predictive biomarkers of this incomplete immune reconstitution have not been established. We aimed to identify a baseline metabolomic signature associated with a poor immunological recovery after antiretroviral therapy (ART) to envisage the underlying mechanistic pathways that influence the treatment response. This was a multicentre, prospective cohort study in ART-naive and a pre-ART low nadir (<200 cells/μl) HIV-infected patients (n = 64). We obtained clinical data and metabolomic profiles for each individual, in which low molecular weight metabolites, lipids and lipoproteins (including particle concentrations and sizes) were measured by NMR spectroscopy. Immunological recovery was defined as reaching CD4 T-cell count at least 250 cells/μl after 36 months of virologically successful ART. We used univariate comparisons, Random Forest test and receiver-operating characteristic curves to identify and evaluate the predictive factors of immunological recovery after treatment. HIV-infected patients with a baseline metabolic pattern characterized by high levels of large high density lipoprotein (HDL) particles, HDL cholesterol and larger sizes of low density lipoprotein particles had a better immunological recovery after treatment. Conversely, patients with high ratios of non-HDL lipoprotein particles did not experience this full recovery. Medium very-low-density lipoprotein particles and glucose increased the classification power of the multivariate model despite not showing any significant differences between the two groups. In HIV-infected patients, a baseline healthier metabolomic profile is related to a better response to ART where the lipoprotein profile, mainly large HDL particles, may play a key role.


Model-Based Inference of Punctuated Molecular Evolution.
In standard models of molecular evolution, DNA sequences evolve through asynchronous substitutions according to Poisson processes with a constant rate (called the molecular clock) or a rate that can vary (relaxed clock). However, DNA sequences can also undergo episodes of fast divergence that will appear as synchronous substitutions affecting several sites simultaneously at the macroevolutionary timescale. Here, we develop a model, which we call the Relaxed Clock with Spikes model, combining basal, clock-like molecular substitutions with episodes of fast divergence called spikes arising at speciation events. Given a multiple sequence alignment and its time-calibrated species phylogeny, our model is able to detect speciation events (including hidden ones) cooccurring with spike events and to estimate the probability and amplitude of these spikes on the phylogeny. We identify the conditions under which spikes can be distinguished from the natural variance of the clock-like component of molecular substitutions and from variations of the clock. We apply the method to genes underlying snake venom proteins and identify several spikes at gene-specific locations in the phylogeny. This work should pave the way for analyses relying on whole genomes to inform on modes of species diversification.


Single-Cell Approach to Influenza-Specific CD8+ T Cell Receptor Repertoires Across Different Age Groups, Tissues, and Following Influenza Virus Infection.
CD8+ T cells recognizing antigenic peptides derived from conserved internal viral proteins confer broad protection against distinct influenza viruses. As memory CD8+ T cells change throughout the human lifetime and across tissue compartments, we investigated how T cell receptor (TCR) composition and diversity relate to memory CD8+ T cells across anatomical sites and immunological phases of human life. We used ex vivo peptide-HLA tetramer magnetic enrichment, single-cell multiplex RT-PCR for both the TCR-alpha (TCRα) and TCR-beta (TCRβ) chains, and new TCRdist and grouping of lymphocyte interactions by paratope hotspots (GLIPH) algorithms to compare TCRs directed against the most prominent human influenza epitope, HLA-A*02:01-M158-66 (A2+M158). We dissected memory TCR repertoires directed toward A2+M158 CD8+ T cells within human tissues and compared them to human peripheral blood of young and elderly adults. Furthermore, we compared these memory CD8+ T cell repertoires to A2+M158 CD8+ TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first ex vivo comparative analysis of paired antigen-specific TCR-α/β clonotypes across different tissues and peripheral blood across different age groups. We show that human A2+M158 CD8+ T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2+M158 TCRαβ repertoires reflect A2+M158 TCRαβ clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2+M158 TCRαβ repertoires displayed distinct features only in elderly adults, with large private TCRαβ clonotypes replacing the prominent and public TRBV19/TRAV27 TCRs. Our study provides novel findings on influenza-specific TCRαβ repertoires within human tissues, raises the question of how we can prevent the loss of optimal TCRαβ signatures with aging, and provides important insights into the rational design of T cell-mediated vaccines and immunotherapies.


Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.
The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C-C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF2· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF2· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC(O)-Trp (C-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong "red-shifting" of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV-visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical "HC(O)·" equivalents enables first examples of C-C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally "edited" into biology.


A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain.
Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments. A "two-hit" hyperalgesic priming strategy was used to induce vulnerability to a normally subthreshold challenge with umbellulone, a transient receptor potential ankyrin 1 (TRPA1) activator, in uninjured female and male C57BL/6 mice. Priming (i.e. the first hit) was induced by three consecutive daily episodes of restraint stress; repeated umbellulone was also evaluated for potential priming effects. Sixteen days after the first restraint stress, mice received inhalational umbellulone (i.e. the second hit) to elicit migraine-like pain. Medications currently used for acute or preventive migraine therapy including propranolol (a beta blocker) and sumatriptan (5HT1B/D agonist), as well as olcegepant, an experimental calcitonin gene related peptide (CGRP) receptor antagonist and nor-Binaltorphimine (nor-BNI), an experimental long-acting kappa opioid receptor (KOR) antagonist, were investigated for their efficacy to block priming and prevent or reverse umbellulone-induced allodynia in primed animals. To assess migraine-like pain, cutaneous allodynia was determined by responses to periorbital or hindpaw probing with von Frey filaments. Repeated restraint stress, but not umbellulone exposure, produced transient cutaneous allodynia that resolved within 16 d. Restraint stress produced long-lasting priming that persisted beyond 16 d, as demonstrated by reinstatement of cutaneous allodynia following inhalational umbellulone challenge. Pretreatment with propranolol or nor-BNI prior to restraint stress prevented both transient cutaneous allodynia and priming, demonstrated by a lack of umbellulone-induced cutaneous allodynia. Following establishment of restraint stress priming, olcegepant, but not propranolol or nor-BNI, prevented umbellulone-induced cutaneous allodynia. When administered 1 h after umbellulone, sumatriptan, but not olcegepant, reversed umbellulone-induced cutaneous allodynia in restraint stress-primed rats. We have developed a novel injury-free model with translational relevance that can be used to study mechanisms relevant to migraine-like pain and to evaluate novel acute or preventive treatments. Restraint stress priming induced a state of vulnerability to a subthreshold stimulus that has been referred to as "latent sensitization". The development of latent sensitization could be prevented by blockade of stress pathways with propranolol or with a kappa opioid receptor antagonist. Following establishment of latent sensitization, subthreshold stimulation with umbellulone reinstated cutaneous allodynia, likely from activation of meningeal TRPA1-expressing nociceptors. Accordingly, in restraint stress-primed animals, sumatriptan reversed umbellulone-induced cutaneous allodynia, supporting peripheral sites of action, while propranolol and nor-BNI were not effective. Surprisingly, olcegepant was effective in mice with latent sensitization when given prior to, but not after, umbellulone challenge, suggesting time-dependent contributions of calcitonin gene-related peptide receptor signaling in promoting migraine-like pain in this model. Activation of the calcitonin gene-related peptide receptor participates in initiating, but has a more limited role in maintaining, pain responses, supporting the efficacy of small molecule calcitonin gene-related peptide antagonists as preventive medications. Additionally, the effectiveness of sumatriptan in reversal of established pain thus suggests modulation of additional, non-calcitonin gene-related peptide receptor-mediated nociceptive mechanisms. Kappa opioid receptor antagonists may represent a novel preventive therapy for stress-related migraine.


