A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons.
Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the cytomegalovirus (CMV) and chicken beta-actin (CAG) promoters in a back-to-back configuration with a shared enhancer, and show efficient expression of two proteins simultaneously in DRG neurons. We demonstrate how this is useful for experiments on axonal regeneration, by co-expressing a gene of interest and an axonal marker. Using a farnesylated form of eGFP, which is actively transported along axons, we show superior long-distance labelling of axons of DRG neurons compared with normal eGFP. Additionally, we have efficiently transduced lumbar DRG neurons by injecting the AAV dual promoter vector into the dorsal intrathecal space, which is a less invasive delivery method. In summary, we have developed an AAV dual promoter vector designed for simultaneous expression of a gene of interest and a fluorescent protein to label long-distance axonal projections, which allows specific quantification of axons from transduced neurons after injury.


Computed tomography of corpus luteal cysts.
To describe the computed tomography (CT) features of corpus luteal cysts. We retrospectively identified 10 patients with a diagnosis of corpus luteal cysts established by ultrasound who had also undergone contemporaneous CT. A single attending radiologist, without knowledge of other clinical or radiologic findings, recorded the morphologic features of the cysts based on the CT images. The corpus luteal cyst seen at sonography was visible at CT in all 10 patients. All cysts were unilocular, with a mean density of 25 HU (range, 12 to 45). The mean maximum axial cyst diameter was 2.2 cm (range, 1.4 to 2.9). The mean cyst wall thickness was 3 mm (range, 2 to 4). All cyst walls were crenulated. Cyst wall enhancement was hyperdense in 6 cases, isodense in 3 cases, and hypodense in 1 case. Free fluid was seen in 9 of 10 patients. At CT, corpus luteal cysts are typically less than 3 cm in diameter and are characterized by a thick, crenulated, or hyperdense wall. Recognition of these CT findings should prevent misinterpretation or inappropriate management.


Predictors of hysterectomy as a treatment for menstrual symptoms.
Hysterectomy is a common procedure in Australia; approximately 1 in 5 Australian women undergoing a hysterectomy by the age of 50 for indications such as fibroids, disorders of menstruation (including excessive or irregular menstrual bleeding), and endometriosis. However, little is known about the characteristics of women who have had the procedure or the predictors of hysterectomy as a treatment for menstrual problems. This study of 687 middle-aged Australian women suffering from menstrual symptoms aimed to identify the common health and demographic characteristics that were likely to lead to hysterectomy for the treatment of these problems. A cross-sectional and a prospective cohort study were undertaken as a substudy of the Australian Longitudinal Study on Women's Health (Women's Health Australia). Women from the mid-aged cohort of the Women's Health Australia study who identified having menstrual problems (n = 715) in the 1996 and 1998 surveys or who had undergone a hysterectomy (n = 218) during that time were recruited. A self-administered instrument was mailed to the 933 women in 2000. Data were analyzed using backward logistic regression to identify the characteristics that increased the likelihood of women undergoing hysterectomy for the treatment of menstrual problems. Factors that increased the likelihood of hysterectomy as a treatment for menstrual problems were varied. They included the number of menstrual symptoms experienced (odds ratio [OR], 1.63; p < .005) or conditions diagnosed (such as fibroids or excessive menstrual bleeding; OR, 2.5; p < .0005), a perception that information was available about menstrual problems (OR, 1.16; p < .001), being influenced in the decision making process to elect a treatment option (OR, 1.25; p < .025), and dissatisfaction with the other treatments tried before hysterectomy (OR, 0.63; p < .0005). Hysterectomy seem to be the treatment of choice for women experiencing a number of menstrual problems and less than satisfactory outcomes with other treatment options. Importantly, women appear to be making decisions based on the perception of sufficient information available about their menstrual problems, regardless of whether or not this was actually the case. Women are being influenced in their decision-making process to undergo a hysterectomy by health professionals, such as general practitioners and gynecologists.


Analytic solutions for spectral properties of superstructure, Gaussian-apodized and phase shift gratings with short- or long-period.
The analytic solutions (AS) for the spectral properties of short- and long-period waveguide gratings with the effects of discrete phase shift (PS), Gaussian-apodization (GA) and superstructure are presented in this paper, which are derived from the Fourier mode coupling (FMC) theory proposed recently. The spectral properties include the reflectivity of short-period gratings, and the transmission of long-period gratings. The calculated spectra based on the analytic solutions are achieved and compared with measured cases and that on the transfer matrix (TM) method, in the case of changing grating parameters. The AS-based calculation requires the average time of several milliseconds at common PC, and the AS-based efficiency is improved up to ~6700 times the TM-based one. The comparisons have confirmed that the FMC-based analytic solutions are suitable for the real-time and accurate analyses of some non-uniform waveguide gratings.


Two-fluid temperature-dependent relativistic waves in magnetized streaming pair plasmas.
A relativistic two-fluid temperature-dependent approach for a streaming magnetized pair plasma is considered. Such a scenario corresponds to secondary plasmas created at the polar caps of pulsar magnetospheres. In the model the generalized vorticity rather than the magnetic field is frozen into the fluid. For parallel propagation four transverse modes are found. Two are electromagnetic plasma modes which at high temperature become light waves. The remaining two are Alfvénic modes split into a fast and slow mode. The slow mode is cyclotron two-stream unstable at large wavelengths and is always subluminous. We find that the instability cannot be suppressed by temperature effects in the limit of large (finite) magnetic field. The fast Alfvén mode can be superluminous only at large wavelengths, however it is always subluminous at high temperatures. In this incompressible approximation only the ordinary mode is present for perpendicular propagation. For oblique propagation the dispersion relation is studied for finite and large strong magnetic fields and the results are qualitatively described.


Pneumococci can persistently colonize adult patients with chronic respiratory disease.
Streptococcus pneumoniae plays an important role in causing acute exacerbations in patients with chronic respiratory disease. However, few data are available regarding pneumococcal persistence in adult patients with chronic respiratory diseases. Fifty pneumococci recovered from sputum samples (1995 to 2010) from 13 adult patients with ≥ 3 episodes of acute exacerbation or pneumonia, with the same serotype and pulsed-field gel electrophoresis (PFGE) pattern, were studied. Multilocus sequence typing (MLST) loci, penicillin-binding protein (PBP) genes (pbp2x, pbp1a, pbp2b), and the quinolone-resistant determining regions (QRDRs) of parC, parE, and gyrA were PCR amplified and sequenced. The average time between the first and last episode was 582 days (standard deviation [SD], ± 362). All but two patients received multiple courses of β-lactam treatment, and all persistent strains were resistant to penicillin; however, the PBP sequences were stable over time apart from one variable nucleotide in pbp2x, observed among pneumococci isolated from three patients. In contrast, 7/11 patients treated with fluoroquinolones had fluoroquinolone-resistant pneumococci. In three patients, the initially fluoroquinolone-susceptible strain developed resistance after fluoroquinolone therapy, and in the remaining four patients, the persistent strain was fluoroquinolone resistant from the first episode. QRDR changes involved in fluoroquinolone resistance were frequently observed in persistent strains after fluoroquinolone treatment; however, the PBP sequences and MLST genotypes of these strains were stable over time.


The Nature of the Familial Risk for Psychosis in Bipolar Disorder.
To clarify whether the familial liability to psychosis associated with bipolar disorder (BD) is nonspecific or has a greater effect on risk for psychosis in cases with prominent mood symptoms and/or a remitting course. We examined, in 984 809 offspring raised in intact families in Sweden, born 1980-1996 and followed-up through 2018, by multivariable Cox proportional hazards regression, risk in offspring of parents with BD for 7 psychotic disorders: Psychotic MD (PMD), psychotic BD (PBD), schizoaffective disorder (SAD), acute psychoses, psychosis NOS, delusional disorder (DD) and schizophrenia (SZ). Diagnoses were obtained from national registers. In the offspring of BD parents, the hazard ratios (HR) for these 7 disorders formed an inverted U-shaped curve, rising from 2.98 for PMD, to peak at 4.49 for PBD and 5.25 for SAD, and then declining to a HR of 3.48 for acute psychoses and 3.22 for psychosis NOS, to a low of 2.19 for DD and 2.33 for SZ. A similar pattern of risks was seen in offspring of mothers and fathers affected with BD and in offspring predicted from age at onset in their BD parent. The BD-associated risk for psychosis impacts most strongly on mood disorders, moderately on episodic psychotic syndromes, and least on chronic psychotic disorders. These results support prior clinical studies suggesting a qualitative difference in the familial substrate for psychosis occurring in BD and SZ.


