Selective desorption/ionization of sulfatides by MALDI-MS facilitated using 9-aminoacridine as matrix.
Recently, we used the favorable properties of 9-aminoacridine (9-AA) as matrix for the quantitative analysis of acidic metabolites and glycerophospholipids from extracts of biological materials [Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2007) A shotgun metabolomics approach for rapid analysis of negatively-charged water-soluble cellular metabolites from mouse heart tissue. Anal. Chem. 79: 6629-6640; Sun, G., Yang, K., Zhao, Z., Guan, S., Han, X., and Gross, R.W. (2008) Matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions. Anal. Chem. 80: 7576-7585.] by MALDI-MS. Herein, we extend this discovery and identified the selective desorption/ionization of sulfatides over other examined anionic lipids present in lipid extracts of biological samples by MALDI-MS using 9-AA as matrix. Through this approach, a high throughput method for the quantitative analysis of low to very low abundance sulfatide molecular species directly from crude lipid extracts has been developed. This method possessed a linear dynamic range of over 1,000-fold, a detection limit at the high attomole level, and a reproducibility of approximately 10% deviation. Many potential factors that might affect the quantitation of sulfatide species employing the method were examined and their effects were found to be negligible within experimental error. Collectively, these results demonstrate a powerful high throughput method for the measurement of sulfatides directly from extracts of biological samples, facilitating the study of sulfatide metabolism, trafficking, and homeostasis in health and disease.


Semi-Automated Analysis of Digital Photographs for Monitoring East Antarctic Vegetation.
Climate change is affecting Antarctica and minimally destructive long-term monitoring of its unique ecosystems is vital to detect biodiversity trends, and to understand how change is affecting these communities. The use of automated or semi-automated methods is especially valuable in harsh polar environments, as access is limited and conditions extreme. We assessed moss health and cover at six time points between 2003 and 2014 at two East Antarctic sites. Semi-automatic object-based image analysis (OBIA) was used to classify digital photographs using a set of rules based on digital red, green, blue (RGB) and hue-saturation-intensity (HSI) value thresholds, assigning vegetation to categories of healthy, stressed or moribund moss and lichens. Comparison with traditional visual estimates showed that estimates of percent cover using semi-automated OBIA classification fell within the range of variation determined by visual methods. Overall moss health, as assessed using the mean percentages of healthy, stressed and moribund mosses within quadrats, changed over the 11 years at both sites. A marked increase in stress and decline in health was observed across both sites in 2008, followed by recovery to baseline levels of health by 2014 at one site, but with significantly more stressed or moribund moss remaining within the two communities at the other site. Our results confirm that vegetation cover can be reliably estimated using semi-automated OBIA, providing similar accuracy to visual estimation by experts. The resulting vegetation cover estimates provide a sensitive measure to assess change in vegetation health over time and have informed a conceptual framework for the changing condition of Antarctic mosses. In demonstrating that this method can be used to monitor ground cover vegetation at small scales, we suggest it may also be suitable for other extreme environments where repeat monitoring via images is required.


Rhythm, movement, and autism: using rhythmic rehabilitation research as a model for autism.
Recently, there has been increased focus on movement and sensory abnormalities in autism spectrum disorders (ASD). This has come from research demonstrating cortical and cerebellar differences in autism, with suggestion of early cerebellar dysfunction. As evidence for an extended profile of ASD grows, there are vast implications for treatment and therapy for individuals with autism. Persons with autism are often provided behavioral or cognitive strategies for navigating their environment; however, these strategies do not consider differences in motor functioning. One accommodation that has not yet been explored in the literature is the use of auditory rhythmic cueing to improve motor functioning in ASD. The purpose of this paper is to illustrate the potential impact of auditory rhythmic cueing for motor functioning in persons with ASD. To this effect, we review research on rhythm in motor rehabilitation, draw parallels to motor dysfunction in ASD, and propose a rationale for how rhythmic input can improve sensorimotor functioning, thereby allowing individuals with autism to demonstrate their full cognitive, behavioral, social, and communicative potential.


Tensions in Communication between Children on Antiretroviral Therapy and Their Caregivers: A Qualitative Study in Jinja District, Uganda.
HIV treatment and disclosure guidelines emphasize the importance of communicating diagnosis and treatment to infected children in ways that are appropriate to children's developmental stage and age. Minimal attention, however, has been given to communication challenges confronted by HIV-infected children and their caregivers. This study examined the tensions between children and their caregivers arising from differing perspectives regarding when and what to communicate about antiretroviral therapy (ART). This qualitative study was conducted between November 2011 and December 2012 and involved 29 HIV-infected children aged 8-17 years on ART and their caregivers. Data were collected through observations and in-depth interviews, which took place in homes, treatment centres and post-test clubs. Children and caregivers were sampled from among the 394 HIV-infected children and (their) 393 caregivers who participated in the cross-sectional survey that preceded the qualitative study. ATLAS.ti. Version 7 was used in the management of the qualitative data and in the coding of the emerging themes. The data were then analyzed using content thematic analysis. While the children felt that they were mature enough to know what they were suffering and what the medications were for, the caregivers wanted to delay discussions relating to the children's HIV diagnosis and medication until they felt that the children were mature enough to deal with the information and keep it a secret and this caused a lot of tension. The children employed different tactics including refusing to take the medicines, to find out what they were suffering from and what the medications were for. Children also had their own ideas about when, where and with whom to discuss their HIV condition, ideas that did not necessarily coincide with those of their caregivers, resulting in tensions. Guidelines should take into consideration differing perceptions of maturity when recommending ages at which caregivers should communicate with their children about diagnosis and ART. Health care providers should also encourage caregivers to recognize and respect children's efforts to learn about and manage their condition. Children's questions and expressions of feelings should be treated as openings for communication on these issues.


Addition of choice of complementary therapies to usual care for acute low back pain: a randomized controlled trial.
A randomized controlled trial. To investigate the effectiveness and cost of usual care plus patient choice of acupuncture, chiropractic, or massage therapy (choice) compared with usual care alone in patients with acute low back pain (LBP). Few studies have evaluated care models with facilitated access to and financial coverage for adjunctive complementary and alternative medicine therapies. A total of 444 patients with acute LBP (<21 days) were recruited from 4 clinical sites and randomized into 2 groups: usual care or choice. Outcomes included symptoms (bothersomeness), functional status (Roland), and satisfaction between baseline and 5 weeks, and cost of medical care in the 12 weeks after randomization. After 5 weeks, providing patients with a choice did not yield clinically important reductions in symptoms (median -4, [interquartile range -7, -2] for usual care, and -5 [-7, -3] for choice; P = 0.002) or improvements in functional status (-8 [-13, -2] for usual care, and -9 [-15, -4] for choice; P = 0.15). Although there was a significantly greater satisfaction with care in the choice group, this came at a net increase in costs of 244 dollars per patient. This consisted of a 99 dollars reduction in the average cost to the insurer for medical care but an additional cost of 343 dollars, for an average of 6.0 complementary and alternative medicine treatments per patient. A model of care that offered access to a choice of complementary and alternative medicine therapies for acute LBP did not result in clinically significant improvements in symptom relief or functional restoration. This model was associated with greater patient satisfaction but increased total costs. Future evaluations of this choice model should focus on patients with chronic conditions (including chronic back pain) for which conventional medical care is often costly and of limited benefit.


The human JC polyomavirus (JCPyV): virological background and clinical implications.
JC polyomavirus (JCPyV) was the first of now 12 PyVs detected in humans, when in 1964, PyV particles were revealed by electron microscopy in progressive multifocal leukoencephalopathy (PML) tissues. JCPyV infection is common in 35-70% of the general population, and the virus thereafter persists in the renourinary tract. One third of healthy adults asymptomatically shed JCPyV at approximately 50,000 copies/mL urine. PML is rare having an incidence of <0.3 per 100,000 person years in the general population. This increased to 2.4 per 1000 person years in HIV-AIDS patients without combination antiretroviral therapy (cART). Recently, PML emerged in multiple sclerosis patients treated with natalizumab to 2.13 cases per 1000 patients. Natalizumab blocks α4-integrin-dependent lymphocyte homing to the brain suggesting that not the overall cellular immunodeficiency but local failure of brain immune surveillance is a pivotal factor for PML. Recovering JCPyV-specific immune control, e.g., by starting cART or discontinuing natalizumab, significantly improves PML survival, but is challenged by the immune reconstitution inflammatory syndrome. Important steps of PML pathogenesis are undefined, and antiviral therapies are lacking. New clues might come from molecular and functional profiling of JCPyV and PML pathology and comparison with other replicative pathologies such as granule cell neuronopathy and (meningo-)encephalitis, and non-replicative JCPyV pathology possibly contributing to some malignancies. Given the increasing number of immunologically vulnerable patients, a critical reappraisal of JCPyV infection, replication and disease seems warranted.


