Divergent functions of orthologous NAC transcription factors in wheat and rice.
The wheat GPC-B1 gene located on chromosome 6B is an early regulator of senescence and affects remobilization of protein and minerals to the grain. GPC-B1 is a NAC transcription factor and has a paralogous copy on chromosome 2B in wheat, GPC-B2. The closest rice homolog to both wheat GPC genes is Os07g37920 which is located on rice chromosome 7 and is colinear with GPC-B2. Since rice is a diploid species with a sequenced genome, we initiated the study of Os07g37920 to develop a simpler model to study senescence and mineral remobilization in cereals. We developed eleven independent RNA interference transgenic rice lines (Os07g37920-RNAi) and 10 over-expressing transgenic lines (Os07g37920-OE), but none of them showed differences in senescence. Transgenic Os07g37920-RNAi rice plants had reduced proportions of viable pollen grains and were male-sterile, but were able to produce seeds by cross pollination. Analysis of the flower morphology of the transgenic rice plants showed that anthers failed to dehisce. Transgenic Os07g37920-OE lines showed no sterility or anther dehiscence problems. Os07g37920 transcript levels were higher in stamens compared to leaves and significantly reduced in the transgenic Os07g37920-RNAi plants. Wheat GPC genes showed the opposite transcription profile (higher transcript levels in leaves than in flowers) and plants carrying knock-out mutations of all GPC-1 and GPC-2 genes exhibited delayed senescence but normal anther dehiscence and fertility. These results indicate a functional divergence of the homologous wheat and rice NAC genes and suggest the need for separate studies of the function and targets of these transcription factors in wheat and rice.


Hydrogen-Rich Saline Promotes the Recovery of Renal Function after Ischemia/Reperfusion Injury in Rats via Anti-apoptosis and Anti-inflammation.
Hydrogen is a proven novel antioxidant that selectively reduces hydroxyl radicals. In this study, we investigated the effects of hydrogen-rich saline solution on the prevention of renal injury induced by ischemia/reperfusion (I/R) and on renal function recovery. A rat model of renal I/R injury was induced by 45 min occlusion of the left renal pedicle, followed by 108 h reperfusion. The right kidney was surgically removed. Then, 0.9% NaCl solution (1 ml/kg) or hydrogen-rich saline solution (HRSS; 1 ml/kg) was injected into the abdominal cavity at 4 h intervals. We assessed the influence of HRSS or control saline solution on the recovery of renal function after I/R injury. Kidney tissues were taken at different time points (24, 36, 48, 72, and 108 h after reperfusion) and frozen (-80°C). Kidney cell apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive staining. Additionally, the apoptotic factors (Bcl-2, Bax, caspase-3, caspase-9, and caspase-8) and the pro-inflammatory cytokines (IL-6 and TNF-α) were measured in the kidney tissues. Finally, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were measured. Histological analyses revealed a marked reduction of interstitial congestion, edema and hemorrhage in renal tissue after HRSS treatment compared to saline treatment. After I/R injury, BUN, Cr, Bcl-2, caspase-3, caspase-9, caspase-8, IL-6, and TNF-α were all significantly increased, while Bax expression was decreased. HRSS remarkably reversed these changes. Moreover, BUN and Cr decreased more rapidly in the rats treated with HRSS compared to the rats treated with control saline solution. HRSS showed a protective effect in the prevention of renal injury and could promote renal function recovery after I/R injury in rats. HRSS might partially exert its role through an anti-apoptotic and anti-inflammatory action in kidney cells.


An investigation of multimorbidity measures as risk factors for pneumonia in elderly frail patients admitted to hospital.
To investigate the association of different chronic comorbidities, considered singularly and together in Cumulative Illness Rating Scale (CIRS) indexes, with pneumonia diagnosis in a group of elderly frail hospitalized patients. With a retrospective cohort design, all clinical records of frail (Rockwood ≥ 5) nonterminal patients ≥ 65 years old acutely admitted over a 8-month span in an internal medicine ward were evaluated. Pneumonia status and its categorization (community-acquired, CAP, vs healthcare-associated, HCAP) were defined according to chest radiology findings and validated criteria. Chronic comorbidities, CIRS Comorbidity Score and CIRS Severity Index were collected for each participant through a standardized methodology. Multivariate logistic regression models were applied to assess the association of each comorbid condition or scores with pneumonia. 1199 patients (546 M, median age 81.9, IQR 72.8-87.9 years), of whom 239 with pneumonia (180 CAP, 59 HCAP) were evaluated. CIRS Comorbidity Score was significantly associated with pneumonia, both at an age- and sex-adjusted model and at a multivariate model (OR for each unitary increase 1.03, 95% CI 1.001-1.062, p=0.04), together with provenience from nursing home (OR 1.96, 95% CI 1.41-2.73, p<0.001). Among single comorbidities, only COPD (OR 2.7, 95% CI 1.9-3.6, p<0.001) and dementia (OR 2.3, 95% CI 1.7-3.3, p<0.001) were associated with pneumonia, while stroke, cancer, cardiovascular, chronic liver and kidney disease were not. In a small cohort of elderly frail hospitalized patients, measures of multimorbidity, like CIRS, are significantly associated with the risk of pneumonia. COPD and dementia are the main conditions concurring to define this risk.


Mitochondrial reprogramming through cardiac oxygen sensors in ischaemic heart disease.
Under hypoxic conditions, mitochondria can represent a threat to the cell because of their capacity to generate toxic reactive oxygen species (ROS). However, cardiomyocytes are equipped with an oxygen-sensing pathway that involves prolyl hydroxylase oxygen sensors and hypoxia-inducible factors (HIFs), which induces a tightly regulated programme to keep ischaemic mitochondrial activity under control. The aim of this review is to provide an update on the pathways leading to mitochondrial reprogramming, which occurs in the myocardium during ischaemia, with particular emphasis on those induced by HIF activation. We start by studying the mechanisms of mitochondrial damage during ischaemia and upon reperfusion, highlighting the importance of the formation of the mitochondrial permeability transition pore during reperfusion and its consequences for cardiomyocyte survival. Next, we analyse hypoxia-induced metabolic reprogramming through HIF and its important consequences for mitochondrial bioenergetics, as well as the phenomenon known as the hibernating myocardium. Subsequently, we examine the mechanisms underlying ischaemic preconditioning, focusing, in particular, on those that involve the HIF pathway, such as adenosine signalling, sub-lethal ROS generation, and nitric oxide production. Finally, the role of the mitochondrial uncoupling proteins in ischaemia tolerance is discussed.


Persistent and nonpersistent Listeria monocytogenes contamination in meat and poultry processing plants.
Contamination analysis of persistent and nonpersistent Listeria monocytogenes strains in three meat processing plants and one poultry processing plant were performed in order to identify factors predisposing to or sustaining persistent plant contamination. A total of 596 L. monocytogenes isolates were divided into 47 pulsed-field gel electrophoresis (PFGE) types by combining the restriction enzyme patterns of AscI (42 patterns) and ApaI (38 patterns). Persistent and nonpersistent strains were found in all plants. Nonpersistent PFGE types were found mostly at one sampling site, with the processing environment being the most common location, whereas the persistent strains were found at several sampling sites in most cases. The processing machines were frequently contaminated with persistent L. monocytogenes PFGE types, and it was of concern that surfaces having direct contact with the products were contaminated. The role of the processing machines in sustaining contamination and in contaminating the products appeared to be important because the final product of several processing lines was contaminated with the same L. monocytogenes PFGE type as that found in the processing machine. The proportion of persistent PFGE types in heat-treated products was eight times higher than in the raw products, showing the importance of the persistent PFGE types as contaminants of the final heat-treated products. The contamination status of the processing lines and machines appeared to be influenced by the compartmentalization of the processing line, with poor compartmentalization increasing L. monocytogenes contamination. The separation of raw and post-heat treatment areas seemed especially important in the contamination status of post-heat treatment lines.


On the social science contribution to governmental decision-making.
It is often said that a major impediment to effective government is the lack of research knowledge underlying the development, implementation, and assessment of federal policies and programs. An examination is made here of the causes of (i) some continuing failures in integrating research information with decision-making and (ii) some notable successes of the past two decades in matching research capabilities with policy needs. Six recommendations are made to facilitate and further the progress that has recently been achieved.


Simultaneous determination of apatinib and its four major metabolites in human plasma using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.
Apatinib, also known as YN968D1, is a novel antiangiogenic agent that selectively inhibits vascular endothelial growth factor receptor-2. Currently, apatinib is undergoing phase II/III clinical trials in China for the treatment of solid tumors. Apatinib is extensively metabolized in humans, and its major metabolites in circulation include cis-3-hydroxy-apatinib (M1-1), trans-3-hydroxy-apatinib (M1-2), apatinib-25-N-oxide (M1-6), and cis-3-hydroxy-apatinib-O-glucuronide (M9-2). To investigate the pharmacokinetics of apatinib and its four major metabolites in patients with advanced colorectal cancer, a sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of apatinib, M1-1, M1-2, M1-6, and M9-2 in human plasma. After a simple protein precipitation using acetonitrile as the precipitation solvent, all the analytes and the internal standard vatalanib were separated on a Zorbax Eclipse XDB C(18) column (50 mm × 4.6 mm, 1.8 μm, Agilent) using acetonitrile: 5 mmol/L ammonium acetate with 0.1% formic acid as the mobile phase with gradient elution. A chromatographic total run time of 9 min was achieved. Mass spectrometry detection was conducted through electrospray ionization in positive ion multiple reaction monitoring modes. The method was linear over the concentration range of 3.00-2000 ng/mL for each analyte. The lower limit of quantification for each analyte was 3.00 ng/mL. The intra-assay precision for all the analytes was less than 11.3%, the inter-assay precision was less than 13.8%, and the accuracy was between -5.8% and 3.3%. The validated method was successfully applied to a clinical pharmacokinetic study following oral administration of 500 mg apatinib mesylate in patients with advanced colorectal cancer.