Osteoarthritis Classification Scales: Interobserver Reliability and Arthroscopic Correlation.
Osteoarthritis of the knee is commonly diagnosed and monitored with radiography. However, the reliability of radiographic classification systems for osteoarthritis and the correlation of these classifications with the actual degree of confirmed degeneration of the articular cartilage of the tibiofemoral joint have not been adequately studied. As the Multicenter ACL (anterior cruciate ligament) Revision Study (MARS) Group, we conducted a multicenter, prospective longitudinal cohort study of patients undergoing revision surgery after anterior cruciate ligament reconstruction. We followed 632 patients who underwent radiographic evaluation of the knee (an anteroposterior weight-bearing radiograph, a posteroanterior weight-bearing radiograph made with the knee in 45° of flexion [Rosenberg radiograph], or both) and arthroscopic evaluation of the articular surfaces. Three blinded examiners independently graded radiographic findings according to six commonly used systems-the Kellgren-Lawrence, International Knee Documentation Committee, Fairbank, Brandt et al., Ahlbäck, and Jäger-Wirth classifications. Interobserver reliability was assessed with use of the intraclass correlation coefficient. The association between radiographic classification and arthroscopic findings of tibiofemoral chondral disease was assessed with use of the Spearman correlation coefficient. Overall, 45° posteroanterior flexion weight-bearing radiographs had higher interobserver reliability (intraclass correlation coefficient = 0.63; 95% confidence interval, 0.61 to 0.65) compared with anteroposterior radiographs (intraclass correlation coefficient = 0.55; 95% confidence interval, 0.53 to 0.56). Similarly, the 45° posteroanterior flexion weight-bearing radiographs had higher correlation with arthroscopic findings of chondral disease (Spearman rho = 0.36; 95% confidence interval, 0.32 to 0.39) compared with anteroposterior radiographs (Spearman rho = 0.29; 95% confidence interval, 0.26 to 0.32). With respect to standards for the magnitude of the reliability coefficient and correlation coefficient (Spearman rho), the International Knee Documentation Committee classification demonstrated the best combination of good interobserver reliability and medium correlation with arthroscopic findings. The overall estimates with the six radiographic classification systems demonstrated moderate (anteroposterior radiographs) to good (45° posteroanterior flexion weight-bearing radiographs) interobserver reliability and medium correlation with arthroscopic findings. The International Knee Documentation Committee classification assessed with use of 45° posteroanterior flexion weight-bearing radiographs had the most favorable combination of reliability and correlation. Diagnostic Level I. See Instructions for Authors for a complete description of levels of evidence.


Planning crowd events to achieve high participant satisfaction.
A case study investigation within a large UK university involving semi-structured stakeholder interviews, examined the organisation, coordination, and security within crowd events of various descriptions. Similarities in approaches and priorities emerged with crowd event organisation, primarily attention to safety requirements, in protecting crowd members, venue reputation, and legal obligations. Conversely, attention to and attitudes and beliefs surrounding user experience, crowd comfort and satisfaction, were often based on personal judgment, and appeared to be influenced by budget considerations. The findings suggest a lack of knowledge and usable evidence based guidance for planning crowd events in relation to important aspects affecting participant satisfaction.


Endocannabinoids in the retina: from marijuana to neuroprotection.
The active component of the marijuana plant Cannabis sativa, Delta9-tetrahydrocannabinol (THC), produces numerous beneficial effects, including analgesia, appetite stimulation and nausea reduction, in addition to its psychotropic effects. THC mimics the action of endogenous fatty acid derivatives, referred to as endocannabinoids. The effects of THC and the endocannabinoids are mediated largely by metabotropic receptors that are distributed throughout the nervous and peripheral organ systems. There is great interest in endocannabinoids for their role in neuroplasticity as well as for therapeutic use in numerous conditions, including pain, stroke, cancer, obesity, osteoporosis, fertility, neurodegenerative diseases, multiple sclerosis, glaucoma and inflammatory diseases, among others. However, there has been relatively far less research on this topic in the eye and retina compared with the brain and other organ systems. The purpose of this review is to introduce the "cannabinergic" field to the retinal community. All of the fundamental works on cannabinoids have been performed in non-retinal preparations, necessitating extensive dependence on this literature for background. Happily, the retinal cannabinoid system has much in common with other regions of the central nervous system. For example, there is general agreement that cannabinoids suppress dopamine release and presynaptically reduce transmitter release from cones and bipolar cells. How these effects relate to light and dark adaptations, receptive field formation, temporal properties of ganglion cells or visual perception are unknown. The presence of multiple endocannabinoids, degradative enzymes with their bioactive metabolites, and receptors provides a broad spectrum of opportunities for basic research and to identify targets for therapeutic application to retinal diseases.


Reduced disease severity following therapeutic treatment with angiotensin 1-7 in a mouse model of multiple sclerosis.
Multiple Sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by autoimmune and neurodegenerative pathologies for which there is no cure and no defined etiology. Although several, modestly effective, disease modifying drugs are available to treat MS, there are presently no treatments that offer neuroprotection and prevent clinical progression. Therapies are needed that control immune homeostasis, prevent disease progression, and stimulate regeneration in the CNS. Components of the renin-angiotensin-system (RAS) have recently been identified as chemical mediators in the CNS and in neurological disease. Here we show the beneficial effect of therapeutic treatment with the Mas receptor agonist and metabolite of the protective arm of RAS, angiotensin 1-7 (A(1-7)), in the experimental autoimmune encephalomyelitis (EAE) animal model of MS. Therapeutic treatment with A(1-7) caused a dose-dependent reduction both in clinical disease severity and progression, and was dependent on Mas receptor activation. Further analysis of the most optimal dose of A(1-7) treatment revealed that the reductions in clinical disease course were associated with decreased immune infiltration and demyelination, axonal loss and oxidative stress in the spinal cord. In addition A(1-7) treatment was also associated with increases in circulating alternatively activated monocytes/macrophages.


Profiling Reactive Metabolites via Chemical Trapping and Targeted Mass Spectrometry.
Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.


Myeloid Cells as Targets for Therapy in Solid Tumors.
It is well established that cancer development ensues based on reciprocal interactions between genomically altered neoplastic cells and diverse populations of recruited "host" cells co-opted to support malignant progression. Among the host cells recruited into tumor microenvironments, several subtypes of myeloid cells, including macrophages, monocytes, dendritic cells, and granulocytes contribute to tumor development by providing tumor-promoting factors as well as a spectrum of molecules that suppress cytotoxic activities of T lymphocytes. Based on compelling preclinical data revealing that inhibition of critical myeloid-based programs leads to tumor suppression, novel immune-based therapies and approaches are now entering the clinic for evaluation. This review discusses mechanisms underlying protumorigenic programming of myeloid cells and discusses how targeting of these has potential to attenuate solid tumor progression via the induction and of mobilization CD8 cytotoxic T cell immunity.


Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line.
The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal maculopathies.


Adenosine triphosphate and hemoglobin in vasospastic monkeys.
Adenosine triphosphate (ATP) is a vasoactive compound found in high levels inside erythrocytes that may contribute to vasospasm occurring after subarachnoid hemorrhage (SAH). This study was instituted to test whether ATP causes vasospasm in a monkey model. Thirty-two monkeys were randomized to four groups of eight monkeys each to undergo cerebral angiography at baseline (Day 0) and then at Day 7 after subarachnoid placement of: 1) agarose, 2) ATP in agarose, 3) autologous hemolysate in agarose, or 4) purified human hemoglobin A(0) in agarose. Vasospasm was assessed by comparison of Day 0 and Day 7 angiograms between and within groups and by pathological examination of a subset of perfusion-fixed monkeys. Levels of adenine nucleotides were measured on Day 7 in subarachnoid agarose by high-pressure liquid chromatography. There was significant vasospasm of the right middle cerebral artery in groups given ATP (-28 +/- 7% reduction, paired t-test, p < 0.05), hemolysate (-23 +/- 7%, p < 0.05), or pure hemoglobin (-15 +/- 2%, p < 0.005). Analysis of variance revealed no significant differences between groups in diameters of cerebral arteries on Day 7. Pathological examination showed mild inflammation in the subarachnoid spaces of animals exposed to hemolysate or hemoglobin and less inflammation in those given ATP or agarose. There were no pathological changes in the cerebral arteries of animals in any group. Most of the ATP diffused out of the subarachnoid agarose by Day 7, and levels of adenine nucleotides in subarachnoid agarose were higher on Day 7 in animals exposed to hemoglobin or hemolysate. It is concluded that ATP could contribute to vasospasm occurring after SAH but that further investigations are necessary to determine if levels of ATP adjacent to vasospastic arteries are sufficient to contribute to vasospasm. In addition, no observation was made of severe vasospasm with histopathological changes in the arteries equivalent to that produced by whole blood clot in the subarachnoid space of monkeys. It should be determined whether this is because a single compound, such as ATP or hemoglobin, causes vasospasm, but that placing the compound in agarose alters its delivery and decreases the amount of vasospasm produced, or whether vasospasm is a more complex, multifactorial process.


