Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking.
Myosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments. In this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1). All results favored the peripheral dynamic tethering hypothesis.


Dual enkephalinase inhibitor PL265: a novel topical treatment to alleviate corneal pain and inflammation.
Ocular pain is a core symptom of inflammatory or traumatic disorders affecting the anterior segment. To date, the management of chronic ocular pain remains a therapeutic challenge in ophthalmology. The main endogenous opioids (enkephalins) play a key role in pain control but exhibit only transient analgesic effects due to their rapid degradation. The aim of this study was to explore the antinociceptive and anti-inflammatory effects of topical administration of PL265 (a dual enkephalinase inhibitor) on murine models of corneal pain. On healthy corneas, chronic PL265 topical administration did not alter corneal integrity nor modify corneal mechanical and chemical sensitivity. Then, on murine models of corneal pain, we showed that repeated instillations of PL265 (10 mM) significantly reduced corneal mechanical and chemical hypersensitivity. PL265-induced corneal analgesia was completely antagonized by naloxone methiodide, demonstrating that PL265 antinociceptive effects were mediated by peripheral corneal opioid receptors. Moreover, flow cytometry (quantification of CD11b+ cells) and in vivo confocal microscopy analysis revealed that instillations of PL265 significantly decreased corneal inflammation in a corneal inflammatory pain model. Chronic PL265 topical administration also decreased Iba1 and neuronal injury marker (ATF3) staining in the nucleus of primary sensory neurons of ipsilateral trigeminal ganglion. These results open a new avenue for ocular pain treatment based on the enhancement of endogenous opioid peptides' analgesic effects in tissues of the anterior segment of the eye. Dual enkephalinase inhibitor PL265 seems to be a promising topical treatment for safe and effective alleviation of ocular pain and inflammation.


Caregiving near the end of life: unmet needs and potential solutions.
A key aspect of the role of clinicians caring for patients in the setting of advanced illness focuses on attending to the needs of informal caregivers during the end-of-life period. The purpose of this study was twofold: (1) to complement and enrich existing quantitative findings regarding caregiver burden near the end of life, and (2) to identify potential solutions to caregivers' unmet needs in an effort to assist clinicians in the development of clinical interventions. This qualitative study, using focus groups and content analysis of transcripts, was conducted in a comprehensive cancer center in Washington, DC. Seven focus groups were held: three with recently bereaved caregivers and four with active caregivers of patients with metastatic cancer and an expected survival of 6 to 12 months. Data were stratified into two broad categories: (1) general problems and (2) behaviors/activities that were helpful/would have been helpful in alleviating these problems. Within each of these two categories, five subcategories emerged: medical care (including provision of information, coordination of care, bedside manner, satisfaction with care), quality of life (including well-being, role adjustments), help from others (including practical assistance, social support), positives of caregiving, and unsolicited themes (including job flexibility, impact of the disease on the family, informational needs, relationship with patient). Results suggest caregivers may benefit from more information about patient prognosis and hospice, attention to quality-of-life issues, and enhanced, direct communication with clinicians. Although information of this nature is likely to be known to palliative care clinicians, the specific details and verbal insights provided by caregivers give an important voice to existing quantitative data and may provide more detailed information to assist palliative care clinicians seeking to develop interventions to meet caregiver needs during the period near the end of life.


Fursultiamine, a vitamin B1 derivative, enhances chondroprotective effects of glucosamine hydrochloride and chondroitin sulfate in rabbit experimental osteoarthritis.
The therapeutic effect of glucosamine hydrochloride (GH) and chondroitin sulfate (CS) in combination with fursultiamine, a vitamin B1 derivative, on the development of cartilage lesions was investigated in an animal model of osteoarthritis (OA). The OA model was created by partial medial meniscectomy of the right knee joint (day 0). The rabbits were placed into three experimental groups: operated (OA) rabbits that received placebo treatment, OA rabbits that received GH (1000 mg/kg) + CS (800 mg/kg), and OA rabbits that received GH + CS + fursultiamine (100 mg/kg). Each treatment was initiated on day 3 and continued for 8 weeks. Macroscopic and histologic analyses were performed on the cartilage. The level of MMP-1 in OA cartilage chondrocytes was evaluated by immunohistochemistry. Only the group receiving combined treatment with GH + CS + fursultiamine showed a significant reduction in the severity of macroscopic and histologic lesions on tibial plateau, which is the weight bearing cartilage surface of the tibia, compared with placebo-treated OA rabbits. This treatment group also revealed a small, but significant, decrease in the body weight gain of the rabbits. In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in superficial layer stained positive for MMP-1 compared with unoperated control. Rabbits treated with the GH + CS + fursultiamine revealed a significant reduction in the level of MMP-1. These results suggest that the chondroprotective effect of GH + CS is enhanced by the addition of fursultiamine in experimental OA. This effect was associated with a reduction in the level of MMP-1, which are known to play an important role in the pathophysiology of OA lesions.


Involvement of cyclic GMP in the release of stereotyped behaviour patterns in moths by a peptide hormone.
A peptide hormone, the eclosion hormone, triggers two behavioural patterns--the pre-eclosion and eclosion patterns--when injected into pharate silkmoths. Injection of cyclic nucleotides caused the same behavioural responses with cGMP being 10 to 100 times more potent than cAMP. Exogenous cGMP also acted directly on the isolated nervous system to evoke the characteristic motor programmes. Protection of endogenous cyclic nucleotides by pretreatment of moths with a phosphodiesterase inhibitor, theophylline, markedly enhanced the sensitivity of the moths to the hormone. Injection of partially purified hormone preparations was followed by an increase in nervous system cGMP but not cAMP. The increase preceded the behavioural effectiveness of each dose was correlated with its ability to cause a cGMP increase. It was concluded that the behavioural effects of the eclosion hormone are mediated through an increase in cGMP in the nervous system.


Many unreported crop pests and pathogens are probably already present.
Invasive species threaten global biodiversity, food security and ecosystem function. Such incursions present challenges to agriculture where invasive species cause significant crop damage and require major economic investment to control production losses. Pest risk analysis (PRA) is key to prioritize agricultural biosecurity efforts, but is hampered by incomplete knowledge of current crop pest and pathogen distributions. Here, we develop predictive models of current pest distributions and test these models using new observations at subnational resolution. We apply generalized linear models (GLM) to estimate presence probabilities for 1,739 crop pests in the CABI pest distribution database. We test model predictions for 100 unobserved pest occurrences in the People's Republic of China (PRC), against observations of these pests abstracted from the Chinese literature. This resource has hitherto been omitted from databases on global pest distributions. Finally, we predict occurrences of all unobserved pests globally. Presence probability increases with host presence, presence in neighbouring regions, per capita GDP and global prevalence. Presence probability decreases with mean distance from coast and known host number per pest. The models are good predictors of pest presence in provinces of the PRC, with area under the ROC curve (AUC) values of 0.75-0.76. Large numbers of currently unobserved, but probably present pests (defined here as unreported pests with a predicted presence probability >0.75), are predicted in China, India, southern Brazil and some countries of the former USSR. We show that GLMs can predict presences of pseudoabsent pests at subnational resolution. The Chinese literature has been largely inaccessible to Western academia but contains important information that can support PRA. Prior studies have often assumed that unreported pests in a global distribution database represent a true absence. Our analysis provides a method for quantifying pseudoabsences to enable improved PRA and species distribution modelling.


An evidence assessment tool for ecosystem services and conservation studies.
Reliability of scientific findings is important, especially if they directly impact decision making, such as in environmental management. In the 1990s, assessments of reliability in the medical field resulted in the development of evidence-based practice. Ten years later, evidence-based practice was translated into conservation, but so far no guidelines exist on how to assess the evidence of individual studies. Assessing the evidence of individual studies is essential to appropriately identify and synthesize the confidence in research findings. We develop a tool to assess the strength of evidence of ecosystem services and conservation studies. This tool consists of (1) a hierarchy of evidence, based on the experimental design of studies and (2) a critical-appraisal checklist that identifies the quality of research implementation. The application is illustrated with 13 examples and we suggest further steps to move towards more evidence-based environmental management.


Probing material properties of polymeric surface layers with tapping mode AFM: which cantilever spring constant, tapping amplitude and amplitude set point gives good image contrast and minimal surface damage?
A phase shift between the oscillatory motion and drive motion of an AFM-cantilever used for tapping mode AFM imaging can be related to adhesive and elastic properties of surface layers. In this study it was studied how optimal contrast between hard and soft surface layers can be achieved while minimizing the surface damage. This was investigated by performing classical force-distance measurements while driving the cantilever as in tapping mode imaging. The amplitude and phase response as a function of the average tip-surface separation was recorded. Five different cantilevers with a wide range of spring constants and four different tapping amplitudes were investigated and compared. Based on these experiments it is concluded that too stiff cantilever, high free tapping amplitude and low amplitude set point value often lead to surface damage, while too low spring constant and low free tapping amplitude result in poor phase image contrast. Intermediate values where little surface damage and significant image contrast are obtained were identified. In all cases it was observed that the best image contrast was obtained when the amplitude set point was chosen such that the amplitude during imaging was reduced to approximately 50% of the free amplitude.