ARD1 stabilization of TSC2 suppresses tumorigenesis through the mTOR signaling pathway.
Mammalian target of rapamycin (mTOR) regulates various cellular functions, including tumorigenesis, and is inhibited by the tuberous sclerosis 1 (TSC1)-TSC2 complex. Here, we demonstrate that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby repressing mTOR activity. The inhibition of mTOR by ARD1 inhibits cell proliferation and increases autophagy, thereby inhibiting tumorigenicity. Correlation between ARD1 and TSC2 abundance was apparent in multiple tumor types. Moreover, evaluation of loss of heterozygosity at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as an inhibitor of the mTOR pathway and that dysregulation of the ARD1-TSC2-mTOR axis may contribute to cancer development.


DNA fragmentation induced in human fibroblasts by accelerated (56)fe ions of differing energies.
DNA fragmentation was studied in the fragment size range 0.023-5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with gamma rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/mum, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after gamma irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than gamma rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.


Heat tolerance and aging.
Although children and older adults appear more susceptible to heat stress, the mechanisms responsible for their lower tolerance are not fully understood. Many studies dealing with the effect of age on temperature regulation have concluded that an inadequate sweating response is primarily responsible for the low tolerance of children and the elderly to exercise in the heat. However, the dependence of core temperature on relative exercise intensity and sweat rate on absolute exercise intensity makes it difficult to avoid the confounding effects of exercise on thermoregulation when aerobic power (Vo2max) varies across age groups. When 38 non-acclimatized females, ages 12 to 68 years, exercised at 30-35% Vo2max in the heat, the degree of cardiovascular stability was the primary predictor of tolerance time. Age was not a significant predictor. However, it was evident that individuals at either end of the age continuum were more likely to be at risk. For children this risk was associated with the instability of an immature cardiovascular system; for older women, a marked decrement in aerobic power. Sweat rate added significantly to the prediction of tolerance time for all subjects regardless of age. Whether the decrease in responsiveness of sweating noted for some older individuals is an age related change or a reflection of their lower fitness levels is not known.


Video. Modified single-incision laparoscopic adjustable gastric band.
This video describes a modified single-incision laparoscopic approach for adjustable gastric band placement. The patient was a 28-year-old female with a BMI of 48.75 with no prior surgery but with numerous comorbidities. With the patient placed in a split-leg position and in steep reverse Trendelenburg, a 12-mm optical trocar is placed 12 cm distal to the xiphoid process in the left paramedian location. A Nathanson liver retractor is placed through a midepigastric 5-mm incision. Two 5-mm low-profile trocars are placed next to the 12-mm trocar through separate incisions (this maintains stability of each cannula) and a 5-mm 45 ° laparoscope is used. Using an automated suturing device, a stay suture is placed high on the fundus and externalized for retraction. An articulating band passer dissects the phrenogastric attachments at the angle of His. The 12-mm port is removed and the gastric band is inserted. The GE junction fat pad is excised and the Pars Flaccida membrane is divided using conventional instruments. A second traction suture is placed to retract the lesser curve fat and right crus fat pad. A peritoneal bite is also taken in the left lateral subcostal area such that when this suture is externalized, it acts as a pulley. An articulating 5-mm grasper is used to develop the retrogastric tunnel. Then the band is fed into position and its buckle is locked. Three interrupted sutures are placed to create an anterior gastric plication and a fourth antislippage suture is placed below the band along the lesser curve. The band tubing is externalized and the port is implanted by joining the three working trocar incisions into a single 4.5-cm incision. The patient did well postoperatively with no complications. A modified single incision approach for laparoscopic gastric band placement is feasible and provides patients with improved cosmesis.


Metabolic regulation of collagen I in fibroblasts isolated from normal peritoneum and adhesions by dichloroacetic acid.
We have previously demonstrated that collagen I, a major component of postoperative adhesions, is differentially regulated in fibroblasts isolated from normal human peritoneum and adhesions. Collagen I messenger RNA (mRNA) levels are significantly lower in fibroblasts from normal peritoneum compared with levels from adhesion fibroblasts. This variation is further accentuated by hypoxia. Because adhesions provide a means of supplying oxygen and nutrients to postsurgical ischemic tissue, we sought to examine the role of aerobic metabolism in the differential expression of collagen I. To examine this issue, we used a compound, dichloroacetic acid (DCA), that stimulates pyruvate dehydrogenase, causing pyruvate to be metabolized in the Kreb's cycle rather than converted into lactate, thereby switching anaerobic to aerobic metabolism. Specifically, we have exposed human fibroblast cultures from normal peritoneum and adhesions to DCA (0, 50, and 100 microg/mL) for 24 hours under normal and hypoxic (2% oxygen) conditions. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) of collagen I and beta-actin was performed by using mRNA extracted from all treatment points. Analysis of PCR-amplified products was performed by fractionation over a 2% agarose gel, followed by ethidium bromide staining of DNA bands. A scanning densimeter was used to determine the ratio of intensity of each band relative to beta-actin. Densimetric analysis of gel bands was performed by using the National Institutes of Health image analysis program. Although DCA stimulated peritoneal fibroblast collagen I mRNA expression under normoxic conditions, its expression was reduced during hypoxia. In adhesion fibroblasts, DCA treatment consistently lowered collagen I mRNA expression; this effect was manifested to a greater extent under hypoxic conditions. In summary, these findings confirm that fibroblasts from adhesions are characterized by excessive collagen I production, which is further accentuated by hypoxia. These observations are extended to show the stimulation of oxidative metabolism by DCA increases collagen I production; in contrast DCA inhibits collagen I production by normoxic adhesion fibroblasts as well as under hypoxic conditions in both types of fibroblasts. Thus, regulation of metabolic activity of peritoneal cells may provide a target for future interventions for reduction of postoperative adhesions.


Losartan's affinity to fluid bilayers modulates lipid-cholesterol interactions.
Losartan is an angiotensin II receptor antagonist mainly used for the regulation of high blood pressure. Since it was anticipated that losartan reaches the receptor site via membrane diffusion, the impact of losartan on model membranes has been investigated by small angle X-ray scattering. For this purpose 2-20 mol% losartan was incorporated into dimyristoyl-phosphatidylcholine (DMPC) and palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers and into their binary mixtures with cholesterol in the concentration range of 0 to 40 mol%. Effects of losartan on single component bilayers are alike. Partitioning of losartan into the membranes confers a negative charge to the lipid bilayers that causes the formation of unilamellar vesicles and a reduction of the bilayer thickness by 3-4%. Analysis of the structural data resulted in an estimate for the partial area of losartan, A(Los) ≈ 40 Å(2). In the presence of cholesterol, differences between the effects of losartan on POPC and DMPC are striking. Membrane condensation by cholesterol is retarded by losartan in POPC. This contrasts with DMPC, where an increase of the cholesterol content shifts the partitioning equilibrium of losartan towards the aqueous phase, such that losartan gets depleted from the bilayers from 20 mol% cholesterol onwards. This indicates (i) a chain-saturation dependent competition of losartan with lipid-cholesterol interactions, and (ii) the insolubility of losartan in the liquid ordered phase of PCs. Consequently, losartan's action is more likely to take place in fluid plasma membrane patches rather than in domains rich in cholesterol and saturated lipid species such as in membrane rafts.


A facile one-pot reduction method for the preparation of a SnO/SnO2/GNS composite for high performance lithium ion batteries.
A SnO/SnO2/GNS composite with controlled oxidation states and composition has been prepared through simple one-pot reduction of an EG suspension of SnCl2 and graphene oxide. The as-prepared composite was characterized by XRD, FT-IR, XPS, SEM, TEM and BET. SnO and SnO2 nanoparticles are uniformly distributed on the surface of the graphene. Taking advantage of the high electron conductivity of graphene and the large theoretical capacity of SnO, this SnO/SnO2/GNS composite exhibits high charge/discharge capacity, good cycling stability and good rate capability. A specific discharge capacity of approximately 464.2 mA h g(-1) is retained after being charged/discharged at a current density of 1000 mA g(-1) for 30 cycles.