Impact of Smoking Cessation Interventions Initiated During Hospitalization Among HIV-Infected Smokers.
Smoking is a key determinant of mortality among people living with HIV (PLWH). To better understand the effects of smoking cessation interventions in PLWH, we conducted a pooled analysis of four randomized controlled trials of hospital-initiated smoking interventions conducted through the Consortium of Hospitals Advancing Research on Tobacco (CHART). In each study, cigarette smokers were randomly assigned to usual care or a smoking cessation intervention. The primary outcome was self-reported past 30-day tobacco abstinence at 6-month follow-up. Abstinence rates were compared between PLWH and participants without HIV and by treatment arm, using both complete-case and intention-to-treat analyses. Multivariable logistic regression was used to determine the effect of HIV status on 6-month tobacco abstinence and to determine predictors of smoking cessation within PLWH. Among 5550 hospitalized smokers, there were 202 (3.6%) PLWH. PLWH smoked fewer cigarettes per day and were less likely to be planning to quit than smokers without HIV. At 6 months, cessation rates did not differ between intervention and control groups among PLWH (28.9% vs. 30.5%) or smokers without HIV (36.1% vs. 34.1%). In multivariable regression analysis, HIV status was not significantly associated with smoking cessation at 6 months. Among PLWH, confidence in quitting was the only clinical factor independently associated with smoking cessation (OR 2.0, 95% CI = 1.4 to 2.8, p < .01). HIV status did not alter likelihood of quitting smoking after hospital discharge, whether or not the smoker was offered a tobacco cessation intervention, but power was limited to identify potentially important differences. PLWH had similar quit rates to participants without HIV following a hospital-initiated smoking cessation intervention. The findings suggest that factors specific to HIV infection may not influence response to smoking cessation interventions and that all PLWH would benefit from efforts to assist in quitting smoking. (1) Using "warm handoffs" to link hospitalized smokers with tobacco treatment after discharge: study protocol of a randomized controlled trial: NCT01305928. (2) Web-based smoking cessation intervention that transitions from inpatient to outpatient: NCT01277250. (3) Effectiveness of smoking-cessation interventions for urban hospital patients: NCT01363245. (4) Effectiveness of Post-Discharge Strategies for Hospitalized Smokers (HelpingHAND2): NCT01714323.


Assessment of genetic diversity, population structure, and gene flow of tigers (Panthera tigris tigris) across Nepal's Terai Arc Landscape.
With fewer than 200 tigers (Panthera tigris tigris) left in Nepal, that are generally confined to five protected areas across the Terai Arc Landscape, genetic studies are needed to provide crucial information on diversity and connectivity for devising an effective country-wide tiger conservation strategy. As part of the Nepal Tiger Genome Project, we studied landscape change, genetic variation, population structure, and gene flow of tigers across the Terai Arc Landscape by conducting Nepal's first comprehensive and systematic scat-based, non-invasive genetic survey. Of the 770 scat samples collected opportunistically from five protected areas and six presumed corridors, 412 were tiger (57%). Out of ten microsatellite loci, we retain eight markers that were used in identifying 78 individual tigers. We used this dataset to examine population structure, genetic variation, contemporary gene flow, and potential population bottlenecks of tigers in Nepal. We detected three genetic clusters consistent with three demographic sub-populations and found moderate levels of genetic variation (He = 0.61, AR = 3.51) and genetic differentiation (FST = 0.14) across the landscape. We detected 3-7 migrants, confirming the potential for dispersal-mediated gene flow across the landscape. We found evidence of a bottleneck signature likely caused by large-scale land-use change documented in the last two centuries in the Terai forest. Securing tiger habitat including functional forest corridors is essential to enhance gene flow across the landscape and ensure long-term tiger survival. This requires cooperation among multiple stakeholders and careful conservation planning to prevent detrimental effects of anthropogenic activities on tigers.


Tissue engineering by self-assembly and bio-printing of living cells.
Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion.


MEMS-based optical limiter.
We propose the design of an optical limiter based on a microelectromechanical systems deformable mirror. The design is based on aperturing focused light reflected out of an optically driven deformable mirror, deformed in a parabolic form. We derive an expression for the reflected light intensity, and we show that the reflected light saturates as a function of back illumination light intensity.


Predominance of genotype 1.1 and emergence of genotype 2.2 classical swine fever viruses in north-eastern region of India.
Classical swine fever (CSF) is a highly contagious and the most important disease of pigs worldwide.CSF is enzootic in pig herds in India and continues to cause huge economic losses to pig farmers. Nearly 40% of the total pig population of India is present in the north-eastern (NE) states where pig husbandry plays an important role in the socio-economic development. Pigs reared in the backyards are the only source of livelihood for a majority of poor tribal population in the region. Hardly any CSF vaccination is currently being undertaken in the unorganized pig farming in the NE region due to economic reasons and vaccine unavailability. A thorough understanding of the current epidemiological status of CSF is essential for the effective control of the disease in the NE region. Hence, we carried out molecular characterization of CSFV isolates from field outbreaks during 2011-2012 in the entire north-eastern region of India to establish the genetic groups of prevalent CSF viruses in the region. A total of 17 CSFV isolates obtained from different parts of the NE region were characterized by comparing the sequences of three partial genomic regions of the virus, that is 150 nt of 5' UTR, 190 nt of E2 and 409 nt of NS5B. Of the 17 CSFV isolates, 15 isolates belonged to 1.1 (88.2%) and two isolates (11.8%) belonged to 2.2 subgenogroup. The genogroup 2.2 CSFV were associated with outbreaks in Arunachal Pradesh that shares international borders with Bhutan, Myanmar and China. Genogroup 2.2 CSFV isolated in the present study shared high level of sequence similarity with 2.2 viruses form China, raising the possibility of virus incursion from this region. In summary, we found a continued predominance of 1.1 subgroup and an emergence of 2.2 subgroup CSFV in NE region of India.


Impact of white blood cell count on clinical outcomes in patients treated with aspirin-free ticagrelor monotherapy after percutaneous coronary intervention: insights from the GLOBAL LEADERS trial.
The aim of this study was to investigate the efficacy and safety of ticagrelor monotherapy in patients undergoing percutaneous coronary intervention (PCI) stratified according to the baseline white blood cell (WBC) count. This is a post hoc analysis of the GLOBAL LEADERS trial, a multi-centre, open-label, randomized all-comer trial in patients undergoing PCI, comparing the experimental strategy (23-month ticagrelor monotherapy following 1-month dual anti-platelet therapy [DAPT]) with the reference strategy (12-month aspirin monotherapy following 12-month DAPT). Patients were stratified into two WBC groups, either < or ≥median WBC count of 7.8 × 109 cells/L (lower or higher WBC group, respectively). The primary endpoint was a composite of all-cause mortality or new Q-wave myocardial infarction at 2 years. Of 14 576 patients included in the present study, 7212 patients (49.5%) were classified as the lower WBC group, who had a significantly lower risk of both ischaemic and bleeding outcomes at 2 years. At 2 years, the experimental strategy was associated with a significant lower incidence of the primary endpoint compared with the reference strategy in the lower WBC group [2.8% vs. 4.2%; hazard ratio (HR): 0.67; 95% confidence interval (CI): 0.52-0.86] but not in the higher WBC group (4.8% vs. 4.7%; HR: 1.01; 95% CI: 0.82-1.25; Pinteraction=0.013). There were no significant differences in the risks of Bleeding Academic Research Consortium type 3 or 5 bleeding between two anti-platelet strategies regardless of the WBC groups. Increased WBC counts, which may reflect degree of inflammation, at the time of index procedure may attenuate the anti-ischaemic benefits of ticagrelor monotherapy observed in patients with lower WBC counts.


Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins.
Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.


Resting-State Phase-Amplitude Coupling Between the Human Subthalamic Nucleus and Cortical Activity: A Simultaneous Intracranial and Scalp EEG Study.
It has been suggested that slow oscillations in the subthalamic nucleus (STN) reflect top-down inputs from the medial prefrontal cortex, thus implementing behavior control. It is unclear, however, whether the STN oscillations are related to cortical activity in a bottom-up manner. To assess resting-state subcortico-cortical interactions, we recorded simultaneous scalp electroencephalographic activity and local field potentials in the STN (LFP-STN) in 11 patients with Parkinson's disease implanted with deep brain stimulation electrodes in the on-medication state during rest. We assessed the cross-structural phase-amplitude coupling (PAC) between the STN and cortical activity within a wide frequency range of 1 to 100 Hz. The PAC was dominant between the δ/θ STN phase and β/γ cortical amplitude in most investigated scalp regions and between the δ cortical phase and θ/α STN amplitude in the frontal and temporal regions. The cross-frequency linkage between the slow oscillations of the LFP-STN activity and the amplitude of the scalp-recorded cortical activity at rest was demonstrated, and similar involvement of the left and right STNs in the coupling was observed. Our results suggest that the STN plays a role in both bottom-up and top-down processes within the subcortico-cortical circuitries of the human brain during the resting state. A relative left-right symmetry in the STN-cortex functional linkage was suggested. Practical treatment studies would be necessary to assess whether unilateral stimulation of the STN might be sufficient for treatment of Parkinson's disease.


