Immunohistochemical analysis of bcl-2 expression in transitional cell carcinoma of the bladder.
To evaluate the expression of bcl-2 in transitional cell carcinoma (TCC) of the bladder; to compare bcl-2 expression with clinicopathological findings, p53 immunoreactivity, proliferating cell nuclear antigen (PCNA) expression, 2c deviation index (2cDI), 5c exceeding rate (5cER), and the mean nuclear area (MNA). Cystectomy specimens from 77 patients with untreated, non-metastatic TCC of the bladder were studied. Expression of bcl-2, p53 and PCNA was detected immunohistochemically using the following monoclonal antibodies: bcl-2/124, DO-7 and PC10, respectively. Nuclear DNA content was analysed using static cytometry. Bcl-2 was expressed in 19 (24.7%) of 77 TCCs and in 74 (96.1%) of 77 normal samples of transitional epithelium (taken from normal tissue adjacent to the tumour in each case). In all cases, bcl-2 immunoreactivity was more intense in normal transitional epithelium than in TCC. In normal transitional epitehlium and superficial TCC bcl-2 immunoreactivity was observed at the basal layer, and not at the invasive front. Bcl-2 immunoreactivity was invesely correlated with histological grade and p53 immunoreactivity, and was not correlated with the pT category, disease progression, PCNA expression, 2cDI, 5cER, and the MNA. No significant correlation was found between bcl-2 expression and overall survival. Bcl-2 expression in TCC of the bladder seems to be associated with a less aggressive phenotype and does not play an important role in tumour progression.


Current End-of-Life Care Needs and Care Practices in Acute Care Hospitals.
A descriptive-comparative study was undertaken to examine current end-of-life care needs and practices in hospital. A chart review for all 1,018 persons who died from August 1, 2008 through July 31, 2009 in two full-service Canadian hospitals was conducted. Most decedents were elderly (73.8%) and urbanite (79.5%), and cancer was the most common diagnosis (36.2%). Only 13.8% had CPR performed at some point during this hospitalization and 8.8% had CPR immediately preceding death, with 87.5% having a DNR order and 30.8% providing an advance directive. Most (97.3%) had one or more life-sustaining technologies in use at the time of death. These figures indicate, when compared to those in a similar mid-1990s Canadian study, that impending death is more often openly recognized and addressed. Technologies continue to be routinely but controversially used. The increased rate of end-stage CPR from 2.9% to 8.8% could reflect a 1994+ shift of expected deaths out of hospital.


Molecular eco-systems biology: towards an understanding of community function.
Systems-biology approaches, which are driven by genome sequencing and high-throughput functional genomics data, are revolutionizing single-cell-organism biology. With the advent of various high-throughput techniques that aim to characterize complete microbial ecosystems (metagenomics, meta-transcriptomics and meta-metabolomics), we propose that the time is ripe to consider molecular systems biology at the ecosystem level (eco-systems biology). Here, we discuss the necessary data types that are required to unite molecular microbiology and ecology to develop an understanding of community function and discuss the potential shortcomings of these approaches.


Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns.
An outbreak of R-factor-mediated carbenicillin resistance in Pseudomonas aeruginosa in burned patients in March 1969 was followed by a second outbreak 6 months later. No R-factor-carrying P. aeruginosa strains were detected in the intervening period but R-factor-determined lactamase was commonly encountered, particularly in Klebsiella aerogenes strains. A comparison of the molecular properties of the R factors in pseudomonads from the first and second phases with those in the Klebsiella strains from the intervening period showed them to be very closely related. A single R-factor type therefore may have been maintained in the Burns Unit between the two Pseudomonas outbreaks as a plasmid conferring resistance to ampicillin in K. aerogenes.


Evaluation of the NOD/SCID xenograft model for glucocorticoid-regulated gene expression in childhood B-cell precursor acute lymphoblastic leukemia.
Glucocorticoids such as prednisolone and dexamethasone are critical drugs used in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL), and response to glucocorticoids is highly predictive of outcome. The NOD/SCID xenograft mouse model of ALL is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Cells from a glucocorticoid-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. When highly engrafted the mice were randomized into groups of 4 to receive dexamethasone 15 mg/kg by intraperitoneal injection or vehicle control. Leukemia cells were harvested from mice spleens at 0, 8, 24 or 48 hours thereafter, and gene expression analyzed on Illumina WG-6_V3 chips, comparing all groups to time 0 hours. The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes, with fewer observed at the 24 and 48 hour timepoints, and with minimal changes seen across the time-matched controls. When compared to publicly available datasets of glucocorticoid-induced gene expression from an in vitro cell line study and from an in vivo study of patients with ALL, at the level of pathways, expression changes in the 8 hour xenograft samples showed a similar response to patients treated with glucocorticoids. Replicate analysis revealed that at the 8 hour timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of > 0.9. However at other timepoints with less signal very poor recovery scores were obtained with 3 replicates. The NOD/SCID xenograft mouse model provides a reproducible experimental system in which to investigate clinically-relevant mechanisms of drug-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates.


Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage.
Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.


Resistant hypertension and obstructive sleep apnea.
Hypertension (HTN) is a modifiable, highly prevalent risk factor for cardiovascular morbidity and renal dysfunction worldwide. In the United States, HTN affects one in three adults, contributes to one out of every seven deaths and to nearly half of all cardiovascular disease-related deaths. HTN is considered resistant when the blood pressure remains above goal despite lifestyle modification and administration of three antihypertensive agents of different classes including a diuretic. Large population-based studies have suggested that obstructive sleep apnea (OSA) is a risk factor for resistant HTN. The mechanism proposed is a pattern of intermittent hypoxia associated with hyperaldosteronism, increased sympathetic tone, endothelial dysfunction, and inflammation. In this review we discuss the association between OSA and resistant HTN, the physiologic mechanisms linking OSA with resistant HTN, and the effect of continuous positive airway pressure therapy (CPAP) on blood pressure in patients with resistant HTN. While the reduction in blood pressure with CPAP is usually modest in patients with OSA, a decrease of only a few mmHg in blood pressure can significantly reduce cardiovascular risk. Patients presenting to a center specializing in management of hypertension should be screened and treated for OSA as a potentially modifiable risk factor.


Patients' preferences for therapy in advanced epithelial ovarian cancer: development, testing, and application of a bedside decision instrument.
The objective of this study was to determine a patient's preference for treatment in the poor prognostic situation of advanced epithelial ovarian cancer. A standardized method of information transfer was developed and pretested for feasibility, acceptability, comprehension, interobserver reliability, and validity. The Decision Board (DB) describes to the patient who has completed surgery for suboptimally debulked ovarian cancer two treatment options, their potential side effects, and possible outcomes. The psychometric properties of the DB were determined in 37 volunteers without cancer and 11 women following first-line chemotherapy for ovarian cancer. The Board was then administered to 12 patients with stage IIIc and IV ovarian cancer at the point of deciding future chemotherapy. The feasibility of presenting poor prognostic information, patient comprehension in an emotionally charged situation, and her treatment choice were determined. Observation of patient-physician interviews revealed that survival information is not usually discussed on the initial consultation. Using the DB, it was feasible to provide survival information to 98% of the healthy women. Comprehension was high, with 96% of the questions being correctly answered. Interobserver reliability was high (kappa = 1.00). The construct that hypothesized that understanding information is a determinant of choice was verified, as 95.2% of women had predictable shifts in expressed preference. The strength of preference in healthy women was statistically significantly higher for Plan B (paclitaxel-cisplatin) than for Plan A (cisplatin-cyclophosphamide) (P < 0.001). The method of presenting survival information (median survival versus percentage survival at 3.5 years) influenced choice (P < 0.04). At the point of decision making, 33% of patients with advanced ovarian cancer chose Plan A and 67% chose Plan B. All volunteers and patients stated that they wanted to be fully informed of the choices of therapy for their disease and to be involved in treatment decisions. The DB is a reliable and valid method for sharing information about advanced ovarian cancer with patients. At the point of deciding first-line chemotherapy in this poor prognostic situation, patients still value survival more highly than the quality of life during chemotherapy.


Lifecycle evidence requirements for high-risk implantable medical devices: a European perspective.
The new European Union (EU) Regulations on medical devices and on in vitro diagnostics provide manufacturers and Notified Bodies with new tools to improve pre-market and post-market clinical evidence generation especially for high-risk products but fail to indicate what type of clinical evidence is appropriate at each stage of the whole lifecycle of medical devices. In this paper we address: i) the appropriate level and timing of clinical evidence throughout the lifecycle of high-risk implantable medical devices; and ii) how the clinical evidence generation ecosystem could be adapted to optimize patient access. The European regulatory and health technology assessment (HTA) contexts are reviewed, in relation to the lifecycle of high-risk medical devices and clinical evidence generation recommended by international network or endorsed by regulatory and HTA agencies in different jurisdictions. Four stages are relevant for clinical evidence generation: i) pre-clinical, pre-market; ii) clinical, pre-market; iii) diffusion, post-market; and iv) obsolescence & replacement, post-market. Each stage has its own evaluation needs and specific studies are recommended to generate the appropriate evidence. Effective lifecycle planning requires anticipation of what evidence will be needed at each stage.