Familial dysalbuminaemic hyperthyroxinaemia interferes with current free thyroid hormone immunoassay methods.
Familial dysalbuminaemic hyperthyroxinaemia (FDH), most commonly due to an Arginine to Histidine mutation at residue 218 (R218H) in the albumin gene, causes artefactual elevation of free thyroid hormones in euthyroid individuals. We have evaluated the susceptibility of most current free thyroid hormone immunoassay methods used in the United Kingdom, Europe and Far East to interference by R218H FDH. Different, one- and two-step immunoassay methods were tested, measuring free T4 (FT4) and free T3 (FT3) in 37 individuals with genetically proven R218H FDH. With the exception of Ortho VITROS, FT4 measurements were raised in all assays, with greatest to lowest susceptibility to interference being Beckman ACCESS > Roche ELECSYS > FUJIREBIO Lumipulse > Siemens CENTAUR > Abbott ARCHITECT > Perkin-Elmer DELFIA. Five different assays recorded high FT3 levels, with the Siemens CENTAUR method measuring high FT3 values in up to 30% of cases. However, depending on the assay method, FT4 measurements were unexpectedly normal in some, genetically confirmed, affected relatives of index FDH cases. All FT4 immunoassays evaluated are prone to interference by R218H FDH, with their varying susceptibility not being related to assay architecture but likely due to differing assay conditions or buffer composition. Added susceptibility of many FT3 assays to measurement interference, resulting in high FT4 and FT3 with non-suppressed TSH levels, raises the possibility of R218H FDH being misdiagnosed as resistance to thyroid hormone beta or TSH-secreting pituitary tumour, potentially leading to unnecessary investigation and inappropriate treatment.


Clinical outcomes and cost-effectiveness of brief guided parent-delivered cognitive behavioural therapy and solution-focused brief therapy for treatment of childhood anxiety disorders: a randomised controlled trial.
Half of all lifetime anxiety disorders emerge before age 12 years; however, access to evidence-based psychological therapies for affected children is poor. We aimed to compare the clinical outcomes and cost-effectiveness of two brief psychological treatments for children with anxiety referred to routine child mental health settings. We hypothesised that brief guided parent-delivered cognitive behavioural therapy (CBT) would be associated with better clinical outcomes than solution-focused brief therapy and would be cost-effective. We did this randomised controlled trial at four National Health Service primary child and mental health services in Oxfordshire, UK. Children aged 5-12 years referred for anxiety difficulties were randomly allocated (1:1), via a secure online minimisation tool, to receive brief guided parent-delivered CBT or solution-focused brief therapy, with minimisation for age, sex, anxiety severity, and level of parental anxiety. The allocation sequence was not accessible to the researcher enrolling participants or to study assessors. Research staff who obtained outcome measurements were masked to group allocation and clinical staff who delivered the intervention did not measure outcomes. The primary outcome was recovery, on the basis of Clinical Global Impressions of Improvement (CGI-I). Parents recorded patient-level resource use. Quality-adjusted life-years (QALYs) for use in cost-utility analysis were derived from the Child Health Utility 9D. Assessments were done at baseline (before randomisation), after treatment (primary endpoint), and 6 months after treatment completion. We did analysis by intention to treat. This trial is registered with the ISCRTN registry, number ISRCTN07627865. Between March 23, 2012, and March 31, 2014, we randomly assigned 136 patients to receive brief guided parent-delivered CBT (n=68) or solution-focused brief therapy (n=68). At the primary endpoint assessment (June, 2012, to September, 2014), 40 (59%) children in the brief guided parent-delivered CBT group versus 47 (69%) children in the solution-focused brief therapy group had an improvement of much or very much in CGI-I score, with no significant differences between groups in either clinical (CGI-I: relative risk 1·01, 95% CI 0·86-1·19; p=0·95) or economic (QALY: mean difference 0·006, -0·009 to 0·02; p=0·42) outcome measures. However, brief guided parent-delivered CBT was associated with lower costs (mean difference -£448; 95% CI -934 to 37; p=0·070) and, taking into account sampling uncertainty, was likely to represent a cost-effective use of resources compared with solution-focused brief therapy. No treatment-related or trial-related adverse events were reported in either group. Our findings show no evidence of clinical superiority of brief guided parent-delivered CBT. However, guided parent-delivered CBT is likely to be a cost-effective alternative to solution-focused brief therapy and might be considered as a first-line treatment for children with anxiety problems. National Institute for Health Research.


Effects of different periodization models on rate of force development and power ability of the upper extremity.
The purpose of our study was to compare the effects of 2 different periodization models on strength and power variables under dynamic and static conditions in the bench press. Participants of the experimental groups were male sport students experienced in weight training (age: 23.98 +/- 3.14 yr). Subjects were tested for the 1 repetition maximum (1RM) in the bench press, maximal movement velocity (Vmax) in the bench press throw (16.9 kg), maximal voluntary contraction (MVC), and maximal rate of force development (MRFD) in 90 degrees elbow and shoulder angle in the isometric bench press. According to their 1RM, subjects were parallelized and assigned to 1 of either 2 training groups: strength-power periodization (SPP, n = 13) or daily undulating periodization (DUP, n = 14). Subjects trained for 14 weeks, 3 days per week. In the strength-power sessions, both groups were instructed to lift the weight as explosively as possible. In addition, a control group (n = 13) was used for comparison. One repetition maximum and Vmax improved significantly through training (p <or= 0.05), with no significant changes in MVC and MRFD. Experimental groups showed no significant group differences in any variable. The results indicate that, in short-term training using previously trained subjects, no differences in 1RM and power are seen between DUP and SPP. As used in our undulating regime, additional training in strength endurance could lead to exhaustion effects and furthermore does not provide an adequate training stimulus for power because of its low training intensity. In spite of this, according to the present findings, it has no negative effect on the application of a neural stimulus that is needed for a strength-power session if adequate regeneration time between workouts is guaranteed.


Sildenafil improves vascular endothelial function in patients with cystic fibrosis.
Cystic fibrosis (CF), characterized by defective CFTR function, is associated with multiple systemic complications, including vascular dysfunction. Sildenafil, a phosphodiesterase type 5 inhibitor, not only enhances nitric oxide (NO) metabolism but has been shown to improve CFTR functionality as well. Thus, sildenafil has been proposed as a therapy to improve vascular health in CF; however, its potential therapeutic role has yet to be determined. We sought to investigate the effect of sildenafil on endothelial function in patients with CF. Patients with CF completed a randomized, double-blind, placebo-controlled, crossover study with an acute dose of sildenafil (50 mg) or placebo followed by a 4-wk open-label extension with sildenafil (20 mg/day). Flow-mediated dilation (FMD) was used to evaluate endothelial function before and after treatments. In addition, phosphorylated endothelial NO synthase (pNOS3) and total NOS3 protein expression was determined from endothelial cells that were exposed to plasma from the patients before and after 4 wk of sildenafil treatment. No changes ( P ≥ 0.110) in endothelial function were observed after the acute dose of sildenafil. However, FMD significantly ( P = 0.029) increased after 4 wk of treatment (∆FMD: 1.5 ± 2.2%). Moreover, pNOS3 protein expression significantly ( P = 0.013) increased after 4 wk of treatment (∆pNOS3: 0.31 ± 0.39 arbitrary units) and was associated ( r = 0.593, P = 0.033) with the change in FMD. These data suggest that 4 wk of sildenafil treatment can improve vascular endothelial function in patients with CF, likely through an increase in NOS3 phosphorylation. NEW & NOTEWORTHY Findings from the present study demonstrate, for the first time, significant improvement of endothelial function in patients with cystic fibrosis treated with sildenafil that is associated with greater phosphorylation of endothelial nitric oxide synthase. These results support the use of sildenafil as a potential novel therapy for this patient population.


