Speech-like and non-speech lip kinematics and coordination in aphasia.
In addition to the well-known linguistic processing impairments in aphasia, oro-motor skills and articulatory implementation of speech segments are reported to be compromised to some degree in most types of aphasia. This study aimed to identify differences in the characteristics and coordination of lip movements in the production of a bilabial closure gesture between speech-like and non-speech tasks in individuals with aphasia and healthy control subjects. Upper and lower lip movement data were collected for a speech-like and a non-speech task using an AG 100 EMMA system from five individuals with aphasia and five age- and gender-matched control subjects. Each task was produced at two rate conditions (normal and fast), and in a familiar and a less familiar manner. Single articulator kinematic parameters (peak velocity, amplitude, duration and cyclic spatio-temporal index) and multi-articulator coordination indices (average relative phase and variability of relative phase) were measured to characterize lip movements. The results showed that when the two lips had similar task goals (bilabial closure) in speech-like and non-speech task, kinematic and coordination characteristics were not found to be different. However, when changes in rate were imposed on the bilabial gesture, only speech-like task showed functional adaptations, indicated by a greater decrease in amplitude and duration at fast rates. In terms of group differences, individuals with aphasia showed smaller amplitudes and longer movement durations for upper lip, higher spatio-temporal variability for both lips, and higher variability in lip coordination than the control speakers. Rate was an important factor in distinguishing the two groups, and individuals with aphasia were limited in implementing the rate changes. The findings support the notion of subtle but robust differences in motor control characteristics between individuals with aphasia and the control participants, even in the context of producing bilabial closing gestures for a relatively simple speech-like task. The findings also highlight the functional differences between speech-like and non-speech tasks, despite a common movement coordination goal for bilabial closure.


Health-related quality of life among men with HIV infection: effects of social support, coping, and depression.
In a prospective cohort study, baseline data were collected on 226 males with HIV infection attending three infectious disease clinics in a southern state. As a result of advances in HIV treatment, understanding the association between psychosocial factors and health-related quality of life has become an important area of study. The purpose of this study was to assess the total effects of social support and coping as well as the direct and indirect effects of these factors through depression on health-related quality of life. Subjects were interviewed and answered the following standardized questionnaires: The HIV Cost and Services Utilization Study tool, Coping with HIV Questionnaire, Social Support Appraisals Scale, and Centers for Epidemiological Studies Depression instrument. Path models with strictly ordered relationships were fitted to study the effects of the psychosocial variables on each quality of life subscale. We found that coping and social support had total effects on some, but not all dimensions of health-related quality of life, whereas depression was associated with all dimensions of health-related quality of life. Furthermore, the effects of both social support and coping were mainly through the intermediate variable, depression. In the era of highly active antiretroviral therapy (HAART), when quality of life issues are of paramount importance, strategies to improve social support, coping, and particularly, depressive symptoms are strongly encouraged.


Role of S-Palmitoylation by ZDHHC13 in Mitochondrial function and Metabolism in Liver.
Palmitoyltransferase (PAT) catalyses protein S-palmitoylation which adds 16-carbon palmitate to specific cysteines and contributes to various biological functions. We previously reported that in mice, deficiency of Zdhhc13, a member of the PAT family, causes severe phenotypes including amyloidosis, alopecia, and osteoporosis. Here, we show that Zdhhc13 deficiency results in abnormal liver function, lipid abnormalities, and hypermetabolism. To elucidate the molecular mechanisms underlying these disease phenotypes, we applied a site-specific quantitative approach integrating an alkylating resin-assisted capture and mass spectrometry-based label-free strategy for studying the liver S-palmitoylome. We identified 2,190 S-palmitoylated peptides corresponding to 883 S-palmitoylated proteins. After normalization using the membrane proteome with TMT10-plex labelling, 400 (31%) of S-palmitoylation sites on 254 proteins were down-regulated in Zdhhc13-deficient mice, representing potential ZDHHC13 substrates. Among these, lipid metabolism and mitochondrial dysfunction proteins were overrepresented. MCAT and CTNND1 were confirmed to be specific ZDHHC13 substrates. Furthermore, we found impaired mitochondrial function in hepatocytes of Zdhhc13-deficient mice and Zdhhc13-knockdown Hep1-6 cells. These results indicate that ZDHHC13 is an important regulator of mitochondrial activity. Collectively, our study allows for a systematic view of S-palmitoylation for identification of ZDHHC13 substrates and demonstrates the role of ZDHHC13 in mitochondrial function and metabolism in liver.