Three faces of entropy for complex systems: Information, thermodynamics, and the maximum entropy principle.
There are at least three distinct ways to conceptualize entropy: entropy as an extensive thermodynamic quantity of physical systems (Clausius, Boltzmann, Gibbs), entropy as a measure for information production of ergodic sources (Shannon), and entropy as a means for statistical inference on multinomial processes (Jaynes maximum entropy principle). Even though these notions represent fundamentally different concepts, the functional form of the entropy for thermodynamic systems in equilibrium, for ergodic sources in information theory, and for independent sampling processes in statistical systems, is degenerate, H(p)=-∑_{i}p_{i}logp_{i}. For many complex systems, which are typically history-dependent, nonergodic, and nonmultinomial, this is no longer the case. Here we show that for such processes, the three entropy concepts lead to different functional forms of entropy, which we will refer to as S_{EXT} for extensive entropy, S_{IT} for the source information rate in information theory, and S_{MEP} for the entropy functional that appears in the so-called maximum entropy principle, which characterizes the most likely observable distribution functions of a system. We explicitly compute these three entropy functionals for three concrete examples: for Pólya urn processes, which are simple self-reinforcing processes, for sample-space-reducing (SSR) processes, which are simple history dependent processes that are associated with power-law statistics, and finally for multinomial mixture processes.


Prospective analysis of strategies for diagnosing renovascular hypertension.
Renovascular hypertension is a potentially curable form of high blood pressure. However, it is unclear how best to select patients who are likely to have renovascular hypertension, what diagnostic strategy to use in these selected patients, and how to predict the hemodynamic significance of a renal artery stenosis. We determined the prevalence of renovascular hypertension in adults who exhibited suggestive clinical features. In these clinically selected patients, we then determined the test characteristics of various diagnostic and potential screening tests. Renovascular hypertension was diagnosed if correction of renal artery stenosis resulted in decreased blood pressure. Of the 66 hypertensive adults evaluated, 11 (16.7%) had renovascular hypertension. Captopril-stimulated peripheral renin activity detected renovascular hypertension with 73% sensitivity, 72% specificity, 38% positive predictive value, and 92% negative predictive value. Less optimal combinations of sensitivity and specificity were found for differential glomerular filtration rate renography, differential effective renal plasma flow renography, and selective renal vein renin ratios, each performed after a single dose of captopril. Intravenous digital subtraction renal angiography detected all patients with renovascular hypertension and was normal in 71% of patients with essential hypertension. To evaluate potential screening tests for renovascular hypertension, we calculated predictive values applied to a low prevalence population. If the observed sensitivities and specificities apply to a population with 5% prevalence of renovascular hypertension, captopril-stimulated peripheral renin would have a positive predictive value of 12% and a negative predictive value of 98%. In 16 patients with known renal artery stenosis, neither the captopril-stimulated renal vein renin ratio nor captopril-stimulated differential renography accurately predicted blood pressure response to correction of the stenosis. We conclude that clinical criteria can identify a subgroup with 16.7% prevalence of renovascular hypertension. In this high prevalence group, intravenous digital subtraction renal angiography will identify virtually all patients with renovascular hypertension, and a normal study will be sufficient to exclude renovascular hypertension. In unselected hypertensive patients, screening with captopril-stimulated peripheral renin activity may be the most useful and efficient procedure for identification of patients with renovascular hypertension. Functional tests do not accurately predict the hemodynamic significance of a renal artery stenosis.


Multisensory visual-auditory object recognition in humans: a high-density electrical mapping study.
Multisensory object-recognition processes were investigated by examining the combined influence of visual and auditory inputs upon object identification--in this case, pictures and vocalizations of animals. Behaviorally, subjects were significantly faster and more accurate at identifying targets when the picture and vocalization were matched (i.e. from the same animal), than when the target was represented in only one sensory modality. This behavioral enhancement was accompanied by a modulation of the evoked potential in the latency range and general topographic region of the visual evoked N1 component, which is associated with early feature processing in the ventral visual stream. High-density topographic mapping and dipole modeling of this multisensory effect were consistent with generators in lateral occipito-temporal cortices, suggesting that auditory inputs were modulating processing in regions of the lateral occipital cortices. Both the timing and scalp topography of this modulation suggests that there are multisensory effects during what is considered to be a relatively early stage of visual object-recognition processes, and that this modulation occurs in regions of the visual system that have traditionally been held to be unisensory processing areas. Multisensory inputs also modulated the visual 'selection-negativity', an attention dependent component of the evoked potential this is usually evoked when subjects selectively attend to a particular feature of a visual stimulus.


Rare Manifestation of Digoxin Toxicity: Right Bundle Branch Block.
A 76-year-old female, with medical history significant for systolic congestive heart failure, who presented to the emergency department with lethargy and abdominal pain with diarrhea for the past 3 weeks. Due to hypotension, the patient received multiple boluses of isotonic saline and was started on norepinephrine. Laboratories were significant for severe digoxin toxicity (29 ng/mL), in setting of acute kidney injury. Electrocardiogram (EKG) revealed a new right bundle branch block (RBBB). She was given Digibind and her repeat digoxin level was 20 ng/mL. Repeat EKG showed resolved RBBB. This case identifies that patients with digoxin toxicity are at risk for RBBB. This is a rare finding and is not commonly recognized. Emergency medicine physicians are often the first to encounter patients with digoxin toxicity and need to be aware of such EKG findings.


Implementation of four strategies in Dutch day-care centres to stimulate young children's fruit and vegetable consumption.
The day-care setting is an ideal place to encourage children's fruit and vegetable (=F&V) eating. Whereas many studies have focused on the effectiveness to increase F&V consumption, little is known about how to successfully implement effective strategies in daily practice. This study aimed to investigate how day-care professionals evaluated the implementation of a self-chosen strategy to support children's F&V eating. Thirteen day-care locations chose one out of five promising strategies and implemented this strategy for 10-12 weeks. Before (N = 98) and after the study (N = 49), day-care professionals completed a questionnaire to assess their implementation experiences, the impact on children's F&V eating as well as their future intention to use the strategy (on a 5-point scale). Parents (N = 152) completed a short questionnaire at the end of the study to capture their experiences and potential transfer effects to the home situation. Results showed that acceptability, appropriateness, feasibility and sustainability of the strategies were generally satisfactory (scores ≥3.5 on a 5-point scale), but the strategy of cooking scored less favourable on appropriateness and sustained implementation. Children's willingness to taste F&V varieties (3.4 ± 0.7 vs. 2.8 ± 0.8; p < 0.001) and eating pleasure for vegetables (3.4 ± 0.6 vs. 3.1 ± 0.8; p = 0.01) increased, whereas children's F&V consumption did not change (p > 0.14). Parents valued the day-cares' efforts to encourage children's F&V consumption and a small group (∼20%) experienced positive effects at home. This study shows that implementing a self-chosen F&V strategy at the day-care is acceptable, appropriate and feasible for day-care professionals and has potential to positively impact children's F&V eating behaviour. Future research should investigate the effects of long-term implementation on children's eating behaviour and examine how structural implementation can be further supported.


Analysis pipelines for cancer genome sequencing in mice.
Mouse models of human cancer have transformed our ability to link genetics, molecular mechanisms and phenotypes. Both reverse and forward genetics in mice are currently gaining momentum through advances in next-generation sequencing (NGS). Methodologies to analyze sequencing data were, however, developed for humans and hence do not account for species-specific differences in genome structures and experimental setups. Here, we describe standardized computational pipelines specifically tailored to the analysis of mouse genomic data. We present novel tools and workflows for the detection of different alteration types, including single-nucleotide variants (SNVs), small insertions and deletions (indels), copy-number variations (CNVs), loss of heterozygosity (LOH) and complex rearrangements, such as in chromothripsis. Workflows have been extensively validated and cross-compared using multiple methodologies. We also give step-by-step guidance on the execution of individual analysis types, provide advice on data interpretation and make the complete code available online. The protocol takes 2-7 d, depending on the desired analyses.