A review of the autonomic nervous system and exploration of diagnoses associated with reflex sympathetic dystrophy.
The hand therapy literature about reflex sympathetic dystrophy (RSD) tends to discuss this phenomenon in terms of traumatic hand or upper-extremity injury etiology. Hand therapists' bibliographies run the risk of being inbred and delimited to orthopedic and neurologic sources. Nonetheless, this well-developed body of knowledge identifies cardinal signs and symptoms, stages, and clinical types of RSD. Therapy guidelines have been well detailed in accordance with pathophysiologic explanations. The purpose of this article is to provide an overview of the autonomic nervous system (ANS), to briefly consider the history of the term "sympathetic," and to explore familiar and less well-known predisposing events and associated diagnoses. Appreciating the gamut of diagnoses associated with RSD may promote additional opportunities for hand therapist collaboration in the care for this complicated and challenging diagnostic group.


Polarization-independent broad-band nearly perfect absorbers in the visible regime.
Polarization-independent broad-band absorbers in the visible regime are theoretically investigated. The absorbers are three-layered structures consisting of a lossy dielectric grating on top of a low-loss dielectric layer and a substrate of the same lossy dielectric placed at the bottom. Enhanced absorption in the underlying structure is attained over a broad range of frequency for both TE and TM polarizations. In particular, a nearly perfect absorbance (over 99.6%) is achieved at λ ≈ 600 nm, around which the absorption spectra show a substantial overlap between two polarizations. The enhanced absorption is attributed to cavity resonance and its hybridization with a weakly bound surface wave. This feature is illustrated with the electric field patterns and time-averaged power loss density associated with the resonances.


A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis.
Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.


Autostereoscopic 3D Display with Long Visualization Depth Using Referential Viewing Area-Based Integral Photography.
We developed an autostereoscopic display for distant viewing of 3D computer graphics (CG) images without using special viewing glasses or tracking devices. The images are created by employing referential viewing area-based CG image generation and pixel distribution algorithm for integral photography (IP) and integral videography (IV) imaging. CG image rendering is used to generate IP/IV elemental images. The images can be viewed from each viewpoint within a referential viewing area and the elemental images are reconstructed from rendered CG images by pixel redistribution and compensation method. The elemental images are projected onto a screen that is placed at the same referential viewing distance from the lens array as in the image rendering. Photographic film is used to record the elemental images through each lens. The method enables 3D images with a long visualization depth to be viewed from relatively long distances without any apparent influence from deviated or distorted lenses in the array. We succeeded in creating an actual autostereoscopic images with an image depth of several meters in front of and behind the display that appear to have 3D even when viewed from a distance.


Feeding in the dark: lateral-line-mediated prey detection in the peacock cichlid Aulonocara stuartgranti.
The cranial lateral line canal system of teleost fishes is morphologically diverse and is characterized by four patterns. One of these, widened lateral line canals, has evolved convergently in a wide range of teleosts, including the Lake Malawi peacock cichlids (Aulonocara), and has been attributed to its role in prey detection. The ability to study Aulonocara in the laboratory provides an opportunity to test the hypothesis that their reported ability to feed on invertebrate prey living in sandy substrates in their natural habitat is the result of lateral-line-mediated prey detection. The goal of this study was to determine whether Aulonocara stuartgranti could detect hydrodynamic stimuli generated by tethered brine shrimp (visualized using digital particle image velocimetry) under light and dark conditions, with and without treatment with cobalt chloride, which is known to temporarily inactivate the lateral line system. Fish were presented with six pairs of tethered live and dead adult brine shrimp and feeding behavior was recorded with HD digital video. Results demonstrate that A. stuartgranti: (1) uses the same swimming/feeding strategy as they do in the field; (2) detects and consumes invertebrate prey in the dark using its lateral line system; (3) alters prey detection behavior when feeding on the same prey under light and dark conditions, suggesting the involvement of multiple sensory modalities; and (4) after treatment with cobalt chloride, exhibits a reduction in their ability to detect hydrodynamic stimuli produced by prey, especially in the dark, thus demonstrating the role of the lateral line system in prey detection.


Should we test for Helicobacter pylori before treating gastroesophageal reflux disease?
Gastroesophageal reflux disease (GERD) is a common problem in childhood. The cause is uncertain but because the incidence of GERD is increasing in developed countries and the prevalence of Helicobacter pylori is decreasing, it has been suggested that this infection protects against GERD. Observational data from 95 children, however, suggest that H. pylori eradication does not have a deleterious effect on GERD and this is supported by randomized controlled trials in adults. H. pylori eradication may also reduce the efficacy of proton pump inhibitor therapy in infected patients. There are no data from children but inferences from randomized controlled trials in adults suggest this effect is likely to be modest and of uncertain clinical significance. H. pylori is an important risk factor for distal gastric adenocarcinoma. It is likely that treating the infection in childhood will prevent pre-malignant changes associated with H. pylori from developing in the future. A meta-analysis of four randomized controlled trials suggest that there is a statistically significant impact on healing of chronic gastritis after one year compared with placebo (RR of chronic gastritis: 0.27; 95% CI 0.23 to 0.32). H. pylori eradication is therefore recommended in children with GERD that are having an endoscopy. However, when the diagnosis of GERD is being made clinically or by pH monitoring, it is not necessary to screen for H. pylori.


The Birmingham Rehabilitation Uptake Maximisation Study (BRUM). Home-based compared with hospital-based cardiac rehabilitation in a multi-ethnic population: cost-effectiveness and patient adherence.
To evaluate the relative effectiveness and cost-effectiveness of a home-based programme of cardiac rehabilitation using the Heart Manual, with centre-based programmes. It also sought to explore the reasons for non-adherence to cardiac rehabilitation programmes. An individually randomised trial, with minimisation for age, gender, ethnicity, initial diagnosis and hospital of recruitment. Participants were followed up after 6, 12 and 24 months by questionnaire and clinical assessment. Individual semistructured interviews were undertaken in the homes of a purposive sample of patients who did not adhere to their allocated programme, and focus groups were undertaken with groups of patients who adhered to the programmes. Four hospitals in predominantly inner-city, multi-ethnic, socio-economically deprived areas of the West Midlands in England, for 2 years from 1 February 2002. A total of 525 patients who had experienced a myocardial infarction (MI) or coronary revascularisation within the previous 12 weeks. All the rehabilitation programmes included exercise, relaxation, education and lifestyle counselling. All patients were seen by a cardiac rehabilitation nurse prior to hospital discharge and provided with information about their condition and counselling about risk factor modification. The four centre-based programmes varied in length from nine sessions at weekly intervals of education, relaxation and circuit training to 24 individualised sessions over 12 weeks of mainly walking, fixed cycling and rowing with group-based education. The home-based programme consisted of an appropriate version of the Heart Manual, home visits and telephone contact. The Heart Manual was introduced to patients on an individual basis, either in hospital or on a home visit. Home visits by a nurse took place at approximately 1, 6 and 12 weeks after recruitment, with a telephone call at 3 weeks. At the final visit, patients were encouraged to maintain their lifestyle changes and to continue with their exercise programme. Where needed, follow-up was made by a rehabilitation nurse who spoke Punjabi. An audiotape of an abridged version of the Heart Manual in Punjabi accompanied the manual for patients with a limited command of English. Primary outcomes were smoking cessation, blood pressure, total and high-density lipoprotein cholesterol, exercise capacity measured by the incremental shuttle walking test and psychological status measured by the Hospital Anxiety and Depression Scale (HADS). Secondary outcomes included self-reported diet, physical activity, cardiac symptoms and quality of life. Health service resource use and costs of rehabilitation programmes from health service and societal perspectives were also measured. Adherence to the physical activity element of the rehabilitation programmes was measured by questionnaire 6, 9 and 12 weeks. No clinically or statistically significant differences were found in any of the primary or secondary outcome measures between the home- and centre-based groups. Significant improvements in total cholesterol, smoking prevalence, the HADS anxiety score, self-reported physical activity and diet were seen in both arms between baseline and the 6-month follow-up. Five or more contacts with a cardiac rehabilitation nurse were received by 96% of home-based participants, whereas only 56% of centre-based participants attended this many rehabilitation classes. The direct rehabilitation costs to the health service were significantly higher for the home-based programme (mean cost 198 pounds versus 157 pounds for the centre-based programme), but when patient costs were included the mean cost of the centre-based arm rose to 182 pounds. Patients' reasons for not taking up or adhering to cardiac rehabilitation were multifactorial and very individual. Other health problems limited some patients' ability to exercise. Most non-adherers found some aspects of their cardiac rehabilitation programme helpful. Many had adapted advice on rehabilitation and were continuing to exercise in other ways and had made lifestyle changes, particularly to their diet. The home-based patients' lack of motivation to exercise on their own at home was a major factor in non-adherence. The focus groups revealed little diversity of views among patients from each programme. Patients in the hospital programme enjoyed the camaraderie of group exercise and the home-based patients valued the wealth of information and advice in the Heart Manual. A home-based cardiac rehabilitation programme for low- to moderate-risk patients does not produce inferior outcomes compared with the traditional centre-based programmes. With the level of home visiting in this trial, the home-based programme was more costly to the health service, but with the difference in costs borne by patients attending centre-based programmes. Different reasons were given by home and hospital cardiac rehabilitation patients for not taking up or adhering to cardiac rehabilitation, with home-based patients often citing a lack of motivation to exercise at home. Social characteristics, individual patient needs and the location of cardiac rehabilitation programmes need to be taken into account in programme design to maximise participation. Research is recommended into cardiac rehabilitation in patients from ethnic minority groups; measurement tools to assess physical activity and dietary change; evaluating the Heart Manual in patients who decline centre-based cardiac rehabilitation; the implementation of home-based programmes in the UK; and strategies that sustain physical activity in the long term.