Walking for transportation in Hong Kong Chinese urban elders: a cross-sectional study on what destinations matter and when.
Walking for transport can contribute to the accrual of health-enhancing levels of physical activity in elders. Identifying destinations and environmental conditions that facilitate this type of walking has public health significance. However, most findings are limited to Western, low-density locations, while a larger proportion of the global population resides in ultra-dense Asian metropolises. We investigated relationships of within-neighborhood objectively-measured destination categories and environmental attributes with walking for transport in 484 elders from an ultra-dense metropolis (Hong Kong). We estimated relationships of diversity (number of different types) and prevalence of within-neighborhood destination categories (environmental audits of 400 m buffers surrounding residential addresses) with transport-related walking (interviewer-administered questionnaire) in 484 Chinese-speaking elders able to walk unassisted and living in 32 neighborhoods stratified by socio-economic status and transport-related walkability. We examined the moderating effects of safety and pedestrian infrastructure-related neighborhood attributes on destination-walking associations. Participants reported on average 569 and 254 min/week of overall and within-neighborhood walking for transport, respectively. The prevalence of public transit points and diversity of recreational destinations were positively related to overall walking for transport. The presence of a health clinic/service and place of worship, higher diversity in recreational destinations, and greater prevalence of non-food retails and services, food/grocery stores, and restaurants in the neighborhood were predictive of more within-neighborhood walking for transport. Neighborhood safety-related aspects moderated the relationship of overall walking for transport with the prevalence of public transit points, this being positive only in safe locations. Similar moderating effects of safety-related attributes were observed for the relationships of within-neighborhood walking for transport with diversity of recreational and entertainment destinations. Pedestrian-infrastructure attributes acted as moderators of associations of within-neighborhood walking for transport with prevalence of commercial destination categories. Composite destinations indices consisting of destination categories related to the specific measures of walking were positively associated with walking for transport. The availability of both non-commercial and commercial destinations may promote within-neighborhood walking for transport, while recreational facilities and public transit points may facilitate overall walking for transport. However, destination-rich areas need to also provide adequate levels of personal safety and a physically-unchallenging pedestrian network.


mTOR has distinct functions in generating versus sustaining humoral immunity.
Little is known about the role of mTOR signaling in plasma cell differentiation and function. Furthermore, for reasons not understood, mTOR inhibition reverses antibody-associated disease in a murine model of systemic lupus erythematosus. Here, we have demonstrated that induced B lineage-specific deletion of the gene encoding RAPTOR, an essential signaling adaptor for rapamycin-sensitive mTOR complex 1 (mTORC1), abrogated the generation of antibody-secreting plasma cells in mice. Acute treatment with rapamycin recapitulated the effects of RAPTOR deficiency, and both strategies led to the ablation of newly formed plasma cells in the spleen and bone marrow while also obliterating preexisting germinal centers. Surprisingly, although perturbing mTOR activity caused a profound decline in serum antibodies that were specific for exogenous antigen or DNA, frequencies of long-lived bone marrow plasma cells were unaffected. Instead, mTORC1 inhibition led to decreased expression of immunoglobulin-binding protein (BiP) and other factors needed for robust protein synthesis. Consequently, blockade of antibody synthesis was rapidly reversed after termination of rapamycin treatment. We conclude that mTOR signaling plays critical but diverse roles in early and late phases of antibody responses and plasma cell differentiation.


Improving dynamic susceptibility contrast MRI measurement of quantitative cerebral blood flow using corrections for partial volume and nonlinear contrast relaxivity: A xenon computed tomographic comparative study.
To test whether dynamic susceptibility contrast MRI-based CBF measurements are improved with arterial input function (AIF) partial volume (PV) and nonlinear contrast relaxivity correction, using a gold-standard CBF method, xenon computed tomography (xeCT). Eighteen patients with cerebrovascular disease underwent xeCT and MRI within 36 h. PV was measured as the ratio of the area under the AIF and the venous output function (VOF) concentration curves. A correction was applied to account for the nonlinear relaxivity of bulk blood (BB). Mean CBF was measured with both techniques and regression analyses both within and between patients were performed. Mean xeCT CBF was 43.3 +/- 13.7 mL/100g/min (mean +/- SD). BB correction decreased CBF by a factor of 4.7 +/- 0.4, but did not affect precision. The least-biased CBF measurement was with BB but without PV correction (45.8 +/- 17.2 mL/100 g/min, coefficient of variation [COV] = 32%). Precision improved with PV correction, although absolute CBF was mildly underestimated (34.3 +/- 10.8 mL/100 g/min, COV = 27%). Between patients correlation was moderate even with both corrections (R = 0.53). Corrections for AIF PV and nonlinear BB relaxivity improve bolus MRI-based CBF maps. However, there remain challenges given the moderate between-patient correlation, which limit diagnostic confidence of such measurements in individual patients.


Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase lambda.
We previously reported that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro. The purpose of this study was to investigate the molecular structural relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol lambda (full-length, i.e. intact pol lambda including the BRCA1 C- terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol lambda inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 microm. The compound did not influence the activities of replicative pols such as alpha, delta, and epsilon. It had no effect on pol beta activity either, although the three-dimensional structure of pol beta is thought to be highly similar to that of pol lambda. Compound 13 did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted from its N-terminal region. MALDI-TOF MS analysis demonstrated that compound 13 bound selectively to the N-terminal domain of pol lambda, but did not bind to the C-terminal region. Based on these results, the pol lambda-inhibitory mechanism of compound 13 is discussed.