Chronic kidney injury in patients after cardiac catheterisation or percutaneous coronary intervention: a comparison of radial and femoral approaches (from the British Columbia Cardiac and Renal Registries).
Acute kidney injury (AKI) is a well-recognised complication of cardiac catheterisation and percutaneous coronary intervention (PCI). However, the incidence of chronic kidney disease (CKD) after catheterisation and PCI has not been fully evaluated. A number of risk factors have been implicated in the development of AKI following cardiac catheterisation. Transradial access could lead to a lower incidence of CKD after catheterisation or PCI because of less catheter contact with aortic atheroma, and reduced potential for atheroembolism. To determine the incidence of CKD onset and its association with arterial access in patients after cardiac catheterisation or PCI. Linkages between the British Columbia (BC) Cardiac Registry (N=69 214) patients who underwent catheterisation or PCI between 1999 and 2005 and the BC Renal Database were determined. Within 6 months after the cardiac procedure 0.4% of patients developed dialysis dependency, 0.2% in the transradial versus 0.4% in the transfemoral group (p<0.0001); 0.3% of patients developed stage 4 or 5 CKD, 0.1% in the transradial versus 0.4% in the transfemoral group (p<0.0001); 0.9% of patients developed new CKD, 0.2% in the transradial versus 1.2% in the transfemoral group (p<0.0001). After adjusting for baseline characteristics the femoral access site had an OR of 4.36 (95% CI 2.48 to 7.66) for the development of the composite end point of new dialysis, new stage 4 or 5 CKD or new CKD. In this large database of current practice coronary catheterisation and PCI, the incidence of CKD onset within 6 months of the procedure was 0.9%. The transradial access site is associated with less CKD than the femoral approach.


Characterizing factors influencing calibration and optical property determination in quantitative reflectance spectroscopy to improve standardization.
The combination of reflectance and fluorescence spectroscopy allows the determination of tissue optical properties and the calculation of the intrinsic fluorescence in vivo. These parameters can discriminate between tissues and may allow the discrimination of malignant from benign tissue. While this approach has significant clinical potential, the lack of standardization and quality assessment prevents the upscaling of research. Investigate which factors influence device calibration and tissue optical property determination. Improve system quality assessment and allow upscaling of the clinical research using multidiameter single fiber reflectance/singe fiber fluorescence spectroscopy. Two studies, one phantom based on uniform calibrations and skin measurements and a clinical study including clinical calibrations. The first validates the effect of factors under identical conditions and the effect of calibration quality on the optical property determination of skin. The second shows the effect of different system configurations and the performance of the system and probe over an extended period. Phantom calibrations showed stability over a period of 20 weeks except for a 16-week-old intralipid phantom which showed a significant difference (at least p = 0.0032) for all five probes evaluated. For clinical calibrations, only the fiber tree had a significant influence (probe 4: p < 0.000001 and probe 5: p = 0.00038) on the calibration quality. Interestingly, no degradation of probe performance was detected over a period of 21 months despite the exposure to stress during clinical measurements. Calibration quality affected μs' and the power law scattering exponent, but the degree of the influence was different per fiber. Intralipid phantom quality and fiber tree performance are the main factors influencing the calibration quality. Probe and user performance did not show any effect, which makes the upscaling of research to multicenter trials easier. A high-quality assessment procedure should be implemented to track changes during clinical trials.


Developing climate-smart restoration: Can plant microbiomes be hardened against heat waves?
Heat waves are increasing in frequency and intensity, presenting a challenge for the already difficult practice of ecological restoration. We investigated whether pre-heating locally sourced rhizosphere soil (inoculum) could acclimatize plants to a field-imposed heat wave in a restoration setting. Soil heating in the laboratory caused a marked shift in rhizosphere bacterial community composition, accompanied by an increase in species evenness. Furthermore, pre-heated rhizosphere soil reduced plant height, number of leaves, and shoot mass of the C4 grass, blue grama (Bouteloua gracilis), and it reduced the shoot mass of the C3 grass, Arizona fescue (Festuca arizonica) in the glasshouse. Following transplantation and the application of a field heat wave, pre-heated inoculum did not influence heat wave survival for either plant species. However, there were strong species-level responses to the field heat wave. For instance, heat wave survivorship was over four times higher in blue grama (92%) than in Arizona fescue (22%). These results suggest that the use of C4 seeds may be preferable for sites exhibiting high heat wave risk. Further research is needed to understand whether inocula are more effective in highly degraded soil in comparison with partially degraded soils.


A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe.
In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase) or DNA damage repair (in G2). Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase ε (Cdc20) are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype) is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular response to inhibition of DNA replication initiation is distinct from blocking DNA replication elongation, and this difference can be exploited to identify mutants specifically defective in DNA replication initiation.


The angiogenic pathway "vascular endothelial growth factor/flk-1(KDR)-receptor" in rheumatoid arthritis and osteoarthritis.
Active angiogenesis, together with an up-regulation of angiogenic factors, is evident in the synovium of both rheumatoid arthritis (RA) and osteoarthritis (OA). The present study assessed, by immunohistochemistry, the microvessel density in the synovium of these arthritides and in normal controls, in relation to the expression of the angiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) and the apoptosis-related proteins bcl-2 and p53. More importantly, using the novel 11B5 MAb, the activated "VEGF/flk-1(KDR)-receptor" microvessel density was assessed. VEGF expression in fibroblasts was diffuse in both RA and OA. Diffuse PD-ECGF expression of fibroblasts was noted in all cases of RA, while fibroblast reactivity was focal in the OA material. The standard microvessel density (sMVD), as assessed with the anti-CD31 monoclonal antibody (MAb), was higher in RA (64+/-12) and in OA (65+/-16) than in normal tissues (52+/-8; p=0.008 and 0.0004, respectively). The activated microvessel density (aMVD), assessed with the 11B5 MAb, was significantly higher in RA (29+/-10) than in OA (17+/-4; p<0.0001) and than in normal tissues (14+/-2; p<0.0001). The "activation ratio" (aMVD/sMVD) was statistically higher in RA (0.46+/-0.17) than in OA and normal synovial tissues, the latter two having a similar ratio (0.28+/-0.08 and 0.26+/-0.03, respectively). Cytoplasmic bcl-2 expression was frequent in the synovial cells of OA, but rare in RA. Nuclear p53 protein accumulation was never observed. It is suggested that the angiogenic pathway VEGF/flk-1(KDR) may play an important role in the pathogenesis of RA and OA. Thus, failure of VEGF/flk-1(KDR) activation, in the presence of increased VEGF expression, may indicate a synovium with an impaired capacity to establish a viable vasculature, consistent with the degenerative nature of OA. On the other hand, the activated angiogenesis in RA shows a functional, still pathologically up-regulated VEGF/flk-1(KDR) pathway. Whether restoration of an impaired VEGF/flk-1(KDR) pathway in OA, or inhibition of this in RA, would prove of therapeutic importance requires further investigation.


An analysis of the clinical practice of emergency medicine in public emergency departments in Kenya.
To describe the case mix, interventions, procedures and management of patients in public emergency departments (ED) in Kenya. An observational study over 24 h, of patients who presented to 15 public ED during the 3-month period from 1 October to 31 December 2010. The study was conducted across Kenya in two national referral hospitals, five secondary level hospitals and eight primary level hospitals. All patients presenting alive to the ED during the 24-h study period that were seen by a doctor or clinical officer were included in the study. A data collection form was completed by the primary investigator at the time of the initial ED consultation documenting patient demographics, presenting complaints, investigations ordered, procedures done, initial diagnosis and outcome of ED consultation. Data on 1887 patient presentations were described. Adults (≥13 years) accounted for the majority (70%) of patients. Two peak age groups, 0-9 and 20-29 years, accounted for 27% and 25% of patients, respectively. Respiratory and trauma presentations each accounted for 21% of presentations, with a wide spread of other presentations. Over half (58%) of the patients were investigated in the department. 385 patients received immediate treatment in the ED before discharge. Fewer than one in three patients admitted or transferred to specialist units received any therapy in the ED. ED in Kenya provide care to an undifferentiated patient population yet most of the immediate therapy is provided only to patients with minor conditions who are subsequently discharged. Sicker patients have to await transfer to wards or specialist units to start receiving treatment.