Eighty-five per cent of what? Discrepancies in the weight cut-off for anorexia nervosa substantially affect the prevalence of underweight.
DSM-IV cites <85% of expected body weight (EBW) as a guideline for the diagnosis of anorexia nervosa (AN) but does not require a specific method for calculating EBW. The purpose of the present study was to determine the degree to which weight cut-off calculations vary across studies, and to evaluate whether differential cut-offs lead to discrepancies in the prevalence of individuals who are eligible for the AN diagnosis. Two coders independently recorded the EBW calculation methods from 99 studies that either (a) compared individuals with AN to those with subclinical eating disorders or (b) conducted AN treatment trials. Each weight cut-off was applied to a nationally representative (n=12001) and treatment-seeking (n=189) sample to determine the impact of EBW calculation on the proportion who met the AN weight criterion. Coders identified 10 different EBW methods, each of which produced different weight cut-offs for the diagnosis of AN. Although only 0.23% of the national sample met the lowest cut-off, this number increased 43-fold to 10.10% under the highest cut-off. Similarly, only 48.1% of treatment seekers met the lowest cut-off, whereas 89.4% met the highest. There is considerable variance across studies in the determination of the AN weight cut-off. Discrepancies substantially affect the proportion of individuals who are eligible for diagnosis, treatment and insurance reimbursement. However, differences may not be fully appreciated because the ubiquitous citation of the 85% criterion creates a sense of false consensus.


Selective motor control of the lower extremities in children with cerebral palsy: inter-rater reliability of two tests.
The purpose of this study was to examine the inter-rater reliability of two tests measuring selective motor control (SMC) of the lower extremities in children with cerebral palsy (CP). Two testers independently assessed 21 children (13 boys, eight girls; mean age 6 years 5 months, SD 12 months) with spastic CP (14 unilateral and seven bilateral) using the Boyd and Graham SMC test (with an existing protocol) and the modified Trost SMC test (with a newly developed protocol). Inter-rater reliability was analysed using Cohen's Kappa. For the Boyd and Graham SMC test for ankle dorsiflexion, Kappa was 0.55 (95% CI = 0.36-0.74). For the modified Trost SMC test for ankle dorsiflexion, knee extension, hip abduction and hip flexion, Kappas were 0.65 (0.47-0.84), 0.69 (0.49-0.88), 0.57 (0.37-0.78) and 0.71 (0.51-0.91), respectively. The SMC tests showed moderate (Boyd and Graham SMC test) to good (modified Trost SMC test) inter-rater reliability.


Endothelial Progenitor Cells: New Targets for Therapeutics for Inflammatory Conditions With High Cardiovascular Risk.
Over the past decade, we have witnessed an exponential growth of interest into the role of endothelial progenitor cells (EPCs) in cardiovascular disease. While the major thinking revolves around EPC angiogenic repair properties, we have used a hypothesis-driven approach to discover disease-related defects in their characteristics and based on these findings, have identified opportunities for functional enhancement, which offer an exciting avenue for translation into clinical intervention. In this review, we focus on two groups; circulating myeloid angiogenic cells (MACs) and late outgrowth endothelial colony forming cells (ECFCs), and will discuss the unique properties and defects of each population, as new insights have been gained into the potential function of each sub-type using current techniques and multiomic technology. We will discuss their role in inflammatory disorders and alterations in mitochondrial function. In addition, we share key insights into the glycocalyx, and propose this network of membrane-bound proteoglycans and glycoproteins, covering the endothelium warrants further investigation in order to clarify its significance in ECFC regulation of vascularization and angiogenesis and ultimately for potential translational therapeutic aspects.


Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.
In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.


IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells.
Smooth muscle responds to IP3-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors (IP3Rs). This release may propagate as Ca2+ waves. The Ca2+ signal emanating from IP3 generation may be amplified by its activating further Ca2+ release from ryanodine receptors (RyRs) in the process of Ca2+-induced Ca2+ release (CICR). Evidence for this proposal has relied largely on the use of blocking drugs such as ryanodine, tetracaine and dantrolene, reportedly specific inhibitors of RyRs. Here we have examined whether or not Ca2+ released via IP3Rs subsequently activates RyRs. In addition, the specificity of the blocking agents has been assessed by determining the extent of their ability to block IP3-mediated Ca2+ release under conditions in which RyRs were not activated. IP3-evoked Ca2+ release and Ca2+ waves did not require or activate RyRs. However, the RyR blocking drugs inhibited IP3-mediated Ca2+ signals at concentrations thought to be selective for RyRs. In single colonic smooth muscle cells, voltage clamped in the whole cell configuration, carbachol (CCh) evoked propagating Ca2+ waves which were not inhibited by ryanodine when the sarcolemma potential was -70 mV. At -20 mV, at which potential the SR Ca2+ content was increased and RyRs activated, ryanodine inhibited the Ca2+ waves. Photolysed caged IP3 increased [Ca2+]c; ryanodine, by itself, did not reduce the IP3-evoked [Ca2+]c increase when the sarcolemma potential was maintained at -70 mV. However, after activation of RyRs by caffeine, in the continued presence of ryanodine, the IP3-evoked [Ca2+]c increase was inhibited. In other experiments, RyRs were activated (as evidenced by the occurrence of spontaneous transient outward currents) by depolarizing the sarcolemma to -20 mV and again ryanodine was effective in inhibiting IP3-evoked Ca2+ increase. Thus while ineffective by itself, ryanodine inhibited IP3-evoked Ca2+ increases, presumably by causing persistent opening of the channel and depleting the SR of Ca2+, after RyRs were activated. These experiments establish that IP3-evoked Ca2+ release and Ca2+ waves do not activate RyRs; had they done so ryanodine would have inhibited the Ca2+ increase. However, under conditions where ryanodine was ineffective against the IP3-evoked Ca2+ transient (i.e. when RyRs were not activated, e.g. at a membrane potential of -70 mV) tetracaine and dantrolene each blocked IP3-evoked Ca2+ increases. The results show that although IP3-mediated Ca2+ release does not activate RyRs, RyR blockers can inhibit IP3-mediated Ca2+ signals.


Sit-and-wait strategies in dynamic visual search.
The role of memory in visual search has lately become a controversial issue. Horowitz and Wolfe (1998) observed that performance in a visual search task was little affected by whether the stimuli were static or randomly relocated every 111 ms. Because a memory-based mechanism, such as inhibition of return, would be of no use in the dynamic condition, Horowitz and Wolfe concluded that memory is likewise not involved in the static condition. However, Horowitz and Wolfe could not effectively rule out the possibility that observers adopted a different strategy in the dynamic condition than in the static condition. That is, in the dynamic condition observers may have attended to a subregion of the display and waited for the target to appear there (sit-and-wait strategy). This hypothesis is supported by experimental data showing that performance in their dynamic condition does not differ from performance in another dynamic condition in which observers are forced to adopt a sit-and-wait strategy by being presented with a limited region of the display only.


Responses of a dominant temperate grassland plant (Leymus chinensis) to elevated carbon dioxide and nitrogen addition in China.
The impact of elevated atmospheric CO(2) concentration on plant communities is varied and strongly dependent on the dominant species response, as well as nutrient conditions. Responses of a dominant species (Leymus chinensis) to elevated CO(2) and N application were examined with open-top chambers in a typical temperate grassland in northern China for 3 yr. The significant effect of elevated CO(2) on L. chinensis growth was mainly reflected in the higher photosynthetic rates, increased leaf number, larger shoot and root biomass, and higher root/shoot (R/S) ratio. Enhancement of root biomass induced by elevated CO(2) was larger (40%) than that of shoot biomass (9%). In contrast, N application had a significant impact on most growth indices examined in this study, which was reflected in the enhanced aboveground growth and depressed belowground growth. Nitrogen application significantly reduced the R/S ratio by an average of 40%. Nitrogen addition significantly enhanced the proportion of senescent biomass and decreased the proportion of green leaf biomass under elevated CO(2). There were no CO(2) x N interactions on most of the measured variables, except on photosynthetic rate and the proportion of aboveground biomass. Plant growth variables showed high interannual variation. These results indicate that belowground growth of L. chinensis is more sensitive to elevated CO(2) than is the aboveground. Aboveground growth of L. chinensis is much more sensitive to N application than to CO(2) enrichment. Therefore, the effect of elevated CO(2) on L. chinensis steppe is more likely to be underestimated if only aboveground parts are considered.