Psychosocial impact of alternative management policies for low-grade cervical abnormalities: results from the TOMBOLA randomised controlled trial.
Large numbers of women who participate in cervical screening require follow-up for minor cytological abnormalities. Little is known about the psychological consequences of alternative management policies for these women. We compared, over 30-months, psychosocial outcomes of two policies: cytological surveillance (repeat cervical cytology tests in primary care) and a hospital-based colposcopy examination. Women attending for a routine cytology test within the UK NHS Cervical Screening Programmes were eligible to participate. 3399 women, aged 20-59 years, with low-grade abnormal cytology, were randomised to cytological surveillance (six-monthly tests; n = 1703) or initial colposcopy with biopsies and/or subsequent treatment based on colposcopic and histological findings (n = 1696). At 12, 18, 24 and 30-months post-recruitment, women completed the Hospital Anxiety and Depression Scale (HADS). A subgroup (n = 2354) completed the Impact of Event Scale (IES) six weeks after the colposcopy episode or first surveillance cytology test. Primary outcomes were percentages over the entire follow-up period of significant depression (≥ 8) and significant anxiety (≥ 11; "30-month percentages"). Secondary outcomes were point prevalences of significant depression, significant anxiety and procedure-related distress (≥ 9). Outcomes were compared between arms by calculating fully-adjusted odds ratios (ORs) for initial colposcopy versus cytological surveillance. There was no significant difference in 30-month percentages of significant depression (OR = 0.99, 95% CI 0.80-1.21) or anxiety (OR = 0.97, 95% CI 0.81-1.16) between arms. At the six-week assessment, anxiety and distress, but not depression, were significantly less common in the initial colposcopy arm (anxiety: 7.9% vs 13.4%; OR = 0.55, 95% CI 0.38-0.81; distress: 30.6% vs 39.3%, OR = 0.67 95% CI 0.54-0.84). Neither anxiety nor depression differed between arms at subsequent time-points. There was no difference in the longer-term psychosocial impact of management policies based on cytological surveillance or initial colposcopy. Policy-makers, clinicians, and women themselves can be reassured that neither management policy has a significantly greater psychosocial cost. Controlled-Trials.com ISRCTN 34841617.


Effect of pulsed electromagnetic stimulation on facial nerve regeneration.
To determine if exposure to electromagnetic fields influences regeneration of the transected facial nerve in the rat. The left facial nerve was transected in the tympanic section of the fallopian canal in 24 rats randomly assigned to 2 groups. The cut ends of the facial nerve were reapproximated without sutures within the fallopian canal to maximize the potential for regeneration. Rats in the experimental group (n= 12) were then exposed to pulsed electromagnetic stimulation (0.4 millitesla at 120 Hz) for 4 hours per day, 5 days per week, for 8 weeks. Rats in the control group (n=12) were handled in an identical manner without pulsed electromagnetic stimulation. Four other rats were given sham operations in which all surgical procedures were carried out except for the actual nerve transection. Two of these rats were placed in each group. Nerve regeneration was evaluated using electroneurography (compound action potentials), force of whisker and eyelid movements, and voluntary facial movements before and at 2-week intervals after transection. Histological evaluation was performed at 10 weeks after transection. Each dependent variable was analyzed using a 2-way analysis of variance with 1 between variable (groups) and 1 within repeated measures variable (days after transection). Statistical analysis indicated that N1 (the negative deflection of depolarization phase of the muscle and/or nerve fibers) area, N1 amplitude, and N1 duration, as well as absolute amplitude of the compound action potentials, were all significantly greater 2 weeks after transection in the experimental than in the control group of rats. The force of eye and whisker movements after electrical stimulation was statistically greater in the experimental group of rats 4 weeks after transection. Voluntary eye movements in the experimental group were significantly better at 5 and 10 weeks, while whisker movements were better at 3 and 10 weeks. There was no statistical difference between the 2 groups for any histological variable. Results of this study indicate that pulsed electromagnetic stimulation enhances early regeneration of the transected facial nerve in rats.


British Columbia Healthy Connections Project process evaluation: a mixed methods protocol to describe the implementation and delivery of the Nurse-Family Partnership in Canada.
The Nurse-Family Partnership is a home visitation program for first-time, socially and economically disadvantaged mothers. The effectiveness of this public health intervention has been well established in the United States; however, whether the same beneficial outcomes will be obtained within the Canadian context is unknown. As part of the British Columbia Healthy Connections Project, which includes a trial comparing Nurse-Family Partnership's effectiveness with existing services in British Columbia, we are conducting a process evaluation to describe and explain how the intervention is implemented and delivered across five regional Health Authorities. A convergent parallel mixed methods research design will be used to address the process evaluation objectives. The principles of interpretive description will guide all sampling, data collection and analytic decisions in the qualitative component of the study. The full population of public health nurses and supervisors (n = 71) will discuss their experiences of implementing and delivering the program in interviews (or focus groups). Managers (n = 5-15) responsible for this portfolio will also be interviewed annually. Fidelity reports with quantitative data on the reach and the dose of the intervention will be collected and analyzed. Summaries of team meetings and supervisory sessions will be analyzed. Data will be used to compare, corroborate and explain results and variances across the five regional Health Authorities. The process evaluation results will be of immediate instrumental use to the program implementers to inform intervention delivery. Findings will contribute to the emerging body of evidence surrounding: 1) professional nurse home visitation practice issues; 2) best practices for meeting the needs of families living in rural and remote communities; 3) a deeper understanding of how health and social issues such as mental health problems including substance misuse and exposure to intimate partner violence affect a young mother's capacity to parent; and 4) strategies to support professionals from the primary care, public health and child welfare sectors to work collaboratively to meet the needs of children and families who are at risk or experiencing maltreatment.


Evidence for an eye-movement contribution to normal foveal crowding.
Along with contour interaction, inaccurate and imprecise eye movements and attention have been suggested to contribute to poorer acuity for "crowded" versus uncrowded targets. To investigate the role of eye movements in foveal crowding, we compared percent correct letter identification for short and long lines of near-threshold letters with different separations. Five normal observers read short (4 to 6 letters) and long (10 to 12 letters) lines of near-threshold, Sloan letters with edge-to-edge letter separations of 0.5, 1, and 2 letter spaces. Percent correct letter identification for the 2 to 4 interior letters in short strings and the 8 to 10 interior letters in long strings was compared with a no-crowding condition. Letter identification was significantly worse than the no-crowding condition for long letter strings with a separation of 1 letter space and for both long and short letter strings with a separation of 0.5 letter spaces. Observers more often reported the incorrect number of letters in long than in short letter strings, even for a separation of 2 letter spaces. Similar results were obtained during straight-ahead gaze and while viewing in 30 to 40 degrees left gaze, where two of the five observers exhibited an increase in horizontal fixational instability. We argue that lower percent correct letter identification and more frequent errors in reporting the number of letters in long compared with short letter strings reflect an eye-movement contribution to foveal crowding.


