Discrepancy between consensus recommendations and actual community use of adjuvant chemotherapy in women with breast cancer.
Although the efficacy of adjuvant chemotherapy in prolonging survival for women with breast cancer has been well documented, limited population-based information is available on the actual use of chemotherapy. To examine the relationship between age and chemotherapy use. Cohort study. New Mexico. 5101 women 20 years of age or older receiving a diagnosis of stage I, stage II, or stage IIIA breast cancer from 1991 through 1997. Pattern of chemotherapy use by age; logistic regression analysis to generate the odds and probabilities of receiving chemotherapy; and sensitivity analysis to estimate potential effects of unmeasured confounders. Overall, 29% of women received chemotherapy. The rate of chemotherapy use for women with stage I, stage II, or stage IIIA breast cancer was 11%, 47%, and 68%, respectively. Across all tumor stages, the use of chemotherapy decreased substantially with increasing age (P < 0.001). Overall, 66% of women younger than 45 years of age received chemotherapy compared with 44% of women between 50 and 54 years of age, 31% of women between 55 and 59 years of age, and 18% of women between 60 and 64 years of age. The decreasing pattern of chemotherapy use with age continued after adjustment for prognostic factors and was relatively insensitive to changes in unmeasured factors. There is considerable discrepancy between the 1990 National Institutes of Health Consensus Conference recommendations for chemotherapy administration in women with breast cancer and the actual use of chemotherapy in the community. The decrease in use with age may relate to the decreasing efficacy of chemotherapy with age, as reported in clinical trials. Outcomes studies should address whether the recommendations are overly aggressive or whether practicing oncologists are too conservative in their use of chemotherapy.


Callosal atrophy in multiple sclerosis is related to cognitive speed.
Long-term changes regarding corpus callosum area (CCA) and information processing speed in cognitive and sensory-motor tasks have rarely been studied in multiple sclerosis (MS). Information processing speed in cognitive (Symbol Digit Modalities Test, SDMT), sensory (visual and auditory reaction time) and motor (finger-tapping speed, FT; right and left hand) tasks as well as auditory inter-hemispheric transfer (verbal dichotic listening, VDL) was related to CCA, measured by MRI at baseline and at follow-up after nine years in 22 patients with MS. Possible confounding by demographic (age, gender and education), clinical (symptom onset, duration, severity of disease) and relative brain volume (RBV) as well as T2 lesion load was taken into account. The smaller the CCA at baseline, the slower was SDMT performance at baseline. In a similar way, CCA at follow-up was associated with poor SDMT result at follow-up. Furthermore, the higher the annual rate of change in CCA, the poorer was performance in VDL on the left ear and the more pronounced was the right ear advantage. A positive relationship between performance in VDL right ear and annual rate of change in RBV was also seen. Sensory-motor tests were not significantly associated with CCA. T2 lesion load at baseline was associated with FT performance at baseline. Demographic, clinical and radiological (RBV and T2 lesion load) characteristics did not confound the significant relation between CCA and SDMT. CCA unlike RBV and T2 lesion load was associated with SDMT, which indicated a marked cognitive rather than perceptual-motor component.


Hearing the voices of children: self-reported information on children's experiences during research procedures: a study protocol.
In paediatric research, there is a tension between what you can ask from a child and what is needed for the development of evidence-based treatments. To find an optimal balance in conducting clinical research and protecting the child, it is necessary to have empirical data on children's experiences. Until now, there are scarce empirical data on the experiences from the perspective of the child. In this manuscript, we describe the protocol of a two-phase study measuring children's self-reported experiences during research procedures. In the first phase of our study, we aim to interview approximately 40 children (6-18 years) about their self-reported experiences during research procedures. In the second phase, we will develop a questionnaire to measure children's experiences during research procedures in a quantitative way. We will use the interview outcomes for the development of this questionnaire. Next, we will measure the experiences of children during seven research procedures with this questionnaire. A one-month follow-up is conducted to investigate the emotional impact of the research procedures on the children. Children will be recruited from different research studies in three academic children's hospitals in the Netherlands. The ethics committee of the VU University medical center evaluated both studies and indicated that there was no risk/discomfort associated, stating that both phases are exempt from getting approval under the Dutch Law. Dissemination of results will occur by conference presentations and peer-reviewed publications. The findings of our project can help Institutional Review Boards and paediatric researchers when evaluating the discomforts of research procedures described in study protocols or when designing a study. Information on experiences of children involved in previous studies may also help children and parents in future research with their decision-making about participation in clinical research, or parts thereof.


Cytomegalovirus replicates in differentiated but not in undifferentiated human embryonal carcinoma cells.
To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.


Novel approaches in colorectal endoscopy: what do we need biopsies for?
Magnification chromoendoscopy, narrow band imaging (NBI) and confocal endomicroscopy can all provide accurate assessment of small and diminutive colonic lesions for neoplastic change that approaches the accuracy of standard histopathology. It is likely that there will be a move to use these techniques in clinical practice for small and particularly diminutive, non-depressed lesions in the near future. Non-neoplastic lesions would be left in situ, and neoplastic lesions resected and disposed of without histopathological assessment. Histopathology would be reserved for larger lesions, indeterminate lesions or lesions where invasion was suspected. There are potentially significant cost savings and patient benefits, with a focussing of histopathological expertise on higher risk lesions, particularly in the era of bowel cancer screening. These techniques may also help target biopsies in colitis surveillance, removing the need for large numbers of random samples. However, in order to convince patients, histopathologists and those funding healthcare of the validity of this approach, further trial data will be needed, with an accreditation process for endoscopists wishing to take on this responsibility.


Teeth: what radiologists should know.
Disease of the teeth and their support structures is common and frequently seen at imaging of the head and neck. Recognition of dental disease by the interpreting radiologist has the potential to alter the course of patient care, such as when periapical disease is identified as the cause of sinusitis or pericoronitis is identified as the cause of deep neck infection. Furthermore, incidental recognition of carious lesions in both children and adults who are undergoing CT for other reasons may alert the patient and care team of the need for a dental consultation. In fact, most of the images of dental and periodontal conditions that are used in this article were obtained from CT studies that were performed to investigate other problems. Familiarity with the imaging appearance of common dental conditions, such as hyperdontia and hypodontia, tooth trauma, periodontal disease, caries, periapical disease, odontogenic sinusitis, and deep neck infections, allows the radiologist to render a timely, confident, and specific diagnosis of dental abnormalities, even when such findings are unexpected.


Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma.
Although expression of non-protein-coding RNA (ncRNA) can be altered in human cancers, their functional relevance is unknown. Ultraconserved regions are noncoding genomic segments that are 100% conserved across humans, mice, and rats. Conservation of gene sequences across species may indicate an essential functional role, and therefore we evaluated the expression of ultraconserved RNAs (ucRNA) in hepatocellular cancer (HCC). The global expression of ucRNAs was analyzed with a custom microarray. Expression was verified in cell lines by real-time PCR or in tissues by in situ hybridization using tissue microarrays. Cellular ucRNA expression was modulated with siRNAs, and the effects on global gene expression and growth of human and murine HCC cells were evaluated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells compared with nonmalignant hepatocytes. Among these ucRNAs, the greatest change was noted for ultraconserved element 338 (uc.338), which was dramatically increased in human HCC compared with noncancerous adjacent tissues. Although uc.338 is partially located within the poly(rC) binding protein 2 (PCBP2) gene, the transcribed ncRNA encoding uc.338 is expressed independently of PCBP2 and was cloned as a 590-bp RNA gene, termed TUC338. Functional gene annotation analysis indicated predominant effects on genes involved in cell growth. These effects were experimentally demonstrated in both human and murine cells. siRNA to TUC338 decreased both anchorage-dependent and anchorage-independent growth of HCC cells. These studies identify a critical role for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.


Profiling surgical staplers: effect of staple height, buttress, and overlap on staple line failure.
Few studies have been designed to assess the performance of surgical staplers. In this study, we analyzed the effect of staple height, buttressing, and overlapping of staple lines on staple line failure. Staple lines created on fresh porcine small bowel segments ex vivo were tested for leak pressure by insufflating air into the bowel under water and recording pressure at failure. Three separate experiments were done and included staple height (white, 2.5 mm, n = 16; blue, 3.5 mm, n = 16; green, 4.1 mm, n = 16; one half of them buttressed); the absence (n = 12) or presence (n = 12) of an overlap in 3.5-mm staple lines; and the absence (n = 14) or presence (n = 11) of buttresses in 3.5-mm overlapping staple lines. Data are reported in median values and ranges; nonparametric tests were used for data analysis. In the porcine small bowel, leak pressure was related to staple height; green loads had the worst profile (35 mm Hg, range 19-105) compared with the blue (79 mm Hg, range 9-177), and white (108 mm Hg, range 28-280) loads (P = .006). Buttressing uniformly improved leak pressure for all staple loads (P <.0001). No significant difference was found between lines with overlapping (59 mm Hg, range 32-121) and those without (42 mm Hg, range 22-75; P = .162). Buttressing also improved the leak pressure of overlapping staple lines from 65 mm Hg (range 47-121) to 93 mm Hg (range 75-187; P = .0014). Great variability was found in the leak pressures among the different applications of the same stapler. Staple height is an important determinant of leak pressure. The presence of an overlap did not affect leak pressure; in fact, a trend toward improvement was seen with overlapping staple lines. Buttressing improved all types of staple lines.