Comparing Health Care Resource Use between Implant and Autologous Reconstruction of the Irradiated Breast: A National Claims-Based Assessment.
In the debate on reconstruction of the irradiated breast, there is little information on associated health care resource use. Nationwide data were used to examine health care resource use associated with implant and autologous reconstruction. It was hypothesized that failure rates would contribute the most to higher average cumulative cost with either reconstruction method. From the 2009 to 2013 MarketScan Commercial Claims and Encounters database, irradiated breast cancer patients who underwent implant or autologous reconstruction were selected. In a 24-month follow-up period, the cumulative costs of health care services used were tallied and described. Regression models stratified by reconstruction method were then used to estimate the influence of failure on cumulative cost of reconstruction. There were 2964 study patients. Most (78 percent) underwent implant reconstruction. The unadjusted mean costs for implant and autologous reconstructions were $22,868 and $30,527, respectively. Thirty-two percent of implant reconstructions failed, compared with 5 percent of autologous cases. Twelve percent of the implant reconstructions had two or more failures and required subsequent autologous reconstruction. The cost of implant reconstruction failure requiring a flap was $47,214, and the cost for autologous failures was $48,344. In aggregate, failures constituted more than 20 percent of the cumulative costs of implant reconstruction compared with less than 5 percent for autologous reconstruction. More than one in 10 patients who had implant reconstruction in the setting of radiation therapy to the breast eventually required a flap for failure. These findings make a case for autologous reconstruction being primarily considered in irradiated patients who have this option available.


Anticarbamylated protein antibodies are associated with long-term disability and increased disease activity in patients with early inflammatory arthritis: results from the Norfolk Arthritis Register.
Anticarbamylated protein (anti-CarP) antibodies are a novel family of autoantibodies recently identified in patients with inflammatory arthritis. The aim of this study was to investigate their association with long-term outcomes of disability and disease activity over 20 years' follow-up in a cohort of patients with inflammatory polyarthritis (IP). Norfolk Arthritis Register recruited adults with recent-onset swelling of ≥2 joints for ≥4 weeks from 1990 to 2009. At baseline, Health Assessment Questionnaire (HAQ) and 28 joint disease activity scores (DAS28) were obtained, and C reactive protein, rheumatoid factor (RF), anticitrullinated protein antibodies (ACPA) and anti-CarP antibodies were measured. Further HAQ scores and DAS28 were obtained at regular intervals over 20 years. Generalised estimating equations were used to test the association between anti-CarP antibody status and longitudinal HAQ and DAS28 scores; adjusting for age, gender, smoking status, year of inclusion and ACPA status. Analyses were repeated in subgroups stratified by ACPA status. The relative association of RF, ACPA and anti-CarP antibodies with HAQ and DAS28 scores was investigated using a random effects model. 1995 patients were included; 1310 (66%) were female. Anti-CarP antibodies were significantly associated with more disability and higher disease activity, HAQ multivariate β-coefficient (95% CI) 0.12 (0.02 to 0.21), and these associations remained significant in the ACPA-negative subgroups. The associations of RF, ACPA and anti-CarP antibodies were found to be additive in the random effects model. Anti-CarP antibodies are associated with increased disability and higher disease activity in patients with IP. Our results suggest that measurement of anti-CarP antibodies may be useful in identifying ACPA-negative patients with worse long-term outcomes. Further, anti-CarP antibody status provided additional information about RF and ACPA.


Association Between Frailty and Time Alive and At Home After Cancer Surgery Among Older Adults: A Population-Based Analysis.
Although frailty is known to impact short-term postoperative outcomes, its long-term impact is unknown. This study examined the association between frailty and remaining alive and at home after cancer surgery among older adults. Adults aged ≥70 years undergoing cancer resection were included in this population-based retrospective cohort study using linked administrative datasets in Ontario, Canada. The probability of remaining alive and at home in the 5 years after cancer resection was evaluated using Kaplan-Meier methods. Extended Cox regression with time-varying effects examined the association between frailty and remaining alive and at home. Of 82,037 patients, 6,443 (7.9%) had preoperative frailty. With median follow-up of 47 months (interquartile range, 23-81 months), patients with frailty had a significantly lower probability of remaining alive and at home 5 years after cancer surgery compared with those without frailty (39.1% [95% CI, 37.8%-40.4%] vs 62.5% [95% CI, 62.1%-63.9%]). After adjusting for age, sex, rural living, material deprivation, immigration status, cancer type, surgical procedure intensity, year of surgery, and receipt of perioperative therapy, frailty remained associated with increased hazards of not remaining alive and at home. This increase was highest 31 to 90 days after surgery (hazard ratio [HR], 2.00 [95% CI, 1.78-2.24]) and remained significantly elevated beyond 1 year after surgery (HR, 1.56 [95% CI, 1.48-1.64]). This pattern was observed across cancer sites, including those requiring low-intensity surgery (breast and melanoma). Preoperative frailty was independently associated with a decreased probability of remaining alive and at home after cancer surgery among older adults. This relationship persisted over time for all cancer types beyond short-term mortality and the initial postoperative period. Frailty assessment may be useful for all candidates for cancer surgery, and these data can be used when counseling, selecting, and preparing patients for surgery.


miR-9 controls the timing of neurogenesis through the direct inhibition of antagonistic factors.
The timing of commitment and cell-cycle exit within progenitor populations during neurogenesis is a fundamental decision that impacts both the number and identity of neurons produced during development. We show here that microRNA-9 plays a key role in this process through the direct inhibition of targets with antagonistic functions. Across the ventricular zone of the developing zebrafish hindbrain, miR-9 expression occurs at a range of commitment stages. Abrogating miR-9 function transiently delays cell-cycle exit, leading to the increased generation of late-born neuronal populations. Target protection analyses in vivo identify the progenitor-promoting genes her6 and zic5 and the cell-cycle exit-promoting gene elavl3/HuC as sequential targets of miR-9 as neurogenesis proceeds. We propose that miR-9 activity generates an ambivalent progenitor state poised to respond to both progenitor maintenance and commitment cues, which may be necessary to adjust neuronal production to local extrinsic signals during late embryogenesis.


[Drug prescription patterns in patients attended at a middle- and high-complexity level institution].
Determining prescription patterns for outpatient medication authorised for patients affiliated to an EPS and assisted by medium- and high-level complexity IPS. This was a cross-sectional study where medication prescription was evaluated in 331 second- and third-level complexity hospitals from 27 Colombian departments during 2006. 38 863 prescriptions for 3 663 patients' medication were analysed. 61,4 % came from third-level complexity hospitals, mainly for patients affiliated to contribution-based regimes. Average prescribed medication per person was 2,2 (2,1-2,2 95 % CI), the percentage of antibiotics formulated by prescription was 29,2 % (28,7-29,6 95 % CI), essential prescribed medicines accounted for 64,2 % (63,7-64,6 95 % CI) and injectable medicines was 22,1 % (21,7-25,5 %). More than half the medications (62,1 %; 61,5-62,7 95 % CI) were in ATC groups such as anti-infectious agents, immunomodulating agents and medications for the alimentary and metabolic tract. DU90% consisted of 64 medications, medication consumption being 8,39 daily de-fined doses (DDD)/1 000 patients, costing 5 216 Colombian pesos per DDD. This analysis led to identifying medications whose formulation frequency did not correlate with an epidemiologic profile as immunomudulator and growth-hormone (somatotropin) agents. There were differences in the frequency and quantity of DDD medications authorised by type of affiliation which could thus be providing evidence of obstacles to the population having access to drugs/medicaments.


Effects of Ginkgo biloba on exhaled nasal nitric oxide during normobaric hypoxia in humans.
Ginkgo biloba, an extract of the ginkgo tree, may prevent or lessen symptoms of acute mountain sickness in humans. The mechanism of this effect is poorly understood. One hypothesis is that ginkgo alters nitric oxide (NO) metabolism, possibly by scavenging NO or altering nitric oxide synthase expression and thereby lessening the vasodilatory effects of NO. To date, an effect of Ginkgo biloba on NO metabolism has not been demonstrated in humans. We measured exhaled nasal NO output in humans (n = 9) during normoxia and then during acute normobaric hypoxia (goal oxyhemoglobin saturation 75% to 85%) before and after administration of a standardized extract of Ginkgo biloba (120 mg twice daily for 5 days). Oxygen saturation, heart rate, and minute ventilation were similar before and after Ginkgo biloba administration. Exhaled nasal NO output was increased during normoxia following ginkgo (p < 0.02) and reduced during normobaric hypoxia both before (p < 0.02) and following (p < 0.003) ginkgo. Exhaled nasal NO output during normobaric hypoxia was lowest following ginkgo (p < 0.003). We conclude that Ginkgo biloba increases exhaled nasal NO output during normoxia and enhances reduced exhaled nasal NO output during normobaric hypoxia. Our results suggest that Ginkgo biloba may act to reduce AMS through an effect on NO metabolism.


Mechanisms of assembly and genome packaging in an RNA virus revealed by high-resolution cryo-EM.
Cowpea mosaic virus is a plant-infecting member of the Picornavirales and is of major interest in the development of biotechnology applications. Despite the availability of >100 crystal structures of Picornavirales capsids, relatively little is known about the mechanisms of capsid assembly and genome encapsidation. Here we have determined cryo-electron microscopy reconstructions for the wild-type virus and an empty virus-like particle, to 3.4 Å and 3.0 Å resolution, respectively, and built de novo atomic models of their capsids. These new structures reveal the C-terminal region of the small coat protein subunit, which is essential for virus assembly and which was missing from previously determined crystal structures, as well as residues that bind to the viral genome. These observations allow us to develop a new model for genome encapsidation and capsid assembly.