Life-threatening infections in children in Europe (the EUCLIDS Project): a prospective cohort study.
Sepsis and severe focal infections represent a substantial disease burden in children admitted to hospital. We aimed to understand the burden of disease and outcomes in children with life-threatening bacterial infections in Europe. The European Union Childhood Life-threatening Infectious Disease Study (EUCLIDS) was a prospective, multicentre, cohort study done in six countries in Europe. Patients aged 1 month to 18 years with sepsis (or suspected sepsis) or severe focal infections, admitted to 98 participating hospitals in the UK, Austria, Germany, Lithuania, Spain, and the Netherlands were prospectively recruited between July 1, 2012, and Dec 31, 2015. To assess disease burden and outcomes, we collected demographic and clinical data using a secured web-based platform and obtained microbiological data using locally available clinical diagnostic procedures. 2844 patients were recruited and included in the analysis. 1512 (53·2%) of 2841 patients were male and median age was 39·1 months (IQR 12·4-93·9). 1229 (43·2%) patients had sepsis and 1615 (56·8%) had severe focal infections. Patients diagnosed with sepsis had a median age of 27·6 months (IQR 9·0-80·2), whereas those diagnosed with severe focal infections had a median age of 46·5 months (15·8-100·4; p<0·0001). Of 2844 patients in the entire cohort, the main clinical syndromes were pneumonia (511 [18·0%] patients), CNS infection (469 [16·5%]), and skin and soft tissue infection (247 [8·7%]). The causal microorganism was identified in 1359 (47·8%) children, with the most prevalent ones being Neisseria meningitidis (in 259 [9·1%] patients), followed by Staphylococcus aureus (in 222 [7·8%]), Streptococcus pneumoniae (in 219 [7·7%]), and group A streptococcus (in 162 [5·7%]). 1070 (37·6%) patients required admission to a paediatric intensive care unit. Of 2469 patients with outcome data, 57 (2·2%) deaths occurred: seven were in patients with severe focal infections and 50 in those with sepsis. Mortality in children admitted to hospital for sepsis or severe focal infections is low in Europe. The disease burden is mainly in children younger than 5 years and is largely due to vaccine-preventable meningococcal and pneumococcal infections. Despite the availability and application of clinical procedures for microbiological diagnosis, the causative organism remained unidentified in approximately 50% of patients. European Union's Seventh Framework programme.


Tumor location is an independent predictive factor for distant metastasis and metastatic sites of rectal adenocarcinoma in patients receiving total mesorectal excision.
Background and Objectives: To evaluate the predictive factor for and patterns of distant metastasis in patients with rectal adenocarcinoma receiving total mesorectal excision (TME). Methods: We enrolled 217 consecutive patients who had histologically confirmed rectal adenocarcinoma and underwent surgery at Taipei Medical University- Wanfang Hospital between January 2000 and December 2014. TME was performed in all patients undergoing a sphincter-sparing procedure or abdominal perineal resection of rectal cancer. We performed univariate and multivariate Cox regression analyses of the distant metastasis rate in all patients to evaluate predictive factors. Overall survival (OS) rates were calculated using the Kaplan-Meier method, and Kaplan-Meier survival curves were compared using the log-rank test. Results: A multivariate Cox regression analysis of the distant metastasis rate in patients with rectal adenocarcinoma identified tumor locations and American Joint Committee on Cancer (AJCC) stages as prognostic risk factors. The adjusted hazard ratios (aHRs) of distant metastasis for the upper-third, middle-third, and AJCC stage I-II cancers were 0.08 (95% CI, 0.01-0.69; p = 0.021), 0.41 (95% CI, 0.15-0.99; p = 0.047), and 0.20 (95% CI, 0.10-0.66; p = 0.008), respectively. The 5-year lung metastasis rates among patients with upper-, middle-, and lower-third rectal cancers were 0%, 3.37%, and 13.33%, respectively (log-rank, p = 0.001), and the 5-year liver metastasis rates among patients with upper-, middle-, and lower-third rectal cancers were 2.12%, 9.10%, and 11.76%, respectively (log-rank, p = 0.096). The 5-year OS rates also differed with different rectal adenocarcinoma locations. The 5-year OS rates for upper, middle, and lower rectal cancers were 96%, 86%, and 64%, respectively (log-rank, p < 0.001). Conclusion: A poor OS rate and high lung or liver metastasis rate were observed in distal rectal adenocarcinoma. Longer intensive surveillance of the chest, abdomen, and pelvis after TME in distal rectal adenocarcinoma could be necessary.


The working hours of hospital staff nurses and patient safety.
The use of extended work shifts and overtime has escalated as hospitals cope with a shortage of registered nurses (RNs). Little is known, however, about the prevalence of these extended work periods and their effects on patient safety. Logbooks completed by 393 hospital staff nurses revealed that participants usually worked longer than scheduled and that approximately 40 percent of the 5,317 work shifts they logged exceeded twelve hours. The risks of making an error were significantly increased when work shifts were longer than twelve hours, when nurses worked overtime, or when they worked more than forty hours per week.


Aqueous flow in humans after adrenalectomy.
This study was performed to determine if the circadian rhythm of aqueous humor formation and the aqueous humor suppressing effect of beta-adrenergic antagonists can occur in the absence of adrenally derived epinephrine. Twenty-one human subjects who had undergone bilateral adrenalectomy were studied during a 28-hour period. The study was divided into four time periods as follows: morning 1 (8 AM to noon), afternoon (noon to 4 PM), night (midnight to 6 AM), and morning 2 (8 AM to noon). At 6:45 AM before the morning 2 measurements, one drop of 0.5% timolol was applied to one eye and one drop of placebo (artificial tears) was applied to the other eye. Topical fluorescein and a scanning fluorophotometer were used to measure the rate of aqueous humor flow. Twenty normal controls were studied in a similar fashion but did not undergo the morning 2 measurement. In the subjects lacking adrenals, the daytime rates of aqueous flow were 3.17 +/- 0.78 microliters/min (mean +/- SD) and 3.16 +/- 0.67 microliters/min for the morning 1 and afternoon periods, respectively. The rates in daytime periods were not significantly different from each other (P = 0.699). The rate of aqueous flow for the night period was 1.37 +/- 0.37 microliters/min, a 57% reduction from both morning 1 and afternoon periods (P < 0.001). The morning, afternoon, and night rates of flow in normal controls were not significantly different from the rates in subjects lacking adrenals. For the morning 2 period, the aqueous flow was 2.74 +/- 0.54 microliters/min for the placebo-treated eye and 1.77 +/- 0.38 microliters/min for the timolol-treated eye. The rate of aqueous flow was reduced (35%) in the timolol-treated eye when compared to the fellow placebo-treated eye (P < 0.001). The timolol-treated eye also showed a 26% reduction in intraocular pressure when compared to the fellow placebo-treated eye (P < 0.001). The study demonstrates that both the circadian rhythm of aqueous flow and the daytime response to timolol persist in the absence of the adrenal glands.