Exploratory scoping of the literature on factors that influence oral health workforce planning and management in developing countries.
The purpose of this study was to scope the literature that exists about factors influencing oral health workforce planning and management in developing countries (DCs). The Arksey and O'Malley method for conducting a scoping review was used. A replicable search strategy was applied, using three databases. Factors influencing oral health workforce planning and management in DCs identified in the eligible articles were charted. Four thousand citations were identified; 41 papers were included for review. Most included papers were situational analyses. Factors identified were as follows: lack of data, focus on the restorative rather than preventive care in practitioner education, recent increase in number of dental schools (mostly private) and dentistry students, privatization of dental care services which has little impact on care maldistribution, and debates about skill mix and scope of practice. Oral health workforce management in the eligible studies has a bias towards dentist-led systems. Due to a lack of country-specific oral health related data in developing or least developed countries (LDCs), oral health workforce planning often relies on data and modelling from other countries. Approaches to oral health workforce management and planning in developing or LDCs are often characterized by approaches to increase numbers of dentists, thus not ameliorating maldistribution of service accessibility. Governments appear to be reducing support for public and preventative oral healthcare, favouring growth in privatized dental services. Changes to professional education are necessary to trigger a paradigm shift to the preventive approach and to improve relationships between different oral healthcare provider roles. This needs to be premised on greater appreciation of preventive care in health systems and funding models.


Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase.
CDC25 dual-specificity phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In human cells, CDC25A and C are involved in the control of G1/S and G2/M respectively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidence for an interaction between CDC25 phosphatases and members of the 14-3-3 protein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that phosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phosphatases and 14-3-3 proteins. We show that in the two-hybrid assay all three splice variants of CDC25B interact similarly and strongly with 14-3-3eta, beta and zeta proteins, but poorly with epsilon and Theta. In vitro, CDC25B interacts at a low level with 14-3-3beta, epsilon, zeta, eta, and Theta isoforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a specific, strong interaction between CDC25B and 14-3-3zeta and eta isoforms is revealed by a deletion of 288 residues in the amino-terminal region of CDC25B. This interaction requires the integrity of Ser 323, although it is independent of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is different from that with CDC25C. We propose that, in addition to a low affinity binding site that is available for all 14-3-3 isoforms, post-translational modification of CDC25B in vivo exposes a high-affinity binding site that is specific for the zeta and eta14-3-3 isoforms.


Photodynamic inactivation of trypsin by the aminophylline-riboflavin system: involvement of hydroxyl radical.
Riboflavin finds ubiquitous occurrence in plants and animals and functions as a coenzyme participating in various oxidation-reduction reactions during the course of metabolism. Photosensitized riboflavin generates reactive oxygen species (ROS). Aminophylline is an antiasthmatic drug and a known phosphodiesterase inhibitor. In this study we examined the effect of photoilluminated riboflavin on aminophylline using trypsin as the target molecule. The possible loss of trypsin activity due to autolysis was assayed after incubation in fluorescent light. Changes in trypsin activity caused by photoilluminated riboflavin alone and with aminophylline were monitored as functions of concentration and time. These effects were also analyzed by SDS-PAGE to visualize protein degradation. Spectra of riboflavin, alone and with aminophylline, under different conditions were taken to monitor the structural changes for elucidating the possible reaction mechanism involved. Free radical scavengers were also included in some experiments. Aminophylline alone is not known to posses any photosensitizing characteristics. However, in the presence of riboflavin and fluorescent light, aminophylline caused inactivation and fragmentation of trypsin. This fragmentation was found to be concentration dependent and was mediated by ROS. In all cases, thiourea, a scavenger of hydroxyl radicals, was most effective in scavenging the damaging effect of the riboflavin-aminophylline combination. Based on our results we suggest that photoilluminated riboflavin generates the singlet and triplet excited states that, upon energy transfer, generate (1)O(2). and (3)O(2). oxygen. These activated oxygen species probably attack aminophylline leading to its oxidation, generating hydroxyl radicals which presumably cause inactivation and fragmentation of trypsin.


Gasless, transaxillary robotic neck dissection: the technique and evidence.
Robotic neck dissection (ND) for the thyroid cancer with lateral neck metastasis case is one of the emerging surgical techniques, and has been highlighted as minimally invasive surgical technique recently. Gasless, transaxillary robotic approach enabled the surgeon to do meticulous and precise dissection in the complex procedure, such as ND, eventually showed favorable surgical safety and outcomes, as well as superior functional and cosmetic benefits. In this article, the author would like to introduce the detailed techniques and secure evidences for the gasless, transaxillary robotic ND.


Biochemical determinants of 5-fluorouracil response in vivo. The role of deoxyuridylate pool expansion.
5-Fluorodeoxyuridine monophosphate (FdUMP), the active metabolite of 5-fluorouracil (5-FU), is a tight-binding inhibitor of thymidylate synthetase, the enzyme which converts dUMP to TMP. Newly developed assays for FdUMP and dUMP were utilized to assess the competitive roles played by these nucleotides in determining the inhibition of TMP synthesis in mice bearing the P1534 ascites tumor. After 5-FU administration, levels of FdUMP reached a dose-dependent peak within 6 h in the ascites tumor and in bone marrow, and declined thereafter in a biphasic manner with an initial t 1/2 of 6 h and a final t 1/2 of 7-9 days. In duodenal mucosa, FdUMP levels were 1.8-2-fold higher than in the other tissues, but elimination was much more rapid. Simultaneous with the fall in FdUMP a progressive accumulation of the competitive substrate dUMP was observed in each tissue after 5-FU; and peak dUMP levels coincided with recovery of thymidylate synthesis, as determined by the incorporation of [3H]deoxyuridine into DNA. In vitro experiments with partially purifed thymidylate synthetase revealed and initial competitive interaction of dUMP and FdUMP, which, at high concentrations of dUMP was capable of markedly slowing the rate of irreversible inactivation of enzyme by FdUMP. These studies were found to be quantitatively consistent with a two-phase model of enzyme inactivation involving an initial competition between dUMP and FdUMP, with subsequent irreversible inactivation of enzyme by covalent linkage to the inhibitor. Recovery of thymidylate synthesis after 5-FU appears to result from both a fall in intracellular levels of inhibitor and a progressive accumulation of the competitive substrate dUMP.


A dedicated surveillance network for congenital toxoplasmosis in Greece, 2006-2009: assessment of the results.
Toxoplasmosis is caused by infection with the protozoan parasite Toxoplasma gondii. Acute infections in pregnant women may be transmitted to the fetus and cause severe illness. The purpose of this study was to establish a dedicated surveillance network (DSN) for congenital toxoplasmosis (CT) in Greece, in order to assess the birth prevalence of CT. A DSN of thirty clinicians was established for reporting CT cases from hospitals throughout Greece. The clinicians were selected on the basis that there was a high possibility the suspected cases would be referred to them from district hospitals or private clinics. Suspected cases of CT were reported on a monthly basis with a zero reporting card during a surveillance period from April 2006 to December 2009. A questionnaire was sent for any suspected case to record information including demographic parameters, clinical signs and symptoms and laboratory results. Serological and molecular confirmation of cases was performed by the Pasteur Hellenic Institute. All newborns suspected of CT received treatment and were serologically and clinically followed up for one year. The monthly response rate reached 100%, although only after reminders sent to 65% of the participant physicians. Sixty-three suspected CT cases were recorded by the DSN during the study period including fourteen confirmed and seven probable cases. Ten cases (47.6%) presented with symptoms at birth. Chorioretinitis was the most prominent manifestation, occurring in five symptomatic CT cases (50%). No other symptoms appeared by the end of the one year clinical follow up. No case was recorded by the existing surveillance system of the Hellenic Center of Disease Control and Prevention (HCDCP) during the same time period. Birth prevalence was estimated at 0.45, 0.51 and 0.51 per 10,000 births for 2007, 2008 and 2009 respectively. The incidence rate of symptomatic CT at birth was estimated at 0.10 cases per 10,000 births per year in Greece (for the period 2007-2009). The DSN for CT proved to be more sensitive than the classical notification system, easy in application and very efficient in reporting rare diseases such as CT. Similar DSNs could be used to provide useful information on other rare diseases.


Interleukin-4 induced down-regulation of skin homing receptor expression by human viral-specific CD8 T cells may contribute to atopic risk of cutaneous infection.
Factors controlling the expression of cutaneous lymphocyte-associated antigen (CLA) by T cells are poorly understood, but data from murine and human CD4(+) T cell systems have suggested that cytokines play an important role. However, there are no data examining the influence of cytokines on the expression of CLA by human antigen-specific CD8(+) T cells. Peripheral blood mononuclear cells (PBMC) were isolated from 10 HLA-A*0201-positive healthy individuals. Using HLA-peptide tetrameric complexes refolded with immunodominant peptides from Epstein-Barr virus (EBV), cytomegalovirus (CMV) and influenza A virus, we investigated the temporal associations of CLA expression by viral-specific CD8(+) T cells following stimulation with antigen. Ex vivo influenza matrix-specific CD8(+) T cells expressed significantly (P < 0.05) greater levels of CLA than EBV BMLF1 and CMV pp65-specific CD8(+) T cells (mean 9.7% influenza matrix versus 1.4% BMLF1 versus 1.1% pp65) and these differences were sustained on culture. However, regardless of viral specificity, interleukin (IL)-12 and IL-4 induced significant (P < 0.05) dose-dependent up-regulation and down-regulation of CLA expression, respectively, with IL-4 showing a dominant negative effect. In many cases, IL-4 resulted in complete abrogation of detectable CLA expression by the viral-specific CD8(+) T cells. Overall these data demonstrate that CLA expression by human viral-specific CD8(+) T cells is highly dynamic and that IL-4 causes significant down-regulation. Disorders associated with a type 2 cytokine shift may reduce the efficiency of skin homing by viral-specific CD8(+) T cells. Furthermore, the ability to modify the local and systemic microenvironment may offer novel therapeutic strategies that influence tissue-specific T cell homing.


Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling and is highly expressed in human atherosclerotic lesions.
Repressor activator protein 1 (Rap1) is essential for maintaining telomere length and structural integrity, but it also exerts other non-telomeric functions. The present study tested the hypothesis that Rap1 is released into the cytoplasm and induces production of pro-inflammatory cytokines via nuclear factor kappa B (NFκB) signaling in macrophages, a cell type involved in the development and progression of atherosclerotic lesions. Western blotting analysis confirmed that Rap1 was present in the cytoplasm of differentiated human monocytic leukemia cells (THP-1, a macrophage-like cell line). Co-immunoprecipitation assay revealed a direct interaction between Rap1 and I kappa B kinase (IKK). Knockdown of Rap1 suppressed lipopolysaccharide-mediated activation of NFκB, and phosphorylation of inhibitor of kappa B α (IκBα) and p65 in THP-1 macrophages. The reduction of NFκB activity was paralleled by a decreased production of NFκB-dependent pro-inflammatory cytokines and an increased expression of IκBα (native NFκB inhibitor) in various macrophage models with pro-inflammatory phenotype, including THP-1, mouse peritoneal macrophages and bone marrow-derived M1 macrophages. These changes were observed selectively in pro-inflammatory macrophages but not in bone marrow-derived M2 macrophages (with an anti-inflammatory phenotype), mouse lung endothelial cells, human umbilical vein endothelial cells or human aortic smooth muscle cells. Immunostaining revealed that Rap1 was localized mainly in macrophage-rich areas in human atherosclerotic plaques and that the presence of Rap1 was positively correlated with the advancement of the disease process. In pro-inflammatory macrophages, Rap1 promotes cytokine production via NFκB activation favoring a pro-inflammatory environment which may contribute to the development and progression of atherosclerosis.


Retinal arterial occlusions in the young: systemic associations in Indian population.
To determine the systemic associations in retinal arterial occlusions (RAO) in young Indian individuals less than 40 years of age. Case records of 32 patients (35 eyes) of less than 40 years, with non-traumatic RAO were analysed. All patients underwent detailed ophthalmic and systemic evaluation including hemogram, lipid profile, coagulation profile, vasculitis screening, carotid Doppler, echocardiogram. In the study 21 were males and 11 were females. The age ranged from 11-39 years (Mean 27.6 ± 8.43). Nine (28%) patients were below 20 years of age. Among 35 eyes, 28 (80%) had central retinal artery occlusion (CRAO), three (8.6%) had branch retinal artery occlusion (BRAO), two (5.7%) each had cilio-retinal (CLAO) and hemi-retinal artery occlusion (HRAO). Vision ranged from no perception of light to 20/20. On systemic evaluation, in 21 (65.6%) patients a hypercoagulable state was responsible for the RAO. Conditions leading to a hypercoagulable state included hyperhomocysteinemia (21.9%), hyperlipidemia (15.6%), anticardiolipin antibody (6.2%), antiphospholipid antibody (6.2%), polycythemia, thrombocytosis, protein S deficiency, use of oral contraceptives and renal disorder (3.1% each). Six (18.7%) patients had cardiac valvular defects. Vasculitis screening was positive in three (9.4%) patients. Two (6.2%) had isolated systemic hypertension. In two (6.2%) patients no abnormality could be detected. The systemic associations of RAOs in the Indian population were distinctly different from those reported in the Western population. Hyperhomocysteinemia was the commonest association found. Whereas associations reported in the Western population such as cardiac abnormalities, coagulation disorders, hemoglobinopathies and oral contraceptive use were uncommon.


Achromatic optical compensator-modulator.
An optical compensator is described which is achromatized in a broad spectral range. It consists of two pairs of birefringent plates of different materials under oblique incidence rotated around the normal to the plates. The phase shift is achromatized by adjusting the plate thicknesses. An electrooptic modulator based on this design is also described. Instead of mechanically rotating the system to introduce phase shifts the optic axis is rotated by applying an electric field.


Novel aspects of the molecular mechanisms controlling insulin secretion.
Pancreatic beta-cells secrete insulin by Ca(2+)-dependent exocytosis of secretory granules. beta-cell exocytosis involves SNARE (soluble NSF-attachment protein receptor) proteins similar to those controlling neurotransmitter release and depends on the close association of L-type Ca(2+) channels and granules. In most cases, the secretory granules fuse individually but there is ultrastructural and biophysical evidence of multivesicular exocytosis. Estimates of the secretory rate in beta-cells in intact islets indicate a release rate of approximately 15 granules per beta-cell per second, 100-fold higher than that observed in biochemical assays. Single-vesicle capacitance measurements reveal that the diameter of the fusion pore connecting the granule lumen with the exterior is approximately 1.4 nm. This is considerably smaller than the size of insulin and membrane fusion is therefore not obligatorily associated with release of the cargo, a feature that may contribute to the different rates of secretion detected by the biochemical and biophysical measurements. However, small molecules like ATP and GABA, which are stored together with insulin in the granules, are small enough to be released via the narrow fusion pore, which accordingly functions as a molecular sieve. We finally consider the possibility that defective fusion pore expansion accounts for the decrease in insulin secretion observed in pathophysiological states including long-term exposure to lipids.


Tubular reabsorption of pepsinogen isozymogens in man studied by the inhibition of tubular protein reabsorption with dibasic amino acids.
1. The fractional clearances of pepsinogen A (PGA), pepsinogen C (PGC) and the main PGA isozymogens, i.e. PGA-3, PGA-4 and PGA-5, were measured in 13 healthy male volunteers before and during blockade of tubular protein reabsorption by intravenous infusion of either L-arginine hydrochloride (n = 8; 0.5 g h-1 kg-1 body weight) or an equimolar amount of L-lysine hydrochloride (n = 5; 0.44 g h-1 kg-1 body weight). Glomerular filtration rate was measured by a radioisotope method. 2. The fractional baseline clearance of PGC (1 +/- 1%) was lower than that of PGA (20 +/- 10%). In addition, the fractional clearance of the PGA isozymogens appeared to be different: the fractional clearance of PGA-5 (7 +/- 3%) was lower than that of PGA-4 (18 +/- 9%), and the fractional clearance of PGA-4 was lower than that of PGA-3 (30 +/- 10%). These differences in fractional clearance between PGA isozymogens decreased during infusion of both arginine and lysine. 3. Pepsinogens are freely filtered proteins. It can therefore be concluded that the differences in fractional clearance between PGA isozymogens imply differences in tubular reabsorption. This is remarkable as PGA isozymogens are proteins with an almost identical amino acid sequence and electric charge. The disappearance of the differences in tubular reabsorption during arginine and lysine infusion suggests that PGA isozymogens differ in affinity for negatively charged binding sites in the tubular cell membrane. In order to explain the low fractional clearance of PGC compared with that of PGA and the less marked effect of arginine or lysine infusion on the fractional clearance of PGC, an additional PGC-specific binding site has to be postulated.


Dispensation of long-acting β agonists with or without inhaled corticosteroids, and risk of asthma-related hospitalisation: a population-based study.
The role of long-acting β-agonists (LABA) added to inhaled corticosteroids (ICS) in the management of asthma is extensively debated. We thought to assess the risk of asthma-related hospitalisation in individuals who regularly filled prescriptions for ICS+LABA compared to those who regularly filled prescriptions for ICS alone or LABA alone, and compared to those who did not regularly fill such medications. Using administrative health databases of the province of British Columbia (BC), Canada, from 1997 to 2012, we conducted a nested case-control analysis of a cohort of asthma patients. Cases were defined as those who experienced asthma-related hospitalisation after the first year of their entry into the cohort. For each case, up to 20 controls were matched based on age, sex, date of cohort entry, and several measures of asthma severity. We categorised individuals as regularly exposed, irregularly exposed, or non-exposed to ICS alone, LABA alone, or ICS+LABA based on dispensation records in the past 12 months. The primary outcome measures were the rate ratio (RR) of the asthma-related hospitalisation among categories of regular exposure. 3319 cases were matched to 43 023 controls. The RR for regular dispensation of ICS+LABA was 1.14 (95% CI 0.93 to 1.41) compared with regular dispensation of ICS alone and 0.45 (95% CI 0.29 to 0.70) compared with regular dispensation of LABA alone. Those who regularly dispensed LABA had to dispense an ICS for at least three quarters of a year to reduce their risk to that of those who did not dispense LABA. Regular dispensation of ICS+LABA was not associated with an increased risk of asthma-related hospitalisation compared with regular dispensation of ICS alone. Adherence to ICS in patients who regularly receive ICS+LABA seems to be an important factor in the prevention of adverse asthma-related outcomes.