Methadone prescribing under supervised consumption on premises: a Scottish clinician's perspective on prescribing practice.
Specialist services have increased their capacity considerably in recent years to initiate and/or provide ongoing treatment for drug misuse. Consequently, methadone prescribing has substantially increased and over 17,000 patients are currently receiving methadone. Clinical guidance promotes consumption on the premises (COP) initially to ensure patients take medication as prescribed and also to prevent diversion. Diversion poses two risks: the patient may remain under-treated and continue illicit heroin use; diverted drugs put others at risk. However, COP can be restrictive. Current UK guidance is vague and not evidence-based recommending 'around three months subject to assessment of compliance and individual circumstances'. The overall aim of this study was to describe clinical practice regarding prescribing methadone under COP in Scotland, and reasons for this. Design and Methods. A structured, postal questionnaire was sent to all lead clinicians in specialist drug treatment centres in Scotland in 2009 (n=42). The questionnaire explored current practice, influence of supervision on retention, views of best practice and contingency management. The response rate was 76% (n=32). Clinicians usually supervise for a minimum of three months with patient-centred criteria used to determine when to reduce or stop COP. Employment, clinical stability, family support and concerns for the safety of children in the home influenced decision making. Contingency management approaches to supervision were widely used (62%). In Scotland, clinicians' practice is variable and individualised to patients, but generally cautious regarding relaxing supervision conditions. This may reflect the lack of evidence-based guidance.


Pathology of labial salivary gland cellular aggregates in Sjögren's syndrome.
Sjögren's syndrome (SS) is a chronic autoimmune inflammatory disease in which lymphocytic infiltration of the lacrimal and salivary glands is the hallmark of disease diagnosis. The present study was conducted to identify developmental features of labial salivary gland histopathology to permit earlier diagnosis of SS patients with borderline biopsies. Control subjects were chosen on the basis of clinical presentation consistent with SS, but whose biopsies did not meet current focus score criteria. Intraglandular connective tissue and diffuse cellular infiltration were significantly greater in SS patients than in controls. Glands in both groups had small cellular aggregates (10 to 50 cells/100 microns2), but those in SS patients were more numerous, larger, and contained more lymphocytes, plasma cells, and active fibroblasts. The large inflammatory foci characteristic of SS appeared to be formed by the enlargement and merging of these aggregates. This suggested aggregate formation was the earliest stage of pathology. Therefore, a comprehensive analysis of these cellular aggregates in borderline or negative biopsies could identify at-risk patients and lead to earlier diagnosis and intervention in the disease.


Bénard-Marangoni Dendrites upon Evaporation of a Reactive ZnO Nanofluid Droplet: Effect of Substrate Chemistry.
Evaporation of a particle laden sessile drop can lead to complex surface patterns with structural hierarchy. Most commonly, the dispersed particles are inert. We have recently reported that when the sessile drop contains reactive ZnO nanoparticles, solidified Bénard-Marangoni (BM) cells with dendritic micromorphology were formed in the residual surface pattern from in situ-generated nanoclusters. Here, we report the effect of substrate chemistry on the residual pattern from the evaporation of nanofluids containing ZnO particles dispersed in a mixture of cyclohexane and isobutylamine, by comparing three different substrates: glass, silicon, and hydrophilized silicon. In particular, we performed a quantitative analysis of the BM cell size, distribution, and the cell morphological characteristics via the fractal dimension analysis. We find that the size dimension λBM of the dendritic Bénard-Marangoni cells varied on the different substrates, attributed to their different hydrophilicity and affinity for water molecules, evident from the different polar components γP in their surface free energy from the Owens-Wendt analysis. The average BM cell size was the smallest for the glass substrate (λBM = 289 μm) and comparable for the unmodified and UV/ozone-treated silicon wafers (with λBM = 466 and 423 μm, respectively). The fractal dimension analysis provided a quantitative description of the BM cells with complex structural hierarchy, highlighting the differences in the geometric features of the surface patterns resulting from different substrate chemistry. We also found that the fractal dimensions depended on the BM cell size, attributing it to two different regimes: the growing fractals and the maturing fractals.


A mutation in the small heat-shock protein HSPB1 leading to distal hereditary motor neuronopathy disrupts neurofilament assembly and the axonal transport of specific cellular cargoes.
Distal hereditary motor neuronopathies (dHMNs) are a clinically and genetically heterogeneous group of disorders in which motor neurons selectively undergo age-dependant degeneration. Mutations in the small heat-shock protein HSPB1 (HSP27) are responsible for one form of dHMN. In this study, we have analysed the effect of expressing a form of mutant HSPB1 in primary neuronal cells in culture. Mutant (P182L) but not wild-type HSPB1 led to the formation of insoluble intracellular aggregates and to the sequestration in the cytoplasm of selective cellular components, including neurofilament middle chain subunit (NF-M) and p150 dynactin. These findings suggest a possible pathogenic mechanism for HSPB1 whereby the mutation may lead to preferential motor neuron loss by disrupting selective components essential for axonal structure and transport.


Comparative effects of amlodipine and benazepril on left atrial pressure in dogs with experimentally-induced mitral valve regurgitation.
One of the purposes of treatment for dogs with mitral regurgitation (MR) is lowering left atrial pressure (LAP). There has been few study of the amlodipine in dogs with MR and amlodipine's effect on LAP has not been fully evaluated in a quantitative manner because of difficulties in directly measuring LAP. The objective of our study was to compare the short-term effects of amlodipine (0.2 mg/kg PO q12h) vs benazepril (0.5 mg/kg PO q12h), on LAP and echocardiographic parameters in five beagle dogs with experimentally-induced MR. LAP of eight dogs that has own control were measured using radiotelemetry system at baseline and again on days 1, 2, 3, 4, 5, 6, 7 of the drug administration. Mean LAP decreased significantly after amlodipine (11.20 ± 4.19 mmHg vs 14.61 ± 3.81 mmHg at baseline, p < .01) but not after benazepril treatment (13.19 ± 3.47 mmHg, p > .05). LAP was lower after 7 days of amlodipine treatment than after 7 days of benazepril treatment. Significant reduction was seen for the first time 4 days after the administration amlodipine. The rate of the maximal area of the regurgitant jet signals to the left atrium area (ARJ/LAA) of the amlodipine treatment was significantly lower (p < .05) after 7 days compared to baseline. Other echocardiographic parameters did not change significantly. LAP was significantly decreased after amlodipine treatment in dogs with surgically-induced MR but not after benazepril treatment. Although this study did not focus on adverse effects, amlodipine may be an effective drug for helping the patients with acute onset of severe MR, such as rupture of chordae tendinae or end stage patients were the LAP is likely to be elevated. Additional studies in clinical patients with degenerative mitral valve disease and acute chordal rupture are warranted because the blood-pressure lowering effects of amlodipine can decrease renal perfusion and this can further activate the RAAS.


Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.
Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.


Pituitary dysfunction after traumatic brain injury: are there definitive data in children?
In the past decade, several studies in adults and children have described the risk of pituitary dysfunction after traumatic brain injury (TBI). As a result, an international consensus statement recommended follow-up on the survivors. This paper reviews published studies regarding hypopituitarism after TBI in children and compares their results. The prevalence of hypopituitarism ranges from 5% to 57%. Growth hormone (GH) and ACTH deficiency are the most common, followed by gonadotropins and thyroid-stimulating hormone. Paediatric studies have failed to identify risk factors for developing hypopituitarism, and therefore we have no tools to restrict screening in severe TBI. In addition, the present review highlights the lack of a unified follow-up and the fact that unrecognised pituitary dysfunction is frequent in paediatric population. The effect of hormonal replacement in patient recovery is important enough to consider baseline screening and reassessment between 6 and 12 months after TBI. Medical community should be aware of the risk of pituitary dysfunction in these patients, given the high prevalence of endocrine dysfunction already reported in the studies. Longer prospective studies are needed to uncover the natural course of pituitary dysfunction, and new studies should be designed to test the benefit of hormonal replacement in metabolic, cognitive and functional outcome in these patients.


Oral manifestations of connective tissue disease and novel therapeutic approaches.
Connective tissue diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren syndrome (SS) have presented many difficulties both in their diagnosis and treatment. Known causes for this difficulty include uncertainty of disease etiology, the multitude of clinical presentations, the unpredictable disease course, and the variable cell types, soluble mediators, and tissue factors that are believed to play a role in the pathogenesis of connective tissue diseases. The characteristic oral findings seen with these specific connective tissue diseases may assist with more swift diagnostic capability. Additionally, the recent use of biologics may redefine the success rate in the treatment and management of the disease. In this review we describe the oral manifestations associated with SLE, SSc, and SS and review the novel biologic drugs used to treat these conditions.