Temperament and character in women with postpartum depression.
To investigate whether women with postpartum depression differ in personality traits from healthy postpartum women, healthy controls from the normal Swedish population and non-postpartum women with major depression. Forty-five women with postpartum depression were compared with 62 healthy postpartum women, 62 age-matched, healthy, non-postpartum women from a normal sample and 74 non-postpartum women with major depression from a clinical sample. The edinburgh postnatal depression scale was used in order to screen for postpartum depression. A clinical diagnostic interview was done including a rating with the Montgomery-Asberg depression rating scale. Personality i.e. temperament and character was measured by the temperament and character inventory. Harm avoidance (HA) was higher (p < 0.001) and self-directedness (SD) scored lower (p < 0.001) in women with postpartum depression compared to healthy postpartum women. These differences were the most important differences between these two groups. Women with postpartum depression scored lower (p = 0.001) in cooperativeness (CO) and higher (p = 0.019) in self-transcendence (ST) compared to healthy postpartum women. Women with postpartum depression scored overall similar to women with major depression. High HA and low SD can be seen as vulnerability factors for developing a depression and especially in a stressful situation as childbirth.


Prevalence of toxin producing strains of Staphylococcus aureus in a pediatric burns unit.
The aims of the study were to determine the sites in a pediatric burns unit that were contaminated with Staphylococcus aureus. Samples from the environment in bedrooms and the common room were taken monthly for 6 months using blood agar for total counts and Baird-Parker agar for S. aureus. The air was sampled using an air-sampling device and settle plates. Hard and soft surfaces including bed, blanket, sofa, chair, taps, bathtub, soft toys, locker and cupboard in the same rooms were sampled using contact plates. Swabs were taken from staff monthly for 3 months. S. aureus isolates were tested for production of enterotoxins A-D and toxic shock syndrome toxin-1 using a reverse passive latex agglutination test. The results showed that S. aureus was recovered more frequently using settle plates than using the air sampler. All surfaces sampled were contaminated with S. aureus and contamination was greatest in frequently occupied rooms. A variety of toxin producing isolates were found with enterotoxin C isolates, either alone or in combination with TSST-1 (toxic shock syndrome toxin-1) dominant. The staff were transiently colonised with S. aureus strains with a different toxin production pattern. The results show that airborne transmission may be a route for infection by S. aureus and is responsible for contaminating the environment.


A study on the coordinative versatility of new N,S-donor macrocyclic ligands: XAFS, and Cu2+ complexation thermodynamics in solution.
We investigated, both in the solid state and in aqueous solution, the coordination environment and stability behavior of four macrocyclic ligands (three N(2)S(2) and one N(3)S(2)) and of the corresponding Cu(II) complexes. The structural characterization in the solid state of the copper derivatives was performed by X-Ray Absorption Spectroscopy. Copper is found to be 4-fold coordinated with a CuN(2)S(2) environment with different Cu-S distances depending on the size of the macrocyclic ring. The EXAFS technique has indicated that nitrogen and sulfur atoms are more preferable to oxygen atoms as donor systems, without the evidence of coordination of the carboxylic moieties to copper in the first shell. The joint EXAFS and XANES study of the copper(II) complex with the N(3)S(2) ligand confirms the 4-fold coordination with an additional, long Cu-N interaction. The Cu(2+) complexation constants for one ligand were determined in aqueous solution. The results indicate that the species [CuL], although isolated in the solid state, is not the most abundant at the pH of blood serum. Instead, at pH 7.4 the protonated [Cu(HL)](+) species was found to be the most relevant. The behaviour of the copper complexes in the presence of the strong copper chelating bioagent human serum albumin was also examined in order to gain information on the stability of these compounds in biological fluids.


Role of substrate and calcium in neurite retraction of leech neurons following depolarization.
The aim of these experiments was to analyze how depolarization influences neurite outgrowth in leech neurons and what role the substrate and Ca2+ play in this response. Neurons in culture were exposed to 60 mM extracellular K+ for 30 min, which induced retraction of a subset of neurites growing on extracellular matrix substrate (ECM), a response comparable to that observed after electrical stimulation (Grumbacher-Reinert and Nicholls, 1992). After normal medium had been restored, the neurites continued to retract for about 1 hr to approximately 80% of the total starting neurite length. Retraction was reversible and regrowth began after the cells had been in normal medium for about 3 hr. Similar depolarization-induced neurite retraction was observed in both Retzius and anterior pagoda cells. Retraction was inhibited by raised extracellular Mg2+, suggesting a mechanism dependent on calcium. The effect of high K+ on neurite outgrowth was also influenced by the substrate on which the cells were plated. Cells plated on concanavalin A (ConA) did not retract but continued to extend processes during exposure to high K+. To understand the different behavior of cells grown on ECM and ConA, the morphology of growth cones was analyzed by scanning electron microscopy. The growth cones of cells grown on ECM and exposed to high K+ revealed retraction of lamellipodial and filopodial structures. On ConA, however, no differences were observed between growth cones of cells exposed to high K+ and those of control cells. These results demonstrate the importance of substrate molecules in the responses of growth cones to depolarization and therefore in the differentiation of neurons.


Optoelectronic oscillator stabilized to an intra-loop Fabry-Perot cavity by a dual servo system.
We report construction and characterization of an optoelectronic oscillator (OEO), which is stabilized to an intra-loop Fabry-Perot cavity by a dual servo system. In addition to providing strong mode selection and increasing the Q factor by adding significant loop length, the cavity serves as a stable frequency reference. In order to fully exploit the stability we employ a dual servo system. Carrier frequency is locked to the cavity mode by using Pound-Drever-Hall technique. The OEO loop length is adjusted by comparing the phase difference between the carrier-sideband beat signals at upstream and downstream sides of the cavity so that the OEO mode spacing is commensurate with the free spectral range of the cavity. This dual servo system and additional stabilizations of the seed laser frequency to a cesium transition and the laser power result in an order of magnitude improvement in OEO frequency stability over a previous work using a free-running Fabry-Perot cavity. Long term Allan deviation of the OEO is 6 x 10(-8). It represents 4 x 10(-4)of the cavity resonance linewidth. We also discuss possibility of relating the OEO frequency to an atomic transition as an absolute frequency reference.