Subtrochanteric fracture: the effect of cerclage wire on fracture reduction and outcome.
Subtrochanteric neck of femur fractures are a challenge to treat due to anatomical and biomechanical factors. Poor reduction, varus deformity, nonunion and return to theatre risks are high. A cerclage wire can augment an intramedullary nail to help fracture reduction and construct stability. Concerns exist regarding the use of cerclage wire on fracture zone vascularity. The aim of this study was to assess the benefits and adverse outcomes associated with the use of cerclage wiring. A 7-year retrospective review of all subtrochanteric fractures at a Level 1 trauma centre was performed. Pathological fractures, those associated with bisphosphonate use and segmental fractures were excluded. A clinical and radiographic review was performed. Our primary outcome measure was a composite of the major complications of this surgery, defined as either return to theatre for fixation failure, nonunion or implant failure. Fracture displacement, angulation and quality of reduction were measured as secondary outcome measures. Specific complications of the use of cerclage wiring were also reported. One hundred and thirty four cases met the inclusion criteria for primary outcome. Reduction was achieved closed in 51.9% (n=70), open in 33.3% (n=45) and open with cerclage wire in 14.8% (n=20). Overall there were a total of 13 (9.7%) major complications. No cases with cerclage wire had a return to theatre. If cerclage wire was not used the major complication rate was 11.4%. Fracture displacement (11.0mm vs. 7.69mm) and distraction were related to return to theatre (p<0.05). Cerclage wire use improved fracture displacement (3.2mm vs. 8.8mm), angulation and quality of reduction (p<0.05). Anatomical reduction is the key to success of subtrochanteric fractures. Cerclage wire use results in better fracture reduction. Some subtrochanteric fractures can be successfully treated with indirect reduction alone. If fractures cannot be reduced closed, reduction should be achieved by open methods. If a fracture is opened, a cerclage wire should be used, if the fracture pattern allows.


A review of selection-based tests of abiotic surrogates for species representation.
Because conservation planners typically lack data on where species occur, environmental surrogates--including geophysical settings and climate types--have been used to prioritize sites within a planning area. We reviewed 622 evaluations of the effectiveness of abiotic surrogates in representing species in 19 study areas. Sites selected using abiotic surrogates represented more species than an equal number of randomly selected sites in 43% of tests (55% for plants) and on average improved on random selection of sites by about 8% (21% for plants). Environmental diversity (ED) (42% median improvement on random selection) and biotically informed clusters showed promising results and merit additional testing. We suggest 4 ways to improve performance of abiotic surrogates. First, analysts should consider a broad spectrum of candidate variables to define surrogates, including rarely used variables related to geographic separation, distance from coast, hydrology, and within-site abiotic diversity. Second, abiotic surrogates should be defined at fine thematic resolution. Third, sites (the landscape units prioritized within a planning area) should be small enough to ensure that surrogates reflect species' environments and to produce prioritizations that match the spatial resolution of conservation decisions. Fourth, if species inventories are available for some planning units, planners should define surrogates based on the abiotic variables that most influence species turnover in the planning area. Although species inventories increase the cost of using abiotic surrogates, a modest number of inventories could provide the data needed to select variables and evaluate surrogates. Additional tests of nonclimate abiotic surrogates are needed to evaluate the utility of conserving nature's stage as a strategy for conservation planning in the face of climate change.


Partial squeeze film levitation modulates fingertip friction.
When touched, a glass plate excited with ultrasonic transverse waves feels notably more slippery than it does at rest. To study this phenomenon, we use frustrated total internal reflection to image the asperities of the skin that are in intimate contact with a glass plate. We observed that the load at the interface is shared between the elastic compression of the asperities of the skin and a squeeze film of air. Stroboscopic investigation reveals that the time evolution of the interfacial gap is partially out of phase with the plate vibration. Taken together, these results suggest that the skin bounces against the vibrating plate but that the bounces are cushioned by a squeeze film of air that does not have time to escape the interfacial separation. This behavior results in dynamic levitation, in which the average number of asperities in intimate contact is reduced, thereby reducing friction. This improved understanding of the physics of friction reduction provides key guidelines for designing interfaces that can dynamically modulate friction with soft materials and biological tissues, such as human fingertips.


Kilo-scale droplet generation in three-dimensional monolithic elastomer device (3D MED).
Droplet-based microfluidics has led to transformational new approaches in diverse areas including materials synthesis and high-throughput biological assays. However, the translation of droplet microfluidics technology into commercial applications requires scale-up of droplet generation from the laboratory (<10 mL h(-1)) to the industrial (>1 L h(-1)) scale. To address this challenge, we develop a three-dimensional monolithic elastomer device (3D MED) for mass production of monodisperse emulsion droplets. Using double-sided imprinting, 3D microchannels are formed in a single elastomer piece that has 1000 parallel flow focusing generators (k-FFGs). Compared to previous work that parallelizes droplet generation, the 3D MED eliminates the needs for alignment and bonding of multiple pieces and thus makes it possible to achieve the high flow rates and pressure necessary for the kilo-scale generation of droplets. Using this approach, we demonstrate mass production of water-in-oil (W/O) emulsion droplets at production rates as high as 1.5 L h(-1) (>30 billion 45 μm diameter droplets per hour), with a coefficient of variation of droplet diameter of only 6.6%. Because of the simplicity, robustness, and manufacturability of our 3D MED architecture, it is well suited to bridge the gap between the continuously growing library of promising microfluidic technologies to generate microparticles that have been demonstrated in laboratory settings and their successful application in industry.


The DICS repository: module-assisted analysis of disease-related gene lists.
The DICS database is a dynamic web repository of computationally predicted functional modules from the human protein-protein interaction network. It provides references to the CORUM, DrugBank, KEGG and Reactome pathway databases. DICS can be accessed for retrieving sets of overlapping modules and protein complexes that are significantly enriched in a gene list, thereby providing valuable information about the functional context. Supplementary information on datasets and methods is available on the web server http://mips.gsf.de/proj/dics.


Histomorphologic evaluation of extracorporeal shock wave therapy of the fourth metatarsal bone and the origin of the suspensory ligament in horses without lameness.
To determine via histologic examination and scintigraphy the effect of focused extracorporeal shock wave therapy (ESWT) on normal bone and the bone-ligament interface in horses. 6 horses without lameness. Origins of the suspensory ligament at the metacarpus (35-mm probe depth) and fourth metatarsal bone (5-mm probe depth) were treated twice (days 0 and 16) with 2,000 shocks (energy flux density, 0.15 mJ/mm2). One forelimb and 1 hind limb were randomly treated, and the contralateral limbs served as nontreated controls. Bone scans were performed on days -1 (before ESWT), 3, 16, and 19. Histomorphologic studies of control and treated tissues were performed on day 30. ESWT significantly increased the number of osteoblasts but caused no damage to associated soft tissue structures and did not induce cortical microfractures. A significant correlation between osteoblast numbers and radiopharmaceutical uptake was noticed on lateral views of the hind limb on days 3 and 16 and on caudal views of the forelimb on day 3. Results suggested that ESWT has the potential to increase osteoblast numbers in horses. The correlation between increased osteoblast numbers and radio-pharmaceutical uptake 3 days and 16 days after the first ESWT suggested that stimulation of osteogenesis occurred soon after ESWT. No damage to bone or the bone-ligament interface should occur at the settings used in this study, and ESWT can therefore be administered safely in horses.


Simulating interactive effects of symbiotic nitrogen fixation, carbon dioxide elevation, and climatic change on legume growth.
The underlying mechanisms of interaction between the symbiotic nitrogen-fixation process and main physiological processes, such as assimilation, nutrient allocation, and structural growth, as well as effects of nitrogen fixation on plant responses to global change, are important and still open to more investigation. Appropriate models have not been adequately developed. A dynamic ecophysiological model was developed in this study for a legume plant [Glycine max (L.) Merr.] growing in northern China. The model synthesized symbiotic nitrogen fixation and the main physiological processes under variable atmospheric CO2 concentration and climatic conditions, and emphasized the interactive effects of these processes on seasonal biomass dynamics of the plant. Experimental measurements of ecophysiological quantities obtained in a CO2 enrichment experiment on soybean plants, were used to parameterize and validate the model. The results indicated that the model simulated the experiments with reasonable accuracy. The R2 values between simulations and observations are 0.94, 0.95, and 0.86 for total biomass, green biomass, and nodule biomass, respectively. The simulations for various combinations of atmospheric CO2 concentration, precipitation, and temperature, with or without nitrogen fixation, showed that increasing atmospheric CO2 concentration, precipitation, and efficiency of nitrogen fixation all have positive effects on biomass accumulation. On the other hand, an increased temperature induced lower rates of biomass accumulation under semi-arid conditions. In general, factors with positive effects on plant growth tended to promote each other in the simulation range, except the relationship between CO2 concentration and climatic factors. Because of the enhanced water use efficiency with a higher CO2 concentration, more significant effects of CO2 concentration were associated with a worse (dryer and warmer in this study) climate.