Attenuation of nephrotoxic acute renal failure in the dog with angiotensin-converting enzyme inhibitor (SQ-20,881).
Angiotensin-converting enzyme inhibitor was used in dogs with uranyl nitrate-induced acute renal failure to evaluate (1) a possible protective effect of angiotensin blockade and (2)the role of angiotensin II in the generation of renal failure in this model. Angiotensin-converting enzyme inhibitor treatment attenuated the fall in glomerular filtration rate and renal blood flow during the first 6 hours after injection of the nephrotoxic agent. A protective effect of similar magnitude was observed whether angiotensin-converting enzyme inhibitor treatment preceded, or shortly followed, the administration of uranyl nitrate. This indicates that angiotensin-converting enzyme inhibitor delivery to its intrarenal site of action remains effective after administration of the nephrotoxin. In addition, protection of glomerular filtration rate correlated with sodium and renal solute excretion. However, combined treatment with angiotensin-converting enzyme inhibitor and furosemide enhanced solute excretion but did not further improve the protection of renal function. Finally, the protective effects of angiotensin-converting enzyme inhibitor on renal function and hemodynamics were abolished by intravenous indomethacin. In conclusion, early, continuous blockade of angiotensin II protects partially against th initiation of acute renal failure. These findings support a major pathogenic role for angiotensin II in the generation phase of acute renal failure in this model. Furthermore, they suggest that an imbalance between vasoconstrictive (angiotensin II) and vasodilating factors (prostaglandins) may be operative in the early phase of uranyl nitrate-induced acute renal failure in the dog.


Defining Surrogate Endpoints for Clinical Trials in Severe Falciparum Malaria.
Clinical trials in severe falciparum malaria require a large sample size to detect clinically meaningful differences in mortality. This means few interventions can be evaluated at any time. Using a validated surrogate endpoint for mortality would provide a useful alternative allowing a smaller sample size. Here we evaluate changes in coma score and plasma lactate as surrogate endpoints for mortality in severe falciparum malaria. Three datasets of clinical studies in severe malaria were re-evaluated: studies from Chittagong, Bangladesh (adults), the African 'AQUAMAT' trial comparing artesunate and quinine (children), and the Vietnamese 'AQ' study (adults) comparing artemether with quinine. The absolute change, relative change, slope of the normalization over time, and time to normalization were derived from sequential measurements of plasma lactate and coma score, and validated for their use as surrogate endpoint, including the proportion of treatment effect on mortality explained (PTE) by these surrogate measures. Improvements in lactate concentration or coma scores over the first 24 hours of admission, were strongly prognostic for survival in all datasets. In hyperlactataemic patients in the AQ study (n = 173), lower mortality with artemether compared to quinine closely correlated with faster reduction in plasma lactate concentration, with a high PTE of the relative change in plasma lactate at 8 and 12 hours of 0.81 and 0.75, respectively. In paediatric patients enrolled in the 'AQUAMAT' study with cerebral malaria (n = 785), mortality was lower with artesunate compared to quinine, but this was not associated with faster coma recovery. The relative changes in plasma lactate concentration assessed at 8 or 12 hours after admission are valid surrogate endpoints for severe malaria studies on antimalarial drugs or adjuvant treatments aiming at improving the microcirculation. Measures of coma recovery are not valid surrogate endpoints for mortality.


Terminology and classification of muscle injuries in sport: the Munich consensus statement.
To provide a clear terminology and classification of muscle injuries in order to facilitate effective communication among medical practitioners and development of systematic treatment strategies. Thirty native English-speaking scientists and team doctors of national and first division professional sports teams were asked to complete a questionnaire on muscle injuries to evaluate the currently used terminology of athletic muscle injury. In addition, a consensus meeting of international sports medicine experts was established to develop practical and scientific definitions of muscle injuries as well as a new and comprehensive classification system. The response rate of the survey was 63%. The responses confirmed the marked variability in the use of the terminology relating to muscle injury, with the most obvious inconsistencies for the term strain. In the consensus meeting, practical and systematic terms were defined and established. In addition, a new comprehensive classification system was developed, which differentiates between four types: functional muscle disorders (type 1: overexertion-related and type 2: neuromuscular muscle disorders) describing disorders without macroscopic evidence of fibre tear and structural muscle injuries (type 3: partial tears and type 4: (sub)total tears/tendinous avulsions) with macroscopic evidence of fibre tear, that is, structural damage. Subclassifications are presented for each type. A consistent English terminology as well as a comprehensive classification system for athletic muscle injuries which is proven in the daily practice are presented. This will help to improve clarity of communication for diagnostic and therapeutic purposes and can serve as the basis for future comparative studies to address the continued lack of systematic information on muscle injuries in the literature. WHAT ARE THE NEW THINGS: Consensus definitions of the terminology which is used in the field of muscle injuries as well as a new comprehensive classification system which clearly defines types of athletic muscle injuries. Expert opinion, Level V.


Desorption of 3,3'-diindolylmethane from imprinted particles: An impact of cross-linker structure on binding capacity and selectivity.
Here, seven cross-linkers (six polar diacrylates or dimethacrylates of different lengths between double bonds, and one aromatic-divinylbenzene) were used to examine the impact of the cross-linker on binding capacity and selectivity of 3,3'-diindolylmethane (DIM) imprinted material. DIM participates in the suppression of viability of human ovarian and human breast cancer cell lines, but has low bioavailability. The investigations of novel imprinted polymer matrices for improvement of DIM release could allow to utilize not only a potency of DIM but also similar alkaloids, which are the important compounds with pharmacological activity. The bulk, thermal radical copolymerization of the cross-linkers in the presence of 3,3'-diindolylmethane (the template) and allylamine (the functional monomer) in dimethyl sulfoxide or in carbon tetrachloride (porogens) was carried out. The binding capacities of imprinted and non-imprinted polymers were compared, and two polymers (these were prepared using ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate as the cross-linkers) with the highest selectivity and binding capacity were selected to desorption test. The desorption profile of polymer prepared using polyethylene glycol dimethacrylate as the cross-linker revealed sustained release of 3,3'-diindolylmethane, and this system was selected for further optimization of the cross-linker amounts. The morphology and structure of the selected particles were analyzed using SEM micrographs, (13)C CP/MAS NMR spectroscopy, and BET measurements. The desorption of 3,3'-diindolylmethane from poly(allylamine-co-polyethylene glycol dimethacrylate) particles was in accordance with pseudo-second-order kinetics and the simplified Higuchi model indicated the diffusion controlled release of 3,3'-diindolylmethane.


Caveolin-1 in the regulation of cell metabolism: a cancer perspective.
Caveolin-1 (CAV1) is an oncogenic membrane protein associated with endocytosis, extracellular matrix organisation, cholesterol distribution, cell migration and signaling. Recent studies reveal that CAV1 is involved in metabolic alterations - a critical strategy adopted by cancer cells to their survival advantage. Consequently, research findings suggest that CAV1, which is altered in several cancer types, influences tumour development or progression by controlling metabolism. Understanding the molecular interplay between CAV1 and metabolism could help uncover druggable metabolic targets or pathways of clinical relevance in cancer therapy. Here we review from a cancer perspective, the findings that CAV1 modulates cell metabolism with a focus on glycolysis, mitochondrial bioenergetics, glutaminolysis, fatty acid metabolism, and autophagy.


Characteristics of Improvised Explosive Device Trauma Casualties in the Gaza Strip and Other Combat Regions: The Israeli Experience.
Low-intensity conflict is characterized in asymmetrical conventional and nonconventional warfare. The use of improvised explosive devices (IEDs) in the Israeli-Palestinian conflict has evolved over the past few decades to include the addition of diesel, biological agents, shrapnel, and nitroglycerin to the explosive content. Due to its nature and mechanism, an IED injury might present as a multidimensional injury, impairing numerous systems and organs. The authors present a case series of 5 Israeli Defense Forces (IDF) soldiers wounded by an IED presenting a typical and similar pattern of burns to their faces, trunks, and limbs, in addition to ocular, ear/nose/throat, and orthopedic injuries. An analysis of the experience in treating the aforementioned injuries is included. Improvement in casualties' burns and traumatic tattoos was observed following debridement, aggressive scrubbing with or without dermabrasion, and conservative local dressing treatment protocol. The authors found a positive correlation between improvement degree and treatment timing. Injury pattern was correlative to the protective gear worn by the soldiers. Wearing protective eye gear and wearing ceramic vests can diminish the extent of IED injuries, while creating typical patterns of injuries to be treated. Based on these experiences, such injuries should be brought to a trauma center as soon as possible. Treating multidimensional trauma should be done in a facility with abilities to treat head injuries, eye injuries, penetrating injuries, blast injuries, and burns. Such specialized disciplines and facilities that have past experience with IEDs and war injuries are able to assess and treat these injuries in a more dedicated manner, resulting in better long-term rehabilitation.


Suspended microchannel resonators with piezoresistive sensors.
Precision frequency detection has enabled the suspended microchannel resonator (SMR) to weigh single living cells, single nanoparticles, and adsorbed protein layers in fluid. To date, the SMR resonance frequency has been determined optically, which requires the use of an external laser and photodiode and cannot be easily arrayed for multiplexed measurements. Here we demonstrate the first electronic detection of SMR resonance frequency by fabricating piezoresistive sensors using ion implantation into single crystal silicon resonators. To validate the piezoresistive SMR, buoyant mass histograms of budding yeast cells and a mixture of 1.6, 2.0, 2.5, and 3.0 µm diameter polystyrene beads are measured. For SMRs designed to weigh micron-sized particles and cells, the mass resolution achieved with piezoresistive detection (∼3.4 fg in a 1 kHz bandwidth) is comparable to what can be achieved by the conventional optical-lever detector. Eliminating the need for expensive and delicate optical components will enable new uses for the SMR in both multiplexed and field deployable applications.