Association between free testosterone levels and anal human papillomavirus types 16/18 infections in a cohort of men who have sex with men.
Human papillomavirus (HPV) types 16 and 18 cause invasive cervical cancer and most invasive anal cancers (IACs). Overall, IAC rates are highest among men who have sex with men (MSM), especially MSM with HIV infection. Testosterone is prescribed for men showing hypogonadism and HIV-related wasting. While there are direct and indirect physiological effects of testosterone in males, its role in anal HPV16/18 infections in men is unknown. Free testosterone (FT) was measured in serum from 340 Multicenter AIDS Cohort Study (MACS) participants who were tested for anal HPV16/18-DNA approximately 36 months later. The effect of log10-transformed current FT level on anal HPV16/18 prevalence was modeled using Poisson regression with robust error variance. Multivariate models controlled for other HPV types, cumulative years of exogenous testosterone use, race, age, lifetime number of receptive anal intercourse partnerships, body mass index, tobacco smoking, HIV-infection and CD4+ T-cell counts among HIV-infected, and blood draw timing. Participants were, on average, 60 (+5.4) years of age, White (86%), and HIV-uninfected (56%); Twenty-four percent tested positive for anal HPV16 and/or 18-DNA (HPV16 prevalence=17.1%, HPV18=9.1%). In adjusted analysis, each half-log10 increase of FT was associated with a 1.9-fold (95% Confidence Interval: 1.11, 3.24) higher HPV16/18 prevalence. Additionally, other Group 1 high-risk HPVs were associated with a 1.56-fold (1.03, 2.37) higher HPV16/18 prevalence. Traditional risk factors for HPV16/18 infection (age, tobacco smoking; lifetime number of sexual partners, including the number of receptive anal intercourse partnerships within 24 months preceding HPV testing) were poorly correlated with one another and not statistically significantly associated with higher prevalence of HPV16/18 infection in unadjusted and adjusted analyses. Higher free testosterone was associated with increased HPV16/18 prevalence measured approximately three years later, independent of sexual behavior and other potential confounders. The mechanisms underlying this association remain unclear and warrant further study.


Use of suboptimal sperm increases the risk of aneuploidy of the sex chromosomes in preimplantation blastocyst embryos.
To compare autosomal and sex chromosome aneuploidy rates of embryos derived from sperm with abnormal and normal parameters. Retrospective cohort study. Assisted reproduction center. Three thousand eight hundred thirty-five embryos generated from 629 couples undergoing IVF. None. Incidence of aneuploidy in the trophectoderm of blastocyst embryos derived from standard IVF embryos and intracytoplasmic (ICSI) males with normal and oligozoospermic semen samples, in couples with donor eggs (mean maternal age, 25.0 years) and their own eggs (mean maternal age, 35.4 years). The rate of sex chromosome aneuploidy was significantly (around threefold) higher in the oligozoospermic group compared with in both control groups (standard vs. ICSI insemination). This applied whether donor (young) or own (older) eggs were used. Significant differences were seen in the oligozoospermic samples for autosomes 1, 2, 11 (own eggs), and 18 (donor eggs) compared with both control groups; however, no significant difference was seen between each of the treatment groups for the overall rate of autosomal aneuploidy. No significant differences were seen between the two control groups (normozoospermic males, standard vs. ICSI insemination) in either of the egg group types for any chromosome pairs. Severe male factor infertility is associated with a significant increase in the occurrence of sex chromosome abnormalities in blastocyst embryos compared with in embryos derived from normal semen samples. Aneuploidy rates in embryos derived from sperm with normal parameters were not significantly different whether ICSI or standard insemination was used to achieve fertilization. These results highlight severe male factor infertility as a possible referral category for preimplantation comprehensive chromosomal screening.


[Cardiovascular risk factors among young subjects with high carotid intima media thickness].
Cardiovascular risk factor (RF) assessment is essential to prevent and predict cardiovascular disease. The presence of RF at early ages, are determinant for the presence of atherosclerosis later in life. To determine the RF profile of young subjects with high carotid intima media thickness (CIMT). We studied 689 subjects (50% women, mean age 36±6 years) from Santiago, Chile. We determined body mass index (BMI), waist circumference, blood pressure, fasting serum lipids, blood glucose and C-reactive protein. CIMT was assessed by ultrasound using an automatic border recognition software. Body mass index, waist circumference, blood pressure and serum lipids were significantly higher among subjects located in the higher CIMT quartile. Also, subjects in the higher quartile of CIMT had a higher prevalence of three or more RF compared with the lower quartile (p = 0.01). Finally, individuals with three or more RF showed three times more risk of being in the higher CIMT quartile, than subjects with no RF (odds ratio = 3.1, p < 0.01). There is a negative influence of cardiovascular RF on CIMT among young subjects.


SCN4B-encoded sodium channel beta4 subunit in congenital long-QT syndrome.
Congenital long-QT syndrome (LQTS) is potentially lethal secondary to malignant ventricular arrhythmias and is caused predominantly by mutations in genes that encode cardiac ion channels. Nearly 25% of patients remain without a genetic diagnosis, and genes that encode cardiac channel regulatory proteins represent attractive candidates. Voltage-gated sodium channels have a pore-forming alpha-subunit associated with 1 or more auxiliary beta-subunits. Four different beta-subunits have been described. All are detectable in cardiac tissue, but none have yet been linked to any heritable arrhythmia syndrome. We present a case of a 21-month-old Mexican-mestizo female with intermittent 2:1 atrioventricular block and a corrected QT interval of 712 ms. Comprehensive open reading frame/splice mutational analysis of the 9 established LQTS-susceptibility genes proved negative, and complete mutational analysis of the 4 Na(vbeta)-subunits revealed a L179F (C535T) missense mutation in SCN4B that cosegregated properly throughout a 3-generation pedigree and was absent in 800 reference alleles. After this discovery, SCN4B was analyzed in 262 genotype-negative LQTS patients (96% white), but no further mutations were found. L179F was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells that contained the stably expressed SCN5A-encoded sodium channel alpha-subunit (hNa(V)1.5). Compared with the wild-type, L179F-beta4 caused an 8-fold (compared with SCN5A alone) and 3-fold (compared with SCN5A + WT-beta4) increase in late sodium current consistent with the molecular/electrophysiological phenotype previously shown for LQTS-associated mutations. We provide the seminal report of SCN4B-encoded Na(vbeta)4 as a novel LQT3-susceptibility gene.


Variations in T-helper 17 and Regulatory T Cells during The Menstrual Cycle in Peripheral Blood of Women with Recurrent Spontaneous Abortion.
Disorders in immune system regulation may result in pregnancy abnormalities such as recurrent spontaneous abortion (RSA). This study aims to determine the ratio of regulatory T (Treg) and T helper (Th) 17 cells in unexplained RSA (URSA) women during proliferative and secretory phases of their menstrual cycles compared to healthy non-pregnant women. In this case control study, 25 women with URSA and 35 healthy, non-pregnant women were enrolled. The percentage of Th17 and Treg cells in participants peripheral blood were determined by flow cytometry. The percentage of Th17 cells and their related cytokines in serum (IL-17A) were higher in the proliferative and secretory phases of the menstrual cycles of URSA women compared to the control women. However, a lower percentage of Treg cells and their related cytokines in serum, transforming growth factor (TGF) β1 and interleukin (IL)-10 were detected in the proliferative but not the secretory phase of the URSA group. The ratio of Th17/CD4+ Treg was higher in the URSA group than the control group. We observed an increased ratio of Th17/CD4+ Treg during the proliferative and secretory phases in URSA women. The imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion.


Association of TMEM18 variants with BMI and waist circumference in children and correlation of mRNA expression in the PFC with body weight in rats.
Genome-wide association studies have shown a strong association of single-nucleotide polymorphisms (SNPs) in the near vicinity of the TMEM18 gene. The effects of the TMEM18-associated variants are more readily observed in children. TMEM18 encodes a 3TM protein, which locates to the nuclear membrane. The functional context of TMEM18 and the effects of its associated variants are as of yet undetermined. To further explore the effects of near-TMEM18 variants, we have genotyped two TMEM18-associated SNPs, rs6548238 and rs4854344, in a cohort of 2352 Greek children (Healthy Growth Study). Included in this study are data on anthropomorphic traits body weight, BMI z-score and waist circumference. Also included are dietary energy and macronutrient intake as measured via 24-h recall interviews. Major alleles of rs6548238 and rs4854344 were significantly associated with an increased risk of obesity (odds ratio = 1.489 (1.161-1.910) and 1.494 (1.165-1.917), respectively), and positively correlated to body weight (P = 0.017, P = 0.010) and waist circumference (P = 0.003, P = 0.003). An association to energy and macronutrient intake was not observed in this cohort. We also correlated food intake and body weight in a food choice model in rats to Tmem18 expression in central regions involved in feeding behavior. We observed a strong positive correlation between TMEM18 expression and body weight in the prefrontal cortex (PFC) (r = 0.5694, P = 0.0003) indicating a potential role for TMEM18 in higher functions related to feeding involving the PFC.