The Effects of Preexercise Caffeinated Coffee Ingestion on Endurance Performance: An Evidence-Based Review.
Endurance athletes commonly ingest caffeine as a means to enhance training intensity and competitive performance. A widely-used source of caffeine is coffee, however conflicting evidence exists regarding the efficacy of coffee in improving endurance performance. In this context, the aims of this evidence-based review were threefold: 1) to evaluate the effects of preexercise coffee on endurance performance, 2) to evaluate the effects of coffee on perceived exertion during endurance performance, and 3) to translate the research into usable information for athletes to make an informed decision regarding the intake of caffeine via coffee as a potential ergogenic aid. Searches of three major databases were performed using terms caffeine and coffee, or coffee-caffeine, and endurance, or aerobic. Included studies (n = 9) evaluated the effects of caffeinated coffee on human subjects, provided the caffeine dose administered, administered caffeine ≥ 45 min before testing, and included a measure of endurance performance (e.g., time trial). Significant improvements in endurance performance were observed in five of nine studies, which were on average 24.2% over controls for time to exhaustion trials, and 3.1% for time to completion trials. Three of six studies found that coffee reduced perceived exertion during performance measures significantly more than control conditions (p < .05). Based on the reviewed studies there is moderate evidence supporting the use of coffee as an ergogenic aid to improve performance in endurance cycling and running. Coffee providing 3-8.1 mg/kg (1.36-3.68 mg/lb) of caffeine may be used as a safe alternative to anhydrous caffeine to improve endurance performance.


Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector.
Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency.


Implementation of recommended trauma system criteria in south-eastern Norway: a cross-sectional hospital survey.
Formalized trauma systems have shown beneficial effects on patient survival and have harvested great recognition among health care professionals. In spite of this, the implementation of trauma systems is challenging and often met with resistance.Recommendations for a national trauma system in Norway were published in 2007. We wanted to assess the level of implementation of these recommendations. A survey of all acute care hospitals that receive severely injured patients in the south-eastern health region of Norway was conducted. A structured questionnaire based on the 2007 national recommendations was used in a telephone interview of hospital trauma personnel between January 17 and 21, 2011. Seventeen trauma system criteria were identified from the recommendations. Nineteen hospitals were included in the study and these received more than 2000 trauma patients annually via their trauma teams. Out of the 17 criteria that had been identified, the hospitals fulfilled a median of 12 criteria. Neither the size of the hospitals nor the distance between the hospitals and the regional trauma centre affected the level of trauma resources available. The hospitals scored lowest on the criteria for transfer of patients to higher level of care and on the training requirements for members of the trauma teams. Our study identifies a major shortcoming in the efforts of regionalizing trauma in our region. The findings indicate that training of personnel and protocols for inter-hospital transfer are the major deficiencies from the national trauma system recommendations. Resources for training of personnel partaking in trauma teams and development of inter-hospital transfer agreements should receive immediate attention.


A rapid method for direct detection of metabolic conversion and magnetization exchange with application to hyperpolarized substrates.
In this work, we present a new MR spectroscopy approach for directly observing nuclear spins that undergo exchange, metabolic conversion, or, generally, any frequency shift during a mixing time. Unlike conventional approaches to observe these processes, such as exchange spectroscopy (EXSY), this rapid approach requires only a single encoding step and thus is readily applicable to hyperpolarized MR in which the magnetization is not replenished after T(1) decay and RF excitations. This method is based on stimulated-echoes and uses phase-sensitive detection in conjunction with precisely chosen echo times in order to separate spins generated during the mixing time from those present prior to mixing. We are calling the method Metabolic Activity Decomposition Stimulated-echo Acquisition Mode or MAD-STEAM. We have validated this approach as well as applied it in vivo to normal mice and a transgenic prostate cancer mouse model for observing pyruvate-lactate conversion, which has been shown to be elevated in numerous tumor types. In this application, it provides an improved measure of cellular metabolism by separating [1-(13)C]-lactate produced in tissue by metabolic conversion from [1-(13)C]-lactate that has flowed into the tissue or is in the blood. Generally, MAD-STEAM can be applied to any system in which spins undergo a frequency shift.


Algorithms, atrophy and Alzheimer's disease: cautionary tales for clinical trials.
Thompson and Holland (2010) highlight a biologically implausible deceleration of atrophy in results previously published in this journal (Hua et al., 2010); the results were derived using tensor based morphometry on images from the Alzheimer's Disease Neuroimaging Initiative. They speculate that bias may have been introduced due to asymmetric interpolation in global image registration, and/or to the use of a statistically defined region of interest. In their reply, Hua et al. (this issue) acknowledge the presence of a bias, but show that it stems largely from an asymmetry in the local image registration algorithm (an asymmetry common to methods in many published studies using nonlinear registration). Hua et al. demonstrate that the bias can largely be removed using a revised symmetric algorithm. This correspondence raises important issues relating to the lack of ground truth against which image analysis methodologies designed to determine atrophy patterns and rates can be assessed; and the increasing importance of striving to avoid potential biases as these techniques become utilised in clinical trials. In the absence of a "gold standard", we discuss a number of steps against which methodologies designed to quantify atrophy from serial scans can be assessed.


Broad domain generality in focal regions of frontal and parietal cortex.
Unlike brain regions that respond selectively to specific kinds of information content, a number of frontal and parietal regions are thought to be domain- and process-general: that is, active during a wide variety of demanding cognitive tasks. However, most previous evidence for this functional generality in humans comes from methods that overestimate activation overlap across tasks. Here we present functional MRI evidence from single-subject analyses for broad functional generality of a specific set of brain regions: the same sets of voxels are engaged across tasks ranging from arithmetic to storing information in working memory, to inhibiting irrelevant information. These regions have a specific topography, often lying directly adjacent to domain-specific regions. Thus, in addition to domain-specific brain regions tailored to solve particular problems of longstanding importance to our species, the human brain also contains a set of functionally general regions that plausibly endow us with the cognitive flexibility necessary to solve novel problems.


The Association of Obesity and Cardiometabolic Traits With Incident HFpEF and HFrEF.
This study evaluated the associations of obesity and cardiometabolic traits with incident heart failure with preserved versus reduced ejection fraction (HFpEF vs. HFrEF). Given known sex differences in HF subtype, we examined men and women separately. Recent studies suggest that obesity confers greater risk of HFpEF versus HFrEF. Contributions of associated metabolic traits to HFpEF are less clear. We studied 22,681 participants from 4 community-based cohorts followed for incident HFpEF versus HFrEF (ejection fraction ≥50% vs. <50%). We evaluated the association of body mass index (BMI) and cardiometabolic traits with incident HF subtype using Cox models. The mean age was 60 ± 13 years, and 53% were women. Over a median follow-up of 12 years, 628 developed incident HFpEF and 835 HFrEF. Greater BMI portended higher risk of HFpEF compared with HFrEF (hazard ratio [HR]: 1.34 per 1-SD increase in BMI; 95% confidence interval [CI]: 1.24 to 1.45 vs. HR: 1.18; 95% CI: 1.10 to 1.27). Similarly, insulin resistance (homeostatic model assessment of insulin resistance) was associated with HFpEF (HR: 1.20 per 1-SD; 95% CI: 1.05 to 1.37), but not HFrEF (HR: 0.99; 95% CI: 0.88 to 1.11; p < 0.05 for difference HFpEF vs. HFrEF). We found that the differential association of BMI with HFpEF versus HFrEF was more pronounced among women (p for difference HFpEF vs. HFrEF = 0.01) when compared with men (p = 0.34). Obesity and related cardiometabolic traits including insulin resistance are more strongly associated with risk of future HFpEF versus HFrEF. The differential risk of HFpEF with obesity seems particularly pronounced among women and may underlie sex differences in HF subtypes.


Phenotype-associated changes in retinal pigment epithelial cell expression of insulin-like growth factor binding proteins.
The objectives of this study were to evaluate retinal pigment epithelial (RPE) cells as a source of insulin-like growth factor binding proteins (IGFBPs) and to characterize biosynthetic changes associated with the cell phenotype and vitreous growth factor stimuli known to be present in fibrocontractive diseases. Early culture-associated changes in RPE phenotype were characterized by indirect immunofluorescence localization and Western blot analysis of cell lysates. IGFBP expression was evaluated by RT-PCR and Northern blot analysis of total RNA preparations. Normal unperturbed RPE are immunoreactive for cytokeratin 18 and negative for cytokeratin 19, vimentin, and alpha-smooth muscle actin (alphaSMA). Early reactive RPE (7 days in culture) express cytokeratin 18, cytokeratin 19, and vimentin. Myofibroblastic RPE (35 days in culture) express cytokeratin 19, vimentin, and alphaSMA. RT-PCR studies revealed that normal RPE can produce IGFBP-2, -3, -4, -5, and -6 but not IGFBP-1. Early reactive and myofibroblastic RPE have detectable levels of message for IGFBP-3, -5, and -6. However, Northern blot analysis suggests that IGFBP-5 is the predominant binding protein produced. Finally, stimulation with biologically relevant quantities of IGF-I and IGF-II had no detectable effects on IGFBP expression. Changes in RPE phenotype are accompanied by dramatic changes in IGFBP expression profile, with IGFBP-5 the predominant binding protein produced by myofibroblastic RPE cells.


Modeling the Electron Transfer Chain in an Artificial Photosynthetic Machine.
The development of efficient artificial leaves relies on the subtle combination of molecular assemblies able to absorb sunlight, converting light energy into electrochemical potential energy and finally transducing it into accessible chemical energy. The electronic design of these charge transfer molecular machines is crucial to build a complex supramolecular architecture for the light energy conversion. Here, we present an ab initio simulation of the whole decay pathways of a recently proposed artificial molecular reaction center. A complete structural and energetic characterization has been carried out with methods based on density functional theory, its time-dependent version, and a broken-symmetry approach. On the basis of our findings we provide a revision of the pathway only indirectly postulated from an experimental point of view, along with unprecedented and significant insights on the electronic and nuclear structure of intramolecular charge-separated states, which are fundamental for the application of this molecular assembly in photoelectrochemical cells. Importantly, we unravel the molecular driving forces of the various charge transfer steps, in particular those leading to the proton-coupled electron transfer final product, highlighting key elements for the future design strategies of such molecular assays.