Teicoplanin-loaded borate bioactive glass implants for treating chronic bone infection in a rabbit tibia osteomyelitis model.
The treatment of chronic osteomyelitis (bone infection) remains a clinical challenge. In this work, pellets composed of a chitosan-bonded mixture of borate bioactive glass particles (<50microm) and teicoplanin powder (antibiotic), were evaluated in vitro and in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model. When immersed in phosphate-buffered saline, the pellets showed sustained release of teicoplanin over 20-30 days, while the bioactive glass converted to hydroxyapatite (HA) within 7 days, eventually forming a porous HA structure. Implantation of the teicoplanin-loaded pellets in a rabbit tibia osteomyelitis model resulted in the detection of teicoplanin in the blood for about 9 days. The implants converted to a bone-like HA graft, and supported the ingrowth of new bone into the tibia defects within 12 weeks of implantation. Microbiological, histological and scanning electron microscopy techniques showed that the implants provided a cure for the bone infection. The results indicate that the teicoplanin-loaded borate bioactive glass implant, combining sustained drug release with the ability to support new bone ingrowth, could provide a method for treating chronic osteomyelitis.


Nitrogen losses in anoxic marine sediments driven by Thioploca-anammox bacterial consortia.
Ninety per cent of marine organic matter burial occurs in continental margin sediments, where a substantial fraction of organic carbon escapes oxidation and enters long-term geologic storage within sedimentary rocks. In such environments, microbial metabolism is limited by the diffusive supply of electron acceptors. One strategy to optimize energy yields in a resource-limited habitat is symbiotic metabolite exchange among microbial associations. Thermodynamic and geochemical considerations indicate that microbial co-metabolisms are likely to play a critical part in sedimentary organic carbon cycling. Yet only one association, between methanotrophic archaea and sulphate-reducing bacteria, has been demonstrated in marine sediments in situ, and little is known of the role of microbial symbiotic interactions in other sedimentary biogeochemical cycles. Here we report in situ molecular and incubation-based evidence for a novel symbiotic consortium between two chemolithotrophic bacteria--anaerobic ammonium-oxidizing (anammox) bacteria and the nitrate-sequestering sulphur-oxidizing Thioploca species--in anoxic sediments of the Soledad basin at the Mexican Pacific margin. A mass balance of benthic solute fluxes and the corresponding nitrogen isotope composition of nitrate and ammonium fluxes indicate that anammox bacteria rely on Thioploca species for the supply of metabolic substrates and account for about 57 ± 21 per cent of the total benthic N2 production. We show that Thioploca-anammox symbiosis intensifies benthic fixed nitrogen losses in anoxic sediments, bypassing diffusion-imposed limitations by efficiently coupling the carbon, nitrogen and sulphur cycles.


Resveratrol suppresses interleukin-1beta-induced inflammatory signaling and apoptosis in human articular chondrocytes: potential for use as a novel nutraceutical for the treatment of osteoarthritis.
Osteoarthritis is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective on pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Resveratrol is a phytoalexin stilbene produced naturally by plants including red grapes, peanuts and various berries. Recent research in various cell models has demonstrated that resveratrol is safe and has potent anti-inflammatory properties. However, its potential for treating arthritic conditions has not been explored. In this study we provide experimental evidence that resveratrol inhibits the expression of VEGF, MMP-3, MMP-9 and COX-2 in human articular chondrocytes stimulated with the pro-inflammatory cytokine IL-1beta. Since these gene products are regulated by the transcription factor NF-kappaB, we investigated the effects of resveratrol on IL-1beta-induced NF-kappaB signaling pathway. Resveratrol, like N-Ac-Leu-Leu-norleucinal (ALLN) suppressed IL-1beta-induced proteasome function and the degradation of IkappaBalpha (an inhibitor of NF-kappaB) without affecting IkappaBalpha kinase activation, IkappaBalpha-phosphorylation or IkappaBalpha-ubiquitination which suppressed nuclear translocation of the p65 subunit of NF-kappaB and its phosphorylation. Furthermore, we observed that resveratrol as well as ALLN inhibited IL-1beta-induced apoptosis, caspase-3 activation and PARP cleavage in human articular chondrocytes. In summary, our results suggest that resveratrol suppresses apoptosis and inflammatory signaling through its actions on the NF-kappaB pathway in human chondrocytes. We propose that resveratrol should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.


Mutations in the GM1 binding site of simian virus 40 VP1 alter receptor usage and cell tropism.
Polyomaviruses are nonenveloped viruses with capsids composed primarily of 72 pentamers of the viral VP1 protein, which forms the outer shell of the capsid and binds to cell surface oligosaccharide receptors. Highly conserved VP1 proteins from closely related polyomaviruses recognize different oligosaccharides. To determine whether amino acid changes restricted to the oligosaccharide binding site are sufficient to determine receptor specificity and how changes in receptor usage affect tropism, we studied the primate polyomavirus simian virus 40 (SV40), which uses the ganglioside GM1 as a receptor that mediates cell binding and entry. Here, we used two sequential genetic screens to isolate and characterize viable SV40 mutants with mutations in the VP1 GM1 binding site. Two of these mutants were completely resistant to GM1 neutralization, were no longer stimulated by incorporation of GM1 into cell membranes, and were unable to bind to GM1 on the cell surface. In addition, these mutant viruses displayed an infection defect in monkey cells with high levels of cell surface GM1. Interestingly, one mutant infected cells with low cell surface GM1 more efficiently than wild-type virus, apparently by utilizing a different ganglioside receptor. Our results indicate that a small number of mutations in the GM1 binding site are sufficient to alter ganglioside usage and change tropism, and they suggest that VP1 divergence is driven primarily by a requirement to accommodate specific receptors. In addition, our results suggest that GM1 binding is required for vacuole formation in permissive monkey CV-1 cells. Further study of these mutants will provide new insight into polyomavirus entry, pathogenesis, and evolution.


Nonaccidental pediatric head injury: diffusion-weighted imaging findings.
Diffusion-weighted imaging (DWI) reveals nonhemorrhagic posttraumatic infarction hours to days before conventional computed tomographic scanning or magnetic resonance imaging (MRI). We evaluated the diagnostic utility of DWI in children with nonaccidental head trauma. The medical records and imaging examinations obtained between January 1998 and May 2000 for all children less than 2 years of age with presumed or suspected nonaccidental head injury were reviewed retrospectively. Twenty children who had undergone DWI within 5 days of presentation were included in the study. Computed tomographic scans, conventional MRI sequences, and DWI combined with apparent diffusion coefficient (ADC) maps were evaluated. Eleven girls and nine boys (median age, 5 mo) were studied. Eighteen children had presumed nonaccidental head trauma, and two children had suspected nonaccidental head trauma. Of the 18 children with presumed nonaccidental trauma, 16 (89%) demonstrated abnormalities on DWI/ADC, as compared with neither of the two children with suspected nonaccidental trauma. In 13 (81%) of 16 positive cases, DWI revealed more extensive brain injury than was demonstrated on conventional MRI sequences or showed injuries not observed on conventional MRI. DWI combined with ADC maps allowed better delineation of the extent of white matter injury. DWI/ADC abnormalities in the nonaccidental head-injured children were likely to involve posterior aspects of the cerebral hemispheres, with relative sparing of the frontal and temporal poles. Severity on DWI correlated significantly with poor outcome (P < 0.005). DWI has broad applications in the early detection of infarction in children with nonaccidental head injury and enhances the sensitivity of conventional MRI. In the patients in this study, early DWI provided an indicator of severity that was more complete than any other imaging modality. The use of DWI may help to identify children at high risk for poor outcome and to guide management decisions.


Mass spectrometry of intact ribosomes.
The ability to maintain intact ribosomes in the mass spectrometer has enabled research into their changes in conformation and interactions. In the mass spectrometer, it is possible to induce dissociation of proteins from the intact ribosome and, in conjunction with atomic structures, to understand the factors governing their release. We have applied this knowledge to interpret the structural basis for release of proteins from ribosomes for which no high resolution structures are available, such as complexes with elongation factor G and ribosomes from yeast. We also describe how improvements in technology and understanding have widened the scope of our research and lead to dramatic improvements in quality and information available from spectra of intact ribosomes.


Fibrin matrices with affinity-based delivery systems and neurotrophic factors promote functional nerve regeneration.
Glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity-based delivery of GDNF or NGF from fibrin-filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity-based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury.


Measurement-based control of a mechanical oscillator at its thermal decoherence rate.
In real-time quantum feedback protocols, the record of a continuous measurement is used to stabilize a desired quantum state. Recent years have seen successful applications of these protocols in a variety of well-isolated micro-systems, including microwave photons and superconducting qubits. However, stabilizing the quantum state of a tangibly massive object, such as a mechanical oscillator, remains very challenging: the main obstacle is environmental decoherence, which places stringent requirements on the timescale in which the state must be measured. Here we describe a position sensor that is capable of resolving the zero-point motion of a solid-state, 4.3-megahertz nanomechanical oscillator in the timescale of its thermal decoherence, a basic requirement for real-time (Markovian) quantum feedback control tasks, such as ground-state preparation. The sensor is based on evanescent optomechanical coupling to a high-Q microcavity, and achieves an imprecision four orders of magnitude below that at the standard quantum limit for a weak continuous position measurement--a 100-fold improvement over previous reports--while maintaining an imprecision-back-action product that is within a factor of five of the Heisenberg uncertainty limit. As a demonstration of its utility, we use the measurement as an error signal with which to feedback cool the oscillator. Using radiation pressure as an actuator, the oscillator is cold damped with high efficiency: from a cryogenic-bath temperature of 4.4 kelvin to an effective value of 1.1 ± 0.1 millikelvin, corresponding to a mean phonon number of 5.3 ± 0.6 (that is, a ground-state probability of 16 per cent). Our results set a new benchmark for the performance of a linear position sensor, and signal the emergence of mechanical oscillators as practical subjects for measurement-based quantum control.