Melatonin influences antioxidant homeostasis and basal metabolism in the BALB/c mouse model.
Melatonin is a hormone with strong antioxidant activity. It is biosynthesized in the pineal gland and serves in the biological signaling and control of the circadian rhythm. Though there is evidence of beneficial effects of melatonin, the substance was not investigated in greater details associated with specific regulation of oxidative stress in organs and tissues. The experiment is based on exposure of BALB/c mice to doses from 10 µg to 1mg of melatonin. Mice were euthanized after one and 24 hours, respectively. Biochemical markers in plasma, ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS) and activity of caspase-3 were examined in selected organs. We confirmed significant alteration in high-density lipoprotein and total cholesterols and glucose. After one day, levels of glucose and high-density lipoprotein cholesterol were decreased, while total cholesterol increased in a dose dependent manner. FRAP values increased in spleen, liver, kidney, heart, and brain indicating a growing potential of low molecular weight antioxidants 24 hours after exposure. However, TBARS values indicating oxidative stress were elevated in heart, kidney, and liver. Despite the antioxidant properties of melatonin, its effect on the organism is more complicated. It influences not only the oxidative homeostasis but also the basal metabolism, as represented by, e.g., cholesterol and glucose. This substance could thus be used for therapeutical purposes such as amelioration of pathologies associated with generation of reactive species or some metabolic dysfunctions.


PLAGL2 regulates actin cytoskeletal architecture and cell migration.
Pleomorphic adenoma gene like-2 (PLAGL2), a member of the PLAG gene family, is a C2H2 zinc finger transcriptional factor that is involved in cellular transformation and apoptosis. In this report, we show that PLAGL2 is associated with the organization of stress fibers and with small guanosine triphosphatase (GTPase) activity. Depletion of PLAGL2 in two different ovarian cancer cell lines, ES-2 and HEY, induced activation of RhoA, whereas activity of Rac1 was suppressed. Organization of actin stress fibers and focal adhesions was significantly promoted by PLAGL2 knockdown in a RhoA-dependent manner. Conversely, exogenous expression of PLAGL2 in MDA-MB-231 cells, a breast cancer cell line, resulted in the activation of Rac1 and the inactivation of RhoA. In addition, PLAGL2 expression induced lamellipodia formation and disruption of stress fiber formation. Finally, we show that CHN1 expression is essential for Rac1 inactivation in PLAGL2-depleted cells. Our results demonstrate a crucial role of PLAGL2 in actin dynamics and give further insight into the role of PLAGL2 in cellular transformation and apoptosis.


Ligand-induced conformational change in transferrins: crystal structure of the open form of the N-terminal half-molecule of human transferrin.
Serum transferrin binds ferric ions in the bloodstream and transports them to cells, where they are released in a process involving receptor-mediated endocytosis. Iron release is believed to be pH dependent and is coupled with a large conformational change. To help define the steps in iron release, we have determined the three-dimensional structure of the iron-free (apo) form of the recombinant N-lobe half-molecule of human serum transferrin (ApoTfN) by X-ray crystallography. Two crystal forms were obtained, form 1 with four molecules in the asymmetric unit and form 2 with two molecules in the asymmetric unit. The structures of both forms were determined by molecular replacement and were refined at 2.2 and 3.2 A resolution, respectively. Final R-factors were 0.203 (free R = 0. 292) for form 1 and 0.217 (free R = 0.312) for form 2. All six copies of the ApoTfN structure are essentially identical. Comparison with the holo form (FeTfN) shows that a large rigid-body domain movement of 63 degrees has occurred in ApoTfN, to give an open binding cleft. The extent of domain opening is the same as in the N-lobe of human lactoferrin, showing that it depends on internal constraints that are conserved in both proteins, and that it is unaffected by the presence or absence of the C-lobe. Although the conformational change is primarily a rigid-body motion, several local adjustments occur. In particular, two iron ligands, Asp 63 and His 249, change conformation to form salt bridges, with Lys 296 and Glu 83, respectively, in the binding cleft of the apo protein. Both salt bridges would have to break for iron coordination to occur. Most importantly, the structure, determined at a pH (5.3) that is close to the pH of physiological iron release, indicates that protonation of His 249 is a key step in iron release.


PARK2 patient neuroprogenitors show increased mitochondrial sensitivity to copper.
Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD.


Deregulated expression of miR-29a-3p, miR-494-3p and miR-660-5p affects sensitivity to tyrosine kinase inhibitors in CML leukemic stem cells.
The development of Imatinib mesylate (IM), which targets the oncogenic BCR-ABL fusion protein, has greatly improved the outcome of Chronic Myeloid Leukemia (CML) patients. However, BCR-ABL-positive progenitors can be detected in CML patients in complete cytogenetic response. Several evidence suggests that CML stem cells are intrinsically resistant to Tyrosine Kinase Inhibitors (TKI), and therefore they represent the most likely candidate responsible for disease relapse.In this work, we investigated the microRNA (miRNA) expression profile of different subpopulations of CML Leukemic Stem Cells (LSCs): Lin-CD34+CD38- and Lin-CD34-CD38- cells. These cell fractions have been previously shown to be endowed with TKI intrinsic resistance. Our analysis identified 33 common deregulated miRNAs in CML LSCs. Among those, 8 miRNAs were deregulated in CML independently from BCR-ABL kinase activity and therefore are likely to be involved in the BCR-ABL-independent resistance to TKI that characterizes CML LSCs. In particular, the up-regulation of miR-29a-3p and miR-660-5p observed in CML LSCs, led to the down-regulation of their respective targets TET2 and EPAS1 and conferred TKI-resistance to CML LSCs in vitro. On the other hand, miR-494-3p down-regulation in CML LSCs, leading to c-MYC up-regulation, was able to decrease TKI-induced apoptosis. These results demonstrate that aberrant miRNA expression in CML LSCs could contribute to the intrinsic TKI-resistance observed in these cell populations, and support the development of novel therapies aimed at targeting aberrantly regulated miRNAs or their targets in order to effectively eradicate CML LSCs.


Polymer-decorated tethered membranes under good- and poor-solvent conditions.
We study tethered membranes grafted by polymer chains on one side. Mean-field and scaling arguments predicting a spontaneous curvature are compared to the results of lattice-based Monte Carlo simulations using the Bond Fluctuation Model, which are carried out for various grafting densities and chain lengths. We show that already slightly overlapping chains bend the membrane significantly. This proves the entropic origin for the bending stiffness, which is of order kT. To understand the membrane curvature under conditions of very small bending stiffness we apply a geometrical model which takes into account the state of chains at the overlap threshold. Applying a thermal solvent model for the grafted chains, we demonstrate that the bending direction of the membrane can be triggered by variation of the solvent quality. This indicates that polymer-decorated membranes may serve as switchable nanoscale devices.


Involvement of calpain-calpastatin in cigarette smoke-induced inhibition of lung endothelial nitric oxide synthase.
We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5-10% CSE for 2-24 h. eNOS gene transcription rate, eNOS messenger ribonucleic acid (mRNA) half-life, and the activity and protein contents of calpain and calpastatin were measured. Incubation of PAEC with CSE caused significant decreases in eNOS gene transcription and calpain activity and an increase in calpastatin protein content. eNOS mRNA half-life was not significantly altered by CSE. To investigate whether CSE-induced inhibition of eNOS gene expression is caused by decreased calpain activity due to an increase in calpastatin protein content, we cloned calpastatin gene from PAEC and constructed adenovirus vectors containing calpastatin. Overexpression of calpastatin mimics the inhibitory effects of CSE on calpain activity and on the activity, protein, and mRNA of eNOS. The cell-permeable calpain inhibitor, calpastatin peptide, inhibits acetylcholine-induced endothelium-dependent relaxation of the pulmonary artery. Incubation of PAEC with an antisense oligodeoxyribonucleotide of calpastatin prevented CSE-induced increases in calpastatin protein and CSE-induced decreases in calpain activity, eNOS gene transcription, activity and protein content of eNOS, and NO release. These results indicate that CSE-induced inhibition of eNOS expression in PAEC is caused by calpain inhibition due to an increase in calpastatin protein content.


Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling.
The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.


Educational level and its relationship with body height and popliteal height in Chilean male workers.
A secular trend in body height has been experienced in many nations and populations, hypothesized to be the result of better living conditions. Educational level has been shown to be closely associated with body height. This study examined the changes in body height and popliteal height in a group of adult Chilean male workers by age cohort and the relationship of these with educational level. The body heights and popliteal heights of 1404 male workers from the Valparaíso and Metropolitan regions of Chile were measured in 2016. The sample was grouped by level of education (primary, secondary, technical and university) and age (21-30, 31-40 and 41-50 years). Robust ANOVA and post-hoc analyses using a one-step modified M-estimation of location were conducted based on bootstrap resampling. Both body height and popliteal height increased from the older to the younger age cohort. The largest increase was from the 41-50 to the 21-30 group, with a 1.1% increase in body height and 1.7% increase in popliteal height. When educational level was introduced into the analysis there was a marked increase in both body height and popliteal height for each cohort, but only in primary- and secondary-educated workers. Despite showing an overall increase in body height and popliteal height, younger workers with the highest levels of education showed fewer differences between them than did older workers with less education. The differences were larger in the older than in the younger cohorts. Similarly, this trend was less clear in workers with higher levels of education (technical and university), probably because of a dilution effect caused by increased access to higher education by workers in the lower income quintiles.