Blood pressure variability predicts cardiovascular events independently of traditional cardiovascular risk factors and target organ damage: a LIFE substudy.
Assessment of antihypertensive treatment is normally based on the mean value of a number of blood pressure (BP) measurements. However, it is uncertain whether high in-treatment visit-to-visit BP variability may be harmful in hypertensive patients with left ventricular hypertrophy (LVH). In 8505 patients randomized to losartan vs. atenolol-based treatment in the LIFE study, we tested whether BP variability assessed as SD and range for BP6-24 months measured at 6, 12, 18 and 24 months of treatment was associated with target organ damage (TOD) defined by LVH on ECG and urine albumin/creatinine ratio at 24 months, and predicted the composite endpoint (CEP) of cardiovascular death, nonfatal myocardial infarction (MI) or stroke occurring after 24 months (CEP = 630 events). In multiple regression models adjusted for mean BP6-24 months and treatment allocation, neither high BP6-24 months SD nor wide range were related to TOD at 24 months, except for a weak association between Sokolow-Lyon voltage and DBP6-24 months SD and range (both β = 0.04, P < 0.01). Independently of mean BP6-24 months, treatment allocation, TOD and baseline characteristics in Cox regression models, CEP after 24 months was associated with DBP6-24 months SD [hazard ratio per 1 mmHg increase1.04, 95% confidence interval (95% CI) 1.01-1.06, P = 0.005], range (hazard ratio 1.02, 95% CI 1.01-1.03, P = 0.004), SBP6-24 months SD (hazard ratio 1.01, 95% CI 0.99-1.02, P = 0.07) and range (hazard ratio 1.006, 95% CI 1.001-1.01, P = 0.04). Adjusted for the same factors, stroke was associated with DBP6-24 months SD (hazard ratio 1.06, 95% CI 1.02-1.10, P = 0.001), range (hazard ratio 1.03, 95% CI 1.01-1.04, P = 0.001), SBP6-24 months SD (hazard ratio 1.02, 95% CI 1.002-1.04, P = 0.04) and range (hazard ratio 1.008, 95% CI 1.001-1.02, P = 0.05), but MI was not. In LIFE patients, higher in-treatment BP6-24 months variability was independently of mean BP6-24 months associated with later CEP and stroke, but not with MI or TOD after 24 months.


A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?
Quantification of biofluid cytokines is a rapidly growing area of translational research. However, comparability across the expanding number of available assay platforms for detection of the same proteins remains to be determined. We aimed to directly compare a panel of commonly measured cytokines in plasma of typically aging adults across two high sensitivity quantification platforms, Meso Scale Discovery high performance electrochemiluminiscence (HPE) and single-molecule immunosorbent assays (Simoa) by Quanterix. 57 community-dwelling older adults completed a blood draw, neuropsychological assessment, and brain MRI as part of a healthy brain aging study. Plasma samples from the same draw dates were analyzed for IL-10, IP-10, IL-6, TNFα, and IL-1β on HPE and Simoa, separately. Reliable detectability (coefficient of variance (CV) < 20% and outliers 3 interquartiles above the median removed), intra-assay precision, absolute concentrations, reproducibility across platforms, and concurrent associations with external variables of interest (e.g., demographics, peripheral markers of vascular health, and brain health) were examined. The proportion of cytokines reliably measured on HPE (87.7-93.0%) and Simoa (75.4-93.0%) did not differ (ps > 0.32), with the exception of IL-1β which was only reliably measured using Simoa (68.4%). On average, CVs were acceptable at <8% across both platforms. Absolute measured concentrations were higher using Simoa for IL-10, IL-6, and TNFα (ps < 0.05). HPE and Simoa shared only small-to-moderate proportions of variance with one another on the same cytokine proteins (range: r = 0.26 for IL-10 to r = 0.64 for IL-6), though platform agreement did not dependent on cytokine concentrations. Cytokine ratios within each platform demonstrated similar relative patterns of up- and down-regulation across HPE and Simoa, though still significantly differed (ps < 0.001). Supporting concurrent validity, all 95% confidence intervals of the correlations between cytokines and external variables overlapped between the two platforms. Moreover, most associations were in expected directions and consistently so across platforms (e.g., IL-6 and TNFα), though with several notable exceptions for IP-10 and IL-10. HPE and Simoa showed comparable detectability and intra-assay precision measuring a panel of commonly examined cytokine proteins, with the exception of IL-1β which was not reliably detected on HPE. However, Simoa demonstrated overall higher concentrations and the two platforms did not show agreement when directly compared against one another. Relative cytokine ratios and associations demonstrated similar patterns across platforms. Absolute cytokine concentrations may not be directly comparable across platforms, may be analyte dependent, and interpretation may be best limited to discussion of relative associations.


Effects of bottom-up and top-down intervention principles in emergent literacy in children at risk of developmental dyslexia: a longitudinal study.
This longitudinal study focused on the effects of two different principles of intervention in children at risk of developing dyslexia from 5 to 8 years old. The children were selected on the basis of a background questionnaire given to parents and preschool teachers, with cognitive and functional magnetic resonance imaging results substantiating group differences in neuropsychological processes associated with phonology, orthography, and phoneme-grapheme correspondence (i.e., alphabetic principle). The two principles of intervention were bottom-up (BU), "from sound to meaning", and top-down (TD), "from meaning to sound." Thus, four subgroups were established: risk/BU, risk/TD, control/BU, and control/TD. Computer-based training took place for 2 months every spring, and cognitive assessments were performed each fall of the project period. Measures of preliteracy skills for reading and spelling were phonological awareness, working memory, verbal learning, and letter knowledge. Literacy skills were assessed by word reading and spelling. At project end the control group scored significantly above age norm, whereas the risk group scored within the norm. In the at-risk group, training based on the BU principle had the strongest effects on phonological awareness and working memory scores, whereas training based on the TD principle had the strongest effects on verbal learning, letter knowledge, and literacy scores. It was concluded that appropriate, specific, data-based intervention starting in preschool can mitigate literacy impairment and that interventions should contain BU training for preliteracy skills and TD training for literacy training.