A Multiplatform Metabolomics Approach to Characterize Plasma Levels of Phenylalanine and Tyrosine in Phenylketonuria.
Different pathophysiological mechanisms have been described in phenylketonuria (PKU) but the indirect metabolic consequences of metabolic disorders caused by elevated Phe or low Tyr concentrations remain partially unknown. We used a multiplatform metabolomics approach to evaluate the metabolic signature associated with Phe and Tyr. We prospectively included 10 PKU adult patients and matched controls. We analysed the metabolome profile using GC-MS (urine), amino-acid analyzer (urine and plasma) and nuclear magnetic resonance spectroscopy (urine). We performed a multivariate analysis from the metabolome (after exclusion of Phe, Tyr and directly derived metabolites) to explain plasma Phe and Tyr concentrations, and the clinical status. Finally, we performed a univariate analysis of the most discriminant metabolites and we identified the associated metabolic pathways. We obtained a metabolic pattern from 118 metabolites and we built excellent multivariate models to explain Phe, Tyr concentrations and PKU diagnosis. Common metabolites of these models were identified: Gln, Arg, succinate and alpha aminobutyric acid. Univariate analysis showed an inverse correlation between Arg, alpha aminobutyric acid and Phe and a positive correlation between Arg, succinate, Gln and Tyr (p < 0.0003). Thus, we highlighted the following pathways: Arg and Pro, Ala, Asp and Glu metabolism. We obtain a specific metabolic signature related to Tyr and Phe concentrations. We confirmed the involvement of different pathophysiological mechanisms previously described in PKU such as protein synthesis, energetic metabolism and oxidative stress. The metabolomics approach is relevant to explore PKU pathogenesis.


Physical performance analysis: A new approach to assessing free-living physical activity in musculoskeletal pain and mobility-limited populations.
Accurate measurement of physical performance in individuals with musculoskeletal pain is essential. Accelerometry is a powerful tool for this purpose, yet the current methods designed to evaluate energy expenditure are not optimized for this population. The goal of this study is to empirically derive a method of accelerometry analysis specifically for musculoskeletal pain populations. We extracted data from 6,796 participants in the 2003-4 National Health and Nutrition Examination Survey (NHANES) including: 7-day accelerometry, health and pain questionnaires, and anthropomorphics. Custom macros were used for data processing, complex survey regression analyses, model selection, and statistical adjustment. After controlling for a multitude of variables that influence physical activity, we investigated whether distinct accelerometry profiles accompany pain in different locations of the body; and we identified the intensity intervals that best characterized these profiles. Unique accelerometry profiles were observed for pain in different body regions, logically clustering together based on proximity. Based on this, the following novel intervals (counts/minute) were identified and defined: Performance Sedentary (PSE) = 1-100, Performance Light 1 (PL1) = 101-350, Performance Light 2 (PL2) = 351-800, Performance Light 3 (PL3) = 801-2500, and Performance Moderate/Vigorous (PMV) = 2501-30000. The refinement of accelerometry signals into these new intervals, including 3 distinct ranges that fit inside the established light activity range, best captures alterations in real-life physical performance as a result of regional pain. These new accelerometry intervals provide a model for objective measurement of real-life physical performance in people with pain and musculoskeletal disorders, with many potential uses. They may be used to better evaluate the relationship between pain and daily physical function, monitor musculoskeletal disease progression, gauge disease severity, inform exercise prescription, and quantify the functional impact of treatments. Based on these findings, we recommend that future studies of pain and musculoskeletal disorders analyze accelerometry output based on these new "physical performance" intervals.


The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors.
T cells express either of the two forms of antigen-specific receptors, the alpha/beta and gamma/delta heterodimers. Their structure closely resembles that of immunoglobulins, and the variable part of the receptor molecule is created by somatic assembly of variable, diversity, and joining regions. The genetic structure of T cell receptor (TCR) genes and their rearrangement in T cell development have been elucidated in great detail in recent years. The human genes for the gamma and beta subunits are located on the short and long arms of chromosome 7, respectively, whereas the delta- and alpha-chain genes are located in tandem on the centromeric half of the long arm of chromosome 14. Expression of either alpha/beta or gamma/delta TCR complexes on T cells in the developing thymus is likely to proceed in an ordered fashion and results in the appearance of distinct T cell subpopulations. The process of DNA rearrangements required for the generation of functional variable region genes also predisposes lymphoid cells to aberrant DNA rearrangements, which can be detected as chromosomal abnormalities such as translocations and inversions. Molecular analysis of such aberrant rearrangements has shown that rearranging loci are fused to loci unrelated to antigen receptor genes. Furthermore, the breakpoint structures represent nonproductive intermediates in the hierarchy of physiological rearrangements. Accordingly, T cell tumors arising early in T cell development often carry chromosomal abnormalities involving the delta-chain locus, whereas tumors generated later in T cell development tend to show aberrations in the alpha-chain gene. This pattern seems to reflect the stage-specific accessibility of TCR loci for rearrangement by the recombinase machinery. This enzyme is guided by specific recombination signals that can sometimes also be found at the site of breakage on the participating locus in chromosomal abnormalities. Although some features of the mechanism of aberrant rearrangements are known, their biological consequences are less well understood. However, molecular analysis of the mechanism of chromosomal aberrations in T cell tumors suggests that their biological consequences may vary. Firm evidence for the pathogenic significance is missing for most of these lesions. This provides a challenge to molecular immunology to determine how chromosomal abnormalities are involved in tumor pathogenesis.


Development and validation of a risk calculator predicting postoperative respiratory failure.
Postoperative respiratory failure (PRF) (requiring mechanical ventilation > 48 h after surgery or unplanned intubation within 30 days of surgery) is associated with significant morbidity and mortality. The objective of this study was to identify preoperative factors associated with an increased risk of PRF and subsequently develop and validate a risk calculator. The American College of Surgeons National Surgical Quality Improvement Program (NSQIP), a multicenter, prospective data set (2007-2008), was used. The 2007 data set (n = 211,410) served as the training set and the 2008 data set (n = 257,385) as the validation set. In the training set, 6,531 patients (3.1%) developed PRF. Patients who developed PRF had a significantly higher 30-day mortality (25.62% vs 0.98%, P < .0001). On multivariate logistic regression analysis, five preoperative predictors of PRF were identified: type of surgery, emergency case, dependent functional status, preoperative sepsis, and higher American Society of Anesthesiologists (ASA) class. The risk model based on the training data set was subsequently validated on the validation data set. The model performance was very similar between the training and the validation data sets (c-statistic, 0.894 and 0.897, respectively). The high c-statistics (area under the receiver operating characteristic curve) indicate excellent predictive performance. The risk model was used to develop an interactive risk calculator. Preoperative variables associated with increased risk of PRF include type of surgery, emergency case, dependent functional status, sepsis, and higher ASA class. The validated risk calculator provides a risk estimate of PRF and is anticipated to aid in surgical decision making and informed patient consent.


A method to audit and score implementation of knowledge translation (KT) interventions in large health regions - an observational pilot study using rectal cancer surgery in Ontario.
Across Ontario, since the year 2006 various knowledge translation (KT) interventions designed to improve the quality of rectal cancer surgery have been implemented by the provincial cancer agency or by individual researchers. Ontario is divided administratively into 14 health regions. We piloted a method to audit and score for each region of the province the KT interventions implemented to improve the quality of rectal cancer surgery. We interviewed stakeholders to audit KT interventions used in respective regions over years 2006 to 2014. Results were summarized into narrative and visual forms. Using a modified Delphi approach, KT experts reviewed these data and then, for each region, scored implementation of KT interventions using a 20-item KT Signature Assessment Tool. Scores could range from 20 to 100 with higher scores commensurate with greater KT intervention implementation. There were thirty interviews. KT experts produced scores for each region that were bimodally distributed, with an average score for 2 regions of 78 (range 73-83) and for 12 regions of 30.5 (range 22-38). Our methods efficiently identified two groups with similar KT Signature scores. Two regions had relatively high scores reflecting numerous KT interventions and the use of sustained iterative approaches in addition to those encouraged by the provincial cancer agency, while 12 regions had relatively low scores reflecting minimal activities outside of those encouraged by the provincial cancer agency. These groupings will be used for future comparative quantitative analyses to help determine if higher KT signature scores correlate with improved measures for quality of rectal cancer surgery.


Hybrid ultra-short Yb:YAG ceramic master-oscillator high-power fiber amplifier.
We demonstrated a hybrid ceramic master-oscillator high-power fiber amplifier with a diode-pumped Yb:YAG ceramic laser as the seeding oscillator, which was passively mode-locked at 103.29 MHz repetition rate around 1031 nm by using a semiconductor saturable absorption mirror, and a two-stage double-clad photonic crystal fiber amplifier, which power-scaled the ceramic laser oscillator up to an average power of 303 W. The amplified pulses were further compressed to 237 and 418 fs at 50 and 150 W output powers, respectively. The compressed pulses exhibited about 0.05% deviation from the Gaussian fit, implying that the high-power fiber amplification induced neither observable temporal and spectral distortion nor significant nonlinear de-chirping of the chirped pulses.