Research Methods in Healthcare Epidemiology and Antimicrobial Stewardship.
Research in Healthcare Epidemiology and Antimicrobial Stewardship (HE&AS) is rapidly expanding with the involvement of researchers from varied countries and backgrounds. Researchers must use scientific methods that will provide the strongest evidence to advance healthcare epidemiology, but there are limited resources for information on specific aspects of HE&AS research or easy ways to access examples of studies using specific methods with HE&AS. In response to this need, the SHEA Research Committee has developed a series of white papers on research methods in HE&AS. The objective of this series is to promote rigorous healthcare epidemiology research by summarizing critical components, practical considerations, and pitfalls of commonly used research methods. Infect Control Hosp Epidemiol 2016;37:627-628.


Elastic reversible valves on centrifugal microfluidic platforms.
Reversible valves on centrifugal microfluidic platforms facilitate the automation of bioanalytical assays, especially of those requiring a series of steps (such as incubation) in a single reaction chamber. In this study, we present fixed elastic reversible (FER) valves and tunable elastic reversible (TER) valves that are easy to fabricate, implement and control. In the FER valve the compression of an elastic barrier/patch against a microchamber's outlet prevents the release of liquid. The valve sealing pressure was determined by adjusting the engraving depth of the valve-seat at which the elastic patch was located, this allows to set the sealing pressure during disc fabrication. In the TER valve, the patch compression value and sealing pressure is controlled by the penetration depth of a plastic screw into the valve-seat. The ER valves prevent liquid flow until the centrifugal force overcomes their sealing pressure. Moreover, at a constant spin speed, turning the screw of a TER valve reduces its sealing pressure and opens the valve. Therefore, the TER valve allows for controlling of the liquid transfer volume at various spin speeds. The FER and TER valves' behavior is mathematically described and equations for the prediction of their operation under centrifugal forces are provided. As a point-of-care (POC) application of ER valves, we have developed a microfluidic disc with a series of TER valves and peptide microarrays for automated multiplexed detection of five different proteins from a single serum sample.


Interactions between calcium release pathways: multiple messengers and multiple stores.
The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles. In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.


Intravenous lidocaine is as effective as epidural bupivacaine in reducing ileus duration, hospital stay, and pain after open colon resection: a randomized clinical trial.
Both postoperative epidural analgesia and intravenous (IV) infusion of local anesthetic have been shown to shorten ileus duration and hospital stay after colon surgery when compared with the use of systemic narcotics alone. However, they have not been compared directly with each other. Prospective, randomized clinical trial was conducted comparing the 2 treatments in open colon surgery patients. Before induction of general anesthesia, patients were randomized either to epidural analgesia (bupivacaine 0.125% and hydromorphone 6 microg/mL were started at 10 mL/hr within 1 hr of the end of surgery) or IV lidocaine (1 mg/min in patients < 70 kg, 2 mg/min in patients > or = 70 kg). Markers of return of bowel function, length of stay, postoperative pain scores, systemic analgesic requirements, and adverse events were recorded and compared between the 2 groups in an intent-to-treat analysis. Study enrollment took place from April 2005 to July 2006. Twenty-two patients were randomized to IV lidocaine therapy and 20 patients to epidural therapy. No statistically significant differences were found between groups in time to return of bowel function or hospital length of stay. The median pain score difference was not statistically significant. No statistically significant differences were found in pain scores for any specific postoperative day or in analgesic consumption. No differences were observed between groups in terms of return of bowel function, duration of hospital stay, and postoperative pain control, suggesting that IV infusion of local anesthetic may be an effective alternative to epidural therapy in patients in whom epidural anesthesia is contraindicated or not desired.


Mono-L-arginine-containing compounds dilate piglet pial arterioles via an endothelium-derived relaxing factor-like substance.
We determined the effects of mono-L-arginine-containing compounds on pial arterioles of anesthetized piglets. A closed cranial window was implanted, and the diameter of one pial arteriole was determined by intravital microscopy. Diameter was determined during application of artificial cerebrospinal fluid containing no drugs and during application of 10(-5), 10(-4), 10(-3), and 10(-2) M L-arginine (ARG), L-arginine ethyl ester (AEE), N alpha-benzoyl-L-arginine (NBA), N alpha-benzoyl-L-arginine ester ethyl (BAEE), and L-citrulline (CIT). Initial diameters were 100-200 microns. All of these compounds dilated arterioles, but the threshold concentration needed to elicit dilation varied: 10(-5) M for NBA (n = 5), 10(-3) M for AEE (n = 9) and BAEE (n = 6), and 10(-2) M for ARG (n = 6) and CIT (n = 4). Maximal responses were 15 +/- 8% for CIT, 17 +/- 4% for ARG, 19 +/- 8% for BAEE, 28 +/- 5% for NBA, and 27 +/- 6% for AEE. Indomethacin pretreatment (5 mg/kg i.v.) did not change arteriolar responses to AEE, NBA, and BAEE. However, coadministration of methylene blue (0.5 x 10(-4) M or 0.5 x 10(-3) M) abolished dilation to 10(-3) M AEE or BAEE and attenuated dilation to 10(-5) M NBA. In addition, coadministration of hemoglobin (0.4 x 10(-4) M) abolished dilation to AEE, BAEE, or NBA. Last, intravenous (5 mg/kg) and coadministration (10(-3) M) of NG-methyl-L-arginine blocked dilation to NBA or AEE. We conclude that mono-L-arginine-containing compounds produce pial arteriolar dilation in piglets, possibly involving an endothelium-derived relaxing factor.


Bombesin, neuromedin B and neuromedin C selectively depress superficial dorsal horn neurones in the cat spinal cord.
The effects of bombesin and related peptides on functionally identified single dorsal horn neurones were studied using iontophoresis and extracellular recording in the anaesthetized and spinalized cat. Bombesin selectively depressed superficial dorsal horn neurones (in laminae I-III). The depression was of spontaneous activity as well as of synaptically elicited responses to natural stimulation of the cutaneous receptive field. Bombesin preferentially depressed neurones that responded to noxious stimulation of the cutaneous receptive field. Naloxone, bicuculline and caffeine failed to block the depression by bombesin, suggesting that the effect of the peptide may be direct and not through the indirect activation of an inhibitory system mediated by opioids, by gamma-aminobutyric acid (GABA) or by purines, respectively. Iontophoretic application of neuromedin B (n = 3) and neuromedin C (GRP-10) (n = 7) induced a similar depression to that observed with bombesin. These results provide physiological evidence that a bombesin-like peptide may play a role in the mediation or the modulation of sensory transmission in the superficial dorsal horn of the spinal cord.


Change in moderate alcohol consumption and quality of life: evidence from 2 population-based cohorts.
Although the association of moderate alcohol consumption with specific disorders, such as cardiovascular disease and cancers, has been well documented, the evidence of the broader impact of alcohol consumption on health-related quality of life is less clear. Our objective was to examine the association of drinking patterns with changes in physical and mental well-being across populations. We conducted a multilevel analysis with multivariate responses in the population-representative FAMILY Cohort in the Hong Kong Special Administrative Region, China, to examine the association between alcohol drinking patterns across 2 waves (2009-2013) (i.e., quitters, initiators, persistent drinkers, persistent former drinkers and lifetime abstainers) and changes in physical and mental well-being (Physical and Mental Component Summary of the 12-Item Short Form Health Survey [SF-12]). Analyses were stratified by sex. We validated findings using a nationally representative cohort in the United States, the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC, 2001-2005). In the FAMILY Cohort (n = 10 386; median follow-up 2.3 yr), the change in mental well-being was more favourable in female quitters than in lifetime abstainers (β = 1.44, 95% confidence interval [CI] 0.43 to 2.45; mean score change of +2.0 for quitters and +0.02 for lifetime abstainers). This association was validated in the NESARC (n = 31 079; median follow-up 3.1 yr) (β = 0.83, 95% CI 0.08 to 1.58; mean score change of -1.1 for quitters and -1.6 for lifetime abstainers). The change in mental well-being was more favourable in female quitters, approaching the level of mental well-being of lifetime abstainers within 4 years of quitting in both Chinese and American populations.