The relationship between magnesium and ambulatory blood pressure, augmentation index, pulse wave velocity, total peripheral resistance, and cardiac output in essential hypertensive patients.
Magnesium levels have been shown to be associated with elevated blood pressure (BP), endothelial dysfunction, insulin resistance, vascular calcification, inflammation, and atherosclerosis. It was also demonstrated that patients with hypertension have increased inflammation, insulin resistance, and endothelial dysfunction. However, the relationship between magnesium, ambulatory BPs, and central hemodynamic parameters were not evaluated extensively. Serum magnesium levels, ambulatory blood pressures, augmentation index (Aix), pulse wave velocity, total peripheral resistances, and cardiac output were measured for all patients. In total, 184 essential hypertension patients were enrolled. In univariate analysis, magnesium levels were correlated with hemoglobin (r = +0.155; P = .037), albumin (r = +0.180; P = .018), pulse pressure (daytime; r = -0.170; P = .021), pulse pressure (24-hour; r = -0.156; P = .035), Aix (daytime; r = -0.223; P = .002), Aix (nighttime; r = -0.169; P = .022), and Aix (24-hour; r = -0.247; P = .001). In regression analysis, magnesium levels were independently and conversely associated with daytime Aix (P < .0001), nighttime Aix (P = .019), and 24-hour Aix (P < .0001). We suggest that magnesium levels were associated with Aix but not with total peripheral resistances, pulse wave velocity, cardiac output, and central BPs. The unique mechanisms related with magnesium and Aix but not shared by other central parameters needs to be determined.


A highly accurate platform for clone-specific mutation discovery enables the study of active mutational processes.
Bulk whole genome sequencing (WGS) enables the analysis of tumor evolution but, because of depth limitations, can only identify old mutational events. The discovery of current mutational processes for predicting the tumor's evolutionary trajectory requires dense sequencing of individual clones or single cells. Such studies, however, are inherently problematic because of the discovery of excessive false positive (FP) mutations when sequencing picogram quantities of DNA. Data pooling to increase the confidence in the discovered mutations, moves the discovery back in the past to a common ancestor. Here we report a robust WGS and analysis pipeline (DigiPico/MutLX) that virtually eliminates all F results while retaining an excellent proportion of true positives. Using our method, we identified, for the first time, a hyper-mutation (kataegis) event in a group of ∼30 cancer cells from a recurrent ovarian carcinoma. This was unidentifiable from the bulk WGS data. Overall, we propose DigiPico/MutLX method as a powerful framework for the identification of clone-specific variants at an unprecedented accuracy.


The interaction between Pseudomonas aeruginosa and the corneal epithelium. An electron microscopic study.
The interaction between Pseudomonas aeruginosa and the rabbit corneal epithelium during the first hour after inoculation was studied using scanning and transmission electron microscopy. Fifteen minutes after inoculation, adherent bacteria were found on damaged or exposed basal epithelial cells at the edge of an epithelial defect, and this adherence was the result of an interaction between the bacterial and epithelial cell membranes. Thirty minutes after inoculation, the adherent bacteria began to penetrate the epithelial cells by the formation of "pockets" surrounding the organisms, and after an additional 15 minutes the bacteria further penetrated the cells as the pockets began to fill in with cellular material. One hour after inoculation, only rare bacteria were seen on the cell surfaces or within the margins of the epithelial defect. Transmission electron microscopy, however, revealed many intracellular bacteria or bacteria that had migrated between the basal epithelium and corneal stroma. Therefore, it appears that the initiating events in Pseudomonas corneal ulceration involve adherence to the damaged or exposed basal epithelial cells by an interaction between the bacterial and cellular membranes, after which the organisms are engulfed by the epithelial cell and reach the corneal stroma by a process of transcellular migration.


High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment.
The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H(+) and e(-) transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba(3)-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O(2)-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu(B) atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe(a3) and Cu(B) atoms that is best modeled as peroxide. The structure of ba(3)-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba(3)-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.


Hypertension and occupation among seniors.
Little is known about statistical associations between occupation and hypertension for persons more than age 65. We analyzed cross-sectional data from the 2004 Health and Retirement Survey. We drew 12 gender-specific sub-samples (age 65+; age 70+; age 75+) who reported at least 1 year or 10 years tenure in the same occupation. For 65+ with at least 1 year tenure, n = 3645 men and n = 3644 women. Hypertension was self-reported based on physician diagnosis. Logistic regressions controlled for demographic variables and risk factors. After adjusting for control variables, including education, race, income, smoking, drinking, body mass, and number of comorbidities, the following occupations were significantly (P < 0.05) more likely than managers to report hypertension in at least two of the six sub-samples for women: professionals, salespeople, private household cleaning service workers, and personal service workers. Statistically significant occupations in at least two of the six sub-samples for men included: salespersons, personal service workers, mechanics, construction trades, precisions production workers, and operators. Current and especially pre-retirement occupations were risk factors for hypertension among seniors.


Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse.
Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.


p38 MAPK priming boosts VSMC proliferation and arteriogenesis by promoting PGC1α-dependent mitochondrial dynamics.
Vascular smooth muscle cell (VSMC) proliferation is essential for arteriogenesis to restore blood flow after artery occlusion, but the mechanisms underlying this response remain unclear. Based on our previous findings showing increased VSMC proliferation in the neonatal aorta of mice lacking the protease MT4-MMP, we aimed at discovering new players in this process. We demonstrate that MT4-MMP absence boosted VSMC proliferation in vitro in response to PDGF-BB in a cell-autonomous manner through enhanced p38 MAPK activity. Increased phospho-p38 in basal MT4-MMP-null VSMCs augmented the rate of mitochondrial degradation by promoting mitochondrial morphological changes through the co-activator PGC1α as demonstrated in PGC1α-/- VSMCs. We tested the in vivo implications of this pathway in a novel conditional mouse line for selective MT4-MMP deletion in VSMCs and in mice pre-treated with the p38 MAPK activator anisomycin. Priming of p38 MAPK activity in vivo by the absence of the protease MT4-MMP or by anisomycin treatment led to enhanced arteriogenesis and improved flow recovery after femoral artery occlusion. These findings may open new therapeutic opportunities for peripheral vascular diseases.


Reducing explicit and implicit outgroup prejudice via direct and extended contact: The mediating role of self-disclosure and intergroup anxiety.
In 4 studies, the authors investigated mediators of the effect of cross-group friendship. In Study 1, cross-group friendship among White elementary school children predicted more positive explicit outgroup attitude toward South Asians, mediated by self-disclosure and intergroup anxiety. In Study 2, cross-group friendship and extended contact among White and South Asian high school students positively predicted explicit outgroup attitude, mediated by self-disclosure and intergroup anxiety. Study 3 replicated these findings in a larger independent sample. In all 3 studies, exposure to the outgroup positively predicted implicit outgroup attitude. Study 4 further showed that self-disclosure improved explicit outgroup attitude via empathy, importance of contact, and intergroup trust. The authors discuss the theoretical and practical implications of these findings, which argue for the inclusion of self-disclosure as a key component of social interventions to reduce prejudice.


Child Conduct Problems across Home and School Contexts: A Person-Centered Approach.
To examine patterns of conduct problems across the home and school context, we used latent class analysis to analyze primary caregivers' and teachers' ratings on the conduct problems subscale of the Strengths and Difficulties Questionnaire (R. Goodman, 1997) in the Family Life Project (N = 1,292), a prospective study of child development in rural and small town contexts. We found a similar four-class solution at five and seven years of age. In decreasing prevalence, the following classes were identified: (1) low symptoms reported by both informants (low cross-context); (2) high parent-reported symptoms, low teacher-reported symptoms (home context); (3) low parent-reported symptoms, moderate teacher-reported symptoms (school context); and (4) high symptoms reported by both informants (high cross-context). Classes exhibited stability from age five to age seven: children were more likely to remain in the same class than to transition to a different class, and longitudinal stability was especially high for children in the low cross-context class at age 5. A number of child and family characteristics measured in early childhood (executive function, verbal ability, poverty-related risk, sensitive parenting, and parental depressive symptoms) were associated with class membership at age five and age seven, but were generally not associated with longitudinal transitions between classes.