Diagnostic tools for animal diseases.
While traditional testing methods are still widely used in veterinary diagnostic laboratories exciting new technologies, such as biosensors and microarray techniques, are being developed. Nucleic acid diagnostic techniques such as polymerase chain reaction (PCR) have become routine diagnostic tools in veterinary laboratories not only to make specific typing determinations but also to rapidly screen large numbers of samples during disease outbreaks. In addition, nanotechnologies, although not yet implemented in veterinary laboratories, hold the promise of screening for numerous pathogens in a single assay. Other biotechnologies are likely to be widely used in the future as they can improve diagnostic capabilities while reducing the time and perhaps, the costs, associated with conventional technologies. This paper describes some of these new technologies and concludes that although a lot of developmental work is still required, biotechnology and its applications hold great promise for improving the speed and accuracy of diagnostics for veterinary pathogens.


No association between variation of the FOXP3 gene and common type 1 diabetes in the Sardinian population.
Mutations of the forkhead/winged helix transcription factor FOXP3 gene on chromosome Xp11.23 cause a rare recessive monogenic disorder called IPEX (immune dysregulation, polyendocrinopathy, including type 1 diabetes, enteropathy, and X-linked syndrome). FOXP3 is necessary for the differentiation of a key immune suppressive subset of T-cells, the CD4+CD25+ regulatory T-cells. Previously, we reported a significant male-female bias in the common, multifactorial form of type 1 diabetes in Sardinia and evidence of linkage of chromosome Xp11 to the disease. These findings indicate that FOXP3 is a prime functional and positional candidate locus for the common form of type 1 diabetes. In the present study, we initially scanned 82 kb of the FOXP3 region for common polymorphisms, including sequencing all of the coding and functionally relevant portions of the gene in 64 Sardinian individuals. Then the most informative polymorphisms in 418 type 1 diabetic families and in 268 male case and 326 male control subjects were sequentially genotyped and tested for disease association. There is no evidence that variants in the FOXP3 regions analyzed are associated with type 1 diabetes and account for the male-female bias observed in Sardinia. Our data indicate that allelic variation in or near the coding regions of the FOXP3 gene does not have a major role in the inherited susceptibility to the common form of type 1 diabetes.


Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model.
Much evidence indicates that women have a higher risk of developing Alzheimer's disease (AD) than do men. The reason for this gender difference is unclear. We hypothesize that estrogen deficiency in the brains of women with AD may be a key risk factor. In rapidly acquired postmortem brains from women with AD, we found greatly reduced estrogen levels compared with those from age- and gender-matched normal control subjects; AD and control subjects had comparably low levels of serum estrogen. We examined the onset and severity of AD pathology associated with estrogen depletion by using a gene-based approach, by crossing the estrogen-synthesizing enzyme aromatase gene knockout mice with APP23 transgenic mice, a mouse model of AD, to produce estrogen-deficient APP23 mice. Compared with APP23 transgenic control mice, estrogen-deficient APP23 mice exhibited greatly reduced brain estrogen and early-onset and increased beta amyloid peptide (Abeta) deposition. These mice also exhibited increased Abeta production, and microglia cultures prepared from the brains of these mice were impaired in Abeta clearance/degradation. In contrast, ovariectomized APP23 mice exhibited plaque pathology similar to that observed in the APP23 transgenic control mice. Our results indicate that estrogen depletion in the brain may be a significant risk factor for developing AD neuropathology.


Perception of sound and gravity by TMC1 and TMC2.
Central to our ability to hear and sense gravity is a cellular process known as mechanotransduction, which is initiated by the opening of mechanosensitive cation channels located near the tips of the stereocilia of auditory and vestibular inner ear hair cells. The molecular identity of the mechanotransduction channels has eluded researchers despite intensive investigations over the years. In this issue of the JCI, Kawashima et al. report their results obtained using mice with targeted deletion of both transmembrane channel-like 1 (Tmc1) and Tmc2. The use of inner ear hair cells isolated from these mice provided a nearly perfect system for testing the mechanotransduction channels without disrupting functions of other accessory proteins needed in the complicated molecular apparatus, and it allowed the authors to show that the proteins encoded by these genes are integral components of the mechanotransduction complex.


TOAST Subtypes of Ischemic Stroke and Its Risk Factors: A Hospital-Based Study at Cipto Mangunkusumo Hospital, Indonesia.
Stroke is a leading cause of death and disability, with ischemic stroke as the highest prevalent cases in Indonesia. Ischemic stroke can be classified further into five subtypes according to TOAST classification. Numerous studies have revealed that stroke risk factor has variable correlation with different stroke subtype. Currently, there is no data regarding this phenomenon in Indonesia. The aim of study is to identify characteristic of ischemic stroke subtypes and the risk factors in TOAST classification. A retrospective, cross-sectional study of patients diagnosed with ischemic stroke at Cipto Mangunkusumo Hospital from January till December 2016. Demographic data, ischemic stroke subtypes, risk factors, and other relevant data were documented. Bivariate and multivariate analysis was done using SPSS 23. 235 recorded data patients were included. Large artery atherosclerosis (LAA) was the most prevalent stroke subtypes at 59,6%, followed with small vessel disease (SVD) at 26,7%, undetermined etiology at 9,8%, cardioembolism (CE) at 2,1%, and other determined etiology at 0,9%. Hypertension was the most common vascular risk factor. However, it was only significant in SVD (p=0,023) and undetermined etiology subtypes (p<0,001). Significant risk factor in LAA was diabetes (55%; p=0,016) while in CE subtypes was atrial fibrillation (60%;p<0,001). In multivariate analyses, hypertension (OR 3; 95% CI 1,12-8,05) was the only variable that was related to SVD while in CE it was atrial fibrillation (OR 113,5; 95% CI 13,6-946,5). LAA was the most common stroke ischemic subtypes. Associated risk factor in LAA was diabetes while in SVD and undetermined etiology subtypes it was hypertension. Atrial fibrillation was associated with cardioembolism.


A sexually dimorphic nucleus in the quail preoptic area.
The cytoarchitectural analysis of the preoptic-anterior hypothalamic region of the Japanese quail reveals a sexual dimorphism in the total volume of the medial preoptic nucleus (significantly larger in males than in females). Different nuclei of the region (dorsal preopticus, suprachiasmaticus) do not show any statistically significant difference. The sex-related difference is more consistent comparing the distribution of dark volume. This last is due to a larger number of cells containing high amount of Nissl's substance in male than in female. Present findings represent the first example of sexual dimorphism in the avian hypothalamus.


Down-regulation of mannose receptors on macrophages after infection with Leishmania donovani.
Macrophages express a mannose-specific endocytosis receptor that binds and internalizes mannose-terminated glycoproteins. Infection of mouse peritoneal macrophages with Leishmania donovani resulted in a decrease in mannose-receptor activity. With 125I-labelled beta-glucuronidase as ligand, a 2-fold decrease in uptake rate was observed in infected cells, with no change in Kuptake. Cell-surface binding of 125I-mannose-BSA was diminished 2.5-fold after infection. The decrease in ligand binding appeared to be due to a decrease in the number of sites, with no change in affinity. Elimination of parasites from infected cells by treatment with neoglycoprotein-conjugated methotrexate resulted in an increase in receptor number. Cycloheximide suppressed the drug-treatment-mediated rise in receptor number in infected macrophages. A decrease in receptor activity was also observed in liver Kupffer cells isolated from parasite-infected mice. Binding of ligand by another carbohydrate receptor, the mannose 6-phosphate receptor, was not altered by infection. Phagocytosis of yeast cells was also not altered. These results suggest that mannose receptor synthesis in macrophages is specifically suppressed after infection with Leishmania parasites.


Effects of the COVID-19 Pandemic on Outpatient Providers in the United States.
During the COVID-19 pandemic, there is concern that social distancing, fear of contagion, quarantining of providers, cancellation of elective procedures, media coverage about the pandemic, and other factors drastically reduced physician visits, putting severe financial strain on outpatient providers, and having unknown ramifications for health outcomes. We estimate the effect of the pandemic on utilization of outpatient services. Using 2010-2020 data from a national dataset, the Outpatient Influenza-like Illness Surveillance Network, we estimate the difference in outpatient care utilization during the time period of the COVID-19 pandemic versus the same weeks in prior years. Our findings indicate that the pandemic started to reduce visits during the week of March 15-21. The effect on visits grew until reaching a peak during the week of April 5-11, 2020, when the pandemic reduced the total number of outpatient visits per provider by 70% relative to the same week in prior years. We find negative effects of the pandemic on visits for non-flu symptoms as well as on visits for flu symptoms, but the magnitudes of these latter effects tended to be larger in magnitude. The pandemic's impact declined over time, and by the week of June 28 to July 4, 2020, there was no longer any difference in total visits per provider relative to the same week in prior years. Despite the resurgence of COVID-19 in June and July, we still find no effects on total visits when our data end in July 26 to August 1, 2020. Our findings show that one by-product of the COVID-19 pandemic in the United States is a large decline in the use of outpatient care which peaked around the week of April 5-11. Total outpatient visits rebounded completely and remain stable as of July 26 to August 1, 2020.


Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.
beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.


Cancer-related changes and low-to-moderate exposure to welding fumes: A longitudinal study.
This study tested for an association between early cancer-related biomarkers and low-to-moderate exposure to fumes from welding mild steel. Male, non-smoking participants from southern Sweden were recruited and examined (N=338, 171 welders and 167 controls); of these, 78 welders and 96 controls were examined on two occasions six years apart. Exposure to welding fumes was evaluated by measuring respirable dust, welding years, and cumulative exposure. DNA methylation of CpG sites within the cancer-related genes AHRR, F2RL3, and B3GNTL1 was measured by pyrosequencing and relative mitochondrial DNA copy number and telomere length were measured by qPCR in whole-blood samples. Multivariate models were used for longitudinal analysis. Median exposure to respirable dust was 0.7 mg/m3 at both timepoints, adjusted for use of personal protective equipment. Compared with controls, welders showed a significant decrease over time in DNA methylation of B3GNTL1 CpG1 and CpG4 [adjusted for age, body mass index, and smoking: β=-0.66, standard error (SE)=0.28; β=-0.48, SE=0.24, respectively]. In addition, exposure to respirable dust and cumulative exposure was associated with a decrease in methylation of F2RL3 CpG2 among all welders (adjusted β=-0.67, SE=0.23 and β=-0.03, SE=0.02, respectively). No significant associations were found for AHRR, mitochondrial DNA copy number, or telomere length. Low-to-moderate exposure to welding fumes was associated with a small effect on selected early epigenetic biomarkers of cancer. The direction of the methylation pattern (lower methylation of specific CpG sites) indicates early lung cancer-related changes associated with mild steel welding.


Assessment of two hemispherical and hemispherical-conical miniature sources used in electronic brachytherapy using Monte Carlo Simulation.
Since the heart of the electronic brachytherapy system is a tube of a miniature x-ray and due to the increasing use of electronic brachytherapy, there is an urgent need for acquiring knowledge about the X-ray spectrum produced, and distribution of x-ray dose. This study aimed to assess the optimal target thickness (TT), the X-ray source spectrum, and the absorbed dose of two miniature sources of hemispherical and hemispherical-conical used in electronic brachytherapy systems, through a Monte Carlo simulation. Considering the advantages of MCNPX Code (2.6.0), two input files corresponding to the characteristics of the investigated miniature sources were prepared for this code and then were used for simulation. The optimal thickness (OT) of gold and tungsten targets was determined for the energy levels of 40, 45, and 50 kilo-electron-volts. In this study, the values of the size of the optimal thickness of 0.92, 1.01 and 1.06 μ for gold target and values of 0.99, 1.08 and 1.34 μ for tungsten target were obtained for energies 40, 45 and 50 keV that using these values, the optimum thickness of 0.92, X-ray spectrum within and outside targets, axial and radial doses for the used energy were calculated for two miniature sources. It was found that the energy of incident electron, target shape, cross-sectional area of the produced bremsstrahlung, atomic number of materials constituting of the target and output window are the factors with the greatest impacts on the produced X-ray spectrum and the absorbed dose.


Spatial frequency masking in human vision: binocular interactions.
Binocular contrast interactions in human vision were studied psychophysically. Thresholds were obtained for sinewave grating stimulation of the right eye in the presence of simultaneous masking gratings presented to the right eye (monocular masking) or left eye (dichoptic masking). In the first experiment, thresholds were measured at 0.25, 1.0, 4.0, and 16.0 cycle per degree (cpd) as a function of the contrast of masking gratings of identical frequency and phase. Thresholds rose nonmonotonically with masking contrast. At medium and high contrast levels, dichoptic masking was more effective in elevating contrast thresholds than monocular masking, and approached Weber's Law behavior. In the second experiment, spatial frequency tuning functions were obtained for test gratings at five spatial frequencies, by measuring threshold elevation as a function of the spatial frequency of constant-contrast masking gratings. At 1.0, 4.0, and 16.0 cpd, the tuning functions peaked at the test frequencies. The dichoptic tuning functions had a bandwidth of about 1 octave between half-maximum points, narrower than +/- 1 octave bandwidths of the monocular tuning functions. At 0.125 and 0.25 cpd, the tuning functions were broader and exhibited a shift in peak masking to frequencies above the test frequencies.


LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively downregulate PMN azurophilic degranulation.
The eicosanoid lipoxin A4 (LXA4) is biosynthesized in vivo by cells present at inflammatory sites and appears to be an endogenous anti-inflammatory mediator. Further, in the presence of aspirin, the 15-epimer of LXA4 (15-epi-LXA4) is biosynthesized and may mediate some of aspirin's desirable bioactions. LXA4, 15-epi-LXA4, and their stable analogs inhibit inflammation in established animal models, indicating that these compounds may be useful for treating inflammatory disease states. To investigate the cellular mechanisms by which these lipid mediators downregulate inflammation, we investigated whether these eicosanoids could influence receptor-mediated degranulation of human neutrophils, an event thought to play a major causative role in several inflammatory disease states. LXA4, 15-epi-LXA4, and their stable analogs potently (IC50 < 1 nM) and selectively downregulated neutrophil release of azurophilic granule contents but did not affect other neutrophil secretory functions. Thus the cellular basis of action of these natural off-switches to inflammation appears to involve downregulation of neutrophil azurophilic granule release.


Quantifying climate change effects on future forest biomass availability using yield tables improved by mechanistic scaling.
Forests and wood products play a major role in climate change mitigation strategies and the transition from a fossil-based economy to a circular bioeconomy. Accurate estimates of future forest productivity are crucial to predict the carbon sequestration and wood provision potential of forests. Since long, forest managers have used empirical yield tables as a cost-effective and reliable way to predict forest growth. However, recent climate change-induced growth shifts raised doubts about the long-term validity of these yield tables. In this study, we propose a methodology to improve available yield tables of 11 tree species in the Netherlands and Flanders, Belgium. The methodology uses scaling functions derived from climate-sensitive process-based modelling (PBM) that reflect state-of-the-art projections of future growth trends. Combining PBM and stand information from the empirical yield tables for the region of Flanders, we found that for the period 1987-2016 stand productivity has on average increased by 13% compared to 1961-1990. Furthermore, simulations indicate that this positive growth trend is most likely to persist in the coming decades, for all considered species, climate or site conditions. Nonetheless, results showed that local site variability is equally important to consider as the in- or exclusion of the CO2 fertilization effect or different climate projections, when assessing the magnitude of forests' response to climate change. Our projections suggest that incorporating these climate change-related productivity changes lead to a 7% increase in standing stock and a 22% increase in sustainably potentially harvestable woody biomass by 2050. The proposed methodology and resulting estimates of climate-sensitive projections of future woody biomass stocks will facilitate the further incorporation of forests and their products in global and regional strategies for the transition to a climate-smart circular bioeconomy.


Prevalence and Drivers of COVID-19 Vaccine Hesitancy among Czech University Students: National Cross-Sectional Study.
university students are believed to retain the highest levels of health literacy. They are perceived as the opinion leaders within their communities; therefore, their health-related beliefs and attitudes are deemed important for public health campaigns. This study aimed to investigate the COVID-19 vaccine hesitancy drivers among university students in the Czech Republic. a cross-sectional study using a self-administered questionnaire was carried out in the weeks before the unrestricted vaccine deployment to Czech adults. The questionnaire had 21 multiple-choice items stratified in 4 categories; demographic characteristics, COVID-19-related anamnesis and influenza vaccine experience, attitudes towards COVID-19 vaccination, and the possible drivers of COVID-19 vaccine hesitancy suggested by the WHO-SAGE. out of the 1351 included students, 66.8% were females, 84.5% were Czech nationals, and 40.6% enrolled in healthcare programs. The overall COVID-19 vaccine acceptance level was 73.3%, 19.3% of participants were vaccine-resistant, and only 7.4% were vaccine-hesitant. Trust in the pharmaceutical industry, trust in healthcare providers, and perceived knowledge sufficiency predicted higher odds of vaccine acceptance. In contrast, media and social media, personal beliefs, immunity misconception, previous COVID-19 infection, and suspicions about novel vaccines and the local availability predicted higher odds of vaccine hesitancy. The findings of this study predict a fair probability to achieve community immunity (herd immunity) among the target population group. The primary prevention strategies in the Czech Republic need to be culturally sensitive and inclusive for foreign nationals. As one-quarter of the participating students are dependent on vaccine safety data, this study findings support the call for independent studies evaluating the side effects of COVID-19 vaccines.