Accounting for interspecific competition and age structure in demographic analyses of density dependence improves predictions of fluctuations in population size.
Understanding species coexistence has long been a major goal of ecology. Coexistence theory for two competing species posits that intraspecific density dependence should be stronger than interspecific density dependence. Great tits and blue tits are two bird species that compete for food resources and nesting cavities. On the basis of long-term monitoring of these two competing species at sites across Europe, combining observational and manipulative approaches, we show that the strength of density regulation is similar for both species, and that individuals have contrasting abilities to compete depending on their age. For great tits, density regulation is driven mainly by intraspecific competition. In contrast, for blue tits, interspecific competition contributes as much as intraspecific competition, consistent with asymmetric competition between the two species. In addition, including age-specific effects of intra- and interspecific competition in density-dependence models improves predictions of fluctuations in population size by up to three times.


New evidence on the use of fosfomycin for bacteremia and infectious endocarditis.
There is growing concern regarding the increased resistance rates of numerous pathogens and the limited availability of new antibiotics against these pathogens. In this context, fosfomycin is of considerable interest due to its activity against a wide spectrum of these microorganisms. We will review the encouraging data on this issue regarding the use of fosfomycin in treating Gram-negative bacterial infections. We will also cover fosfomycin's role against 2 of the main causal agents of bacteremia and endocarditis worldwide (nosocomial and community-acquired): enterococci, whose growing resistance to glycopeptides and aminoglycosides represents a serious threat, and methicillin-resistant Staphylococcus aureus, whose infection, despite efforts, continues to be associated with high morbidity and mortality and a high risk of complications. Thanks also to its considerable synergistic capacity with various antibiotics, fosfomycin is a tool for extending the therapeutic arsenal against these types of infections.


Why do patients with stroke not receive the recommended amount of active therapy (ReAcT)? Study protocol for a multisite case study investigation.
Increased frequency and intensity of inpatient therapy contributes to improved outcomes for stroke survivors. Differences exist in the amount of therapy provided internationally. In England, Wales and Northern Ireland it is recommended that a minimum of 45 min of each active therapy should be provided at least 5 days a week provided the therapy is appropriate and that the patient can tolerate this. Sentinel Stroke National Audit Programme (2014) data demonstrate this standard is not being achieved for most patients. No research been undertaken to explore how therapists in England manage their practice to meet time-specific therapy recommendations. The ReAcT study aims to develop an in-depth understanding of stroke therapy provision, including how the guideline of 45 min a day of each relevant therapy, is interpreted and implemented by therapists, and how it is experienced by stroke-survivors and their families. A multisite ethnographic case study design in a minimum of six stroke units will include modified process mapping, observations of service organisation, therapy delivery and documentary analysis. Semistructured interviews with therapists and service managers (n=90), and with patients and informal carers (n=60 pairs) will be conducted. Data will be analysed using the Framework approach. The study received a favourable ethical opinion via the National Research Ethics Service (reference number: 14/NW/0266). Participants will provide written informed consent or, where stroke-survivors lack capacity, a consultee declaration will be sought. ReAcT is designed to generate insights into the organisational, professional, social, practical and patient-related factors acting as facilitators or barriers to providing the recommended amount of therapy. Provisional recommendations will be debated in consensus meetings with stakeholders who have not participated in ReAcT case studies or interviews. Final recommendations will be disseminated to therapists, service managers, clinical guideline developers and policymakers and stroke-survivors and informal carers.


Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer.
Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.


Epigenetic activation of immune genes in cervical cancer.
Immune system provides us protection from infectious pathogens and tumors formation during lifetime. Cervical cancer (CC), and its cause, human papillomavirus (HPV) are both challenges for the immune system. We present here evidence of epigenetic activation of immune system genes in CC. Illumina Infinium Human Methylation 450K BeadChip identified genes, which were all significantly hypomethylated in CC tissue versus normal tissue. The GeneMANIA computer program identified a tight network between those genes. The most strongly correlated genes based on their function are immune effectors' process (AIM2, BST2, BTN3A3, and IL12RB1) and response to virus related genes (AIM2, BST2, and IL12RB1). Thus, activation of those genes through demethylation is probably triggered by HPV oncogenes. In conclusion, the immune system of women who do not develop CC is probably activated earlier through DNA demethylation.


Assessing the health status of children entering foster care.
Most research on health problems of children in foster care has been cross-sectional, resulting in overselection of children who have been in care long-term and underrepresentation of children who are in care for a short time. This paper reports on the health of a large cohort of children who had complete health examinations at the time of entry into foster care in a middle-size city during a 2-year period. Results indicate that > 90% of the children had an abnormality in at least one body system, 25% failed the vision screen, and 15% failed the hearing screen. The children were also lighter and shorter than the norm. Mental health screening revealed that 75% had a family history of mental illness or drug or alcohol abuse. Of children older than 3 years of age, 15% admitted to or were suspect for suicidal ideation and 7% for homicidal ideation. Of the children younger than 5 years of age, 23% had abnormal or suspect results on developmental screening examinations. At the time of entry into foster care, 12% of the children required an antibiotic. More than half needed urgent or nonurgent referrals for medical services and, for children > 3 years of age, more than half needed urgent or nonurgent referrals for dental and mental health services. Just 12% of the children required only routine follow-up care. The high prevalence and broad range of health needs of children at the time they enter foster care necessitate the design and implementation of better models of health care delivery for children in foster care.


Transitioning From a Level II to Level I Trauma Center Increases Resident Patient Exposure.
Increased patient exposure has been shown to improve residency training as determined by better patient outcomes. The transition of John Peter Smith Hospital from a level II to a level I trauma center in 2009 provided a unique opportunity to investigate the direct effects of increased patient exposure on residency training in a relatively controlled setting. We evaluated the effect of the transition to a level I trauma center on residency training. In 2014, we examined the annual facility reports and separated the data into 2 groups: level II (2001 to 2008) and level I (2010 to 2013). The primary outcome measures were patient volume, surgical volume, patient acuity, and scholarly activity by the residents. The patient volume in all units increased significantly (p < .05 for all) after the transition to a level I center. The surgical volume increased significantly for the general surgery, orthopedics, and podiatry departments (p < .05 for all) but remained unchanged in the gynecology and oral maxillofacial surgery departments. The volume measures were performed on all 98 residents (100%). Patient acuity and scholarly activity increased by 17% and 52%, respectively; however, the differences in these data were not statistically significant. The scholarly activity per resident was measured for the orthopedic and podiatry departments. For those departments, the total number of residents was 30, and scholarly activity was measured for 100% of them. Overall, resident education improved when the hospital transitioned to a level I trauma center, although certain subspecialties benefited more than did others from this transition.


Is close monitoring in the intensive care unit necessary after elective liver resection?
Many surgical patients are admitted to the intensive care unit (ICU), resulting in an increased demand, and possible waste, of resources. Patients who undergo liver resection are also transferred postoperatively to the ICU. However, this may not be necessary in all cases. This study was designed to assess the necessity of ICU admission. The medical records of 313 patients who underwent liver resections, as performed by a single surgeon from March 2000 to December 2010 were retrospectively reviewed. Among 313 patients, 168 patients (53.7%) were treated in the ICU. 148 patients (88.1%) received only observation during the ICU care. The ICU re-admission and intensive medical treatment significantly correlated with major liver resection (odds ratio [OR], 6.481; P = 0.011), and intraoperative transfusions (OR, 7.108; P = 0.016). Patients who underwent major liver resection and intraoperative transfusion were significantly associated with need for mechanical ventilator care, longer postoperative stays in the ICU and the hospital, and hospital mortality. Most patients admitted to the ICU after major liver resection just received close monitoring. Even though patients underwent major liver resection, patients without receipt of intraoperative transfusion could be sent to the general ward. Duration of ICU/hospital stay, ventilator care and mortality significantly correlated with major liver resection and intraoperative transfusion. Major liver resection and receipt of intraoperative transfusions should be considered indicators for ICU admission.


Reviewing deaths in British and US hospitals: a study of two scales for assessing preventability.
Standardised mortality ratios do not provide accurate measures of preventable mortality. This has generated interest in using case notes to assess the preventable component of mortality. But, different methods of measurement have not been compared. We compared the reliability of two scales for assessing preventability and the correspondence between them. Medical specialists reviewed case notes of patients who had died in hospital, using two instruments: a five-point Likert scale and a continuous (0-100) scale of preventability. To enhance generalisability, we used two different hospital datasets with different types of acute medical patients across different epochs, and in two jurisdictions (UK and USA). We investigated the reliability of measurement and correspondence of preventability estimates across the two scales. Ordinal mixed effects regression methods were used to analyse the Likert scale and to calibrate it against the continuous scale. We report the estimates of the probability a death could have been prevented, accounting for reviewer inconsistency. Correspondence between the two scales was strong; the Likert categories explained most of the variation (76% UK, 73% USA) in the continuous scale. Measurement reliability was low, but similar across the two instruments in each dataset (intraclass correlation: 0.27, UK; 0.23, USA). Adjusting for the inconsistency of reviewer judgements reduced the proportion of cases with high preventability, such that the proportion of all deaths judged probably or definitely preventable on the balance of probability was less than 1%. The correspondence is high between a Likert and a continuous scale, although the low reliability of both would suggest careful measurement design would be needed to use either scale. Few to no cases are above the threshold when using a balance of probability approach to determining a preventable death, and in any case, there is little evidence supporting anything more than an ordinal correspondence between these reviewer estimates of probability and the true probability. Thus, it would be more defensible to use them as an ordinal measure of the quality of care received by patients who died in the hospital.