High-dose antioxidants for central serous chorioretinopathy; the randomized placebo-controlled study.
To determine the efficacy of high-dose antioxidants in the acute stage of central serous chorioretinopathy (CSC). This was a randomized placebo-controlled study. The patients with acute CSC (onset within 6 weeks) were randomized to receive either high-dose antioxidant tablets (study group A) or placebo tablets (control group B) for 3 months or until the complete resolution of subretinal fluid. After 3 months, additional treatment with laser or photodynamic therapy (PDT) was considered if any fluorescein leakage persisted. The outcomes measured were the changes in visual acuity (VA) and central macular thickness (CMT), the number of patients with subretinal fluid at each follow-up time, the number of patients with fluorescein leakage at the end of the 3rd month and patients who received additional treatments. Fifty-one of 58 patients (88%) completed the follow-up criteria. The baseline demographic data were comparable in both groups. At the end of the 3rd month, the VA and CMT showed no statistical difference between the groups but the patients in group A has less fluorescein leakage and additional treatments than in group B (p = 0.027 and 0.03). The high-dose antioxidants for acute CSC did not show any benefits in VA and CMT. However, the drugs might decrease the chance for fluorescein leakage and additional treatments at the end of the 3rd month.


Charge Catastrophe and Dielectric Breakdown During Exposure of Organic Thin Films to Low-Energy Electron Radiation.
The effects of exposure to ionizing radiation are central in many areas of science and technology, including medicine and biology. Absorption of UV and soft-x-ray photons releases photoelectrons, followed by a cascade of lower energy secondary electrons with energies down to 0 eV. While these low energy electrons give rise to most chemical and physical changes, their interactions with soft materials are not well studied or understood. Here, we use a low energy electron microscope to expose thin organic resist films to electrons in the range 0-50 eV, and to analyze the energy distribution of electrons returned to the vacuum. We observe surface charging that depends strongly and nonlinearly on electron energy and electron beam current, abruptly switching sign during exposure. Charging can even be sufficiently severe to induce dielectric breakdown across the film. We provide a simple but comprehensive theoretical description of these phenomena, identifying the presence of a cusp catastrophe to explain the sudden switching phenomena seen in the experiments. Surprisingly, the films undergo changes at all incident electron energies, starting at ∼0 eV.


Somatostatin analogues for receptor targeted photodynamic therapy.
Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2+ AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate.


Neonatal leukaemia.
Neonatal leukaemia is defined as occurring within the first 28 days of life and most, if not all, cases are congenital. With the exception of Down syndrome-associated transient abnormal myelopoiesis, which is not considered here, neonatal leukaemias are rare. In two-thirds of patients the disease manifests as an acute myeloid leukaemia, frequently with monocytic/monoblastic characteristics. Most other cases are acute lymphoblastic leukaemia, particularly B lineage, but some are mixed phenotype or blastic plasmacytoid dendritic cell neoplasms. The most frequently observed cytogenetic/molecular abnormality is t(4;11)(q21.3;q23.3)/KMT2A-AFF1 followed by t(1;22)(p13.3;q13.1)/RBM15-MKL1 and t(8;16)(p11.2;p13.3)/KAT6A-CREBBP. Common clinical features include prominent hepatosplenomegaly and a high incidence of skin involvement, sometimes in the absence of bone marrow disease. A distinctive feature is the occurrence of spontaneous remission in some cases, particularly in association with t(8;16). In this review, we summarise current knowledge of the clinical, cytogenetic and molecular features of neonatal leukaemia and discuss clinical management of these cases.


Current evidence of the efficacy of cell-based therapies in heart failure.
Heart failure (HF) is the major cause of mortality worldwide. For more than a decade, cell-based therapies have been developed as treatment for heart disease as an alternative to current therapies. Trials and systematic reviews have assessed the safety and efficacy of cell therapies in a diverse number of participants and clinical settings. The present study collated and synthesized evidence from all systematic reviews related to cell-based therapies and HF. A total of 11 systematic reviews were identified through searches of electronic databases up to June 2014. We set out to answer 2 key questions on the efficacy of cell therapies in HF: (1) What is the overall effect of cell therapies on primary outcomes such as left ventricular ejection fraction (LVEF) and mortality? (2) How important is it to define the clinical setting and length of follow-up when assessing cell therapies and HF? There seems to be enough evidence to suggest that cell therapies have a moderate, long-lasting effect on LVEF, but the reduction on the risk of mortality observed by some systematic reviews needs to be confirmed in larger, statistically powered clinical trials. Additionally, and in order to strengthen conclusions, it is important to assess clinical evidence for defined clinical settings and to standardize the length of follow-up when comparing outcome data across several trials and systematic reviews.


Ecological fragmentation effects in mouse lemurs and small mammals in northwestern Madagascar.
Habitat loss and fragmentation are major ecological forces threatening animal communities across the globe. These issues are especially true in Madagascar, where forest loss is ongoing. We examined the effects of forest fragmentation on the distribution and abundance of sympatric, endemic gray, and golden-brown mouse lemurs (Microcebus murinus and Microcebus ravelobensis), the endemic western tuft-tailed rat (Eliurus myoxinus), and the invasive black rat (Rattus rattus) in two regions in northwestern Madagascar. We used systematic capture procedures in 40 forest fragments and four continuous forest sites which differed in size, shape, and degree of isolation. With a trapping effort of 11,567 trap nights during two dry seasons (2017-2018), we captured 929 individuals (432 M. ravelobensis, 196 M. murinus, 116 E. myoxinus, and 185 R. rattus). We examined the influence of study region, forest type (fragment vs. continuous), forest size, forest shape, the proportion of 50-m forest edge and distance to continuous forest on the abundance and interaction of the four species. Responses to fragmentation differed strongly between species, but no interaction could be detected between the abundance of the different species. Thus competition within and between native and invasive species may not be regulating abundances in these regions. On the contrary, the abundance of M. ravelobensis and E. myoxinus differed significantly between study regions and was negatively affected by fragmentation. In contrast, there was no evidence of an impact of fragmentation on the abundance of M. murinus. Finally, the invasive R. rattus responded positively to the increasing distance to the continuous forest. In conclusion, the response of small Malagasy mammals to forest fragmentation varies largely between species, and fragmentation effects need to be examined at a species-specific level to fully understand their ecological dynamics and complexity.


Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.
Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 18.104.22.168), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.


How COVID-19 vaccine supply chains emerged in the midst of a pandemic.
Many months after COVID-19 vaccines were first authorised for public use, still limited supplies could only partially reduce the devastating loss of life and economic costs caused by the pandemic. Could additional vaccine doses have been manufactured more quickly some other way? Would alternative policy choices have made a difference? This paper provides a simple analytical framework through which to view the contours of the vaccine value chain. It then creates a new database that maps the COVID-19 vaccines of Pfizer/BioNTech, Moderna, AstraZeneca/Oxford, Johnson & Johnson, Novavax and CureVac to the product- and location-specific manufacturing supply chains that emerged in 2020 and 2021. It describes the choppy process through which dozens of other companies at nearly 100 geographically distributed facilities came together to scale up global manufacturing. The paper catalogues major pandemic policy initiatives - such as the United States' Operation Warp Speed - that are likely to have affected the timing and formation of those vaccine supply chains. Given the data, a final section identifies further questions for researchers and policymakers.


Larval parasitoid uses aggregation pheromone of adult hosts in foraging behaviour: a solution to the reliability-detectability problem.
Parasitoids that forage for herbivorous hosts by using infochemicals may have a problem concerning the reliability and detectability of these stimuli: host stimuli are highly reliable but not very detectable at a distance, while stimuli from the host's food are very detectable but generally not very reliable in indicating host presence. One solution to this problem is to learn to link highly detectable stimuli to reliable but not very detectable stimuli. Ample knowledge is available on how associative learning aids foraging parasitoids in the location of suitable microhabitats. However, in this paper we report on another solution to the reliability-detectability problem and present evidence for an essential, but as yet overlooked, aspect of Drosophila parasitoid ecology. For the first time it is shown that a parasitoid of Drosophila larvae spies on the communication system of adult Drosophila flies to locate potential host sites: naive parasitoids strongly respond to a volatile aggregation pheromone that is deposited in the oviposition site by recently mated female flies. Thus, the parasitoids resort to using highly detectable information from a host stage different from the one under attack (i.e. infochemical detour). The function and ecological implications of these findings are discussed.