Melanin-concentrating hormone receptor 1 polymorphisms are associated with components of energy balance in the Complex Diseases in the Newfoundland Population: Environment and Genetics (CODING) study.
The melanin-concentrating hormone receptor 1 (MCHR1) is a G protein-coupled receptor that regulates energy balance and body composition in animal models. Inconsistent effects of MCHR1 polymorphisms on energy homeostasis in humans may partly be attributable to environmental factors. We examined the effect of 4 single nucleotide polymorphisms (rs133073, rs133074, rs9611386, and rs882111) in the MCHR1 gene on body composition as well as energy-related lifestyle factors (diet and physical activity). We also examined the effect of gene-lifestyle interactions on body composition. A total of 1153 participants (248 men and 905 women) from the cross-sectional Complex Diseases in the Newfoundland Population: Environment and Genetics (CODING) study were genotyped by using probe-based chemistry validated assays. Diet and physical activity were estimated by using validated frequency questionnaires, and body composition was assessed by using dual-energy X-ray absorptiometry. Three polymorphisms (rs9611386, rs882111, and rs133073) were associated with differences in body-composition measurements (all P < 0.05). There was an interaction between rs9611386 and carbohydrate intake on total mass and waist circumference (both P ≤ 0.01). There was also an interaction between rs9611386 and body mass index categories (normal weight, overweight, and obese) on energy intakes (P = 0.02). A similar interaction was shown with rs882111 (P = 0.02). Interactions were also observed between each of these polymorphisms (rs9611386, rs882111, and rs133073) and physical activity score on body-composition measurements (all P < 0.05). These findings suggest that polymorphisms in the MCHR1 gene are associated with differences in body composition and interact with physiologic and energy-related lifestyle factors.


Pulmonary hypertension: diagnosis and management.
Pulmonary arterial hypertension is a progressive, symptomatic, and ultimately fatal disorder for which substantial advances in treatment have been made during the past decade. Effective management requires timely recognition and accurate diagnosis of the disorder and appropriate selection among therapeutic alternatives. Despite progress in treatment, obstacles remain that impede the achievement of optimal outcomes. The current article provides an overview of the pathobiologic mechanisms of pulmonary arterial hypertension, including genetic substrates and molecular and cellular mechanisms, and describes the clinical manifestations and classification of pulmonary arterial hypertension. The article also reviews established approaches to evaluation and treatment, with emphasis on the appropriate application of calcium channel blockers, prostacyclin analogues, endothelin receptor antagonists, and phosphodiesterase 5 inhibitors. In addition, the authors discuss unresolved issues that may complicate patient management, such as the clinical importance of mild or exercise-related pulmonary arterial hypertension, and they identify avenues by which treatment may advance in the future through the use of combination treatment, outcomes assessment, and exploration of alternative pharmacologic strategies.


Health insurance and stage at diagnosis of laryngeal cancer: does insurance type predict stage at diagnosis?
To examine whether patients with no insurance or Medicaid are more likely to present with advanced-stage laryngeal cancer. Retrospective cohort study from the National Cancer Database, 1996-2003. Hospital-based practice. Patients with known insurance status diagnosed as having invasive laryngeal cancer at Commission on Cancer facilities (N = 61 131) were included. Adjusted and unadjusted logistic regression models analyzed the likelihood of presenting at a more advanced stage. Overall stage of laryngeal cancer (early vs advanced) and tumor size (T stage) at diagnosis. Patients with advanced-stage laryngeal cancer at diagnosis were more likely to be uninsured (odds ratio [OR], 1.97; 95% confidence interval [CI], 1.79-2.15) or covered by Medicaid (OR, 2.40; 95% CI, 2.21-2.61) compared with those with private insurance. Similarly, patients were most likely to present with the largest tumors (T4 disease) if they were uninsured (OR, 2.92; 95% CI, 2.60-3.28) or covered by Medicaid (OR, 3.97; 95% CI, 3.56-4.34). Patients who were black, between ages 18 and 56 years, and who resided in zip codes with low proportions of high school graduates or low median household incomes were also more likely to be diagnosed as having advanced disease and/or larger tumors. Individuals lacking insurance or having Medicaid are at greatest risk for presenting with advanced laryngeal cancer. Results for the Medicaid group may be influenced by the postdiagnosis enrollment of uninsured patients. It is important to consider the impact of insurance coverage on stage at diagnosis and associated morbidity, mortality, quality of life, and costs.


The effects of the duration of mobile phone use on heart rate variability parameters in healthy subjects.
This study aimed to estimate the influence of the duration of mobile phone use on heart rate variability (HRV) in healthy individuals. One hundred forty-eight individuals without any established systemic disease and who had undergone 24-h ambulatory ECG monitoring were included in the case-control study. All the individuals had been using mobile phones for more than 10 years. Three-channel 24-h Holter monitoring was performed to derive the mean heart rate, standard deviation of normal NN intervals (SDNN), standard deviation of 5-min (m) mean NN intervals (SDANN), the proportion of NN50 divided by the total number of NNs (pNN50), the root mean square differences of successive NN intervals (RMSSD), high (HF)-, low (LF)-, very low (VLF)-frequency power, total power components, and the LF/HF ratio. Individuals were divided into four groups according to their duration of mobile phone use [no mobile phone use (Control group), <30 min/day (Group 1), 30-60 min/day (Group 2), and >60 min/day (Group 3)]. All the groups had similar features with regard to demographic and clinical characteristics. No significant arrhythmias were observed in any of the groups. The LF/HF ratio was higher, whereas the SDNN, SDANN, RMSSD, and pNN50 values were lower in the study groups than in the control group (p<0.05). No significant differences were identified among groups with respect to heart rate, VLF, and total power values (p>0.05). In this study, it was shown that the duration of mobile phone use may affect the autonomic balance in healthy subjects. The electromagnetic field created by mobile phone use may induce HRV changes in the long term.


Swimming by reciprocal motion at low Reynolds number.
Biological microorganisms swim with flagella and cilia that execute nonreciprocal motions for low Reynolds number (Re) propulsion in viscous fluids. This symmetry requirement is a consequence of Purcell's scallop theorem, which complicates the actuation scheme needed by microswimmers. However, most biomedically important fluids are non-Newtonian where the scallop theorem no longer holds. It should therefore be possible to realize a microswimmer that moves with reciprocal periodic body-shape changes in non-Newtonian fluids. Here we report a symmetric 'micro-scallop', a single-hinge microswimmer that can propel in shear thickening and shear thinning (non-Newtonian) fluids by reciprocal motion at low Re. Excellent agreement between our measurements and both numerical and analytical theoretical predictions indicates that the net propulsion is caused by modulation of the fluid viscosity upon varying the shear rate. This reciprocal swimming mechanism opens new possibilities in designing biomedical microdevices that can propel by a simple actuation scheme in non-Newtonian biological fluids.


Substance P (SP)-neurokinin-1 receptor (NK-1R) alters adipose tissue responses to high-fat diet and insulin action.
Peripheral administration of a specific neurokinin-1 receptor (NK-1R) antagonist to mice leads to reduced weight gain and circulating levels of insulin and leptin after high-fat diet (HFD). Here, we assessed the contribution of substance P (SP) and NK-1R in diet-induced obesity using NK-1R deficient [knockout (KO)] mice and extended our previous findings to show the effects of SP-NK-1R interactions on adipose tissue-associated insulin signaling and glucose metabolic responses. NK-1R KO and wild-type (WT) littermates were fed a HFD for 3 wk, and obesity-associated responses were determined. Compared with WT, NK-1 KO mice show reduced weight gain and circulating levels of leptin and insulin in response to HFD. Adiponectin receptor mRNA levels are higher in mesenteric fat and liver in NK-1 KO animals compared with WT, after HFD. Mesenteric fat from NK-1R KO mice fed with HFD has reduced stress-activated protein kinase/c-Jun N-terminal kinase and protein kinase C activation compared with WT mice. After glucose challenge, NK-1R KO mice remove glucose from the circulation more efficiently than WT and pair-fed controls, suggesting an additional peripheral effect of NK-1R-mediated signaling on glucose metabolism. Glucose uptake experiments in isolated rat adipocytes showed that SP directly inhibits insulin-mediated glucose uptake. Our results further establish a role for SP-NK-1R interactions in adipose tissue responses, specifically as they relate to obesity-associated pathologies such as glucose intolerance and insulin resistance. Our results highlight this pathway as an important therapeutic approach for type 2 diabetes.


Reliability of electromyographic activity vs. bite-force from human masticatory muscles.
The reproducibility of electromyographic (EMG) activity in relation to static bite-force from masticatory muscles for a given biting situation is largely unknown. Our aim was to evaluate the reliability of EMG activity in relation to static bite-force in humans. Eighty-four subjects produced five unilateral static bites of different forces at different biting positions on molars and incisors, at two separate sessions, and the surface EMG activities were recorded from temporalis, masseter, and suprahyoid muscles bilaterally. Intraclass correlation coefficients (ICCs) were determined, and an ICC of ≥ 0.60 indicated good reliability of these slopes. The ICCs for jaw-closing muscles during molar biting were: temporalis muscles, ipsilateral 0.58-0.93 and contralateral 0.88-0.91; and masseter muscles, ipsilateral 0.75-0.86 and contralateral 0.69-0.88. The ICCs for jaw-closing muscles during incisor biting were: temporalis muscles, ipsilateral 0.56-0.81 and contralateral 0.34-0.86; and masseter muscles, ipsilateral 0.65-0.78 and contralateral 0.59-0.80. For the suprahyoid muscles the 95% CIs were mostly wide and most included zero. The slopes of the EMG activity vs. bite-force for a given biting situation were reliable for temporalis and masseter muscles. These results support the use of these outcome measurements for the estimation and validation of mechanical models of the masticatory system.