The absence of 'minor' risk factors for recurrent venous thromboembolism: a systematic review of negative predictive values and negative likelihood ratios.
The decision to stop anticoagulation after an episode of venous thromboembolism (VTE) depends on the risk of recurrence. A number of recently discussed risk factors that are not routinely used in clinical practice might be useful for the identification of patients with a low risk of recurrence. The aim of this study was to determine the negative predictive value and likelihood ratio of the absence of each of these factors in patients with VTE. We performed a systematic review of the literature, calculated negative predictive values and likelihood ratios, and summarized the available evidence. A negative D-dimer result, non-elevated factor VIII level and non-elevated thrombin generation after discontinuation of anticoagulant treatment, female sex and distal location might be useful in determining a low risk of recurrence in patients with VTE. Each of these factors individually is associated with a negative predictive value for recurrence-free survival in the next 2-3 years of about 90% (with an a priori risk of about 85%), with negative likelihood ratios between 0.8 and 0.5. Some of the risk factors for first VTE event (FV Leiden and prothrombin mutation) are not useful at all for prediction of non-recurrence. None of the risk factors that we identified is strong enough on its own to guide treatment. Combinations of risk factors might be more useful, but additional data on the independence of these factors are required before a prediction rule can be designed.


Phase noise generation in an amplitude limiter using saturation of a fiber-optic parametric amplifier.
Generation of phase noise in an amplitude limiter using saturation of a fiber-optic parametric amplifier is analyzed. The analysis is based on the coupled differential equations for the amplitudes and phases of pump, signal, and idler involved in the four-wave mixing interaction. The equations are linearized about small fluctuations at operation points and are solved, which results in relations between the input and output phase and amplitude noise. These relations are used in deriving expressions of amplitude signal-to-noise ratio (SNR) and variance of phase noise of the output signal in terms of SNRs of input signal and pump. It is shown that optimum pump power exists for minimum output phase noise in the output signal.


First Report of Powdery Mildew Caused by Podosphaera fusca on Herba eupatorii in China.
Herba eupatorii, one of the most important Chinese medicinal herbs, belongs to the Asteraceae family. In June 2012, a previously unknown disease, tentatively identified as powdery mildew, was observed on H. eupatorii growing in Shangqiu, in eastern Henan Province, China. Symptoms began as white mycelium partially covering upper leaf surfaces; as the disease progressed, it spread to cover entire leaf surfaces. The infected leaves became yellow and necrotic at advanced stages of infection. Specimens consisting of infected leaves were maintained at the Plant-Microbe Interaction Laboratory at Shangqiu Normal University. Microscopic observations of the morphology of the fungus revealed oval primary conidia measuring 18 to 27 × 15 to 22 μm. A long unbranched germ tube that germinated laterally from the ends of conidia was observed in some samples. Conidiophores were cylindrical, simple unbranched, and composed of a basal cell with a swollen base and three to six barrel-shaped conidia formed in chains, measuring 112 to 180 × 9 to 12 μm. Mycelial appressoria were nipple-shaped. Chasmothecia were not observed in the collected samples. To verify the identity of the fungus, the internal transcribed spacer (ITS) rDNA was amplified with ITS1 and ITS4 primers (3) and sequenced. The sequences were deposited as GenBank Accession No. JX546297. Comparison with sequences in the GenBank database revealed that the ITS sequence was 100% homologous with the sequence of Podosphaera fusca on Calendula officinalis (AB525914) (2) and Syneilesis palmata (AB040349) (1). The ITS sequence analysis verified that the causal agent was P. fusca, which is reported to be a cosmopolitan powdery mildew fungus, parasitic on numerous plant species in the Asteraceae family. Koch's postulates were completed by inoculating healthy H. eupatorii plants with a conidial suspension (prepared in distilled water) of 105 conidia/ml collected from infected plants. Five plants were sprayed until the suspension ran off the leaves, while five additional plants were sprayed with distilled water as a control. Plants were maintained in a climate cell under the following conditions: day, 24°C, 16 h; night, 20°C, 8 h; 85% humidity. After 10 days, inoculated plants developed symptoms similar to those observed in the field, whereas control plants remained healthy. Further examination showed that the inoculated plants were infected by P. fusca. To our knowledge, this is the first report of P. fusca affecting H. eupatorii in China. Because there are no fungicides labeled for use on this plant, the appearance of powdery mildew caused by P. fusca could result in substantial production loss of H. eupatorii. References: (1) T. Hirata et al. Can. J. Bot. 78:1521, 2000. (2) S. Takamatsu et al. Persoonia 24:38, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.


Influence of surface modification on vitality and differentiation of Caco-2 cells.
It is widely accepted that the functional and morphological differentiation of cells is initiated and determined by the interaction of molecules of the extracellular matrix and adhesion molecules of the cell membrane. To assess the influence of the underlying matrix on the characteristics of cells, enterocyte-like Caco-2 cells were cultivated on substrates commonly used for cell culture as well as on glass coated with hydrophobic layers. Providing the same starting conditions for growth, the parameters investigated on preconfluent Caco-2 cells were the number of adhering cells, the proliferative activity and the degree of differentiation indicated by the expression of three brush border enzymes. Whereas tissue culture treated polystyrene elicited highest rates of adhesion, proliferation, and differentiation, even glass altered the pattern of brush border enzyme expression. The hydrophobic surfaces strongly decreased the adhesion and the proliferation but the surviving cells exhibited a pronounced higher degree of differentiation. Interestingly, each sub-type of hydrophobic matrix triggered a different pattern of brush border enzyme expression. Thus, the development of a certain phenotype of a cell can not only be triggered by certain components of the extracellular matrix but also by artificially prepared surface coatings of the underlying matrix. In the future it seems to be feasible that cells can be programmed by tailoring the surface of the underlying substrate.


Effectiveness of Health Literacy Interventions on COPD Self-Management Outcomes in Outpatient Settings: A Systematic Review.
Chronic obstructive pulmonary disease (COPD) is a chronic progressive lung disease which imposes significant health and economic burdens on societies. Self-management is beneficial in controlling and managing COPD and health literacy (HL) is a major driver of COPD self-management. This review aims to summarize the most recent evidence on the effectiveness of HL driven COPD self-management interventions using randomized controlled trials (RCTs). Eight data bases including Science Citation Index, Academic Search Complete, Social Sciences Citation Index, CINAHL Plus, APA PsycInfo, MEDLINE, Scopus and ScienceDirect were searched to find eligible RCTs assessing the effectiveness of HL interventions on COPD self-management outcomes in outpatient settings between 2008 and February 2020. Ten RCTs met the eligibility criteria. The review found that HL interventions led to moderate improvements in physical activity levels (four out of seven trials) and COPD knowledge (three out of six trials). Surprisingly, none of the RCTs led to significant improvement in medication adherence, which warrants further studies. Furthermore, there were inconclusive findings regarding other COPD self-management outcomes such as smoking cessation, medication adherence, dyspnea, mental health, hospital admissions and health related quality of life.


Bullying victimization and adolescent self-harm: testing hypotheses from general strain theory.
Self-harm is widely recognized as a significant adolescent social problem, and recent research has begun to explore its etiology. Drawing from Agnew's (1992) social psychological strain theory of deviance, this study considers this issue by testing three hypotheses about the effects of traditional and cyber bullying victimization on deliberate self-harm and suicidal ideation. The data come from a school-based survey of adolescents in a rural county of a southeastern state (n = 426); 50% of subjects are female, their mean age was 15 years, and non-Hispanic whites represent 66% of the sample. The analysis revealed that both types of bullying are positively related to self-harm and suicidal ideation, net of controls. Moreover, those relationships are partially mediated by the negative emotions experienced by those who are bullied and partially moderated by features of the adolescent's social environment and self. Regarding the latter, exposure to authoritative parenting and high self-control diminished the harmful effects of bullying victimization on self-harm and suicidal ideation. The article concludes by discussing the implications of these conclusions for future research and for policy efforts designed to reduce self-harm.