Near-field investigations of the landers earthquake sequence, april to july 1992.
The Landers earthquake, which had a moment magnitude (M(w)) of 7.3, was the largest earthquake to strike the contiguous United States in 40 years. This earthquake resulted from the rupture of five major and many minor right-lateral faults near the southern end of the eastern California shear zone, just north of the San Andreas fault. Its M(w) 6.1 preshock and M(w) 6.2 aftershock had their own aftershocks and foreshocks. Surficial geological observations are consistent with local and far-field seismologic observations of the earthquake. Large surficial offsets (as great as 6 meters) and a relatively short rupture length (85 kilometers) are consistent with seismological calculations of a high stress drop (200 bars), which is in turn consistent with an apparently long recurrence interval for these faults.


Outcomes of a Hepatitis C screening program at a large urban VA medical center.
To determine the outcomes of implementing clinical care guidelines for Hepatitis C screening, evaluation, and treatment in a large urban Veterans Affairs Medical Center. Little information exists regarding the actual outcomes of institutional screening programs for Hepatitis C. Retrospective review of all patients tested for Hepatitis C at the Minneapolis Veterans Affairs Medical Center from January 1, 2000 to December 31, 2001. Logistic regression was used to determine factors related to successful referral and treatment. During this period 36,422 unique patients were screened for Hepatitis C virus (HCV) risk factors, resulting in 12,485 HCV enzyme-linked immunoassay antibody tests. HCV antibodies were positive in 681 (5.4%) patients and 520 (4.2%) were HCV-RNA-positive. Of HCV-RNA-positive patients, 430 (83%) were referred, 382 (73%) attended the Hepatitis clinic, and 232 (44.6%) received liver biopsies. Patients referred had significantly fewer comorbidities, known marital status, and greater prior clinic attendance than those not referred. Overall, 124 patients with established fibrosis received antiviral therapy (32% of patients attending clinic or 24% of viremic cohort). White race, fewer major medical problems, and age less than 60 years predicted antiviral treatment. Sustained virologic response occurred in 46 (37%) of treated patients (9% of the viremic cohort). Patients with a sustained virologic response include 17 patients with stage 3 to 4 fibrosis. This screening and referral program resulted in 73% of HCV-RNA-positive patients attending a specialty Hepatitis C clinic and 24% of those most likely to benefit received antiviral therapy. Measures to increase referral, engagement in care, and antiviral treatment are needed.


A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology.
Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.


Impact of the chemical nature and position of spacers on controlling the optical properties of silicon quantum dots.
The unique properties of silicon quantum dots (SQDs), including intriguing optical properties, biocompatibility, and ease of surface modification have made them excellent candidates for a variety of optoelectronic and biomedical applications. Unfortunately, the low quantum efficiency (QE), unstable photoluminescence, and poor colloidal stability of SQDs have hindered their wide applicability. Herein, we report the synthesis of four assemblies of SQDs (1.6-1.8 nm average diameter) functionalized with fluorescein dye through isothiocyanate (-NCS) and carboxylate (COO-) spacers in the benzene ring of the fluorescein to produce the dyads Am-SQD-Fl, DiAm-SQD-Fl, urea-SQD-Fl, and SQD-Fl. The photophysical measurements showed that the spacer played a key role in directing and controlling the optical properties of SQDs dyads, with the isothiocyanate spacer leading to a significant improvement in the QE of the dyad systems up to 65% and extending their photostability for at least one year. The interactions between the SQDs and fluorescein in the dyads Am-SQD-Fl, DiAm-SQD-Fl, and SQD-Fl were found to mainly proceed through photoinduced electron transfer at different rates, while energy transfer was confirmed to be the predominant process in the dyad urea-SQD-Fl. To demonstrate the suitability of the functionalized SQDs for bioimaging applications, the water-soluble dyads were examined for fluorescence imaging of human bone cancerous U2OS cells.


Use of medical face masks versus particulate respirators as a component of personal protective equipment for health care workers in the context of the COVID-19 pandemic.
Currently available evidence supports that the predominant route of human-to-human transmission of the SARS-CoV-2 is through respiratory droplets and/or contact routes. The report by the World Health Organization (WHO) Joint Mission on Coronavirus Disease 2019 (COVID-19) in China supports person-to-person droplet and fomite transmission during close unprotected contact with the vast majority of the investigated infection clusters occurring within families, with a household secondary attack rate varying between 3 and 10%, a finding that is not consistent with airborne transmission. The reproduction number (R0) for the SARS-CoV-2 is estimated to be between 2.2-2.7, compatible with other respiratory viruses associated with a droplet/contact mode of transmission and very different than an airborne virus like measles with a R0 widely cited to be between 12 and 18. Based on the scientific evidence accumulated to date, our view is that SARS-CoV-2 is not spread by the airborne route to any significant extent and the use of particulate respirators offers no advantage over medical masks as a component of personal protective equipment for the routine care of patients with COVID-19 in the health care setting. Moreover, prolonged use of particulate respirators may result in unintended harms. In conjunction with appropriate hand hygiene, personal protective equipment (PPE) used by health care workers caring for patients with COVID-19 must be used with attention to detail and precision of execution to prevent lapses in adherence and active failures in the donning and doffing of the PPE.


siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor.
Poxvirus genome uncoating is a two-step process. First, cytoplasmic viral cores are activated and early viral genes are expressed. Next, cores are disassembled and the genomes released. This second step depends on an early viral factor(s) that has eluded identification for over 40 years. We used a large-scale, high-throughput RNAi screen directed against vaccinia virus (VACV) to identify the VACV AAA+ ATPase D5 as the poxvirus uncoating factor. We show that the ATPase activity of D5 is required for uncoating. Superresolution microscopy suggests that D5 acts directly at viral cores for genome release. Thus, the putative helicase D5 is a multifunctional protein required for genome uncoating and replication. Additionally, in vivo delivery of anti-D5 siRNAs reduced virus production in a mouse model of VACV infection. These results demonstrate the use of virus-targeting RNAi libraries to investigate viral gene function and suggest therapeutic avenues.


Costs of illness of multiple sclerosis in Sweden: a population-based register study of people of working age.
Multiple sclerosis (MS) causes work disability and healthcare resource use, but little is known about the distribution of the associated costs to society. We estimated the cost of illness (COI) of working-aged individuals with MS, from the societal perspective, overall and in different groups. A population-based study was conducted, using data linked from several nationwide registers, on 14,077 individuals with MS, aged 20-64 years and living in Sweden. Prevalence-based direct and indirect costs in 2010 were calculated, including costs for prescription drug use, specialized healthcare, sick leave, and disability pension. The estimated COI of all the MS patients were SEK 3950 million, of which 75% were indirect costs. MS was the main diagnosis for resource use, causing 38% of healthcare costs and 67% of indirect costs. The distribution of costs was skewed, in which less than 25% of the patients accounted for half the total COI. Indirect costs contributed to approximately 75% of the estimated overall COI of MS patients of working age in Sweden. MS was the main diagnosis for more than half of the estimated COI in this patient group. Further studies are needed to gain knowledge on development of costs over time during the MS disease course.


OCTN2-Mediated Acetyl-l-Carnitine Transport in Human Pulmonary Epithelial Cells In Vitro.
The carnitine transporter OCTN2 is associated with asthma and other inflammatory diseases. The aims of this work were (i) to determine carnitine uptake into freshly isolated human alveolar type I (ATI)-like epithelial cells in primary culture, (ii) to compare the kinetics of carnitine uptake between respiratory epithelial in vitro cell models, and (iii) to establish whether any cell line was a suitable model for studies of carnitine transport at the air-blood barrier. Levels of time-dependent [3H]-acetyl-l-carnitine uptake were similar in ATI-like, NCl-H441, and Calu-3 epithelial cells, whereas uptake into A549 cells was ~5 times higher. Uptake inhibition was more pronounced by OCTN2 modulators, such as l-Carnitine and verapamil, in ATI-like primary epithelial cells compared to NCl-H441 and Calu-3 epithelial cells. Our findings suggest that OCTN2 is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium and that none of the tested cell lines are optimal surrogates for primary cells.


The contribution of the palmaris longus muscle to the strength of thumb abduction.
The palmaris longus muscle (PLM) is described as a weak flexor of the wrist and a tensor of the palmar aponeurosis, but not a thumb abductor. The PLM is believed to aid thumb abduction through its insertion onto the thenar eminence. Two groups, both right hand dominant, were selected from 1,200 sampled participants. The first group comprised of 38 subjects with unilateral presence of the PLM and was used to determine the strength of thumb abduction. The second group comprised of 30 subjects, with bilateral presence of the PLM, and it was used to calculate the effects of hand dominance. A significant number of subjects with bilateral absence of the PLM were observed and undocumented. Using a dynamometer in subjects with unilateral presence of the PLM, the force of thumb abduction was significantly greater on the hand with a PLM than the one without it (P = 0.014), irrespective of hand dominance. In the second sample with bilateral PLM, thumb abduction on the dominant hand was 10% stronger than on the nondominant hand and was similar to the universally accepted average of 10% increase in grip strength of the dominant hand. Thus, 10% was deducted from all the dominant hands, and the force of thumb abduction remained greater on the hand with PLM than the hand without it (P = 0.049). The results of this study demonstrated the PLM to be involved in thumb abduction, and the authors therefore recommend that this action of the muscle be universally accepted by anatomists and hand surgeons.


RBOHF2 of barley is required for normal development of penetration resistance to the parasitic fungus Blumeria graminis f. sp. hordei.
Plant respiratory burst oxidase homologs are prominent sources of reactive oxygen species (ROS) in signal transduction and in interaction with microbes. However, the function of respiratory burst oxidase homologue (RBOH) genes in interaction with microbes might differ for certain plant and pathogen species. We produced transgenic barley knock down (KD) for the HvRBOHF2 isoform of NADPH oxidases. Young HvRBOHF2 KD shoots did not show obvious morphological alterations from the wild type but adult HvRBOHF2 KD plants developed fewer tillers, were less fertile, and showed spontaneous cell death in leaf mesophyll. Additionally, HvRBOHF2 KD plants were unable to contain wound-induced cell death. Before developmental failure became obvious, young HvRBOHF2 KD seedlings were much more susceptible to penetration by the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei. Strikingly, the B. graminis f. sp. hordei-induced cell-wall-associated oxidative burst was not substantially attenuated in HvRBOHF2 KD plants but enhanced susceptibility apparently influenced the subcellular site of hydrogen peroxide accumulation. Taken together, misexpression of HvRBOHF2 caused failure of barley to normally develop penetration resistance to B. graminis f. sp. hordei and to control leaf cell death.