Improved frame-based estimation of head motion in PET brain imaging.
Head motion during PET brain imaging can cause significant degradation of image quality. Several authors have proposed ways to compensate for PET brain motion to restore image quality and improve quantitation. Head restraints can reduce movement but are unreliable; thus the need for alternative strategies such as data-driven motion estimation or external motion tracking. Herein, the authors present a data-driven motion estimation method using a preprocessing technique that allows the usage of very short duration frames, thus reducing the intraframe motion problem commonly observed in the multiple frame acquisition method. The list mode data for PET acquisition is uniformly divided into 5-s frames and images are reconstructed without attenuation correction. Interframe motion is estimated using a 3D multiresolution registration algorithm and subsequently compensated for. For this study, the authors used 8 PET brain studies that used F-18 FDG as the tracer and contained minor or no initial motion. After reconstruction and prior to motion estimation, known motion was introduced to each frame to simulate head motion during a PET acquisition. To investigate the trade-off in motion estimation and compensation with respect to frames of different length, the authors summed 5-s frames accordingly to produce 10 and 60 s frames. Summed images generated from the motion-compensated reconstructed frames were then compared to the original PET image reconstruction without motion compensation. The authors found that our method is able to compensate for both gradual and step-like motions using frame times as short as 5 s with a spatial accuracy of 0.2 mm on average. Complex volunteer motion involving all six degrees of freedom was estimated with lower accuracy (0.3 mm on average) than the other types investigated. Preprocessing of 5-s images was necessary for successful image registration. Since their method utilizes nonattenuation corrected frames, it is not susceptible to motion introduced between CT and PET acquisitions. The authors have shown that they can estimate motion for frames with time intervals as short as 5 s using nonattenuation corrected reconstructed FDG PET brain images. Intraframe motion in 60-s frames causes degradation of accuracy to about 2 mm based on the motion type.


Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells.
To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.


CpG hypermethylation of collagen type I alpha 2 contributes to proliferation and migration activity of human bladder cancer.
In our microarray screening of methylated genes in bladder cancer (BC), the collagen type 1 alpha 2 (COL1A2) gene was the most up-regulated among the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC. We subjected a bladder cancer cell line (BOY) and 67 BC specimens and 10 normal bladder epitheliums (NBEs) to conventional or real-time methylation quantitative polymerase chain reaction (PCR) and to real-time reverse transcriptase (RT)-PCR. We also established a stable COL1A2 transfectant for evaluating cell proliferation and migration activity. After 5-aza-dC treatment, the expression levels of COL1A2 mRNA transcript markedly increased in BOY. Our cell proliferation assays consistently demonstrated growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (p<0.0001). Wound healing assays also showed significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (p=0.0016). We demonstrated by bisulfite DNA sequencing that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index of COL1A2 was significantly higher in the 67 BCs than in the 10 NBEs (p=0.0011). Conversely, COL1A2 mRNA transcript was significantly lower in the BCs than in the NBEs (p=0.0052). The mechanism of COL1A2 down-regulation in BC is through CpG hypermethylation of the promoter region. COL1A2 gene inactivation through CpG hypermethylation may contribute to proliferation and migration activity of BC.


Cell response in bronchoalveolar lavage fluid after sulfur dioxide exposure.
Environmental chamber exposure and bronchoalveolar lavage (BAL) were used to study the dose-response relationship between short-term exposure to sulfur dioxide (SO2) and inflammatory reactions in the human lung as reflected in BAL fluid. Healthy subjects were exposed to 10, 13, 20, or 30 mg/m3 for 20 min. BAL was performed several weeks preexposure and 24 h postexposure. Mast cells, lymphocytes, lysozyme positive macrophages, and the total number of macrophages were significantly increased after SO2 exposure. A dose-dependent increase in the cell response in BAL fluid was observed after exposure to 10-20 mg/m3, but no further increase was detected after 30 mg/m3. Inflammatory cell response was found in BAL fluid at SO2 levels that occur in industrial indoor environments worldwide, and cell response to SO2 was also seen below the short-term exposure limit of Sweden and many other countries (13 mg/m3).


Patients' perceptions and experiences of patient safety in primary care in England.
One of the most remarkable features of patient safety research in primary care is the sparse attention paid to patients' own experiences. To explore patient's perceptions and experiences of patient safety in primary care in England. We conducted a qualitative study in the South of England with an opportunistic sample of 27 primary care users. Information was obtained from four patient focus groups. A thematic content analysis was conducted by three analysts and consensus reached within the research team on the key themes that emerged. Participants' conceptualizations of patient safety referred to high standards of health care delivery within a relationship of trust. Participants identified four main factors that they believed could potentially affect patient safety. These included factors related to (i) the patient (attitudes, behaviours and health literacy); (ii) the health professional (attitudes, behaviours and accuracy of diagnoses); (iii) the relationship between patients and health professionals (communication and trust); and (iv) the health care system (workload, resources, care coordination, accessibility, interdisciplinary teamwork and accuracy of health care records). Confidentiality, continuity of care and treatment-related safety emerged as cross-cutting major threats to patient safety. The exploration of participants' perceptions and experiences allowed the identification of a wide variety of themes that were perceived to impact on patient safety in primary care. The findings of this study could be used to enrich current frameworks that are exclusively based on professional or health care system perspectives.


[Gene therapy using anticancer drug-resistance genes].
Myelosuppression is a major dose-limiting factor in cancer chemotherapy. Introduction of drug-resistance genes into bone marrow cells of cancer patients has been proposed to overcome this limitation. In theory, any gene whose expression protects cells against the toxic effects of chemotherapy should be useful in vivo for this purpose. Among such genes, human multidrug-resistance gene (MDR1) has been studied most extensively for this purpose, and clinical trials of drug-resistance gene therapy have been started in the US for cancer patients who undergo high-dose chemotherapy with autologous hematopoietic stem cell transplantation. In Japan, our clinical protocol of MDR1 gene therapy "A clinical study of drug-resistance gene therapy to improve the efficacy and safety of chemotherapy against breast cancer" has been submitted to the government. To improve the efficacy and safety of this drug-resistance gene therapy, we have constructed a series of MDR1-bicistronic retrovirus vectors using a retrovirus backbone of Harvey murine sarcoma virus and internal ribosome entry site (IRES) from picornavirus to co-express a second gene with the MDR1 gene. MDR1-MGMT bicistronic vectors can be used to protect bone marrow cells of cancer patients from combination chemotherapy with MDR1-related anticancer agents and nitrosoureas. In addition, MDR1-bicistronic retrovirus vectors can be designed to use the MDR1 gene as an in vivo selectable marker to enrich the transduced cells which express therapeutic genes, if disease is curable by the expression of a single-peptide gene in any types of bone marrow cells or peripheral blood cells.


Forced Migration and Changing Livelihoods in the Brazilian Amazon.
Forced migration due to development projects or environmental change impacts livelihoods, as affected households are faced with new-and often less favorable-environmental, social, and economic conditions. This article examines changing livelihood strategies among a population of rural agricultural households displaced by the Belo Monte Dam in the Brazilian Amazon. Using longitudinal data, I find that many households used compensation payments to concentrate income generation efforts on the most lucrative strategies-cacao and cattle production and business or rental income. Poorer households and those that received the least compensation were more likely to continue relying on agricultural wage labor-a less desirable income source associated with not owning land or with persons needing to supplement income with additional work as a day laborer. Results also indicate that the amount of compensation received by most households was sufficient to enable them to make productive investments beyond attaining replacement land and housing. Many households invested in assets such as agricultural infrastructure, cattle, rental houses, or tractors-all of which directly contribute to future income. Displacement compensation, similar to remittances or conditional cash transfers, can therefore act as an important infusion of capital to promote socioeconomic development and poverty reduction.


The effect of oral preexposure prophylaxis on the progression of HIV-1 seroconversion.
To investigate whether oral preexposure prophylaxis (PrEP) alters timing and patterns of seroconversion when PrEP use continues after HIV-1 infection. Retrospective testing of the timing of Fiebig stage HIV-1 seroconversion in the Partners PrEP Study, a randomized placebo-controlled clinical trial of PrEP conducted in Kenya and Uganda. Specimens from 138 seroconverters were collected every 3 months and when HIV-1 infection was suspected based on monthly rapid HIV-1 tests. Progression of seroconversion was compared between randomized groups (PrEP versus placebo) and per-protocol groups (placebo versus PrEP participants with detectable tenofovir during the seroconversion period) using laboratory assessment of Fiebig stage. Delay in site-detection of seroconversion and association with PrEP drug-regimen resistant virus were assessed using logistic regression. Analysis of time to each Fiebig stage used maximum likelihood estimation with a parametric model to accommodate the varying lengths of HIV-infection intervals. There was a significant increase in delayed site detection of infection associated with PrEP (odds ratio = 3.49, P = 0.044). Delay in detection was not associated with increased risk of resistance in the PrEP arm (odds ratio = 0.93, P = 0.95). Estimated time to each Fiebig stage was elongated in seroconverters with evidence of ongoing PrEP use, significantly for only Stage 5 (28 versus 17 days, P = 0.05). Adjusted for Fiebig stage, viral RNA was ∼2/3 log lower in those assigned to PrEP compared with placebo; no differences were found in Architect signal to cut-off at any stage. Ongoing PrEP use in seroconverters may delay detection of infection and elongate seroconversion, although the delay does not increase risk of resistance.


Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae.
The circular, single-stranded DNA genome of a novel circovirus of canaries, tentatively named canary circovirus (CaCV), was cloned and sequenced. Sequence analysis indicated that the genome was 1952 nucleotides (nt) in size and had the potential to encode three viral proteins, including the putative capsid and replication-associated (Rep) proteins. The CaCV genome shared greatest sequence similarity (58.3% nt identity) with the newly characterized columbid circovirus (CoCV) and was more distantly related to the two porcine circovirus strains, PCV1 and PCV2, beak and feather disease virus (BFDV) and a recently isolated goose circovirus (GCV) isolate (46.8-50.9% nt identity). In common with other members of the Circovirus genus, several nt structures and amino acid motifs thought to be implicated in virus replication were identified on the putative viral strand. Phylogenetic analysis of both the capsid and Rep protein-coding regions provided further evidence that CaCV is more closely related to CoCV and BFDV and more distantly related to GCV, PCV1 and PCV2.


Allograft Inflammatory Factor-1 Links T-Cell Activation, Interferon Response, and Macrophage Activation in Chronic Kawasaki Disease Arteritis.
Kawasaki disease (KD) is widely viewed as an acute arteritis. However, our pathologic studies show that chronic coronary arteritis can persist long after disease onset and is closely linked with arterial stenosis. Transcriptome profiling of acute KD arteritis tissues revealed upregulation of T lymphocyte, type I interferon, and allograft inflammatory factor-1 (AIF1) genes. We determined whether these immune responses persist in chronic KD arteritis, and we investigated the role of AIF1 in these responses. Gene expression in chronic KD and childhood control arteries was determined by real-time reverse-transcriptase polymerase chain reaction, and arterial protein expression was determined by immunohistochemistry and immunofluorescence. Allograft inflammatory factor-1 small-interfering ribonucleic acid macrophage treatment was performed to investigate the role of AIF1 in macrophage and T lymphocyte activation. Allograft inflammatory factor-1 protein was highly expressed in stenotic KD arteries and colocalized with the macrophage marker CD68. T lymphocyte and interferon pathway genes were significantly upregulated in chronic KD coronary artery tissues. Alpha interferon-induced macrophage expression of CD80 and major histocompatibility complex class II was dependent on AIF1, and macrophage expression of AIF1 was required for antigen-specific T lymphocyte activation. Allograft inflammatory factor-1, originally identified in posttransplant arterial stenosis, is markedly upregulated in KD stenotic arterial tissues. T lymphocyte and type I interferon responses persist in chronic KD arteritis. Allograft inflammatory factor-1 may play multiple roles linking type I interferon response, macrophage activation, and antigen-specific T lymphocyte activation. These results suggest the likely importance of lymphocyte-myeloid cell cross-talk in the pathogenesis of KD arteritis and can inform selection of new immunotherapies for clinical trials in high-risk KD children.


A full-length recombinant Plasmodium falciparum PfRH5 protein induces inhibitory antibodies that are effective across common PfRH5 genetic variants.
The lack of an effective licensed vaccine remains one of the most significant gaps in the portfolio of tools being developed to eliminate Plasmodium falciparum malaria. Vaccines targeting erythrocyte invasion - an essential step for both parasite development and malaria pathogenesis - have faced the particular challenge of genetic diversity. Immunity-driven balancing selection pressure on parasite invasion proteins often results in the presence of multiple, antigenically distinct, variants within a population, leading to variant-specific immune responses. Such variation makes it difficult to design a vaccine that covers the full range of diversity, and could potentially facilitate the evolution of vaccine-resistant parasite strains. In this study, we investigate the effect of genetic diversity on invasion inhibition by antibodies to a high priority P. falciparum invasion candidate antigen, P. falciparum Reticulocyte Binding Protein Homologue 5 (PfRH5). Previous work has shown that virally delivered PfRH5 can induce antibodies that protect against a wide range of genetic variants. Here, we show that a full-length recombinant PfRH5 protein expressed in mammalian cells is biochemically active, as judged by saturable binding to its receptor, basigin, and is able to induce antibodies that strongly inhibit P. falciparum growth and invasion. Whole genome sequencing of 290 clinical P. falciparum isolates from across the world identifies only five non-synonymous PfRH5 SNPs that are present at frequencies of 10% or more in at least one geographical region. Antibodies raised against the 3D7 variant of PfRH5 were able to inhibit nine different P. falciparum strains, which between them included all of the five most common PfRH5 SNPs in this dataset, with no evidence for strain-specific immunity. We conclude that protein-based PfRH5 vaccines are an urgent priority for human efficacy trials.


Genetic dissection of complex behaviour traits in German Shepherd dogs.
A favourable genetic structure and diversity of behavioural features highlights the potential of dogs for studying the genetic architecture of behaviour traits. However, behaviours are complex traits, which have been shown to be influenced by numerous genetic and non-genetic factors, complicating their analysis. In this study, the genetic contribution to behaviour variation in German Shepherd dogs (GSDs) was analysed using genomic approaches. GSDs were phenotyped for behaviour traits using the established Canine Behavioural Assessment and Research Questionnaire (C-BARQ). Genome-wide association study (GWAS) and regional heritability mapping (RHM) approaches were employed to identify associations between behaviour traits and genetic variants, while accounting for relevant non-genetic factors. By combining these complementary methods we endeavoured to increase the power to detect loci with small effects. Several behavioural traits exhibited moderate heritabilities, with the highest identified for Human-directed playfulness, a trait characterised by positive interactions with humans. We identified several genomic regions associated with one or more of the analysed behaviour traits. Some candidate genes located in these regions were previously linked to behavioural disorders in humans, suggesting a new context for their influence on behaviour characteristics. Overall, the results support dogs as a valuable resource to dissect the genetic architecture of behaviour traits and also highlight the value of focusing on a single breed in order to control for background genetic effects and thus avoid limitations of between-breed analyses.


Long-term functional outcomes and predictors of shunt-dependent hydrocephalus after treatment of ruptured intracranial aneurysms in the BRAT trial: revisiting the clip vs coil debate.
Acute hydrocephalus is a well-known sequela of aneurysmal subarachnoid hemorrhage (SAH). Controversy exists about whether open microsurgical methods serve to reduce shunt dependency compared with endovascular techniques. To determine predictors of shunt-dependent hydrocephalus and functional outcomes after aneurysmal SAH. A total of 471 patients who were part of a prospective, randomized, controlled trial from 2003 to 2007 were retrospectively reviewed. All variables including demographic data, medical history, treatment, imaging, and functional outcomes were included as part of the trial. No additional variables were retrospectively collected. Ultimately, 147 patients (31.2%) required a ventriculoperitoneal shunt (VPS) in our series. Age, dissecting aneurysm type, ruptured vertebrobasilar aneurysm, Fisher grade, Hunt and Hess grade, admission intraventricular hemorrhage, admission intraparenchymal hemorrhage, blood in the fourth ventricle on admission, perioperative ventriculostomy, and hemicraniectomy were significant risk factors (P < .05) associated with shunt-dependent hydrocephalus on univariate analysis. On multivariate analysis, intraventricular hemorrhage and intraparenchymal hemorrhage were independent risk factors for shunt dependency (P < .05). Clipping vs coiling treatment was not statistically associated with VPS after SAH on both univariate and multivariate analyses. Patients who did not receive a VPS at discharge had higher Glasgow Outcome Scale and Barthel Index scores and were more likely to be functionally independent and to return to work 72 months after surgery (P < .05). There is no difference in shunt dependency after SAH among patients treated by endovascular or microsurgical means. Patients in whom shunt-dependent hydrocephalus does not develop after SAH tend to have improved long-term functional outcomes.