Stepping behavior and muscle activity of dairy cattle standing on concrete or rubber flooring for 1 or 3 hours.
The type of flooring in dairy cattle systems influences cows' health and welfare. Although concrete is common, the use of more compressible flooring, such as rubber, is increasing. Cows prefer to stand and walk on rubber surfaces than on concrete; however, it is largely unknown how walking and standing for longer periods of time influence muscle activity and fatigue. Therefore, we used measures of behavior and muscle activity to investigate the potential benefits of providing a rubber flooring surface to dairy cattle. Sixteen lactating Holstein cows were forced to stand on either concrete or rubber flooring for 1 or 3 h in a 2 × 2 crossover design. Surface electromyograms (SEMG) and skin surface temperature were used to evaluate muscle activity, fatigue, and movement of muscle activity between the hind legs. Activity of 2 muscles, the bicep femoris and middle gluteal, was assessed during both static contractions, when cows transferred weight to each hind leg, before and after 1 and 3 h of standing, and dynamic contractions, associated with steps and with shifts in weight without steps. In addition, we evaluated stepping rate, time between each step, feeding behavior, skin surface temperature, and latency to lie down after standing. Standing duration influenced both the behavior and muscle activity of cows. Stepping rate increased with standing time for cows on both flooring types. Static muscle activity parameters of the bicep femoris muscle were higher after 3 h of standing for cows standing on both flooring types (2.3 and 3.6% increases in median amplitude and median power frequency, respectively) compared with the change after 1 h and compared with baseline values before the standing treatment. Flooring type influenced the behavior and muscle activity of the cows, particularly during the first hour of exposure; cows standing on rubber had a higher stepping rate, shorter interval between steps, and higher number of SEMG shifts (muscle activity shifts with or without visible steps) than cows on concrete. There was no difference in skin surface temperature, feeding behavior, or latency to lie down between the treatments. The results show that standing on a rubber flooring caused a different initial behavioral response compared with standing on concrete; however, possible reasons for these changes are unclear. Standing for 3 h resulted in an increase in stepping rate and in some muscle activity parameters; however, the results regarding muscle fatigue in relation to flooring type are inconclusive.


Preparedness for admission of patients with suspected Ebola virus disease in European hospitals: a survey, August-September 2014.
In response to the Ebola virus disease (EVD) outbreak in West Africa, the World Health Organization has advised all nations to prepare for the detection, investigation and management of confirmed and suspected EVD cases in order to prevent further spread through international travel. To gain insights into the state of preparedness of European hospitals, an electronic survey was circulated in August–September 2014 to 984 medical professionals representing 736 hospitals in 40 countries. The survey addressed the willingness and capacity to admit patients with suspected EVD as well as specific preparedness activities in response to the current Ebola crisis. Evaluable responses were received from representatives of 254 (32%) hospitals in 38 countries, mostly tertiary care centres, of which 46% indicated that they would admit patients with suspected EVD. Patient transfer agreements were in place for the majority of hospitals that would not admit patients. Compared with non-admitting hospitals, admitting hospitals were more frequently engaged in various preparedness activities and more often contained basic infrastructural characteristics such as admission rooms and laboratories considered important for infection control, but some gaps and concerns were also identified. The results of this survey help to provide direction towards further preparedness activities and prioritisation thereof.


Single versus multiple courses of antenatal betamethasone and neonatal outcome: a randomized controlled trial.
This study compared the beneficial and adverse neonatal effects of a single versus repeated courses of antenatal betamethasone. Tertiary care hospital Open labeled, randomized controlled trial. Pregnant women (26-33 weeks) at risk of preterm delivery, who received one course of antenatal betamethasone and remained undelivered for 7 days. Those with uncertain gestation, major malformations and frank chorioamnionitis were excluded. Subjects were randomized to receive weekly antenatal betamethasone until 34 weeks or no further betamethasone. Primary: incidence of severe respiratory distress syndrome (RDS). Secondary: incidence of non-severe RDS and other neonatal morbidity; birth weight, length and occipito-frontal circumference (OFC); and, development and growth at 6 mo corrected age. 38 subjects were allocated to each group. Severe RDS was similar in multiple and single course groups (7% vs. 3% respectively, P=0.34), as was incidence of other morbidity. Composite outcome of RDS and or death within 28 days tended to be less in multiple course group (P=0.07). Birth anthropometry was similar in the 2 groups. At 6 mo corrected age (n=44), weight and length were significantly lower in multiple course group (p=0.003 and P=0.007, respectively), whereas OFC was not different (P=0.1). There were no differences vis a vis neurodevelopmental outcomes. A single course of antenatal betamethasone was as efficacious as multiple courses, with respect to prevention of neonatal morbidity. Multiple antenatal betamethasone courses have long-term adverse effects on infant weight and length growth, but not on OFC and neurodevelopment.


The effectiveness of manual therapy in treating cervicogenic dizziness: a systematic review.
[Purpose] This review provides an evaluation of the evidence for the effectiveness of using manual therapy to treat cervicogenic dizziness. [Subjects and Methods] The literature was systematically searched on the May 2, 2016 using the following online databases: Medline, EMBASE, CINAHL and PEDro. This review included randomised controlled trials and compared the efficacy of manual therapy for the treatment of cervicogenic dizziness, compared to other types of intervention. This study measured changes based on dizziness intensity and frequency. [Results] The primary search found 30 articles, but only four articles met the inclusion criteria. Assessment of methodological quality was performed by two researchers using the PEDro scale. The level of evidence was determined using a recognised grading scale. Three out of the four articles were deemed to have high methodological quality, while the fourth was rated as moderate quality. The attributed level of evidence was moderate (level 2). [Conclusion] Manual therapy is potentially effective for managing cervicogenic dizziness. However, due to the heterogeneity of the results and techniques and the low number of studies, further research is recommended to provide conclusive evidence.


Renewing focus on resident education: increased responsibility and ownership in interventional radiology rotations improves the educational experience.
To enhance the educational experience among residents rotating through interventional radiology (IR) by encouraging ownership and responsibility. In May 2006, the authors implemented changes in resident education in IR that included increased clinical responsibilities, structured didactics, and greater hands-on experience, including call. Residents were assigned as first assistants, ownership of cases was encouraged, and assignment to a week on the consult service was instituted to help residents better understand all aspects of IR practice. Additional faculty recruitment and program expansion ensured the same high level of training for the fellowship program. Evaluations were reviewed every year (July 1, 2007-June 30, 2009) for hands-on training, daily teaching, didactic conferences, and overall effectiveness of the clinical service. A graduated scale of 1-5 was used. In 2009, 3 years after the curricular changes were made, the quality of hands-on training, daily case reviews and consults, didactics, and overall education had markedly improved with 89%, 71%, 65%, and 82% of the residents rating these respective aspects of the training as "above expectations" (4 on a scale of 5) or "superior" (5 on a scale of 5) compared with 77%, 23%, 20%, and 60% in 2005-2006. Three years after the changes, the impact of these changes on recruitment patterns also showed improvement, with 28.6% of the class of 2010 pursuing a fellowship in IR. Increasing resident ownership, responsibility, and hands-on experience improves resident education in IR, which, in turn, promotes interest in the field.


The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis.
Recent studies have demonstrated the critical role of IL-10 in susceptibility to cutaneous and visceral leishmaniasis caused by Leishmania major and Leishmania donovani, respectively. To determine whether IL-10 also plays a similar role in the susceptibility and pathogenesis of cutaneous leishmaniasis caused by the New World species, L. mexicana and L. amazonensis, we analyzed their course of infection in IL-10-deficient BALB/c mice and their wild-type counterparts. Although IL-10-deficient mice infected with either L. mexicana or L. amazonensis failed to control the lesion progression, we did observe consistently lower levels of infection in IL-10(-/-) mice compared with wild-type BALB/c mice. We also observed increased IFN-gamma and NO production and higher levels for IL-12p40 and IL-12Rbeta(2) mRNA in cells from IL-10(-/-) mice compared with cells from BALB/c mice. The mRNA levels for IL-4, which increased significantly in both IL-10(-/-) and BALB/c mice, were comparable. When treated with anti-IL-4 mAb, IL-10(-/-) mice resolved the infection more effectively and had significantly fewer parasites in their lesions compared with similarly treated BALB/c mice. These findings suggest that IL-10, although not the dominant mediator of susceptibility of BALB/c mice to infection with L. mexicana and L. amazonensis, does play a significant role in regulating the development of a protective Th1-type response. However, effective resolution of infection with these New World parasites requires neutralization of both IL-4 and IL-10.


Women undergoing assisted fertilisation and high-intensity interval training: a pilot randomised controlled trial.
Overweight and obese women often seek assisted fertilisation. In the obese population, pregnancy rates are 30%-75% below that of normal weight women who undergo assisted fertilisation. We hypothesised that high-intensity interval training (HIT) would improve fertility by improving insulin sensitivity and thus affect the hypothalamic-pituitary-ovarian axis and ovarian androgen production. Our aim was to assess whether HIT prior to assisted fertilisation would increase pregnancy rate. Eighteen overweight and obese women (body mass index>25.0 kg/m2) were randomised to HIT (n=8) or usual care (control, n=10) before assisted fertilisation. HIT was undertaken three times weekly for 10 weeks; two sessions of 4×4 min HIT and one session of 10×1 min HIT. Primary outcome was ongoing pregnancy. Secondary outcomes included insulin sensitivity, reproductive hormones, oxygen uptake and body composition. Four women got pregnant in both the HIT group (50%) and in the control group (44%), no between-group difference (p=0.6). Insulin sensitivity (glucose infusion rate) improved significantly after HIT, from 264.1 mg/m2/min (95% CI 193.9 to 334.4) at baseline to 324.7 mg/m2/min (95% CI 247.2 to 402.2) after 10 weeks (between-group difference, p=0.04). Fasting glucose, visceral fat, waist circumference and VO2peak were significantly improved in the group that undertook HIT. HIT significantly improved insulin sensitivity, VO2peak and abdominal fat. Low statistical power makes it difficult to conclude on whether HIT prior to assisted fertilisation could increase pregnancy rate. Larger trials are needed to determine if improvements in insulin sensitivity are clinically relevant for assisted fertilisation success rates in this population.