Impact of a "Diagonal" Intervention on Uptake of Sexual and Reproductive Health Services by Female Sex Workers in Mozambique: A Mixed-Methods Implementation Study.
Female sex workers (FSWs) have high risks for adverse sexual and reproductive health (SRH) outcomes, yet low access to services. Within an implementation research project enhancing uptake of SRH services by FSWs, we piloted a "diagonal" intervention, which combined strengthening of FSW-targeted services (vertical) with making public health facilities more FSW-friendly (horizontal), and tested its effect. The study applied a convergent parallel mixed-methods design to assess changes in access to SRH services. Results of structured interviews with FSWs pre-intervention (N = 311) and thereafter (N = 404) were compared with the findings of eight post-intervention focus group discussions (FGDs) with FSWs and two with FSW-peer educators (PEs). Marked and statistically significant rises occurred in consistent condom use with all partners (55.3-67.7%), ever use of female condoms (37.9-54.5%), being tested for HIV in the past 6 months (56.0-76.6%), using contraception (84.5-95.4%), ever screened for cervical cancer (0.0-16.9%) and having ≥10 contacts with a PE in the past year (0.5-24.45%). Increases mostly resulted from FSW-targeted outreach, with no rise detected in utilization of public health facilities. FGD participants reported that some facilities had become more FSW-friendly, but barriers such as stock-outs, being asked for bribes and disrespectful treatment persisted. The combination of expanding FSW-targeted SRH services with improving access to the public health services resulted in an overall increased uptake of services, but almost exclusively because of the strengthened targeted (vertical) outreach services. Utilization of public SRH services had not yet increased and many barriers to access remained. Our diagonal approach was thus only successful in its vertical component. Improving access to the general health services remains nevertheless important and further research is needed how to reduce barriers. Ideally, the combination approach should be maintained and more successful approaches to increase utilization of public services should be explored.


Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber.
Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.


Making sense of antisense.
The specific and rational targeting of key genes, identified to be vital to driving cancer growth, has recently led to the successful development of several small molecule and antibody therapeutics. However, despite considerable efforts, antisense oligonucleotides (ASO) have yet to prove their worth as targeted therapies. However, many important genes cannot be readily targeted by antibodies or small molecules, and could be blocked by ASOs. Moreover, the latest generation of ASOs is safe, well tolerated and able to modulate target protein expression both in surrogate and tumour tissue in the clinic. This review will describe the experience acquired with these agents to date and will raise critical issues relevant to the further optimal development of these agents. Future clinical studies need to evaluate combinations of several different ASO targeting multiple key targets, including strategies that reverse functional redundancy of the key target (e.g., targeting several Bcl family members including Bcl-2 and Bcl-x). Approaches to maximise the duration of target blockade yet avert the need for prolonged intravenous infusions, with the consequent risk of line infection and thrombosis, are also needed. These may include slow-release depot subcutaneous formulations. Short interfering (Si) RNA therapeutics, which are now being evaluated in early clinical trials, are also envisioned to impact the future utility of this class of therapeutics. The high manufacture cost of these agents, when compared with small chemical molecules, could however, limit their success unless cost-effective manufacturing processes are developed.


Enhancing condom use experiences among young men to improve correct and consistent condom use: feasibility of a home-based intervention strategy (HIS-UK).
Condoms remain the main protection against sexually transmitted infections (STIs) when used correctly and consistently. Yet, there are many reported barriers to their use such as negative attitudes, reduced sexual pleasure, fit-and-feel problems and erection difficulties. The UK home-based intervention strategy (HIS-UK) is a behaviour change condom promotion intervention for use among young men (aged 16-25 years) designed to increase condom use by enhancing enjoyment of condom-protected intercourse. The objective of this feasibility study was to test HIS-UK for viability, operability and acceptability. Along with an assessment of the recruitment strategy and adherence to the intervention protocol, the study tested the reliability and suitability of a series of behavioural and condom use outcome measures to assess condom use attitudes, motivations, self-efficacy, use experience, errors and problems and fit and feel. The HIS-UK intervention and associated assessment instruments were tested for feasibility using a single-arm, repeated measures design with baseline measurement and two follow-up measurements over 3 months. A 3-month target of 50 young men completing the baseline questionnaire was set. Twenty process and acceptability evaluation interviews with participants and health promotion professionals were conducted post trial. Of the 61 young men who registered for the study, 57 completed the baseline questionnaire and 33 met with the study researcher to receive the HIS-UK condom kit. Twenty-one young men remained for the duration of the study (64% retention). The Cronbach's alpha scores for the condom use outcome measures were 0.84 attitudes, 0.78 self-efficacy, 0.83 use experience, 0.69 errors and problems and 0.75 fit and feel. Participant and health professional feedback indicated strong acceptability of the intervention. The feasibility study demonstrated that our recruitment strategy was appropriate and the target sample size was achieved. Adherence was favourable when compared to other similar studies. The condom use measures tested proved to be fit-for-purpose with good internal consistency. Some further development and subsequent piloting of HIS-UK is required prior to a full randomised controlled trial, including the feasibility of collecting STI biomarkers, and assessment of participant acceptance of randomisation. Research registry, RR2315, 27th March 2017 (retrospectively registered).


Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span.
Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.


Magnetic resonance imaging findings in treated spinal dural arteriovenous fistulas: lack of correlation with clinical outcomes.
Little information is available on follow-up MR imaging after treatment of spinal dural arteriovenous fistulas (DAVFs). The authors studied MR imaging findings in treated spinal DAVFs in relation to clinical outcomes. A retrospective review of patients with spinal DAVFs who had undergone both pre- and postoperative spinal MR imaging was conducted. Postoperative MR images were obtained as routine follow-up studies or because of subjective or objective clinical deterioration. Several pre- and posttreatment MR imaging characteristics were evaluated by 2 neuroradiologists blinded to the clinical outcome. Clinical outcomes of motor, sensory, and urinary function (in relation to the patient's preoperative status) at the time of the postoperative MR imaging were obtained from the clinical record. The chi-square, Fisher exact, and rank-sum tests were performed to correlate imaging findings and changes with clinical outcomes. Thirty-four patients met inclusion criteria. Treatment was surgical in 33 patients and endovascular in 1 patient. Follow-up MR imaging was performed at a mean 168 ± 107 days after treatment. Twenty-seven patients (79.4%) were either clinically stable or improved, and 7 (20.6%) experienced worsening in one or more clinical outcomes. Most patients were found to have improvement of MR imaging changes. However, some degree of persistent spinal cord signal abnormality, enhancement, and swelling was observed in 31 (91.2%), 29 (85.3%), and 18 (52.3%) patients, respectively. Changes in these MR imaging characteristics compared with preoperative MR imaging did not correlate with clinical outcomes (p > 0.05), with the one exception of a significant correlation between change in urinary function and extent of spinal cord contrast enhancement (p = 0.026), a correlation of uncertain importance. Ten of the 34 patients underwent posttreatment digital subtraction angiography, and 3 of these patients had recurrent/residual DAVFs. Worsening of motor function significantly correlated with recurrent/residual DAVF (p = 0.053). Spinal cord abnormalities persist on postoperative MR imaging studies in patients with treated spinal DAVFs, and although they tend to mildly improve with time, these changes may not correlate with clinical outcomes. However, regardless of imaging findings, worsening motor function may correlate with a recurrent or residual DAVF.


A genetically engineered oncolytic adenovirus decoys and lethally traps quiescent cancer stem-like cells in S/G2/M phases.
Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem-like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem-like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem-like cells. We established CD133(+) cancer stem-like cells from human gastric cancer MKN45 and MKN7 cells. We investigated the efficacy of OBP-301 against quiescent cancer stem-like cells. We visualized the treatment dynamics of OBP-301 killing of quiescent cancer stem-like cells in dormant tumor spheres and xenografts using a fluorescent ubiquitination cell-cycle indicator (FUCCI). CD133(+) gastric cancer cells had stemness properties. OBP-301 efficiently killed CD133(+) cancer stem-like cells resistant to chemoradiotherapy. OBP-301 induced cell-cycle mobilization from G0-G1 to S/G2/M phases and subsequent cell death in quiescent CD133(+) cancer stem-like cells by mobilizing cell-cycle-related proteins. FUCCI enabled visualization of quiescent CD133(+) cancer stem-like cells and proliferating CD133(-) non-cancer stem-like cells. Three-dimensional visualization of the cell-cycle behavior in tumor spheres showed that CD133(+) cancer stem-like cells maintained stemness by remaining in G0-G1 phase. We showed that OBP-301 mobilized quiescent cancer stem-like cells in tumor spheres and xenografts into S/G2/M phases where they lost viability and cancer stem-like cell properties and became chemosensitive. Oncolytic adenoviral infection is an effective mechanism of cancer cell killing in solid cancer and can be a new therapeutic paradigm to eliminate quiescent cancer stem-like cells.