Assessment of Quality of Life and Safety in Postmenopausal Breast Cancer Patients Receiving Letrozole as an Early Adjuvant Treatment.
There are few reports from Asian countries about the long-term results of aromatase inhibitor adjuvant treatment for breast cancer. This observational study aimed to evaluate the long-term effects of letrozole in postmenopausal Korean women with operable breast cancer. Self-reported quality of life (QoL) scores were serially assessed for 3 years during adjuvant letrozole treatment using the Korean version of the Functional Assessment of Cancer Therapy-Breast questionnaires (version 3). Changes in bone mineral density (BMD) and serum cholesterol levels were also examined. All 897 patients received the documented informed consent form and completed a baseline questionnaire before treatment. Adjuvant chemotherapy was administered to 684 (76.3%) subjects, and 410 (45.7%) and 396 (44.1%) patients had stage I and II breast cancer, respectively. Each patient completed questionnaires at 3, 6, 12, 18, 24, 30, and 36 months after enrollment. Of 897 patients, 749 (83.5%) completed the study. The dropout rate was 16.5%. The serial trial outcome index, the sum of the physical and functional well-being subscales, increased gradually and significantly from baseline during letrozole treatment (p<0.001). The mean serum cholesterol level increased significantly from 199 to 205 after 36 months (p=0.042). The mean BMD significantly decreased from -0.39 at baseline to -0.87 after 36 months (p<0.001). QoL gradually improved during letrozole treatment. BMD and serum cholesterol level changes were similar to those in Western countries, indicating that adjuvant letrozole treatment is well tolerated in Korean women, with minimal ethnic variation.


A Randomized Trial of the Effect of E-cigarette TV Advertisements on Intentions to Use E-cigarettes.
Adolescents' use of electronic cigarettes (e-cigarettes) and exposure to e-cigarette TV advertising have increased in recent years, despite questions about their safety. The current study tests whether exposure to e-cigarette TV advertisements influences intentions to use e-cigarettes in the future and related attitudes. A parallel-group randomized controlled experiment was conducted and analyzed in 2014 using an online survey with a convenience sample of 3,655 U.S. adolescents aged 13-17 years who had never tried e-cigarettes. Adolescents in the treatment group viewed four e-cigarette TV advertisements. Adolescents in the treatment group reported a greater likelihood of future e-cigarette use compared with the control group. ORs for the treatment group were 1.54 (p=0.001) for trying an e-cigarette soon; 1.43 (p=0.003) for trying an e-cigarette within the next year; and 1.29 (p=0.02) for trying an e-cigarette if a best friend offered one. Adolescents in the treatment group had higher odds of agreeing that e-cigarettes can be used in places where cigarettes are not allowed (OR=1.71, p<0.001); can be used without affecting those around you (OR=1.83, p<0.001); are a safer alternative to cigarettes (OR=1.19, p=0.01); and are less toxic (OR=1.16, p=0.03). Exposure to e-cigarette advertising had relatively large and consistent effects across experimental outcomes. Together with the simultaneous increase in e-cigarette advertising exposure and e-cigarette use among adolescents, findings suggest that e-cigarette advertising is persuading adolescents to try this novel product. This raises concerns that continued unregulated e-cigarette advertising will contribute to potential individual- and population-level harm.


Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant).
A microbial surfactant (biosurfactant) was investigated for its potential to enhance bioavailability and, hence, the biodegradation of octadecane. The rhamnolipid biosurfactant used in this study was extracted from culture supernatants after growth of Pseudomonas aeruginosa ATCC 9027 in phosphate-limited proteose peptone-glucose-ammonium salts medium. Dispersion of octadecane in aqueous solutions was dramatically enhanced by 300 mg of the rhamnolipid biosurfactant per liter, increasing by a factor of more than 4 orders of magnitude, from 0.009 to > 250 mg/liter. The relative enhancement of octadecane dispersion was much greater at low rhamnolipid concentrations than at high concentrations. Rhamnolipid-enhanced octadecane dispersion was found to be dependent on pH and shaking speed. Biodegradation experiments done with an initial octadecane concentration of 1,500 mg/liter showed that 20% of the octadecane was mineralized in 84 h in the presence of 300 mg of rhamnolipid per liter, compared with only 5% octadecane mineralization when no surfactant was present. These results indicate that rhamnolipids may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility.


Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.
N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.


Effectiveness of nature reserve system for conserving tropical forests: a statistical evaluation of Hainan Island, China.
Evaluating the effectiveness of existing nature reserve systems for the conservation of tropical forests is an urgent task to save the remaining biodiversity. Here, we tested the effectiveness of the reserve system on Hainan Island by conducting a three-way comparison of changes in forest area in locations within the reserves, adjacent to the reserves, and far outside of the reserves. We used a general linear model to control for the effects of covariates (historical forest area, elevation, slope, and distance to nearest roads), which may also be correlated with the changes in forest area, to better explain the effectiveness of the reserve system. From 2000 to 2010, the forest area inside Hainan's nature reserve system showed an increase while adjacent unprotected areas and the wider, unprotected landscape both experienced deforestation. However, the simple inside-outside comparisons may overestimate the protective effect of the reserve system. Most nature reserves (>60%) showed increasing fragmentation. And the risk of rapid deforestation remained high at low elevations, where remaining forests tend to be easily logged and converted to commercial plantations. Future conservation efforts should pay more attention to those sites with less challenging environmental conditions.


Magnetic resonance imaging and histology correlation in the neocortex in temporal lobe epilepsy.
To investigate the histopathological correlates of quantitative relaxometry and diffusion tensor imaging (DTI) and to determine their efficacy in epileptogenic lesion detection for preoperative evaluation of focal epilepsy. We correlated quantitative relaxometry and DTI with histological features of neuronal density and morphology in 55 regions of the temporal lobe neocortex, selected from 13 patients who underwent epilepsy surgery. We made use of a validated nonrigid image registration protocol to obtain accurate correspondences between in vivo magnetic resonance imaging and histology images. We found T1 to be a predictor of neuronal density in the neocortical gray matter (GM) using linear mixed effects models with random effects for subjects. Fractional anisotropy (FA) was a predictor of neuronal density of large-caliber neurons only (pyramidal cells, layers 3 and 5). Comparing multivariate to univariate mixed effects models with nested variables demonstrated that employing T1 and FA together provided a significantly better fit than T1 or FA alone in predicting density of large-caliber neurons. Correlations with clinical variables revealed significant positive correlations between neuronal density and age (rs = 0.726, pfwe = 0.021). This study is the first to relate in vivo T1 and FA values to the proportion of neurons in GM. Our results suggest that quantitative T1 mapping and DTI may have a role in preoperative evaluation of focal epilepsy and can be extended to identify GM pathology in a variety of neurological disorders.


Holistic Human-Serving Digitization of Health Care Needs Integrated Automated System-Level Assessment Tools.
Digital health tools, platforms, and artificial intelligence- or machine learning-based clinical decision support systems are increasingly part of health delivery approaches, with an ever-greater degree of system interaction. Critical to the successful deployment of these tools is their functional integration into existing clinical routines and workflows. This depends on system interoperability and on intuitive and safe user interface design. The importance of minimizing emergent workflow stress through human factors research and purposeful design for integration cannot be overstated. Usability of tools in practice is as important as algorithm quality. Regulatory and health technology assessment frameworks recognize the importance of these factors to a certain extent, but their focus remains mainly on the individual product rather than on emergent system and workflow effects. The measurement of performance and user experience has so far been performed in ad hoc, nonstandardized ways by individual actors using their own evaluation approaches. We propose that a standard framework for system-level and holistic evaluation could be built into interacting digital systems to enable systematic and standardized system-wide, multiproduct, postmarket surveillance and technology assessment. Such a system could be made available to developers through regulatory or assessment bodies as an application programming interface and could be a requirement for digital tool certification, just as interoperability is. This would enable health systems and tool developers to collect system-level data directly from real device use cases, enabling the controlled and safe delivery of systematic quality assessment or improvement studies suitable for the complexity and interconnectedness of clinical workflows using developing digital health technologies.