ZnRF3 induces apoptosis of gastric cancer cells by antagonizing Wnt and Hedgehog signaling.
The aim of this paper was to investigate the function an importance of E3-ubiquitin ligase ZnRF3 in the progression of cancer cell growth. A total of 58 patients (44 males and 14 females) were enrolled in the study and their gastric tumors were removed surgically and were staged by the TNM approach. Among these patients, 43 patients died and 15 survived at the time of this study. The tumors and the paracancerous tissues were examined by immunohostochemistry for the expression of ZnRF3. We assessed the expression of ZnRF3 in gastric tumors and paracancerous tissues from our patients and related this to patient survival. A large proportion of malignant cancers of the stomach are gastric adenocarcinoma type. In spite of many studies, the molecular basis for this cancer is still unclear. Deregulated cell proliferative signaling via Wnt/β-catenin and Hedgehog pathways is considered important in the pathogenesis of many cancers including the gastric cancer. Recent studies identified ZnRF3 protein, which is a E3-ubiquitin ligase and which is either deleted or mutated in cancers, to inhibit Wnt signaling. However, the significance of ZnRF3 in the control of gastric cancer and whether it also regulates Hedgehog signaling pathway, is not known. ZnRF3 expression was much higher in tumors from aged patients. Male patients showed higher mortality than the females. Mechanistic studies using normal gastric cells (GES1) and gastric cancer cells (MGC-803) infected with either AdZnRF3 or AdGFP viral vectors, revealed that ZnRF3 overexpression causes significantly more apoptosis and lowered proliferation of cancer cells. ZnRF3 overexpression led to greatly reduced levels of Lgr5, a component of Wnt signaling and also Gli1, a component of Hedgehog signaling. Thus, ZnRF3 negatively influences both the Wnt and Hedgehog proliferative pathways and probably this way it negatively regulates cancer progression. These results suggest the importance of normal ZnRF3 function in checking the progression of cancer cell growth and indicate that a lack of this protein can lead to poorer clinical outcomes for gastric cancer patients. We observed a clear relationship between ZnRF3 expression in paracancerous tissue and tumor size.


Phosphorylation in the plant circadian system.
Circadian regulation is essential for optimum plant performance. In addition to loops and cascades of transcription and translation, the plant circadian clock and its associated signal transduction networks incorporate many post-translational mechanisms. Phosphorylation is a common feature of signal transduction and gene regulation. In this opinion article, we illustrate how phosphorylation events are positioned within the entrainment, functioning, and regulation of the circadian timing system. Phosphorylation regulates protein stability, protein-protein interactions and protein-DNA interactions within the core oscillator. We suggest that phosphorylation provides a potential mechanism for the distribution of circadian timing information within the cell and for the integration of circadian timing information with other signaling pathways.


The relationship between upwelling underwater polarization and attenuation/absorption ratio.
The attenuation coefficient of the water body is not directly retrievable from measurements of unpolarized water-leaving radiance. Based on extensive radiative transfer simulations using the vector radiative transfer code RayXP, it is demonstrated that the underwater degree of linear polarization (DoLP) is closely related to the attenuation-to-absorption ratio (c/a) of the water body, a finding that enables retrieval of the attenuation coefficient from measurements of the Stokes components of the upwelling underwater polarized light field. The relationship between DoLP and the c/a ratio is investigated for the upwelling polarized light field for a complete set of viewing geometries, at several wavelengths in the visible part of the spectrum; for varying compositions of the aquatic environment, whose constituents include phytoplankton, non-algal particles, and color dissolved organic matter (CDOM); and for varying microphysical properties such as the refractive index and the slope of the Junge-type particle size distribution (PSD). Consequently, this study reveals the possibility for retrieval of additional inherent optical properties (IOPs) from air- or space-borne DoLP measurements of the water-leaving radiation.


The exaggerated blood pressure response to exercise in the sub-acute phase after stroke is not affected by aerobic exercise.
The prevalence of an exaggerated exercise blood pressure (BP) response is unknown in patients with subacute stroke, and it is not known whether an aerobic exercise program modulates this response. The authors randomized 53 patients (27 women) with subacute stroke to 12 weeks of twice-weekly aerobic exercise (n = 29) or to usual care without scheduled physical exercise (n = 24). At baseline, 66% of the patients exhibited an exaggerated exercise BP response (peak systolic BP ≥210 mm Hg in men and ≥190 mm Hg in women) during a symptom-limited ergometer exercise test. At follow-up, patients who had been randomized to the exercise program achieved higher peak work rate, but peak systolic BP remained unaltered. Among patients with a recent stroke, it was common to have an exaggerated systolic BP response during exercise. This response was not altered by participation in a 12-week program of aerobic exercise.


The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses.
Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.


Traumatic Stem Fractures in Short Stem THA-A Rare Case Series.
Adequate trauma of a well-fixed total hip prosthesis might cause relevant osseous injuries. Concomitant occult fractures of the implant itself are very rare. We report on two patients admitted to our institution and who were previously treated with similar types of short-stem total hip arthroplasty (THA). Both were unable to walk after an adequate trauma, although the initial admitting hospital misdiagnosed the exact diagnosis. Detailed reexamination later revealed a prosthetic neck fracture of the cement-free stem. Both patients were treated with a stem revision. In THA patient, special attention should be drawn to the implants after relevant trauma. A single examination shortly after trauma seems to be insufficient to diagnose implant-related injuries. A secondary follow-up several weeks after trauma, including at least X-rays, has to be recommended.


Reducing neuroinflammation by delivery of IL-10 encoding lentivirus from multiple-channel bridges.
The spinal cord is unable to regenerate after injury largely due to growth-inhibition by an inflammatory response to the injury that fails to resolve, resulting in secondary damage and cell death. An approach that prevents inhibition by attenuating the inflammatory response and promoting its resolution through the transition of macrophages to anti-inflammatory phenotypes is essential for the creation of a growth permissive microenvironment. Viral gene delivery to induce the expression of anti-inflammatory factors provides the potential to provide localized delivery to alter the host inflammatory response. Initially, we investigated the effect of the biomaterial and viral components of the delivery system to influence the extent of cell infiltration and the phenotype of these cells. Bridge implantation reduces antigen-presenting cell infiltration at day 7, and lentivirus addition to the bridge induces a transient increase in neutrophils in the spinal cord at day 7 and macrophages at day 14. Delivery of a lentivirus encoding IL-10, an anti-inflammatory factor that inhibits immune cell activation and polarizes the macrophage population towards anti-inflammatory phenotypes, reduced neutrophil infiltration at both day 7 and day 28. Though IL-10 lentivirus did not affect macrophages number, it skewed the macrophage population toward an anti-inflammatory M2 phenotype and altered macrophage morphology. Additionally, IL-10 delivery resulted in improved motor function, suggesting reduced secondary damage and increased sparing. Taken together, these results indicate that localized expression of anti-inflammatory factors, such as IL-10, can modulate the inflammatory response following spinal cord injury, and may be a key component of a combinatorial approach that targets the multiple barriers to regeneration and functional recovery.


Gender differences in academic productivity and leadership appointments of physicians throughout academic careers.
Because those selected for leadership in academic medicine often have a record of academic productivity, publication disparities may help explain the gender imbalance in leadership roles. The authors aimed to compare the publication records, academic promotions, and leadership appointments of women and men physicians longitudinally throughout academic careers. In 2007, the authors conducted a retrospective, longitudinal cohort study of all 25 women physicians then employed at Mayo Clinic with ≥20 years of service at Mayo and of 50 male physician controls, matched 2:1 by appointment date and career category, to women. The authors recorded peer-reviewed publications, timing of promotion, and leadership appointments throughout their careers. Women published fewer articles throughout their careers than men (mean [standard deviation] 29.5 [28.8] versus 75.8 [60.3], P = .001). However, after 27 years, women produced a mean of 1.57 more publications annually than men (P < .001). Thirty-three men (66%) achieved an academic rank of professor compared with seven women (28%) (P = .01). Throughout their careers, women held fewer leadership roles than men (P < .001). Nearly half (no. = 11; 44%) of women attained no leadership position, compared with 15 men (30%). Women's publication rates increase and actually exceed those of men in the latter stages of careers, yet women hold fewer leadership positions than men overall, suggesting that academic productivity assessed midcareer may not be an appropriate measure of leadership skills and that factors other than publication record and academic rank should be considered in selecting leaders.


Effects of transdermal estrogen on levels of lipids, lipase activity, and inflammatory markers in men with prostate cancer.
Androgen deprivation therapy (ADT) for prostate cancer is now used in earlier disease stages and as adjuvant treatment. Recognizing and reducing the toxicity of this therapy, including worsened lipid levels and cardiovascular disease (CVD) risks, has become an important clinical concern. Oral estrogen therapy induces hypogonadism and mitigates many side effects of ADT, but has a high thrombosis risk. Transdermal estrogen therapy (TDE) has a lower thrombosis risk than oral estrogen and may improve CVD risk compared with ADT. This prospective pilot study of 18 men with androgen-independent prostate cancer receiving ADT measured effects of TDE on lipid and inflammatory CVD risk factors before and after 8 weeks of TDE (estradiol 0.6 mg/day). During treatment, estradiol levels rose 17-fold; total cholesterol, LDL cholesterol, and apolipoprotein B levels decreased. HDL2 cholesterol increased, with no changes in triglyceride or VLDL cholesterol levels. Dense LDL cholesterol decreased and LDL buoyancy increased in association with a decrease in HL activity. Highly sensitive C-reactive protein levels and other inflammatory markers did not worsen. Compared with ADT, short-term TDE therapy of prostate cancer improves lipid levels without deterioration of CVD-associated inflammatory markers and may, on longer-term follow-up, improve CVD and mortality rates.


Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion.
Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e. endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.


Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca²⁺-permeable AMPA receptors during the incubation of cocaine craving.
Cue-induced cocaine seeking intensifies or incubates after withdrawal from extended access cocaine self-administration, a phenomenon termed incubation of cocaine craving. The expression of incubated craving is mediated by Ca²⁺-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Thus, CP-AMPARs are a potential target for therapeutic intervention, making it important to understand mechanisms that govern their accumulation. Here we used subcellular fractionation and biotinylation of NAc tissue to examine the abundance and distribution of AMPAR subunits, and GluA1 phosphorylation, in the incubation model. We also studied two transmembrane AMPA receptor regulatory proteins (TARPs), γ-2 and γ-4. Our results, together with earlier findings, suggest that some of the new CP-AMPARs are synaptic. These are probably associated with γ-2, but they are loosely tethered to the PSD. Levels of GluA1 phosphorylated at serine 845 (pS845 GluA1) were significantly increased in biotinylated tissue and in an extrasynaptic membrane-enriched fraction. These results suggest that increased synaptic levels of CP-AMPARs may result in part from an increase in pS845 GluA1 in extrasynaptic membranes, given that S845 phosphorylation primes GluA1-containing AMPARs for synaptic insertion and extrasynaptic AMPARs supply the synapse. Some of the new extrasynaptic CP-AMPARs are likely associated with γ-4, rather than γ-2. The maintenance of CP-AMPARs in NAc synapses during withdrawal is accompanied by activation of CaMKII and ERK2 but not CaMKI. Overall, AMPAR plasticity in the incubation model shares some features with better described forms of synaptic plasticity, although the timing of the phenomenon and the persistence of related neuroadaptations are significantly different.


MR imaging of Crohn disease: use of perflubron as a gastrointestinal contrast agent.
This study was undertaken to evaluate the use of perflubron (perfluorooctylbromide) as an oral contrast agent for magnetic resonance (MR) imaging of patients with Crohn disease. MR examinations were performed before and after perflubron administration in 12 patients with documented Crohn disease. Glucagon was administered intramuscularly before the post-perflubron examinations. Each patient also underwent abdominal computed tomography within 48 hours of MR imaging. The imaging studies were analyzed for effectiveness of bowel marking with oral contrast agent, clarity bowel wall visualization, and presence of bowel wall thickening and extraluminal manifestations of Crohn disease such as abscess or fistula formation. Analysis of the imaging studies showed effective marking of the bowel with perflubron and improved bowel wall visualization on postcontrast MR images. Detection of bowel wall thickening and extraluminal complications of Crohn disease was not significantly improved on postcontrast MR images. The authors conclude that perflubron administration effectively marked the bowel and increased the clarity of bowel wall visualization but did not significantly increase the detection of abnormalities related to Crohn disease in the study population.


Indoor air quality in Virginia waterpipe cafes.
A revised indoor air quality law has been implemented in Virginia to protect the public from the harmful effects of secondhand smoke exposure. This legislation contains exemptions that include allowances for smoking in a room that is structurally separated and separately ventilated. The objective of the current study was to examine the impact of this law on air quality in waterpipe cafés, as well as to compare the air quality in these cafés to restaurants that allow cigarette smoking and those where no smoking is permitted. Indoor air quality in 28 venues (17 waterpipe cafés, five cigarette smoking-permitted restaurants and six smoke-free restaurants (five with valid data)) in Virginia was assessed during 4 March to 27 May 2011. Real-time measurements of particulate matter (PM) with 2.5 μm aerodynamic diameter or smaller (PM2.5) were obtained and occupant behaviour/venue characteristics were assessed. The highest mean PM2.5 concentration was observed for waterpipe café smoking rooms (374 μg/m(3), n=17) followed by waterpipe café non-smoking rooms (123 μg/m(3), n=11), cigarette smoking-permitted restaurant smoking rooms (119 μg/m(3), n=5), cigarette smoking-permitted restaurant non-smoking rooms (26 μg/m(3), n=5) and smoke-free restaurants (9 μg/m(3), n=5). Smoking density was positively correlated with PM2.5 across smoking rooms and the smoke-free restaurants. In addition, PM2.5 was positively correlated between smoking and non-smoking rooms of venues. The PM2.5 concentrations observed among the waterpipe cafés sampled here indicated air quality in the waterpipe café smoking rooms was worse than restaurant rooms in which cigarette smoking was permitted, and state-required non-smoking rooms in waterpipe cafés may expose patrons and employees to PM2.5 concentrations above national and international air quality standards. Reducing the health risks of secondhand smoke may require smoke-free establishments in which tobacco smoking sources such as water pipes are, like cigarettes, prohibited.


Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies.
S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 126.96.36.199). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.


A connectivity portfolio effect stabilizes marine reserve performance.
Well-managed and enforced no-take marine reserves generate important larval subsidies to neighboring habitats and thereby contribute to the long-term sustainability of fisheries. However, larval dispersal patterns are variable, which leads to temporal fluctuations in the contribution of a single reserve to the replenishment of local populations. Identifying management strategies that mitigate the uncertainty in larval supply will help ensure the stability of recruitment dynamics and minimize the volatility in fishery catches. Here, we use genetic parentage analysis to show extreme variability in both the dispersal patterns and recruitment contribution of four individual marine reserves across six discrete recruitment cohorts for coral grouper (Plectropomus maculatus) on the Great Barrier Reef. Together, however, the asynchronous contributions from multiple reserves create temporal stability in recruitment via a connectivity portfolio effect. This dampening effect reduces the variability in larval supply from individual reserves by a factor of 1.8, which effectively halves the uncertainty in the recruitment contribution of individual reserves. Thus, not only does the network of four marine reserves generate valuable larval subsidies to neighboring habitats, the aggregate effect of individual reserves mitigates temporal fluctuations in dispersal patterns and the replenishment of local populations. Our results indicate that small networks of marine reserves yield previously unrecognized stabilizing benefits that ensure a consistent larval supply to replenish exploited fish stocks.


A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.
A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.


Progress of fundamental research in Wilms' tumor.
The progress of fundamental research on the histopathological and molecular genetic properties, model systems, growth factor involvement, and tumor markers of clinical nephroblastoma (Wilms' tumor) are reviewed. Histologically, Wilms' tumor (WT) has been found to reveal a disorganized renal developmental process in which blastema and epithelia are randomly interspersed in varying amounts of stroma. Anaplasia is the only criterion for assigning a WT as having an "unfavorable histology." Cytogenetic analysis identified WT genes at chromosome 11p13 (WT1), 11p15 region (WT2), and 16q (WT3). Permanent in vitro WT cell lines and in vivo WT models, such as human xenografts, have been established which provide indefinite sources of tumor material for fundamental, as well as therapy-directed, research. Abnormalities of growth factor (GF) expression in WT indicate that GF may play an important role in WT pathogenesis. A series of monoclonal antibodies was tested in WT by immunohistochemical techniques to identify specific diagnostic and prognostic markers. p53 expression in anaplastic WT is significantly higher than in differentiated WTs, indicating p53 may be a prognostic marker. Although significant progress has been made in the fundamental research, our basic knowledge of this malignancy is still limited. The availability of suitable experimental models, particularly the human xenograft system, offers the opportunity for further study of the cell biological and molecular aspects of WT and its clinical progression.


Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.
In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.


Isolation and characterisation of an unexpected class of insulinotropic peptides in the skin of the frog Agalychnis litodryas.
Skin secretions of the frog Agalychnis litodryas were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse-phase high-performance liquid chromatography (HPLC) yielding 70 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 39-42 (band 1) and fractions 44-46 (band 2) significantly stimulated insulin release by 2-3.5-fold compared with 5.6 mM glucose alone. Pooled fractions in band 1 and band 2 were rechromatographed to reveal 20 homogenous peptide peaks, which elicited significant 1.5-4-fold increases in insulin release. Mass spectrometry analyses indicated molecular masses of between 1649.2 and 4988.9 Da. The two peptides with the greatest insulin-releasing activity were directly subjected to N-terminal amino acid sequence analysis. The sequence of the 3020 Da peptide, called frog skin insulinotropic peptide or FSIP, was determined as AVWKDFLKNIGKAAGKAVLNSVTDMVNE, which has 79% homology with the C-terminal of the 75 amino acid dermaseptin BIV precursor. A partial N-terminal sequence was determined for the 2546.2 Da peptide as MLADVFEKIMGD... These data indicate that the skin secretions of A. litodryas frogs contain biologically active peptides which merit further evaluation as a new class of insulin secretagogues.