Life cycle and immature stages of the arctiid moth, Phoenicoprocta capistrata.
Phoenicoprocta capistrata (Fabricius 1775) (Lepidoptera: Arctiidae) is an arctiid moth reported for the Caribbean and Brazil, whose immature stages and life cycle are unknown. In this study, and for the first time, a host plant is registered and the immature stages and the captivity life cycle are described using a Cuban population. Larvae feed on fowlsfoot, Serjania diversifolia (Jacq.) Radlk (Sapindales: Sapindaceae). One complete cohort was obtained from December of 2004 to February of 2005 and about 57 days lapsed from oviposition to adult emergence. The egg is light green-yellowish and semi-spherical. Most larvae developed through 6 or 7 instars, although there were individuals with 8 instars. The last instar has a cephalic capsule width of 2.04 +/- 0.06 mm (n = 29) irrespective of the number of instars. The cephalic capsule growth curves of the larvae with 6 and 7 instars have different slopes, but both follow a geometric pattern consistent with the Dyar's rule. In each larval molt the setae types and the larvae coloration change. Adult females have two color morphs, one orange-reddish and the other blue. Female descendants of blue and red females differ in the proportion of color morphs, which could indicate the existence of a female-limited polymorphism phenomenon in this species.


Risk factors for the hemolytic uremic syndrome in children infected with Escherichia coli O157:H7: a multivariable analysis.
Escherichia coli O157:H7 is the leading cause of hemolytic uremic syndrome (HUS). Risk factors for development of this complication warrant identification. We enrolled children infected with E. coli O157:H7 within 1 week of the onset of diarrhea in this prospective cohort study. The study was conducted in 5 states over 9.5 years . The primary and secondary outcomes were HUS (hematocrit <30% with smear evidence of hemolysis, platelet count <150 × 10(3)/µL, and serum creatinine concentration > upper limit of normal for age) and oligoanuric HUS. Univariate and multivariable and ordinal multinomial regression analyses were used to test associations between factors apparent during the first week of illness and outcomes. Of the 259 children analyzed, 36 (14%) developed HUS. Univariate analysis demonstrated that children who received antibiotics during the diarrhea phase more frequently developed HUS than those who did not (36% vs 12%; P = .001). The higher rate of HUS was observed across all antibiotic classes used. In multivariable analysis, a higher leukocyte count (adjusted odds ratios [aOR] 1.10; 95% CI, 1.03-1.19), vomiting (aOR 3.05; 95% CI, 1.23-7.56), and exposure to antibiotics (aOR 3.62; 95% CI, 1.23-10.6) during the first week of onset of illness were each independently associated with development of HUS. Multinomial ordinal logistic regression confirmed that initial leukocyte count and antibiotic use were independently associated with HUS and, additionally, these variables were each associated with the development of oligoanuric HUS. Antibiotic use during E. coli O157:H7 infections is associated with a higher rate of subsequent HUS and should be avoided.


Influencing risk behavior of sexually transmitted infection clinic visitors: efficacy of a new methodology of motivational preventive counseling.
A quasi-experimental study was conducted at a Dutch sexually transmitted infection (STI) clinic to compare the effects of educational counseling and motivational interviewing (MI)-based HIV/STI counseling on determinants of condom use and partner notification at 6-month follow-up. It also examined the feasibility of MI-based counseling in a busy real-life clinic. The counseling approaches were historically compared: respondents in the control condition were recruited between April and July 2005, those in the experimental condition between September and December 2005. The study involved 428 participants. These were all high-risk clients of the STI clinic. Their mean age was 33.7 years, and 39.6% were female. The study showed that MI-based counseling had a more positive effect on self-efficacy, intentions to use condoms with casual partners, and long-term condom use with steady partners. It had no adversarial outcomes on other social cognitions or behaviors compared to educational counseling. Furthermore, MI-based counseling is experienced as a more respectful and structured way of counseling. MI-based counseling was relatively easily implemented into the current clinic procedures. In addition to the implementation of the training, neither specialized staff nor additional or longer client visits were needed. However, some nurses indicated that the new method required more personal investment and effort. Limitations of the current study are the low response rates, the high educational level of most participants, and the small sample size regarding partner notification. Nonetheless, we conclude that MI-based counseling was a more effective approach to preventive counseling compared to educational counseling and feasible in the busy real-life setting.