Optimization of Vertical Double-Diffused Metal-Oxide Semiconductor (VDMOS) Power Transistor Structure for Use in High Frequencies and Medical Devices.
Power transistors, such as the vertical, double-diffused, metal-oxide semiconductor (VDMOS), are used extensively in the amplifier circuits of medical devices. The aim of this research was to construct a VDMOS power transistor with an optimized structure to enhance the operation of medical devices. First, boron was implanted in silicon by implanting unclamped inductive switching (UIS) and a Faraday shield. The Faraday shield was implanted in order to replace the gate-field parasitic capacitor on the entry part of the device. Also, implanting the UIS was used in order to decrease the effect of parasitic bipolar junction transistor (BJT) of the VDMOS power transistor. The research tool used in this study was Silvaco software. By decreasing the transistor entry resistance in the optimized VDMOS structure, power losses and noise at the entry of the transistor were decreased, and, by increasing the breakdown voltage, the lifetime of the VDMOS transistor lifetime was increased, which resulted in increasing drain flow and decreasing Ron. This consequently resulted in enhancing the operation of high-frequency medical devices that use transistors, such as Radio Frequency (RF) and electrocardiograph machines.


The eutT gene of Salmonella enterica Encodes an oxygen-labile, metal-containing ATP:corrinoid adenosyltransferase enzyme.
The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP:co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT+ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe2+ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe2+. We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.


PEGylated polyamidoamine dendrimers with bis-aryl hydrazone linkages for enhanced gene delivery.
Surface modification of polyamidoamine (PAMAM) dendrimers with polyethylene glycol (PEG) often results in the decrease in their buffering capacity, which is essential for gene transfer. In this work, bis-aryl hydrazone bond, which possesses protonatable pyridine and amines, was explored as a new linkage for PEGylation of PAMAM dendrimers. PEGylated polyamidoamine (PAMAM) dendrimer G4.0 conjugates with bis-aryl hydrazone (BAH) linkages were synthesized following a two-step procedure: activation of PAMAM dendrimer G4.0 and monofunctional methoxypolyethylene glycol amine (MW=5000 Da) with succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) and succinimidyl 4-formylbenzoate (SFB), respectively, and coupling of SFB-activated PEG to SANH-activated G4.0 to generate PEGylated G4.0 with bis-aryl hydrazone linkages (G4.0-BAH-PEG). It was found that the incorporation of BAH linkages into the vector significantly enhanced the buffering capacity of the vector even with a high degree of PEGylation (42 PEG chains per dendrimer). G4.0-BAH-PEG conjugates could complex with DNA plasmid tightly at low weight ratios and display dramatically improved cytocompatibility. According to gene transfection studies in 293T and HN12 cells, this new vector has been shown to be capable of both transfecting more cells and inducing higher gene expression than the parent dendrimer. This work demonstrates that the use of the BAH linkage in coupling of PEG to the dendrimer helps maintain or increase the buffering capacity of the functionalized dendrimer and results in enhanced transfection.


Regulation of growth and dissemination of a human lymphoma by CD44 splice variants.
CD44 is a polymorphic cell surface glycoprotein, currently proposed to be the principal cell surface receptor for hyaluronan. However, different isoforms of CD44, expressed in human lymphoid tumor cells, appear to have distinct effects on the ability of the cells to attach to hyaluronan-coated surfaces and on their capacity to form tumors in vivo. In the present study, we address the mechanisms that may regulate CD44 isoform-dependent adhesion to hyaluronan. We use a human Burkitt lymphoma, stably transfected with six different alternatively spliced human CD44 isoforms, to determine their potential hyaluronan binding and tumor growth promoting roles. We show that transfectants expressing CD44 splice variants that contain variable exons 6-10, 7-10 and 8-10 adhere to hyaluronan-coated surfaces weakly and that corresponding tumor formation in vivo is delayed with respect to CD44-negative parental cell-derived tumors. Abundant shedding of these three isoforms may play a significant role in determining the rate of tumor development. Transfectants expressing variable exon 3, on the other hand, fail to display CD44-mediated adhesion to hyaluronan, but form bone marrow tumors rapidly following intravenous injection. These observations suggest that different mechanisms regulate CD44-mediated adhesion and tumor growth, and provide evidence that expression of exon v3 may confer novel ligand-binding properties.


[Categorization of adrenergic alpha 1 receptors in detrusor of patients with obstructive BPH. Initial study on experimental animal model].
The action of alpha 1-adrenergic receptor antagonists in ameliorating irritation and obstruction in patients with bladder outlet obstruction (due to Benign Prostatic Hyperplasia-BPH) has been demonstrated. Although it is well known that alpha 1-a receptors are responsible for prostate smooth muscle relaxation, the mechanism by which irritative bladder symptoms are improved is unknown. Different alpha 1 receptor subtypes may be involved. The objective of this study is to investigate the changes in the alpha-adrenergic receptor populations in the obstructed detrusor, and to determine which subtype is proportionally increased in this situation (bladder outlet obstruction). This was an in vivo study in an experimental model: male NZ (New Zealand) rabbits. The bladder neck of one group of rabbits was obstructed surgically using the technique proposed by Levin et al. Subsequent to bladder catherization with a 8Fr catheter, the bladder neck was exposed by means of a low medial abdominal incision. The bladder neck was tied with a 2/0 silk thread below the ureteral orifices. The catheter was taken out at the end of the intervention. The rabbits were sacrificed after 4 weeks using intracardiac pentothal and cystectomy was performed. A control group that did not undergo bladder obstruction were also sacrificed at this time, for the comparative study. Part of the detrusor was used for a pathological study and the rest for a physio-pharmacological study in which the organ was placed in a bath of adrenoceptor agonists (phenylephrine and noradrenaline) and antagonists WB101, AH11101A and BMY7378 (antagonists of the alpha 1a, b, d, respectively). The findings of the pathological study show that the bladder wall was thicker in the rabbits that underwent bladder obstruction. The physio-pharmacological studies demonstrate that the detrusor response to the selective alpha-1 adrenergic agonist was greater in the rabbits that underwent bladder obstruction, however detrusor contractility was decreased (KPSS). With bladder obstruction the alpha 1d receptors were increased. Receptor-binding studies (Malloy et al) aim to differentiate the alpha-adrenoreceptor populations. These studies identify and quantitate the different receptor subtypes in tissue without taking into account their activity. The isometric and physio-pharmacological tests evaluate active receptors, i.e. those that respond to agonist and antagonist stimuli. This enables detrusor activity to be evaluated accurately. The results obtained in this investigational study support the hypothesis that there is a high statistically significant increase in the alpha 1 adrenergic receptors in the obstructed detrusor. Furthermore, in agreement with previous molecular studies, during prostate obstruction alpha 1d is the predominate sub-population in the bladder. These findings may have patho-physiological, clinical and pharmacological implications. If this hypothesis which has been demonstrated in an experimental model, is also demonstrated in studies in humans, pharmacological development should not only be focussed on selective alpha 1a receptor antagonists (prostate) but also on those of alpha 1d, for relieving symptoms in patients with bladder outlet obstruction (BPH and prostatism).


Care of pediatric tracheostomy in the immediate postoperative period and timing of first tube change.
To analyze the safety of a standardized pediatric tracheostomy care protocol in the immediate postoperative period and its impact on tracheostomy related complications. Retrospective case series. Pediatric patients undergoing tracheotomy from February 2010-February 2014. In 2012, a standardized protocol was established regarding postoperative pediatric tracheostomy care. This protocol included securing newly placed tracheostomy tubes using a foam strap with hook and loop fastener rather than twill ties, placing a fresh drain sponge around the tracheostomy tube daily, and performing the first tracheostomy tube change on postoperative day 3 or 4. Outcome measures included rate of skin breakdown and presence of a mature stoma allowing for a safe first tracheostomy tube change. Two types of tracheotomy were performed based on patient age: standard pediatric tracheotomy and adult-style tracheotomy with a Bjork flap. Patients were analyzed separately based on age and the type of tracheotomy performed. Thirty-seven patients in the pre-protocol group and 35 in the post-protocol group were analyzed. The rate of skin breakdown was significantly lower in the post-protocol group (standard: p=0.0048; Bjork flap: p=0.0003). In the post-protocol group, all tube changes were safely accomplished on postoperative day three or four, and the stomas were deemed to be adequately matured to do so in all cases. A standardized postoperative pediatric tracheostomy care protocol resulted in decreased rates of skin breakdown and demonstrated that pediatric tracheostomy tubes can be safely changed as early as 3 days postoperatively.


Human erythrocyte glutathione reductase: pH dependence of kinetic parameters.
Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH, 2',3'-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2',3'-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's for ATP-ribose and 2',5'-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2'-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467'-Glu-472' ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.