Song competition affects monoamine levels in sensory and motor forebrain regions of male Lincoln's sparrows (Melospiza lincolnii).
Male animals often change their behavior in response to the level of competition for mates. Male Lincoln's sparrows (Melospiza lincolnii) modulate their competitive singing over the period of a week as a function of the level of challenge associated with competitors' songs. Differences in song challenge and associated shifts in competitive state should be accompanied by neural changes, potentially in regions that regulate perception and song production. The monoamines mediate neural plasticity in response to environmental cues to achieve shifts in behavioral state. Therefore, using high pressure liquid chromatography with electrochemical detection, we compared levels of monoamines and their metabolites from male Lincoln's sparrows exposed to songs categorized as more or less challenging. We compared levels of norepinephrine and its principal metabolite in two perceptual regions of the auditory telencephalon, the caudomedial nidopallium and the caudomedial mesopallium (CMM), because this chemical is implicated in modulating auditory sensitivity to song. We also measured the levels of dopamine and its principal metabolite in two song control nuclei, area X and the robust nucleus of the arcopallium (RA), because dopamine is implicated in regulating song output. We measured the levels of serotonin and its principal metabolite in all four brain regions because this monoamine is implicated in perception and behavioral output and is found throughout the avian forebrain. After controlling for recent singing, we found that males exposed to more challenging song had higher levels of norepinephrine metabolite in the CMM and lower levels of serotonin in the RA. Collectively, these findings are consistent with norepinephrine in perceptual brain regions and serotonin in song control regions contributing to neuroplasticity that underlies socially-induced changes in behavioral state.


In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells.
Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.


STUDIES ON THE SHWARTZMAN PHENOMENON : I. DETOXIFICATION OF MENINGOCOCCUS CULTURE FILTRATES.
Formalin induces a considerable change in meningococcus culture filtrates. This consists of a marked decrease in toxidty as concerns both the Shwartzman phenomenon and the lethal effect, with relative preservation of the antibody-combining capacity and antigenicity. A similar modification occurs spontaneously in meningococcus culture filtrates on standing. Inasmuch as these changes parallel those occurring in the conversion of diphtheria toxin into toxoid, it is justifiable to consider such altered meningococcus toxin as meningococcus toxoid.


Cannabis and psychosis/schizophrenia: human studies.
The association between cannabis use and psychosis has long been recognized. Recent advances in knowledge about cannabinoid receptor function have renewed interest in this association. Converging lines of evidence suggest that cannabinoids can produce a full range of transient schizophrenia-like positive, negative, and cognitive symptoms in some healthy individuals. Also clear is that in individuals with an established psychotic disorder, cannabinoids can exacerbate symptoms, trigger relapse, and have negative consequences on the course of the illness. The mechanisms by which cannabinoids produce transient psychotic symptoms, while unclear may involve dopamine, GABA, and glutamate neurotransmission. However, only a very small proportion of the general population exposed to cannabinoids develop a psychotic illness. It is likely that cannabis exposure is a "component cause" that interacts with other factors to "cause" schizophrenia or a psychotic disorder, but is neither necessary nor sufficient to do so alone. Nevertheless, in the absence of known causes of schizophrenia, the role of component causes remains important and warrants further study. Dose, duration of exposure, and the age of first exposure to cannabinoids may be important factors, and genetic factors that interact with cannabinoid exposure to moderate or amplify the risk of a psychotic disorder are beginning to be elucidated. The mechanisms by which exposure to cannabinoids increase the risk for developing a psychotic disorder are unknown. However, novel hypotheses including the role of cannabinoids on neurodevelopmental processes relevant to psychotic disorders are being studied.


Persistent microbial dysbiosis in preterm premature rupture of membranes from onset until delivery.
Background. Preterm Premature Rupture of Membranes (PPROM) is a major leading cause of preterm births. While the cause for PPROM remains unidentified, it is anticipated to be due to subclinical infection, since a large proportion of PPROM patients display signs of chorioamnionitis. Since subclinical infections can be facilitated by dysbiosis, our goal was to characterize the vaginal microbiome and amniotic fluid discharge upon PPROM, through latency antibiotic treatment, and until delivery, to detect the presence of pathogens, microbiota alteration, and microbial response to treatment. Methods. Enrolled subjects (15) underwent routine institutional antenatal care for PPROM, including the administration of latency antibiotics. Serial vaginal swabs were obtained from diagnosis of PPROM through delivery and the sequencing of the V3-V5 region of the 16S rRNA gene was performed for all collected samples. Results. The results show that Lactobacilli species were markedly decreased when compared to vaginal swabs collected from uncomplicated pregnancy subjects with a matched gestational time. Prevotella and Peptoniphilus were the most prevalent taxa in PPROM subjects at presentation. The vaginal microbiome of the PPROM subjects varied substantially intra- and inter-subjects. Several taxa were found to be significantly reduced during and after the antibiotic treatment: Weeksella, Lachnospira, Achromobacter, and Pediococcus. In contrast, Peptostreptococcus and Tissierellaceae ph2 displayed a significant increase after the antibiotic treatment. However, the relative abundance of Lactobacillus, Prevotella, and Peptoniphilus was not substantially impacted during the hospitalization of the PPROM subjects. The deficiency of Lactobacillus, and constancy of known pathogenic species, such as Prevotella and Peptoniphilus during and after antibiotics, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches. Discussion. PPROM is responsible for one third of all preterm births. It is thought that subclinical infection is a crucial factor in the pathophysiology of PPROM because 25-40% of patients present signs of chorioamnionitis on amniocentesis. Here we sought to directly assess the bacterial content of the vagina and leaking amniotic fluid of subjects at presentation, throughout treatment and up until delivery, in order to search for common pathogens, microbiota changes, and microbial response to latency antibiotic treatment. We have found that the vaginal microbiome of PPROM subjects is highly variable and displays significant changes to treatment. However, the unchanging deficiency of Lactobacillus, and persistence of known pathogenic species, such as Prevotella and Peptoniphilus from presentation, through antibiotic treatment and up until delivery, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches.


Effect of gingival displacement cord and cordless systems on the closure, displacement, and inflammation of the gingival crevice.
The gingival sulcus should remain open long enough for the impression material to flow into it and completely fill the space provided by the gingival displacement. Impressions with less sulcal width have a higher incidence of voids, tearing of impression materials, and reduction in marginal accuracy. The purpose of this clinical study was to investigate the closure, gingival displacement, and gingival inflammation of the gingival crevice after the use of medicated gingival displacement cord and cordless systems. Gingival sulcus closure was studied in 40 participants. They were divided into 4 groups: 2 cord (Ultrapack, SilTrax AS) and 2 cordless (Expasyl, Traxodent Hemodent paste) methods. The labial surfaces of the maxillary right and left central incisors were evaluated. Gingival sulcus was photographed every 20 seconds from 0 to 180 seconds after the removal of the cord or cordless system. The bleeding index (BI) and gingival index (GI) were measured at day 0, day 1, and day 7. The width of the sulcal orifice was measured at the mid-buccal (MB) and transitional line angle (TLA) on a digital image, using computer software (Photoshop version 7.0; Adobe). Data were analyzed with ANOVA, Tukey honest significant difference (HSD), Kruskal-Wallis, and Mann-Whitney U tests (α=.05). All groups showed a sulcal width greater than 0.22 mm up to 60 seconds after the removal of the displacement materials at the MB and up to 40 seconds at the TLA. Among all groups, Expasyl showed the fastest closure. Gingival displacement in the MB area for the cord group was greater than for the cordless groups. GI and BI indices were larger for the cord group than for the cordless group at days 0 and 1. At up to 60 seconds, the cord and cordless techniques were equally effective. The cord group showed a greater amount of displacement than the cordless group. However, the cordless materials showed reduced frequency of changes to the gingival index.


Endotracheal cuff pressures in the PICU: Incidence of underinflation and overinflation.
While uncuffed endotracheal tubes have been traditionally used in the pediatric intensive care unit (PICU) population, evidence suggests cuffed endotracheal tubes (ETTs) are also safe to use within this population. Nevertheless, risks related to the use of cuffed ETTs increase when guidelines for safe and appropriate use are not followed. The primary goal of this study was to measure the cuff pressure (CP) using a cuff pressure manometer in a group of intubated pediatric subjects and determine the rate of cuff underinflation (<20 cm H20) or overinflation (>30 cm H20). The secondary aim was to determine whether CP was associated to gender, age, ETT size, and PICU length of stay prior to CP measurement. This was a prospective observational study conducted in an urban PICU. Pediatric subjects intubated with cuffed ETTs from 1 April 2017 to 1 May 2017 were included in the study. ETT CPs were measured daily to determine degree of inflation and compared according to gender, age, ETT size, and number of days intubated prior to CP measurement. Descriptive data are expressed as means and standard deviations. A two-sample t test was used to compare groups according to age, gender, and number of days present. And significance was considered with a P < 0.05. Pearson chi test was used to evaluate correlation between CPs and size of the ETT, number of days intubated prior to CP measurement, gender, and age. Twenty pediatric subjects admitted during the study period were included for analysis. Eleven cuff measurements were found to be within normal limits, while 9 cuff measurements were found to be underinflated. No cases of overinflation were found. There were no significant associations between CP and size of the ETT (r = -0.08), number of days intubated prior to CP measurement (r = 0.19), gender (r = 0.09), and age (r = 0.12). Our study suggests that endotracheal cuff underinflation occurs often in the PICU population. Strategies to ensure appropriate ETT CPs are maintained are essential in the intubated pediatric population. Additional studies are necessary to develop interventions and training focused on the use of a cuff pressure manometer to measure CPs in the PICU by respiratory therapists and ensure consistent measurement using inter rater evaluation processes are needed.