RGS2-deficient mice exhibit decreased intraocular pressure and increased retinal ganglion cell survival.
Contractile activity of the trabecular meshwork (TM) and ciliary muscle (CM) influences aqueous humor drainage; however, the mechanisms linking tissue contractility and regulation of aqueous humor drainage are not well understood. Regulator of G Protein Signaling 2 (RGS2), a GTPase-activating protein of the Galphaq family of proteins, plays a critical role in regulation of contractile activity of vascular smooth muscle and in blood pressure homeostasis. To explore a potential role for RGS2 in intraocular pressure (IOP) homeostasis, we evaluated RGS2 knockout (RGS2(-/-)) mice for changes in IOP. IOP was measured using a rebound tonometer in awake male RGS2(-/-) and littermate wild-type mice. Histological and immunofluorescence analyses were performed to evaluate changes in the iridocorneal structure, actomyosin organization in CM and TM, and retinal ganglion cell survival in both central and peripheral retina. In repeated measurements, IOP was found to be consistently lower in the RGS2(-/-) mice compared to littermate wild-type mice. This change in IOP appears to be associated with increased actin filament assembly in the CM, and widening of the Schlemm's canal in the aqueous humor drainage pathway. Furthermore, ganglion cell number in the central retina was found to be significantly higher in the RGS2(-/-) mice relative to wild-type mice. The data suggest that the deficiency of RGS2 decreased IOP, presumably due to increased aqueous humor drainage in association with increased CM contraction. These data indicate a potentially critical role for RGS2 in homeostasis of IOP and for retinal ganglion cell survival.


A genealogical map of the concept of habit.
The notion of information processing has dominated the study of the mind for over six decades. However, before the advent of cognitivism, one of the most prominent theoretical ideas was that of Habit. This is a concept with a rich and complex history, which is again starting to awaken interest, following recent embodied, enactive critiques of computationalist frameworks. We offer here a very brief history of the concept of habit in the form of a genealogical network-map. This serves to provide an overview of the richness of this notion and as a guide for further re-appraisal. We identify 77 thinkers and their influences, and group them into seven schools of thought. Two major trends can be distinguished. One is the associationist trend, starting with the work of Locke and Hume, developed by Hartley, Bain, and Mill to be later absorbed into behaviorism through pioneering animal psychologists (Morgan and Thorndike). This tradition conceived of habits atomistically and as automatisms (a conception later debunked by cognitivism). Another historical trend we have called organicism inherits the legacy of Aristotle and develops along German idealism, French spiritualism, pragmatism, and phenomenology. It feeds into the work of continental psychologists in the early 20th century, influencing important figures such as Merleau-Ponty, Piaget, and Gibson. But it has not yet been taken up by mainstream cognitive neuroscience and psychology. Habits, in this tradition, are seen as ecological, self-organizing structures that relate to a web of predispositions and plastic dependencies both in the agent and in the environment. In addition, they are not conceptualized in opposition to rational, volitional processes, but as transversing a continuum from reflective to embodied intentionality. These are properties that make habit a particularly attractive idea for embodied, enactive perspectives, which can now re-evaluate it in light of dynamical systems theory and complexity research.


High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.
Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.


Seasonal changes in anthropometric and physical characteristics within English academy rugby league players.
Professional rugby league clubs implement training programmes for the development of anthropometric and physical characteristics within an academy programme. However, research that examines seasonal changes in these characteristics is limited. The purpose of the study was to evaluate the seasonal changes in anthropometric and physical characteristics of academy rugby league players by age category (i.e., under 14, 16, 18, 20). Data were collected on 75 players pre- and postseason over a 6-year period (resulting in a total of 195 assessments). Anthropometric (body mass, sum of 4 skinfolds) and physical (10- and 20-m sprint, vertical jump, Yo-Yo intermittent recovery test and 1 repetition maximum squat, bench press, and prone row) measures were collected. The under 14s and 16s showed greater seasonal improvements in body mass (e.g., under 14s = 7.4 ± 4.3% vs. under 20s = 1.2 ± 3.3%) and vertical jump performance than under 18s and under 20s. In contrast, under 18s and under 20s players showed greater seasonal improvements in Yo-Yo performance and 10-m sprint (e.g., under 14s = 1.3 ± 3.9% vs. under 20s = -1.9 ± 1.2%) in comparison to under 14s and under 16s. Seasonal strength improvements were greater for the under 18s compared with under 20s. This study provides comparative data for seasonal changes in anthropometric and physical characteristics within rugby league players aged 13-20 years. Coaches should be aware that seasonal improvements in speed may not exist within younger age categories, until changes in body mass stabilize and consider monitoring changes in other characteristics (e.g., momentum). Large interplayer variability suggests that player development should be considered on an individual and longitudinal basis.


Mapping of transcription start sites of human retina expressed genes.
The proper assembly of the transcriptional initiation machinery is a key regulatory step in the execution of the correct program of mRNA synthesis. The use of alternative transcription start sites (TSSs) provides a mechanism for cell and tissue specific gene regulation. Our knowledge of transcriptional initiation sequences in the human genome is limited despite the availability of the complete genome sequence. While genome wide experimental and bioinformatic approaches are improving our knowledge of TSSs, they lack information concerning genes expressed in a restricted manner or at very low levels, such as tissue specific genes. In this study we describe the mapping of TSSs of genes expressed in human retina. Genes have been selected on the basis of their physiological or developmental role in this tissue. Our work combines in silico analysis of ESTs and known algorithm predictions together with their experimental validation via Cap-finder RACE. We report here the TSSs mapping of 54 retina expressed genes: we retrieved new sequences for 41 genes, some of which contain un-annotated exons. Results can be grouped into five categories, compared to the RefSeq; (i) TSS located in new first exons, (ii) splicing variation of the second exon, (iii) extension of the annotated first exon, (iv) shortening of the annotated first exon, (v) confirmation of previously annotated TSS. In silico and experimental analysis of the transcripts proved to be essential for the ultimate mapping of TSSs. Our results highlight the necessity of a tissue specific approach to complete the existing gene annotation. The new TSSs and transcribed sequences are essential for further exploration of the promoter and other cis-regulatory sequences at the 5'end of genes.