Post-disaster psychosocial services across Europe: the TENTS project.
At present post-disaster activities and plans seem to vary widely. An adequate estimation of the availability of post-disaster psychosocial services across Europe is needed in order to compare them with recently developed evidence-informed psychosocial care guidelines. Here we report on the results of a cross-sectional web-based survey completed in 2008 by two hundred and eighty-six representatives of organizations involved in psychosocial responses to trauma and disaster from thirty-three different countries across Europe. The survey addressed planning and delivery of psychosocial care after disaster, methods of screening and diagnosis, types of interventions used, and other aspects of psychosocial care after trauma. The findings showed that planning and delivery of psychosocial care was inconsistent across Europe. Countries in East Europe seemed to have less central coordination of the post-disaster psychosocial response and fewer post-disaster guidelines that were integrated into specific disaster or contingency plans. Several forms of psychological debriefing, for which there is no evidence of efficacy to date, were still used in several areas particularly in North Europe. East European countries delivered evidence-based interventions for PTSD less frequently, whilst in South- and South-Eastern European countries anxiety suppressing medication such as benzodiazepines were prescribed more frequently to disaster victims than in other areas. Countries across Europe are currently providing sub-optimal psychosocial care for disaster victims. This short report shows that there is an urgent need for some countries to abandon non-effective interventions and others to develop more evidence based and effective services to facilitate the care of those involved in future disasters.


Iron loading exerts synergistic action via a different mechanistic pathway from that of acetaminophen-induced hepatic injury in mice.
Acetaminophen (APAP) overdose is a major cause of drug-induced acute liver failure. In such cases, free iron is released from lysosomes and is transported to mitochondria where it plays a pivotal role in APAP-induced liver injury. We previously reported that ascorbic acid (Asc) markedly mitigates APAP-induced hepatic damage in aldehyde reductase (Akr1a)-knockout (KO) mice that produce about 10% Asc as wild-type (WT) mice. However, the issue of the protective mechanism of Asc in association with the status of iron remains ambiguous. To gain additional insights into this issue, we examined effects of APAP (500 mg/kg) on female KO mice under conditions of iron loading. While the KO mice without AsA supplementation were more sensitive to APAP toxicity than the WT mice, FeSO4 loading (25 mg/kg) to WT mice aggravated the hepatic injury, which was a similar extent to that of the KO mice. Supplementation of Asc (1.5 mg/ml in the drinking water) ameliorated KO mice irrespective of iron status but did not change the iron-mediated increase in the lethality in the WT mice. Hepatic cysteine and glutathione levels declined to similar extents in all mouse groups at 3 h irrespective of the iron status and largely recovered at 18 h after the APAP treatment when liver damage was evident. Asc prominently mitigated APAP toxicity in KO mice irrespective of the iron status but had no effect on the synergistic action of iron and APAP in the WT mice, suggesting that the mechanism for the deteriorating action of loaded iron is different from that of APAP toxicity.


The interplay between macronutrients and sleep: focus on circadian and homeostatic processes.
Sleep disturbances are an emerging risk factor for metabolic diseases, for which the burden is particularly worrying worldwide. The importance of sleep for metabolic health is being increasingly recognized, and not only the amount of sleep plays an important role, but also its quality. In this review, we studied the evidence in the literature on macronutrients and their influence on sleep, focusing on the mechanisms that may lay behind this interaction. In particular, we focused on the effects of macronutrients on circadian and homeostatic processes of sleep in preclinical models, and reviewed the evidence of clinical studies in humans. Given the importance of sleep for health, and the role of circadian biology in healthy sleep, it is important to understand how macronutrients regulate circadian clocks and sleep homeostasis.


Young mother-father dyads and maternal harsh parenting behavior.
This study examined whether the age of parents predicted maternal harsh parenting behavior, specifically whether younger mothers might be at higher risk than older mothers, and which paternal characteristics might be associated with maternal parenting behavior. This study used data from the Fragile Families and Child Wellbeing (FFCW) study. In the present study, the authors examined a subsample of families for which complete data were available on all variables that were used in the analyses (n=1,597). Based on the parents' age at the time of the child's birth, mother-father age-dyad types were classified, and selected paternal factors were used to examine their association with maternal harsh parenting behavior. Psychological aggression, physical aggression, and self-reports of spanking were used as proxies for maternal harsh parenting behavior. Multivariate analyses indicated that adolescent mothers, regardless of how old their partners were, were at higher risk for harsh parenting behavior than older adult mothers. Regarding paternal factors, paternal coercion against mother and the fathers' use of spanking were significantly associated with all three proxies for maternal harsh parenting behavior. Fathers' employment was a risk factor for maternal physical aggression. This study supported findings from previous studies that younger mothers may indeed be at greater risk for harsh parenting behavior. It is critical, therefore, that they acquire appropriate parenting behavior and develop a healthy relationship with their children. Additional studies, both cross-sectional and longitudinal, are needed to involve their partners (i.e., their child's father) in order to shed light on ways of preventing harsh parenting behavior and examining the role of fathers in maternal parenting behavior. The present study calls for more attention to sex education and intervention programs in school and health care settings as important components of prevention services. Practitioners need to better understand the concept of harsh parenting behavior in order to work with young parents and prevent future physical child abuse. Policy makers should support these efforts and research should be done that engages both mothers and fathers and seeks to enhance and modify existing programs for youths.


Lysophosphatidic Acid Initiates Epithelial to Mesenchymal Transition and Induces β-Catenin-mediated Transcription in Epithelial Ovarian Carcinoma.
During tumor progression, epithelial ovarian cancer (EOC) cells undergo epithelial-to-mesenchymal transition (EMT), which influences metastatic success. Mutation-dependent activation of Wnt/β-catenin signaling has been implicated in gain of mesenchymal phenotype and loss of differentiation in several solid tumors; however, similar mutations are rare in most EOC histotypes. Nevertheless, evidence for activated Wnt/β-catenin signaling in EOC has been reported, and immunohistochemical analysis of human EOC tumors demonstrates nuclear staining in all histotypes. This study addresses the hypothesis that the bioactive lipid lysophosphatidic acid (LPA), prevalent in the EOC microenvironment, functions to regulate EMT in EOC. Our results demonstrate that LPA induces loss of junctional β-catenin, stimulates clustering of β1 integrins, and enhances the conformationally active population of surface β1 integrins. Furthermore, LPA treatment initiates nuclear translocation of β-catenin and transcriptional activation of Wnt/β-catenin target genes resulting in gain of mesenchymal marker expression. Together these data suggest that LPA initiates EMT in ovarian tumors through β1-integrin-dependent activation of Wnt/β-catenin signaling, providing a novel mechanism for mutation-independent activation of this pathway in EOC progression.


Association of XRCC3 18067 C>T (Thr241Met) polymorphism with risk of cervical and ovarian cancers: A systematic review and meta-analysis.
The 18067 C>T polymorphism of XRCC3 gene has been considered to be implicated in the development of cervical and ovarian cancers, but the results are inconsistent. Thus, we conducted a meta-analysis to assess the association of XRCC3 18067 C>T polymorphism with risk of cervical and ovarian cancers. All studies on the association of XRCC3 18067 C>T polymorphism with cervical and ovarian cancers risk were retrieved. Finally, a total of 17 studies including 10 studies with 5,637 cases and 10,057 controls on ovarian cancer and 7 studies with 1,112 cases and 1,233 controls on cervical cancer were selected. Overall, pooled results showed that the XRCC3 18067 C>T polymorphism was significantly associated with increased risk of ovarian cancer (TC vs. CC: OR = 0.904, 95% CI = 0.841-0.972, p = 0.006; TT + TC vs. CC: OR = 0.914, 95% CI = 0.853-0.979, p = 0.010) and cervical cancer (TC vs. CC: OR = 1.00, 95% CI = 1.066-1.585, p = 0.009). Further subgroup analysis by ethnicity revealed an increased risk of cervical and ovarian cancer in Asians and Caucasians, respectively. The present meta-analysis inconsistent with the previous meta-analysis suggests that the XRCC3 18067 C>T polymorphism might be implicated in the pathogenesis of cervical and ovarian cancers.


Health Literacy and Health Behaviors Among Adults With Prediabetes, 2016 Behavioral Risk Factor Surveillance System.
Evidence is needed for designing interventions to address health literacy-related issues among adults with prediabetes to reduce their risk of developing type 2 diabetes. This study assessed health literacy and behaviors among US adults with prediabetes and the mediating role of health literacy on health behaviors. We used data from the 2016 Behavioral Risk Factor Surveillance System (BRFSS) (N = 54 344 adults). The BRFSS health literacy module included 3 questions on levels of difficulty in obtaining information, understanding health care providers, and comprehending written information. We defined low health literacy as a response of "somewhat difficult" or "very difficult" to at least 1 of these 3 questions. Respondents self-reported their prediabetes status. We included 3 health behavior indicators available in the BRFSS survey-current smoking, physical inactivity, and inadequate sleep, all measured as binary outcomes (yes/no). We used a path analysis to examine pathways among prediabetes, health literacy, and health behaviors. About 1 in 5 (19.0%) adults with prediabetes had low health literacy. The rates of physical inactivity (31.0% vs 24.6%, P < .001) and inadequate sleep (38.8% vs 33.5%, P < .001) among adults with prediabetes were significantly higher than among adults without prediabetes. The path analysis showed a significant direct effect of prediabetes and health literacy on health behaviors. The indirect effect of prediabetes through health literacy on health behaviors was also significant. BRFSS data from 2016 showed that rates of low health literacy and unhealthy behaviors were higher among adults with prediabetes than among adults without prediabetes. Interventions are needed to assist adults with prediabetes in comprehending, communicating about, and managing health issues to reduce the risk of type 2 diabetes.