New evaluation method for postoperative scar redness.
Even after successful operations, ugly postoperative skin scars are often distressing to patients and their parents. To judge the success of surgical methods and postoperative treatment, postoperative scars should be evaluated using a quantitative system. Height and width are easily measured, but scar redness is not. We have developed a simple and effective method for evaluating scar redness. According to the color definitions employed in computer graphics, each color can be expressed as RGB (red, green or blue) coordinates (r, g, b): 0 < or = r, g, b < or = 10. The degree of scar redness is defined by the following formula: redness score (RS) = (r1 - r0)2 + (g1 - g0)2 + (b1 - b0)2. Here, (r1, g1, b1) = coordinates of the scar color and (r0, g0, b0) = coordinates of the surrounding skin color. RS was evaluated in 59 children (35 males, 24 females; ages 1 month to 12 years old) who had scar redness after congenital cardiac surgery. For each patient, scar color and surrounding skin color was identified on the color sample table. Scar redness was also evaluated by the conventional grading method: 1 = mild, 2 = moderate and 3 = severe. The RS of the colored scars ranged from 4 to 100 (38 +/- 27). By the conventional grading method, 44 scars were grade 1, 15 grade 2 and none grade 3. RS was significantly higher among grade 2 than grade 1 patients, 52 +/- 25 and 33 +/- 27, respectively (P < 0.05). Given its subjectivity, the conventional grading method yields variable data surrounding skin color, moreover, is not considered. Our new evaluation method using RS effectively and accurately defines scar and skin colors, and allows quantitative studies of these factors.


FoxJ1 inhibits African swine fever virus replication and viral S273R protein decreases the expression of FoxJ1 to impair its antiviral effect.
African swine fever (ASF) is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar, which is caused by the African swine fever virus (ASFV). ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health. To date, there is no safe and effective commercial vaccine against ASF. Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV. In the present study, RNA-sequencing, RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages (PAMs) infected by ASFV. RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes (ISGs) induced by poly(dA:dT). FoxJ1 revealed a function to positively regulate innate immune response, therefore, suppressing the replication of ASFV. In addition, Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway. Meanwhile, RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1. Altogether, we determined that FoxJ1 plays an antiviral role against ASFV replication, and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1. Our findings provide new insights into the antiviral function of FoxJ1, which might help design antiviral drugs or vaccines against ASFV infection.


Immune responses of Asian sea bass, Lates calcarifer Bloch, against an inactivated betanodavirus vaccine.
Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.


Conversion of the 2 Cl(-)/1 H+ antiporter ClC-5 in a NO3(-)/H+ antiporter by a single point mutation.
Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.


Terrestrial water fluxes dominated by transpiration.
Renewable fresh water over continents has input from precipitation and losses to the atmosphere through evaporation and transpiration. Global-scale estimates of transpiration from climate models are poorly constrained owing to large uncertainties in stomatal conductance and the lack of catchment-scale measurements required for model calibration, resulting in a range of predictions spanning 20 to 65 per cent of total terrestrial evapotranspiration (14,000 to 41,000 km(3) per year) (refs 1, 2, 3, 4, 5). Here we use the distinct isotope effects of transpiration and evaporation to show that transpiration is by far the largest water flux from Earth's continents, representing 80 to 90 per cent of terrestrial evapotranspiration. On the basis of our analysis of a global data set of large lakes and rivers, we conclude that transpiration recycles 62,000 ± 8,000 km(3) of water per year to the atmosphere, using half of all solar energy absorbed by land surfaces in the process. We also calculate CO2 uptake by terrestrial vegetation by connecting transpiration losses to carbon assimilation using water-use efficiency ratios of plants, and show the global gross primary productivity to be 129 ± 32 gigatonnes of carbon per year, which agrees, within the uncertainty, with previous estimates. The dominance of transpiration water fluxes in continental evapotranspiration suggests that, from the point of view of water resource forecasting, climate model development should prioritize improvements in simulations of biological fluxes rather than physical (evaporation) fluxes.


Labeling of phospholipids in vesicles and human erythrocytes by photoactivable fatty acid derivatives.
The photoactivable glycolipid probes, 2-(4-azido-2-nitrophenoxy)palmitoyl[1-14C]glucosamine (compound A) and 12-(4-azido-2-nitrophenoxy)stearoyl[1-14C]glucosamine (compound B) were synthesized essentially as described before [Iwata, K. K. et al. (1978) Prog. Clin. Biol. Res. 22, 579-589]. These probes were used to label phospholipid vesicles and erythrocyte membranes. A chromatographic method was developed to quantify the individual probe-phospholipid adducts involving both phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. For both membranes as well as for both probes a phospholipid labeling pattern was obtained which appeared to reflect the relative content of fatty acid double bonds in each phospholipid class. The distinct labeling of phosphatidylserine in intact erythrocytes strongly suggested that the probes spontaneously and rapidly redistributed between the two halves of the membrane bilayer. In addition, both probes yielded an extensive labeling of the membrane proteins. Analysis by dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography has indicated that the protein labeling pattern was different, depending on whether the 'shallow' probe (compound A) or 'depth' probe (compound B) were used.


Optimizing decellularization techniques to create a new nerve allograft: an in vitro study using rodent nerve segments.
OBJECTIVE Commercially available processed nerve allografts have been shown to be inferior to autografts in previous animal studies. The authors hypothesized that combining different processing and storage techniques will result in improved nerve ultrastructure preservation, lower immunogenicity, and minimized cellular debris. Different processing protocols were evaluated using chemical detergents, enzymes, and irradiation, with the addition the of enzyme elastase, were used. Additionally, the difference between cold and frozen storage was investigated. The goal of this study was to create an optimized nerve allograft. METHODS Fifty rat nerves were decellularized with modifications of previous protocols and the addition of elastase. Subsequently, the nerve segments were stored at either 4°C or -80°C. Both processed and fresh control nerves were analyzed with confocal microscopy using immunohistochemical staining on the basal lamina (laminin γ-1), Schwann cells (S100 protein), and immunogenicity using major histocompatibility complex-I (MHCI) staining. Morphology of the ultrastructure and amount of cellular debris were analyzed on cross-sections of the nerves stained with toluidine blue and H & E, and by using electron microscopy. RESULTS Nerve ultrastructure was preserved with all decellularization protocols. Storage at -80°C severely altered nerve ultrastructure after any decellularization method. Elastase was found to significantly reduce the immunogenicity and amount of Schwann cells, while maintaining good structural properties. CONCLUSIONS Reduced immunogenicity, diminished cellular debris, and the elimination of Schwann cells was observed when elastase was added to the nerve processing while maintaining ultrastructure. Storage at -80°C after the decellularization process heavily damaged the nerve ultrastructure as compared with cold storage. Further in vivo studies are needed to prove the nerve regenerative capacity of these optimized allografts.


Self-reported hypertension prevalence and income among older adults in Canada and the United States.
Hypertension is one of the most common chronic conditions worldwide. There is strong evidence that low socioeconomic status is associated with elevated rates of blood pressure-related cardiovascular disease. Few studies have examined the association between socioeconomic circumstances and hypertension among people aged 65 years and older. The purpose of this study was to examine the relationship between household income and self-reported hypertension prevalence among persons aged 65 and older in the United States and Canada. Data were obtained from the 2002-2003 Joint Canada/United States Survey of Health for 755 Canadian and 1151 US adults aged 65 and older. Aggregate hypertension prevalence rates in the United States and Canada were generally similar (53.8% versus 48.0%). We found a significant inverse linear relationship between household income and the hypertension prevalence rate in the United States, but no evidence of such a relationship in Canada. In Canada, unlike the United States, the burden of hypertension is approximately equal for socioeconomically advantaged and disadvantaged older adults. It is important to consider these findings in the context of long-term and broader institutional policies. Social disparities and barriers to health care access and primary prevention among non-elderly persons in the United States may play a role in the higher hypertension prevalence rate among low-income older adults.


Validation of the SF-36 in patients with endometriosis.
Endometriosis presents with significant pain as the most common symptom. Generic health measures can allow comparisons across diseases or populations. However, the Medical Outcomes Study Short Form 36 (SF-36) has not been validated for this disease. The goal of this study was to validate the SF-36 (version 2) for endometriosis. Using data from two clinical trials (N = 252 and 198) of treatment for endometriosis, a full complement of psychometric analyses was performed. Additional instruments included a pain visual analog scale (VAS); a physician-completed questionnaire based on patient interview (modified Biberoglu and Behrman--B&B); clinical global impression of change (CGI-C); and patient satisfaction with treatment. Bodily pain (BP) and the Physical Component Summary Score (PCS) were correlated with the pain VAS at baseline and over time and the B&B at baseline and end of study. In addition, those who had the greatest change in BP and PCS also reported the greatest change on CGI-C and patient satisfaction with treatment. Other subscales showed smaller, but significant, correlations with change in the pain VAS, CGI-C, and patient satisfaction with treatment. The SF-36--particularly BP and the PCS--appears to be a valid and responsive measure for endometriosis and its treatment.