Conditioned Medium from Human Amnion-Derived Mesenchymal Stromal/Stem Cells Attenuating the Effects of Cold Ischemia-Reperfusion Injury in an In Vitro Model Using Human Alveolar Epithelial Cells.
The clinical results of lung transplantation (LTx) are still less favorable than other solid organ transplants in both the early and long term. The fragility of the lungs limits the procurement rate and can favor the occurrence of ischemia-reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) with Steen SolutionTM (SS) aims to address problems, and the implementation of EVLP to alleviate the activation of IRI-mediated processes has been achieved using mesenchymal stromal/stem cell (MSC)-based treatments. In this study, we investigated the paracrine effects of human amnion-derived MSCs (hAMSCs) in an in vitro model of lung IRI that includes cold ischemia and normothermic EVLP. We found that SS enriched by a hAMSC-conditioned medium (hAMSC-CM) preserved the viability and delayed the apoptosis of alveolar epithelial cells (A549) through the downregulation of inflammatory factors and the upregulation of antiapoptotic factors. These effects were more evident using the CM of 3D hAMSC cultures, which contained an increased amount of immunosuppressive and growth factors compared to both 2D cultures and encapsulated-hAMSCs. To conclude, we demonstrated an in vitro model of lung IRI and provided evidence that a hAMSC-CM attenuated IRI effects by improving the efficacy of EVLP, leading to strategies for a potential implementation of this technique.


[Frozen-section biopsy in ovarian neoplasm diagnosis: diagnostic correlation according to diameter and weight in tumors of epithelial origin].
Adequate management and treatment of ovarian carcinoma requires a complete surgical staging supported by frozen-section examination. To achieve this goal it is necessary a high level of accuracy. To evaluate the accuracy of frozen-sections in ovarian carcinoma considering the influence of tumor diameter and weight. Retrospective study of frozen-sections performed in patients with ovarian tumors who underwent surgery. Frozen- and permanent-sections were divided into three categories (benign, borderline and malignant) and stratified by diameter (< 10 cm, 10 to 20 cm, > 20 cm) and weight (< 700 g, 700 a 1400 g, > 1400 g). The diagnostic correlation, sensitivity, specificity, predictive values and accuracy of each frozen-section diagnosis were determined. Eight hundred forty two ovarian tumors that underwent frozen-sections between January 1988 and October 1998 were studied. Final diagnosis was 86.7% benign, 2.7% low malignant potential (LMP) and 10.6% malignant. The diagnosis correlation between frozen- and permanent-sections was 98.2%. Misdiagnosis was in epithelial ovarian tumors, particularly in LMP tumors. Sensitivity, specificity, positive- and negative-predictive values and accuracy of the four hundred eighty nine epithelial tumor were 92.6%, 99.2%, 96.7%, 98.2% and 97.9%, respectively. Diagnostic correlation was higher in epithelial ovarian tumors with diameter < 10 cm (98.2% v/s 93.8%) and weight < 700 g (96.9% v/s 88.9%). Diagnostic correlation with permanent-section examination, sensitivity, specificity and predictive values of frozen-sections are high in ovarian tumors. Accurate diagnosis at frozen sections of epithelial ovarian tumors with diameter > 10 cm or weight > 700 g (particularly in LMP tumors) is difficult because of the extensive sampling required. Frozen-sections diagnoses are important to determine the type and extent of surgery performed at the initial operation.


Reliability assessment of a novel cervical spine deformity classification system.
Despite the complexity of cervical spine deformity (CSD) and its significant impact on patient quality of life, there exists no comprehensive classification system. The objective of this study was to develop a novel classification system based on a modified Delphi approach and to characterize the intra- and interobserver reliability of this classification. Based on an extensive literature review and a modified Delphi approach with an expert panel, a CSD classification system was generated. The classification system included a deformity descriptor and 5 modifiers that incorporated sagittal, regional, and global spinopelvic alignment and neurological status. The descriptors included: "C," "CT," and "T" for primary cervical kyphotic deformities with an apex in the cervical spine, cervicothoracic junction, or thoracic spine, respectively; "S" for primary coronal deformity with a coronal Cobb angle ≥ 15°; and "CVJ" for primary craniovertebral junction deformity. The modifiers included C2-7 sagittal vertical axis (SVA), horizontal gaze (chin-brow to vertical angle [CBVA]), T1 slope (TS) minus C2-7 lordosis (TS-CL), myelopathy (modified Japanese Orthopaedic Association [mJOA] scale score), and the Scoliosis Research Society (SRS)-Schwab classification for thoracolumbar deformity. Application of the classification system requires the following: 1) full-length standing posteroanterior (PA) and lateral spine radiographs that include the cervical spine and femoral heads; 2) standing PA and lateral cervical spine radiographs; 3) completed and scored mJOA questionnaire; and 4) a clinical photograph or radiograph that includes the skull for measurement of the CBVA. A series of 10 CSD cases, broadly representative of the classification system, were selected and sufficient radiographic and clinical history to enable classification were assembled. A panel of spinal deformity surgeons was queried to classify each case twice, with a minimum of 1 intervening week. Inter- and intrarater reliability measures were based on calculations of Fleiss k coefficient values. Twenty spinal deformity surgeons participated in this study. Interrater reliability (Fleiss k coefficients) for the deformity descriptor rounds 1 and 2 were 0.489 and 0.280, respectively, and mean intrarater reliability was 0.584. For the modifiers, including the SRS-Schwab components, the interrater (round 1/round 2) and intrarater reliabilities (Fleiss k coefficients) were: C2-7 SVA (0.338/0.412, 0.584), horizontal gaze (0.779/0.430, 0.768), TS-CL (0.721/0.567, 0.720), myelopathy (0.602/0.477, 0.746), SRS-Schwab curve type (0.590/0.433, 0.564), pelvic incidence-lumbar lordosis (0.554/0.386, 0.826), pelvic tilt (0.714/0.627, 0.633), and C7-S1 SVA (0.071/0.064, 0.233), respectively. The parameter with the poorest reliability was the C7-S1 SVA, which may have resulted from differences in interpretation of positive and negative measurements. The proposed classification provides a mechanism to assess CSD within the framework of global spinopelvic malalignment and clinically relevant parameters. The intra- and interobserver reliabilities suggest moderate agreement and serve as the basis for subsequent improvement and study of the proposed classification.