Combined intestinal trefoil factor and epidermal growth factor is prophylactic against indomethacin-induced gastric damage in the rat.
1. The availability of recombinant epidermal growth factor provides a potentially exciting development for the treatment of gastrointestinal ulceration. However, because of its potent mitogenic activity, there is a need for strategies which reduce the dose required. Intestinal trefoil factor stimulates mucosal healing without increasing proliferation. Studies were undertaken to examine the biological effects of rat intestinal trefoil factor and/or human epidermal growth factor upon gastrointestinal epithelial cell functions pertinent to mucosal protection, using two wounding models. 2. The study of epithelial restitution in vitro demonstrated a marked synergistic effect on the rate of migration of the wound edge when intestinal trefoil factor was used in combination with epidermal growth factor. There was no increased cellular proliferation due to the addition of intestinal trefoil factor to the cells when given alone, or to the stimulatory effect of cells treated with epidermal growth factor. In the rat model of gastric ulceration, the presence of both epidermal growth factor and intestinal trefoil factor protected against the development of indomethacin-induced gastric lesions. 3. We conclude that combination therapy of epidermal growth factor with intestinal trefoil factor could provide a more potent, safer approach to the treatment of human gastrointestinal ulceration.


N-terminal pro-brain natriuretic peptide: a potential follow-up biomarker of mandibular advancement device efficacy on cardiac function in obstructive sleep apnea.
Interrelationships between obstructive sleep apnea (OSA) and cardiovascular diseases are now recognized, but some underlying pathophysiological mechanisms remain controversial. Circulating cardiac biomarkers are diagnostic tools that can help understand them, in particular the N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of myocardial stretch, and a potential indicator of subclinical cardiac stress in OSA. Continuous positive airway pressure (CPAP), the first-line treatment of moderate to severe OSA, may be considered as uncomfortable, resulting in poor adherence, and reduced effectiveness. In this case, mandibular advancement devices (MAD) are an effective alternative therapy, more comfortable, and generally well accepted, with higher compliance. To date, few studies have compared the cardiovascular effects of CPAP and MAD. From recent literature reviews, it emerges that both therapies are effective in blood pressure reduction. However, the effects of MAD on other cardiovascular outcomes are conflicting, in particular as regards to its impact on circulating cardiac biomarkers. In a recent ancillary study from a randomized controlled trial, Recoquillon et al concluded that two months of MAD treatment had no effect on NT-proBNP plasma levels in patients with severe OSA. The present discussion analyses this result from a biological, statistical, and analytical standpoint, in light of results from other studies evaluating natriuretic peptides in MAD-treated OSA, with the aim to support further longitudinal studies designed with a high methodological quality.


Characterization of di-4-ANEPPS with nano-black lipid membranes.
We report a platform based on lateral nano-black lipid membranes (nano-BLMs), where electrical measurements and fluorescence microscopy setup are combined, for the calibration of di-4-ANEPPS, a common voltage sensitive dye (VSD). The advantage of this setup is (1) its flexibility in the choice of lipids and applied voltages, (2) its high stability that enables a high voltage (500 mV) application and long-time measurements and (3) its fluorescence microscopy readout, which can be directly correlated with other fluorescence microscopy experiments using VSDs (e.g. membrane potential measurements in living cells). Using this setup, we observed that the calibration curve of di-4-ANEPPS is strongly dependent on the net electric charge of the lipids. The developed setup can be used to calibrate VSDs in different lipid environments in order to better understand their fundamental voltage-sensing mechanism in the future.


Acute effects of photobiomodulation therapy and magnetic field on functional mobility in stroke survivors: a randomized, sham-controlled, triple-blind, crossover, clinical trial.
Identify the optimal energy delivered with a single application of the combination of photobiomodulation therapy (PBMT) combining different light sources (low-level laser therapy-LLLT and light emitting diode therapy-LEDT) and static magnetic field (sMF) in order to determine the acute effects on functional mobility of stroke survivors. Was conducted a randomized, placebo-controlled, crossover, triple-blind, clinical trial (RCT). Twelve patients were recruited, however ten concluded the study, they were randomly treated with four PBMT/sMF energies (sham-0 J, 10 J, 30 J, and 50 J per site irradiated), with 1-week interval washout between treatments. PBMT/sMF were administered after the pre-intervention (baseline) evaluation and the total energy delivered per site at each treatment was determined based on the results of the randomization procedure. PBMT/sMF were administered in direct contact with the skin and applied with slight pressure to nine sites on the knee extensors, six sites on the knee flexors, and two sites on the plantar flexors' muscles in both lower limbs (bilaterally). The primary outcome measure was the 6-min walk test (6MWT) and the secondary outcome was the Timed Up and Go (TUG) test. Significant improvements were found in the 6MWT test using a total energy of 30 J per site compared with sham (0 J) (p < 0.05) and compared with the baseline evaluation (p < 0.01). And in the TUG test significant improvements were also found using a total energy per site of 30 J per site compared to sham (0 J) and baseline (p < 0.05). PBMT with different light sources (laser and LEDs) and wavelengths in combination with sMF with a total energy per site of 30 J has positive acute effects on functional mobility in stroke survivors.