Valuating environmental impacts from ship emissions - The marine perspective.
Shipping is an activity responsible for a range of different pressures affecting the marine environment, air quality and human welfare. The methodology on how ship emissions impact air quality and human health are comparatively well established and used in cost-benefit analysis of policy proposals. However, the knowledge base is not the same for impacts on the marine environment and a coherent environmental and socio-economic impact assessment of shipping has not yet been made. This risk policies to be biased towards air pollution whilst trading off impacts on the marine environment. The aim of the current study was to develop a comprehensive framework on how different pressures from shipping degrade marine ecosystems, air quality and human welfare. A secondary aim was to quantify the societal damage costs of shipping due to the degradation of human welfare in a Baltic Sea case study. By adding knowledge from marine ecotoxicology and life-cycle analysis to the existing knowledge from climate, air pollution and environmental economics we were able to establish a more comprehensive conceptual framework that allows for valuation of environmental impacts from shipping, but it still omits economic values for biological pollution, littering and underwater noise. The results for the Baltic Sea case showed the total annual damage costs of Baltic Sea shipping to be 2.9 billion €2010 (95% CI 2.0-3.9 billion €2010). The damage costs due to impacts on marine eutrophication (768 million €2010) and marine ecotoxicity (582 million €2010) were in the same range as the total damage costs associated with reduced air quality (816 million €2010) and climate change (737 million €2010). The framework and the results from the current study can be used in future socio-economic assessments of ship emissions to prioritize cost efficient measures. The framework can be used globally but the damage costs presented on the marine environment are restricted to emissions on the Baltic Sea and Kattegat region as they are based on willingness to pay studies conducted on citizens around the Baltic Sea where eutrophication and emissions of chemicals are particularly threats to the state of the Baltic Sea.


Real or media-mediated outbreak of coxsackie infections in 2002 in Greece?
The purpose of this study was to provide evidence about the existence of a coxsackie B outbreak in Greece in 2002 by comparing data of laboratory confirmed coxsackie B recent infections in northern Greece between 1998-2001 with data from 2002. The infections were confirmed serologically, using the indirect immunofluorescence method detecting IgM antibodies for coxsackie B1-B6 viruses. Sera from 2701 patients residents of northern Greece who were suspected to be suffering from coxsackie B virus infections were examined: 2056 between 1998 and 2001, and 645 in 2002. The comparison between the results of laboratory confirmed cases and data available at the laboratory between the two periods showed that: The total number of patients examined per year was higher in 2002 (645 versus an annual average of 514 in 1998-2001). The proportion of laboratory confirmed recent infections was lower in 2002 (27.8% versus 32.7%) and the estimated incidence was 0.66/10,000 for 2002 and 0.32-0.84/10,000 for 1998-2001. The age distribution differed: the proportions of cases in children versus cases in adults were reversed in 2002 compared with 1998-2001, with a higher proportion among children in 2002. The difference between the two periods was statistically significant. Children aged 3-5 years were the age group most affected in 2002. Seasonal distribution remained the same for both periods (peaks in spring and autumn). In 2002, three fatal cases occurred in April, but no deaths were reported in 1998-2001. The clinical syndromes involved also differed: cases of respiratory infections, mainly pneumonia, rose from 5.75% to 24.3% in children in 2002 and cases of myopericarditis rose in adults from 13% in 1998-2001 to 29.5% in 2002. The last finding, combined with the involvement of the media (because of the three fatal cases) and the panic in the general public that followed suggested that an outbreak had occurred, but we conclude that there was no outbreak.


An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum.
The presence of Cryptosporidium in drinking water supplies is a significant problem faced by the water industry. Although a variety of methods exist for the detection of waterborne oocysts, water utilities currently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to public health by waterborne Cryptosporidium. In this paper, the development of an infectivity assay for waterborne Cryptosporidium parvum is described. Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the heat shock protein 70 (hsp70) gene. A single infectious oocyst was detected by this experimental procedure. The use of concentrated samples obtained from 250 liters of finished water had no observable effect on the integrity of cell monolayers or on the infectivity of oocysts seeded into the concentrate. Intracellular developmental stages of the parasite were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parvum, while a second pair recognized all species of Cryptosporidium tested. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samples and were compatible with multiplex PCR for the simultaneous detection of C. parvum and Giardia lambia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly, demonstrates the potential of an in vitro infectivity assay.


Cancer chemoprevention by the antioxidant tempol in Atm-deficient mice.
Reactive oxygen species (ROS) are important endogenous etiological agents for DNA damage, and ROS perform critical signaling functions in apoptosis, stress responses and proliferation. The correlation between a lower incidence of cancer in people who consume a diet high in naturally occurring antioxidants and the observed increased ROS in cancerous tissues suggest that antioxidants may be used in cancer chemoprevention. We tested this hypothesis by determining whether the well-described nitroxide antioxidant, tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), acts as a chemopreventative agent in Atm mutant mice, a model of the human cancer prone syndrome ataxia-telangiectasia. Tempol administered continuously via the diet after weaning resulted in an increased lifespan of these mice by prolonging the latency to thymic lymphomas. Tempol treatment reduced ROS, restored mitochondrial membrane potential, reduced tissue oxidative damage and oxidative stress, consistent with antioxidant effects. In addition, this nitroxide lowered weight gain of tumor prone mice without changes in food intake, metabolism or activity level and exhibited an anti-proliferative effect in vitro. Thus, tempol acts as a novel chemopreventative agent in this mouse model of a human cancer prone syndrome, associated with broad antioxidant effects.


Decoding Allosteric Control in Hypoxia-Inducible Factors.
The mammalian family of basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors possess the ability to sense and respond to diverse environmental and physiological cues. These proteins all share a common structural framework, comprising a bHLH domain, two PAS domains, and transcriptional activation or repression domain. To function effectively as transcription factors, members of the family must form dimers, bringing together bHLH segments to create a functional unit that allows for DNA response element binding. The significance of bHLH-PAS family is underscored by their involvement in many major human diseases, offering potential avenues for therapeutic intervention. Notably, the clear identification of ligand-binding cavities within their PAS domains enables the development of targeted small molecules. Two examples are Belzutifan, targeting hypoxia-inducible factor (HIF)-2α, and Tapinarof, targeting the aryl hydrocarbon receptor (AHR), both of which have gained regulatory approval recently. Here, we focus on the HIF subfamily. The crystal structures of all three HIF-α proteins have been elucidated, revealing their bHLH and tandem PAS domains are used to engage their dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1β). A broad range of recent findings point to a shared allosteric modulation mechanism among these proteins, whereby small-molecules at the PAS-B domains exert direct influence over the HIF-α transcriptional functions. As our understanding of the architectural and allosteric mechanisms of bHLH-PAS proteins continues to advance, the possibility of discovering new therapeutic drugs becomes increasingly promising.


Pyruvate dehydrogenase activity in cardiac mitochondria from genetically diabetic mice.
Pyruvate dehydrogenase (PDH) was studied in isolated heart mitochondria from genetically diabetic mice (C57BL/KsJ db/db) with progressive cardiomyopathy. Both the basal activity (active enzyme) and the total activity (the sum of active and inactive enzyme) were determined. In mitochondrial extracts from 8-18-wk-old db/db mice, there was a 73% decrease of basal activity accompanied by a 38% decrease of total activity as compared with controls. The lower basal activity at 8 wk of age suggests an increased conversion of the enzyme into the phosphorylated (inactive) form. Evidence is also given that the conversion of inactive (phosphorylated) enzyme into active (dephosphorylated) enzyme is inhibited in cardiac mitochondria prepared from 8-wk and older db/db mice. These changes coincide with the onset of defective oxidative metabolism and can explain the depressed pyruvate oxidation reported previously.


Microstructure degradation of Ni/CGO anodes for solid oxide fuel cells after long operation time using 3D reconstructions by FIB tomography.
Solid oxide fuel cells (SOFCs) are electrochemical conversion devices, which essentially consist of two porous electrodes separated by a dense, oxide ion conducting electrolyte. The performance and the durability of SOFCs strongly depend on the electrode microstructure. In this paper, the impact of a relatively long exposure time (up to 20 000 h) under realistic operation terms (temperature (T) = 850 °C, current density (J) = 190-250 mA cm-2) in the kinetics of microstructural degradation are investigated for porous nickel (Ni)/ceria gadolinium oxide (CGO) anodes, to understand the microstructural evolution in SOFC cermet anodes. A combined system of Focused Ion Beam (FIB) and Scanning Electron Microscope (SEM) tomography was used to analyze various anode microstructures aged during different operating times (2500 h, 15 000 h and 20 000 h). The methodologies of image acquisition as well as the segmentation and the object recognition were improved, offering a reliable quantification of Ni-grain growth, connectivity, tortuosity factor and triple phase boundary length (TPBL). The representative volume element (RVE) was also studied, and its dependence on aging time was confirmed. To construct a volume that can be accurate and representative for the whole sample, the necessary corresponding 3D reconstruction size was adjusted by increasing operating time, in order to suppress the influence of microstructure variation caused by Ni and CGO agglomeration. Statistically significant 3D microstructural changes were observed in the anode by increasing the operating time, including nickel particle size distribution, changes in connectivity of the ceramic part (CGO) and a significant decrease of contiguous triple phase boundary densities. Additional qualitative observations were done in order to gain a complete insight of the degradation phenomena in nickel based cermet anodes.