A unique cytoplasmic localization of retinoic acid receptor-gamma and its regulations.
Recent evidence suggests that extranuclear action of retinoid receptors is involved in mediating the pleiotropic effects of retinoids. However, whether they reside in the cytoplasm remains elusive. Here, we showed that retinoic acid receptor-gamma (RARgamma) was cytoplasmic in confluent cells, or when cells were released from serum depletion or treated with growth factors. In studying the regulation of RARgamma subcellular localization, we observed that ectopically overexpressed RARgamma was mainly cytoplasmic irrespective of serum concentration and cell density. The cytoplasmic retention of RARgamma was inhibited by ligand retinoic acid (RA). In addition, coexpression of retinoid X receptor-alpha (RXRalpha) resulted in nuclear localization of RARgamma through their heterodimerization. Mutagenesis studies revealed that a C-terminal fragment of RXRalpha potently prevents RA-induced RARgamma nuclear localization and transcriptional function. Furthermore, our results showed that the cytoplasmic retention of RARgamma was due to the presence of its unique N-terminal A/B domain, which was subject to regulation by p38 MAPK-mediated phosphorylation. Deletion or mutation of the N-terminal A/B domain largely impaired its cytoplasmic localization. Together, our data demonstrate that the subcellular localization of RARgamma is regulated by complex interactions among ligand binding, receptor phosphorylation, and receptor dimerizations.


Serial MR imaging of small arterially-enhancing liver lesions in patients with chronic liver disease.
To determine the significance of small arterially-enhancing liver lesions seen with magnetic resonance imaging (MRI) in patients with chronic liver disease. Our institutional review board approved this retrospective study, without requiring informed consent. Over a two-year period, 258 consecutive patients with cirrhosis or chronic hepatitis underwent multiphase three-dimensional, gadolinium-enhanced, breath-hold gradient-echo MRI. From this group, 29 patients underwent at least one follow-up MR study. When a small (< or = 20 mm) arterially-enhancing lesion was detected, the maximum diameter shape, signal intensities (T1-weighted and T2-weighted), and pattern of enhancement were evaluated to assess the associations between the imaging appearance on initial MR exam and subsequent behavior on follow-up imaging. Statistical testing was performed with JMP Statistical Software (SAS, Inc., Cary, NC) and StatXact 7 Statistical Software for Exact Nonparmetric Inference (Cytel, Inc., Cambride, MA). Sixty-five small (< or = 20 mm) arterially-enhancing lesions were detected in 29 patients. Ten of 65 lesions (15%) in nine patients were subsequently proven to represent hepatocellular carcinoma (HCC), while the remaining lesions either disappeared (46) or remained stable in size (9). Of the 10 lesions subsequently proven to represent HCC, eight lesions converted from hypo- or isointense to hyperintense on subsequent T2-weighted MRI (p < 0.001), seven lesions converted from hyper- or isointense to hypointense on subsequent T1-weighted images (p < 0.001), seven lesions demonstrated growth on subsequent MRI exam (mean increase in mean diameter = 1.4 cm), and five lesions subsequently developed rim enhancement that was not initially present. Small, arterially-enhancing lesions detected with MRI have a low likelihood of representing HCC, and MRI follow-up of such lesions is a reasonable approach. Lesions that increase in size, convert to hypointense on subsequent T1W images, convert to hyperintense in T2W images, or develop rim enhancement on follow-up MRI images are concerning and should prompt consideration of intervention.


Impact of medication review, within a shared decision-making framework, on deprescribing in people living in care homes.
The key objectives of this study were to quantify extent of prescribing, reasons for deprescribing, common therapeutic groups of medicines deprescribed and adverse events. A retrospective analysis was carried out on a quality improvement project where 422 care home residents in 20 care homes received a medicines optimisation review with a pharmacist and other members of the healthcare team (general medical practitioner, care home nurse). Data on number, type and cost of medicines were collected. Statistical analysis was performed to test for differences between pharmacist-only review and the pharmacist plus general practitioner (GP), and to identify any correlation between the original number of medicines and the number of medicines stopped. Of the 422 patients reviewed, 298 (70.6%) had at least one medicine stopped with 704 medicines being stopped. This represented 19.5% of the medicines originally prescribed (3602 medicines). There was no statistically significant difference between pharmacist only and pharmacist plus GP in terms of stopping medicines. The main groups of medicines stopped were laxatives, skin products and bone protection. There was weak correlation between the original number of medicines prescribed and the number stopped. This study has shown that medicines optimisation reviews can lead to a reduction in polypharmacy for care home residents through a deprescribing process. Patients' medicine regimens were simplified and optimised while making financial significant savings for the National Health Service.


Drosophila SNAP-29 is an essential SNARE that binds multiple proteins involved in membrane traffic.
Each membrane fusion event along the secretory and endocytic pathways requires a specific set of SNAREs to assemble into a 4-helical coiled-coil, the so-called trans-SNARE complex. Although most SNAREs contribute one helix to the trans-SNARE complex, members of the SNAP-25 family contribute two helixes. We report the characterization of the Drosophila homologue of SNAP-29 (dSNAP-29), which is expressed throughout development. Unlike the other SNAP-25 like proteins in fruit fly (i.e., dSNAP-25 and dSNAP-24), which form SDS-resistant SNARE complexes with their cognate SNAREs, dSNAP-29 does not participate in any SDS-resistant complexes, despite its interaction with dsyntaxin1 and dsyntaxin16 in vitro. Immunofluorescence studies indicated that dSNAP-29 is distributed in various tissues, locating in small intracellular puncta and on the plasma membrane, where it associates with EH domain-containing proteins implicated in the endocytic pathway. Overexpression and RNAi studies suggested that dSNAP-29 mediates an essential process in Drosophila development.


Analysis of the three most common MEFV mutations in 412 patients with familial Mediterranean fever.
Familial Mediterranean fever is an autosomal recessive disease characterized by recurrent attacks of fever and serositis. The disease is caused by mutations in the MEFV gene, presumed to act as a down-regulator of inflammation within the polymorphonuclear cells. To present the results of 412 FMF patients genotyped for three MEFV mutations, M694V, V726A and E148Q. The most frequent mutation, M694V, was detected in 47% of the carrier chromosomes. This mutation, especially common among North African Jewish FMF patients, was not found in any of the Ashkenazi (East European origin) patients. Overall, one of the three mutations was detected in 70% of the carrier chromosomes. M694V/M694V was the most common genotype (27%), followed by M694V/V726A (16%). The full genotype could be assessed in 57% of the patients, and one disease-causing mutation in an additional 26%. Only one patient with the E148Q/E148Q genotype was detected despite a high carrier rate for this mutation in the Jewish population, a finding consistent with a low penetrance of this genotype. The M694V/M694V genotype was observed in 15 patients with amyloidosis compared to 4 amyloidosis patients with other genotypes (P < 0.0001). Because of low penetrance and as yet other undetermined reasons, mutation analysis of the most common MEFV mutations supports a clinical diagnosis in only about 60% of patients with definite FMF.