Corneal biomechanics and biomechanically corrected intraocular pressure in primary open-angle glaucoma, ocular hypertension and controls.
To compare the biomechanically corrected intraocular pressure (IOP) estimate (bIOP) provided by the Corvis-ST with Goldmann applanation tonometry (GAT-IOP) in patients with high-tension and normal-tension primary open-angle glaucoma (POAG; HTG and NTG), ocular hypertension (OHT) and controls. Moreover, we compared dynamic corneal response parameters (DCRs) of the Corvis-ST in POAG, OHT and controls, evaluated the correlation between global visual field parameters mean deviation and pattern SD (MD and PSD) and DCRs in the POAG group. 156 eyes of 156 patients were included in this prospective, single-centre, observational study, namely 41 HTG and 33 NTG, 45 OHT cases and 37 controls. Central corneal thickness (CCT), GAT-IOP and bIOP were measured, GAT-IOP was also adjusted for CCT (GATAdj). DCRs provided by Corvis-ST were evaluated, MD and PSD were recorded by 24-2 full-threshold visual field. To evaluate the difference in DCRs between OHT, HTG and NTG, a general linear model was used with sex, medications and group as fixed factors and bIOP and age as covariates. There was a significant difference between GAT-IOP, GATAdj and bIOP in NTG and HTG, OHT and controls. NTG corneas were significantly softer and more deformable compared with controls, OHT and HTG as demonstrated by significantly lower values of stiffness parameters A1 and highest concavity and higher values of inverse concave radius (all p<0.05). There was a significant correlation (p<0.05) between MD, PSD and many DCRs with POAG patients with softer or more compliant corneas more likely to show visual field defects. Corneal biomechanics might be a significant confounding factor for IOP measurement that should be considered in clinical decision-making. The abnormality of corneal biomechanics in NTG and the significant correlation with visual field parameters might suggest a new risk factor for the development or progression of NTG.


Increasing Prevalence of Artemisinin-Resistant HRP2-Negative Malaria in Eritrea.
Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments. In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria. We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests. The emergence and spread of P. falciparum lineages with both Pfkelch13-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.).


An update on cannabis research.
A symposium of over 125 scientists, held in August 1984 at the campus of Oxford University, considered the latest developments concerning cannabis research. Evidence on the mode of tetrahydrocannabinol action on the central nervous system indicates that acetylcholine turnover in the hippocampus through a GABA-ergic mechanism is of major importance, though the role of the dopaminergic or serotoninergic mechanism and involvement of prostaglandins and c-AMP is not ruled out. The use of cannabis causes prominent and predictable effects on the heart, including increased work-load, increased plasma volume and postural hypotension, which could impose threats to the cannabis users with hypertension, cerebrovascular disease or coronary arteriosclerosis. Cannabis or tetrahydrocannabinol has damaging effects on the endocrine functions in both male and female of all animal species tested. Among possible mechanisms of action, it is suggested that tetrahydrocannabinol disrupts gonadal functions by depriving the testicular cells of their energy reserves by inhibition of cellular energetics, and that it stimulates androgen-binding protein secretion, which may account for oligospermia seen in chronic cannabis smokers. In addition to these direct effects on gonads, tetrahydrocannabinol interferes with hormonal secretions from the pituitary, including luteinizing hormones, follicle-stimulating hormones and prolactin. Research findings indicate that maternal and paternal exposure to cannabinoids can influence developmental and reproductive functions in the offspring, but it is difficult to separate possible teratogenic effects from subsequent gametotoxic and mutagenic potentials of cannabinoids.


Glaucocalyxin A induces G2/M cell cycle arrest and apoptosis through the PI3K/Akt pathway in human bladder cancer cells.
Glaucocalyxin A (GLA), a major component isolated from Rabdosia japonica, has been proven to show anti-bacterial and anti-tumor biological characteristics according to previous studies. However, its potential effect on bladder cancer remains unknown. The present research aims to investigate the underlying mechanism in treating bladder cancer in vivo and in vitro. Cell proliferation was analyzed by CCK-8 assay and colony formation. Flow cytometry was used to measure the cell cycle distribution and apoptosis. The expressions of the cell cycle and apoptosis-related proteins were detected by western blotting and immunofluorescence staining. Meanwhile, the in vivo study was performed to evaluate the anti-tumor effect on a UMUC3 subcutaneous tumor of NOD/SCID mice model. GLA suppressed colony-formation ability, triggered G2/M arrest and promoted apoptosis of UMUC3 cells in a dose-dependent manner. Furthermore, western blotting showed that GLA downregulated the expressions of PI3K p85, p-Akt, Bcl-2, CDK1, Cyclin B1 whereas upregulated the levels of PTEN, Bax, Cleaved Caspase-3. In vivo, GLA at a dosage of 20 mg/kg significantly inhibited tumor growth compared with the control group by intraperitoneal injection. These results suggested that GLA-related G2/M arrest and apoptosis in UMUC3 cells were mediated by a suppressed PI3K/Akt signaling pathway, which regulated p21Waf1/Cip1 as well as intrinsic caspase cascade. Collectively, our observations could help to develop new drugs targeting the PI3K/Akt pathway for the treatment of bladder cancer.


Technical recommendations for clinical translation of renal MRI: a consensus project of the Cooperation in Science and Technology Action PARENCHIMA.
The potential of renal MRI biomarkers has been increasingly recognised, but clinical translation requires more standardisation. The PARENCHIMA consensus project aims to develop and apply a process for generating technical recommendations on renal MRI. A task force was formed in July 2018 focused on five methods. A draft process for attaining consensus was distributed publicly for consultation and finalised at an open meeting (Prague, October 2018). Four expert panels completed surveys between October 2018 and March 2019, discussed results and refined the surveys at a face-to-face meeting (Aarhus, March 2019) and completed a second round (May 2019). A seven-stage process was defined: (1) formation of expert panels; (2) definition of the context of use; (3) literature review; (4) collection and comparison of MRI protocols; (5) consensus generation by an approximate Delphi method; (6) reporting of results in vendor-neutral and vendor-specific terms; (7) ongoing review and updating. Application of the process resulted in 166 consensus statements. The process generated meaningful technical recommendations across very different MRI methods, while allowing for improvement and refinement as open issues are resolved. The results are likely to be widely supported by the renal MRI community and thereby promote more harmonisation.


Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.
We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.


Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.
Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.


Polarization resolved classification of winter road condition in the near-infrared region.
Three different configurations utilizing polarized short-wave infrared light to classify winter road conditions have been investigated. In the first configuration, polarized broadband light was detected in the specular and backward directions, and the quotient between the detected intensities was used as the classification parameter. Best results were obtained for the SS-configuration. This sensor was shown to be able to distinguish between the smooth road conditions of water and ice from the diffuse road conditions of snow and dry asphalt with a probability of wrong classification as low as 7%. The second sensor configuration was a pure backward architecture utilizing polarized light with two distinct wavelengths. This configuration was shown to be effective for the important problem of distinguishing water from ice with a probability of wrong classification of only 1.5%. The third configuration was a combination of the two previous ones. This combined sensor utilizing bispectral illumination and bidirectional detection resulted in a probability of wrong classification as low as 2% among all four surfaces.


Towards early detection of retinoblastoma.
Survival rates for retinoblastoma (RB) in a region of South Africa (SA) of only 50% reflect the high frequency of late presentation, the simple reason for which is lack of effective screening. Early detection of suspected RB would significantly reduce this unacceptably high mortality rate. The SA health system has the expertise to manage a child with RB well. The issue at stake is timely referral of the affected child to one of the specialist treatment centres. Until universal screening with digital imaging becomes a reality, the red reflex test should be mandatory at discharge from all neonatal services and at all subsequent routine health supervision visits. Most RBs would then be detected early.


Constructing and evaluating a validity argument for the final-year ward simulation exercise.
The authors report final-year ward simulation data from the University of Dundee Medical School. Faculty who designed this assessment intend for the final score to represent an individual senior medical student's level of clinical performance. The results are included in each student's portfolio as one source of evidence of the student's capability as a practitioner, professional, and scholar. Our purpose in conducting this study was to illustrate how assessment designers who are creating assessments to evaluate clinical performance might develop propositions and then collect and examine various sources of evidence to construct and evaluate a validity argument. The data were from all 154 medical students who were in their final year of study at the University of Dundee Medical School in the 2010-2011 academic year. To the best of our knowledge, this is the first report on an analysis of senior medical students' clinical performance while they were taking responsibility for the management of a simulated ward. Using multi-facet Rasch measurement and a generalizability theory approach, we examined various sources of validity evidence that the medical school faculty have gathered for a set of six propositions needed to support their use of scores as measures of students' clinical ability. Based on our analysis of the evidence, we would conclude that, by and large, the propositions appear to be sound, and the evidence seems to support their proposed score interpretation. Given the body of evidence collected thus far, their intended interpretation seems defensible.