Influenza A virus infection dysregulates the expression of microRNA-22 and its targets; CD147 and HDAC4, in epithelium of asthmatics.
Specific microRNAs (miRNAs) play essential roles in airway remodeling in asthma. Infection with influenza A virus (IAV) may also magnify pre-existing airway remodeling leading to asthma exacerbation. However, these events remain to be fully defined. We investigated the expression of miRNAs with diverse functions including proliferation (miR-20a), differentiation (miR-22) or innate/adaptive immune responses (miR-132) in primary bronchial epithelial cells (pBECs) of asthmatics following infection with the H1N1 strain of IAV. pBECs from subjects (n = 5) with severe asthma and non-asthmatics were cultured as submerged monolayers or at the air-liquid-interface (ALI) conditions and incubated with IAV H1N1 (MOI 5) for up to 24 h. Isolated miRNAs were subjected to Taqman miRNAs assays. We confirmed miRNA targets using a specific mimic and antagomir. Taqman mRNAs assays and immunoblotting were used to assess expression of target genes and proteins, respectively. At baseline, these miRNAs were expressed at the same level in pBECs of asthmatics and non-asthmatics. After 24 h of infection, miR-22 expression increased significantly which was associated with the suppression of CD147 mRNA and HDAC4 mRNA and protein expression in pBECs from non-asthmatics, cultured in ALI. In contrast, miR-22 remained unchanged while CD147 expression increased and HDAC4 remained unaffected in cells from asthmatics. IAV H1N1 mediated increases in SP1 and c-Myc transcription factors may underpin the induction of CD147 in asthmatics. The different profile of miR-22 expression in differentiated epithelial cells from non-asthmatics may indicate a self-defense mechanism against aberrant epithelial responses through suppressing CD147 and HDAC4, which is compromised in epithelial cells of asthmatics.


Melanomas Associated With Blue Nevi or Mimicking Cellular Blue Nevi: Clinical, Pathologic, and Molecular Study of 11 Cases Displaying a High Frequency of GNA11 Mutations, BAP1 Expression Loss, and a Predilection for the Scalp.
Melanomas associated with blue nevi (MABN) or mimicking cellular blue nevi (MMCBN) represent exceptional variants of malignant cutaneous melanocytic tumors. Uveal and leptomeningeal melanomas frequently have somatic mutations of GNAQ or GNA11, which are believed to be early driver mutations. In uveal melanomas, monosomy 3, linked to the BAP1 gene, is an adverse prognostic factor. We have studied the clinical, histologic, BAP1 expression profile, and molecular data of 11 cases of MABN/MMCBN and 24 cellular blue nevi. Most of the cases of MABN/MMCBN occurred on the scalps of adult patients and presented as rapidly growing nodules, typically >1 cm, often arising at the site of a preexisting melanocytic lesion. The MABN/MMCBN were composed of dense nests of large dermal atypical melanocytes, in some cases lying adjacent to a blue nevus. Four patients developed metastatic disease, and 2 died from their disease. A GNA11 mutation was found in 8/11 cases and a GNAQ mutation in 1 case. Seven of 11 cases showed loss of nuclear BAP1 immunohistochemical (IHC) expression in the malignant component, sparing the adjacent nevus. Array comparative genomic hybridization revealed recurrent deletions of chromosomes 1p, 3p, 4q, 6q, 8p, 16q, and 17q and recurrent gains of chromosomes 6p, 8q, and 21q. The 24 cases of cellular blue nevi frequently occurred on the sacrum, had GNAQ mutations, and showed normal positive IHC staining for BAP1. These results underscore overlapping features in all blue-like malignant melanocytic tumors. Loss of BAP1 IHC expression was restricted to melanomas, including all metastatic cases.


Integration of a foreign gene into a native complex does not impair fitness in an experimental model of lateral gene transfer.
Lateral gene transfer (LGT) is a central force in microbial evolution. The observation that genes encoding subunits of complexes exhibit relatively compatible phylogenies, suggesting vertical descent, can be explained by different evolutionary scenarios. On the one hand, the failure of a new gene product to correctly interact with preexisting protein subunits can make its acquisition neutral-a theory termed the "complexity hypothesis." On the other hand, foreign subunit-encoding genes may reduce the fitness of the new host by disrupting the stoichiometric balance between complex subunits, resulting in purifying selection against gene retention. We previously showed in a model LGT system that overexpression of an orthologous subunit was neutral due to lack of interaction with host subunits. Here, we examine a case where the foreign protein is more similar to its native orthologs, by expressing the RNA polymerase β subunit (RpoB) of Bacillus subtilis in Escherichia coli. The foreign subunit is shown by coimmunoprecipitation to interact with the host subunits, and to form novel, nonspecific interactions. Nevertheless, the host did not incur any fitness disadvantage, as measured by its growth. We conclude that LGT of complex subunits may be neutral even when the transferred subunit can integrate into the host complex and that this neutrality can be a fertile ground for selective forces once the environment changes.


Validation of an automated system for measuring anxiety-related behaviours in the elevated plus maze.
The elevated plus maze (EPM) is one of the most widely used and established tests to assess anxiety-related behaviours and has been validated for use in both mice and rats. Although relatively quick and simple to conduct, there always exists the potential for observer bias during data collection. The KinderScientific EPM system uses a series of apparatus-embedded photobeams to collect spatiotemporal measures such as the amount of time spent in each zone of the maze (centre, open and closed arms), and the frequency of arm entries. Risk assessment behaviours, such as head dips and protected stretches, are also measured which represents a unique feature of this system over other automated EPM systems. We compared observer derived spatiotemporal and risk assessment measurements with automated generated data to test the reliability and accuracy of the automated system. Data were manually collected using different zone entry/exit criteria (2 vs. 4 paws). Automated data were generated using both the default zone map provided with the system and a user-modified zone map. We show that the automated EPM provides accurate and reliable measurements of both spatiotemporal and risk assessment behaviours. In addition, we show that the default zone map overestimated visually observed arm entries while our modified zone map generated data comparable to manually generated data using a 4 paws open arm entry criteria which is most consistently used to define arm entry in the literature. The KinderScientific automated EPM system represents a reliable tool for collection of a wide range of anxiety-related behavioural measures.


Infective Endocarditis: Identification of Catalase-Negative, Gram-Positive Cocci from Blood Cultures by Partial 16S rRNA Gene Analysis and by Vitek 2 Examination.
Streptococci, enterococci and Streptococcus-like bacteria are frequent etiologic agents of infective endocarditis and correct species identification can be a laboratory challenge. Viridans streptococci (VS) not seldomly cause contamination of blood cultures. Vitek 2 and partial sequencing of the 16S rRNA gene were applied in order to compare the results of both methods. STRAINS ORIGINATED FROM TWO GROUPS OF PATIENTS: 149 strains from patients with infective endocarditis and 181 strains assessed as blood culture contaminants. Of the 330 strains, based on partial 16S rRNA gene sequencing results, 251 (76%) were VS strains, 10 (3%) were pyogenic streptococcal strains, 54 (16%) were E. faecalis strains and 15 (5%) strains belonged to a group of miscellaneous catalase-negative, Gram-positive cocci. Among VS strains, respectively, 220 (87,6%) and 31 (12,3%) obtained agreeing and non-agreeing identifications with the two methods with respect to allocation to the same VS group. Non-agreeing species identification mostly occurred among strains in the contaminant group, while for endocarditis strains notably fewer disagreeing results were observed.Only 67 of 150 strains in the mitis group strains obtained identical species identifications by the two methods. Most VS strains belonging to the groups of salivarius, anginosus, and mutans obtained agreeing species identifications with the two methods, while this only was the case for 13 of the 21 bovis strains. Pyogenic strains (n=10), Enterococcus faecalis strains (n=54) and a miscellaneous group of catalase-negative, Gram-positive cocci (n=15) seemed well identified by both methods, except that disagreements in identifications in the miscellaneous group of strains occurred for 6 of 15 strains.


Influence of the Hole Transporting Layer on the Thermal Stability of Inverted Organic Photovoltaics Using Accelerated-Heat Lifetime Protocols.
High power conversion efficiency (PCE) inverted organic photovoltaics (OPVs) usually use thermally evaporated MoO3 as a hole transporting layer (HTL). Despite the high PCE values reported, stability investigations are still limited and the exact degradation mechanisms of inverted OPVs using thermally evaporated MoO3 HTL remain unclear under different environmental stress factors. In this study, we monitor the accelerated lifetime performance under the ISOS-D-2 protocol (heat conditions 65 °C) of nonencapsulated inverted OPVs based on the thiophene-based active layer materials poly(3-hexylthiophene) (P3HT), poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl]] (PTB7), and thieno[3,2-b]thiophene-diketopyrrolopyrrole (DPPTTT) blended with [6,6]-phenyl C71-butyric acid methyl ester (PC[70]BM). The presented investigation of degradation mechanisms focus on optimized P3HT:PC[70]BM-based inverted OPVs. Specifically, we present a systematic study on the thermal stability of inverted P3HT:PC[70]BM OPVs using solution-processed poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and evaporated MoO3 HTL. Using a series of measurements and reverse engineering methods, we report that the P3HT:PC[70]BM/MoO3 interface is the main origin of failure of the P3HT:PC[70]BM-based inverted OPVs under intense heat conditions, a trend that is also observed for the other two thiophene-based polymers used in this study.