T cell repertoire diversity is required for relapses in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.
Comparison of TCRalphabeta repertoires of myelin oligodendrocyte glycoprotein (MOG)-specific T lymphocytes in C57BL/6 and TdT-deficient littermates (TdT(-/-)) generated during experimental autoimmune encephalomyelitis (EAE) highlights a link between a diversified TCRalphabeta repertoire and EAE relapses. At the onset of the disease, the EAE-severity is identical in TdT(+/-) and TdT(-/-) mice and the neuropathologic public MOG-specific T cell repertoires express closely similar public Valpha-Jalpha and Vbeta-Jbeta rearrangements in both strains. However, whereas TdT(+/+) and TdT(+/-) mice undergo successive EAE relapses, TdT(-/-) mice recover definitively and the lack of relapses does not stem from dominant regulatory mechanisms. During the first relapse of the disease in TdT(+/-) mice, new public Valpha-Jalpha and Vbeta-Jbeta rearrangements emerge that are distinct from those detected at the onset of the disease. Most of these rearrangements contain N additions and are found in CNS-infiltrating T lymphocytes. Furthermore, CD4(+) T splenocytes bearing these rearrangements proliferate to the immunodominant epitope of MOG and not to other immunodominant epitopes of proteolipid protein and myelin basic protein autoantigens, excluding epitope spreading to these myelin proteins. Thus, in addition to epitope spreading, a novel mechanism involving TCRalphabeta repertoire diversification contributes to autoimmune progression.


Efficacy and adverse events of antimuscarinics for treating overactive bladder: network meta-analyses.
Millions of people worldwide experience overactive bladder (OAB), and antimuscarinics are the pharmacologic treatment of choice. Several conventional meta-analyses have been published, but they fail to quantify efficacy and adverse events across drugs, dosages, formulations, and pharmaceutical forms. To perform two network meta-analyses summarizing the efficacy and adverse events of antimuscarinics in the treatment of OAB. Medline and Scopus searches, previous systematic reviews, conference abstracts, book chapters, and the reference lists of relevant articles were searched. Trialists were contacted. Eligible studies were randomized trials that compared at least one antimuscarinic for treating OAB with placebo or with another antimuscarinic, and that reported efficacy and/or adverse event outcomes. Efficacy was assessed for six outcomes (perception of cure or improvement, urgency episodes per 24h, leakage episodes per 24h, urgency incontinence episodes per 24h, micturitions per 24h, and nocturia episodes per 24h). Adverse events were assessed in seven categories according to the Common Terminology Criteria for Adverse Events. Across all outcomes, a summary efficacy and an adverse event score were computed. Two authors independently extracted data. For the comparison of the efficacy, 76 trials enrolling 38 662 patients were included; for adverse events, 90 trials enrolling 39 919 patients were included. In the subset of studies reporting on treatments and dosages as used in clinical practice, 40 mg/d trospium chloride, 100mg/g per day oxybutynin topical gel, and 4 mg/d fesoterodine had the best efficacy, while higher dosages of orally administered oxybutynin and propiverine had the least favorable relationship of efficacy and adverse events. This is the first study allowing trade-offs between efficacy and adverse events of various drugs and dosages in the treatment of patients with OAB. Differences among the various antimuscarinics call for careful, patient-centered management in which regimen changes should be considered.


Parental care and clutch sizes in North and South American birds.
The evolutionary causes of small clutch sizes in tropical and Southern Hemisphere regions are poorly understood. Alexander Skutch proposed 50 years ago that higher nest predation in the south constrains the rate at which parent birds can deliver food to young and thereby constrains clutch size by limiting the number of young that parents can feed. This hypothesis for explaining differences in clutch size and parental behaviors between latitudes has remained untested. Here, a detailed study of bird species in Arizona and Argentina shows that Skutch's hypothesis explains clutch size variation within North and South America. However, neither Skutch's hypothesis nor two major alternatives explain differences between latitudes.


A cycling cis-Golgi protein mediates endosome-to-Golgi traffic.
Toxins can invade cells by using a direct endosome-to-Golgi endocytic pathway that bypasses late endosomes/prelysosomes. This is also a route used by endogenous proteins, including GPP130, which is an integral membrane protein retrieved via the bypass pathway from endosomes to its steady-state location in the cis-Golgi. An RNA interference-based test revealed that GPP130 was required for efficient exit of Shiga toxin B-fragment from endosomes en route to the Golgi apparatus. Furthermore, two proteins whose Golgi targeting depends on endosome-to-Golgi retrieval in the bypass pathway accumulated in early/recycling endosomes in the absence of GPP130. GPP130 activity seemed specific to bypass pathway trafficking because the targeting of other tested proteins, including those retrieved to the Golgi via the more conventional late endosome route, was unaltered. Thus, a distally cycling Golgi protein mediates exit from endosomes and thereby underlies Shiga toxin invasion and retrieval-based targeting of other cycling Golgi proteins.


Modulation of natural killer cell function by alpha-adrenoreceptor-coupled signalling.
Our previous work has shown that α-adrenoreceptor (α-AR)-coupled signaling modulates T lymphocyte function. Here, we investigate the expression of α₁- and α₂-ARs in natural killer (NK) cells and roles of the two subtypes of α-ARs and their coupled signals in modulation of NK cell function. NK cells were purified by Ficoll-Isopaque one-step gradient centrifugation and in discontinuous Percoll density gradients from splenic cells of rats. The mRNA expressions of α₁-ARs and α₂-ARs in NK cells were measured by reverse transcription-polymerase chain reaction (RT-PCR). Flow cytometry was employed to detect the cytotoxicity of NK cells. NK cells expressed both α₁-AR and α₂-AR mRNAs. Phenylephrine, a selective α₁-AR agonist, increased the cytotoxicity of NK cells. This effect of phenylephrine was reduced by corynanthine, a selective α₁-AR antagonist, and was blocked by PLC inhibitor U-73122, but not by PKA inhibitor H-89. Clonidine, a selective α₂-AR agonist, also enhanced the cytotoxicity of NK cells. This action of clonidine was blocked by α₂-AR antagonist yohimbine or by PKA inhibitor H-89, but not by PLC inhibitor U-73122. NK cells express α₁- and α₂-ARs. Activation of the either subtype of α-ARs augments NK cell function. This action of α₁-ARs is transduced by PLC, while α₂-AR effect is mediated by PKA signaling.