Obinutuzumab is Effective in Chronic Lymphocytic Leukemia and Rheumatoid Arthritis After Rituximab Failure: A Case Report.
BACKGROUND Chronic lymphocytic leukemia (CLL) is the most common leukemia affecting older adults. As such, many of these patients suffer from co-existing disease states, and the provider must take these comorbidities into account when determining a treatment regimen. The widespread use of monoclonal antibodies (mAbs) has drastically changed the treatment landscape of multiple diseases, ranging from leukemia to autoimmune conditions such as rheumatoid arthritis. CASE REPORT We present the case of a patient who had progression of his CLL and rheumatoid symptoms on rituximab therapy, and was subsequently treated with the second-generation anti-CD20 antibody obinutuzumab. Obinutuzumab therapy was associated with simultaneous sustained remission of both disease states, allowing for discontinuation of all other disease-modifying anti-rheumatic drugs (DMARDs), and prolonged remission of his CLL. CONCLUSIONS While anti-CD20 antibodies have a clear role in the treatment of leukemia and inflammatory conditions, the success of obinutuzumab in RA has not been fully evaluated. We present this case as further evidence of the strong role of anti-CD 20 therapy in multiple conditions, and the unique opportunity for control of simultaneous disease states through targeted inhibition of shared common pathways.


Vitamin E deficiency induced neurological disease in common variable immunodeficiency: two cases and a review of the literature of vitamin E deficiency.
Vitamin E deficiency causes a neurological disorder characterised by sensory loss, ataxia and retinitis pigmentosa due to free radical mediated neuronal damage. Symptomatic vitamin E deficiency has been reported in genetic defects of the vitamin E transport protein and in malabsorption complicating cholestasis, abetalipoproteinaemia, celiac disease, cystic fibrosis and small bowel resection. There are no reports to date of vitamin E deficiency in patients with primary immunodeficiencies. We describe two CVID patients with the associated enteropathy who developed neurological disease because of vitamin E deficiency, suggesting a possible predisposition to developing this complication. We recommend that all CVID patients with evidence of an enteropathy be screened for vitamin E deficiency, as early detection and consequent treatment may prevent, halt or reverse the neurological sequelae.


Plasmonic waveguides based on symmetric and asymmetric T-shaped structures.
We describe the fabrication and characterization of plasmonic waveguides based on a periodic one-dimensional array of symmetric and asymmetric T-shaped structures. The devices are fabricated in a polymer resin using conventional 3D printing and subsequently overcoated with ~500 nm of Au. Using terahertz (THz) time-domain spectroscopy, we systematically measure the guided-wave transmission properties of the devices as a function of the different geometrical parameters. Through these measurements, we find that the resonance frequency associated with the lowest order mode depends primarily on the structure height and the cap width and appears to be independent of its lateral width. We also perform numerical simulations using the same geometrical parameters and find excellent agreement between experiment and simulation. We fabricate a waveguide in which the lateral width of the T-shaped structures is tapered in a linear fashion. While the spectrum of this device is similar to one without tapering, we observe relatively little reduction in the mode size, even as the structure width is reduced by a factor of eight.


HSulf-1 deficiency dictates a metabolic reprograming of glycolysis and TCA cycle in ovarian cancer.
Warburg effect has emerged as a potential hallmark of many cancers. However, the molecular mechanisms that led to this metabolic state of aerobic glycolysis, particularly in ovarian cancer (OVCA) have not been completely elucidated. HSulf-1 predominantly functions by limiting the bioavailability of heparan binding growth factors and hence their downstream signaling. Here we report that HSulf-1, a known putative tumor suppressor, is a negative regulator of glycolysis. Silencing of HSulf-1 expression in OV202 cell line increased glucose uptake and lactate production by upregulating glycolytic genes such as Glut1, HKII, LDHA, as well as metabolites. Conversely, HSulf-1 overexpression in TOV21G cells resulted in the down regulation of glycolytic enzymes and reduced glycolytic phenotype, supporting the role of HSulf-1 loss in enhanced aerobic glycolysis. HSulf-1 deficiency mediated glycolytic enhancement also resulted in increased inhibitory phosphorylation of pyruvate dehydrogenase (PDH) thus blocking the entry of glucose flux into TCA cycle. Consistent with this, metabolomic and isotope tracer analysis showed reduced glucose flux into TCA cycle. Moreover, HSulf-1 loss is associated with lower oxygen consumption rate (OCR) and impaired mitochondrial function. Mechanistically, lack of HSulf-1 promotes c-Myc induction through HB-EGF-mediated p-ERK activation. Pharmacological inhibition of c-Myc reduced HB-EGF induced glycolytic enzymes implicating a major role of c-Myc in loss of HSulf-1 mediated altered glycolytic pathway in OVCA. Similarly, PG545 treatment, an agent that binds to heparan binding growth factors and sequesters growth factors away from their ligand also blocked HB-EGF signaling and reduced glucose uptake in vivo in HSulf-1 deficient cells.


Cross-term selective, two-pulse correlation measurements by phase-shifted parallel modulation for analysis of a multi-photon process.
We propose a new modulation technique for phase-sensitive detection to extract a cross-term signal appearing in multi-pulse experiments and demonstrate the selective detection of cross-term signal on two-pulse correlation measurements. This modulation technique can be widely applied to the selective detection in multi-beam laser experiments such as two-pulse correlation, auto-correlation, and double-resonance experiments, because the selectivity can be realized by using a conventional single-frequency mechanical chopper and 2f phase-sensitive detection. The effectiveness of the proposed technique was confirmed by two-pulse correlation measurements of two-photon-excited fluorescence from rhodamine 6G. In addition to the selective observation of a correlation peak, the technique succeeded in observing a weak correlation background. By combining asymmetric, two-pulse correlation measurements with the proposed technique, it was clarified that the background was produced by a three-photon process and assigned to fluorescence depletion caused by an up-conversion process. The results indicate that the cross-term selective, two-pulse correlation method that is based on the phase-shifted parallel modulation technique is powerful tool to find and analyze a high-order optical event buried in a low-order optical event.


Focused ion beam manipulation and ultramicroscopy of unprepared cells.
The focused ion beam (FIB) technique of nanomachining combined with simultaneous scanning electron microscopy (SEM) was used for submicron manipulation and imaging of unprepared (fresh) cells to demonstrate the potentiality of the FIB/SEM technique for ultramicroscopic studies. Sectioning at the nanoscale level was successfully performed by means of ion beam-driven milling operations that reveal the ultrastructure of fresh yeast cells. The FIB/SEM has many advantages over other ultramicroscopy techniques already applied for unprepared/fresh biological samples.


Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.
The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.


Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation.
We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-gamma inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.


Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli.
Iron-based nanoparticles have been proposed for an increasing number of biomedical or environmental applications although in vitro toxicity has been observed. The aim of this study was to understand the relationship between the redox state of iron-based nanoparticles and their cytotoxicity toward a Gram-negative bacterium, Escherichia coli. While chemically stable nanoparticles (gammaFe2O3) have no apparent cytotoxicity, nanoparticles containing ferrous and, particularly, zerovalent iron are cytotoxic. The cytotoxic effects appear to be associated principally with an oxidative stress as demonstrated using a mutant strain of E. coli completely devoid of superoxide dismutase activity. This stress can result from the generation of reactive oxygen species with the interplay of oxygen with reduced iron species (Fe(II) and/or Fe(0)) or from the disturbance of the electronic and/or ionic transport chains due to the strong affinity of the nanoparticles for the cell membrane.


Primaquine ineligibility in anti-relapse therapy of Plasmodium vivax malaria: the problem of G6PD deficiency and cytochrome P-450 2D6 polymorphisms.
The hypnozoite reservoir of Plasmodium vivax represents both the greatest obstacle and opportunity for ultimately eradicating this species. It is silent and cannot be diagnosed until it awakens and provokes a clinical attack with attendant morbidity, risk of mortality, and opportunities for onward transmission. The only licensed drug that kills hypnozoites is primaquine, which attacks the hypnozoite reservoir but imposes serious obstacles in doing so-at hypnozoitocidal doses, it invariably causes a threatening acute haemolytic anaemia in patients having an inborn deficiency in glucose-6-phosphate dehydrogenase (G6PD), affecting about 8% of people living in malaria endemic nations. That problem excludes a large number of people from safe and effective treatment of the latent stage of vivax malaria: the G6PD deficient, pregnant or lactating women, and young infants. These groups were estimated to comprise 14.3% of populations resident in the 95 countries with endemic vivax malaria. Another important obstacle regarding primaquine in the business of killing hypnozoites is its apparent metabolism to an active metabolite exclusively via cytochrome P-450 isozyme 2D6 (CYP2D6). Natural polymorphisms of this allele create genotypes expressing impaired enzymes that occur in over 20% of people living in Southeast Asia, where more than half of P. vivax infections occur globally. Taken together, the estimated frequencies of these primaquine ineligibles due to G6PD toxicity or impaired CYP2D6 activity composed over 35% of the populations at risk of vivax malaria. Much more detailed work is needed to refine these estimates, derive probabilities of error for them, and improve their ethnographic granularity in order to inform control and elimination strategy and tactics.