Minor depression in primary care.
Minor depression is defined as a mood disturbance of at least 2 weeks' duration, with between two and five symptoms of depression, including depressed mood, diminished interest, weight change, sleep disturbance, psychomotor changes, fatigue, feelings of worthlessness, poor concentration, and recurrent thoughts of death. Patients with this condition may have fewer vegetative symptoms (appetite, diurnal mood variation) and more subjective symptoms (self-blame, worry, irritability, lethargy). Minor depressive disorder is more prevalent in primary care than major depressive disorder. Failure to adequately treat this condition may have far-reaching impact on the health, functional status, quality of life, and cost of care for patients who have it. The notion that minor depression requires minor treatment is misleading. Cognitive-behavioral modes of therapy and selective serotonin reuptake inhibitor antidepressants have demonstrated efficacy for primary care patients who have minor depression.


In what ways are adolescents who engage in self-harm or experience thoughts of self-harm different in terms of help-seeking, communication and coping strategies?
The purpose of this study was to ascertain whether adolescents who deliberately harmed themselves or had thoughts of self-harm differed from other adolescents in terms of help-seeking, communication and coping strategies. The participants were 6020 15-16 year-old school pupils who were surveyed using an anonymous self-report questionnaire. Adolescents with one or more episodes of deliberate self-harm (DSH) in the previous year were more likely to identify themselves as having serious problems than other adolescents. However, a substantial proportion of adolescents with either DSH or thoughts of self-harm did not identify themselves as having serious problems. Adolescents with DSH were most likely to feel the need for help but not try to get any; they were less able to talk to family members and teachers and had fewer categories of people who they were able to talk to. Like other adolescents, those with DSH or thoughts of self-harm were more likely to seek and receive help from their friends than from other sources. They differed from other adolescents in terms of coping strategies they reported employing when faced with difficulties, showing less focus on problems and more avoidant behaviours. The findings have important implications for preventive strategies, including educational programmes on emotional health and coping, and for the clinical care of adolescents identified as at risk or having self-harmed.


Identifying parameters to improve the reproducibility of transient gene expression in High Five cells.
Virus-free, transient gene expression (TGE) in High Five cells was recently presented as an efficient protein production method. However, published TGE protocols have not been standardized to a general protocol. Therefore, reproducibility and implementation of the method in other labs remains difficult. The aim of this study is to analyse the parameters determining the reproducibility of the TGE in insect cells. Here, we identified that using linear 40 kDa PEI instead of 25 kDa PEI was one of the most important aspects to improve TGE. Furthermore, DNA amount, DNA:PEI ratio, growth phase of the cells before transfection, passage number, the origin of the High-Five cell isolates and the type of cultivation medium were considered. Interestingly, a correlation of the passage number to the DNA content of single cells (ploidy) and to the transfection efficacy could be shown. The optimal conditions for critical parameters were used to establish a robust TGE method. Finally, we compared the achieved product yields in High Five cells using our improved TGE method with both the baculoviral expression system and TGE in the mammalian HEK293-6E cell line. In conclusion, the presented robust TGE protocol in High Five cells is easy to establish and produces ample amounts of high-quality recombinant protein, bridging the gap in expression level of this method to the well-established mammalian TGE in HEK293 cells as well as to the baculoviral expression vector system (BEVS).


Cytoprotective and proangiogenic activity of ex-vivo netrin-1 transgene overexpression protects the heart against ischemia/reperfusion injury.
In continuation of a previous work that transgene expression of sonic hedgehog promoted neo-vascularization via netrin-1 release, the current study was aimed at assessing the anti-apoptotic and pro-angiogenic role of netrin-1 transgene overexpression in the ischemic myocardium. pLP-Adeno-X ViralTrak vectors containing netrin-1 cDNA amplified from rat mesenchymal stem cells (Ad-netrin) or without a therapeutic gene (Ad-null) were constructed and transfected into HEK-293 cells to produce Ad-netrin and Ad-null vectors. Sca-1(+)-like cells were isolated and propagated in vitro and were successfully transduced with Ad-netrin transduced Sca-1(+) cells ((Net)Sca-1(+)) and Ad-null transduced Sca-1(+) cells ((Null)Sca-1(+)). Overexpression of netrin-1 in (Net)Sca-1(+) was confirmed by reverse transcription-polymerase chain reaction and western blot. Neonatal cardiomyocytes and rat endothelial cells expressed netrin-1 specific receptor Uncoordinated-5b and the conditioned medium from (Net)Sca-1(+) cells was protective for both the cell types against oxidant stress. For in vivo studies, the rat model of myocardial ischemia/reperfusion injury was developed in female Wistar rats by left anterior descending coronary artery occlusion for 45 min followed by reperfusion. The animals were grouped to receive 70 μL of Dulbecco's modified Eagle's medium without cells (group-1), containing 2×10(6) (Null)Sca-1(+) cells (group-2) and (Net)Sca-1(+) cells (group-3). (Net)Sca-1(+) cells significantly reduced ischemia/reperfusion injury in the heart and preserved the global heart function in group-3 (P<0.05 vs. groups-1 and group-2). Ex-vivo netrin-1 overexpression in the heart increased NOS activity in the heart. Blood vessel density was significantly higher in group-3 (P<0.05 vs. controls). We concluded that netrin-1 decreased apoptosis in cardiomyocytes and endothelial cells via activation of Akt. Netrin-1 transgene expression was proangiogenic and effectively reduced ischemia/reperfusion injury to preserve global heart function.


Characterization of polysaccharide fractions in mulberry fruit and assessment of their antioxidant and hypoglycemic activities in vitro.
The present study aimed to characterize and investigate the polysaccharide fractions of mulberry fruit, and their antioxidant and hypoglycemic activities. Mulberry fruit polysaccharides (MFP) were extracted by using hot water as the solvent and fractioned by using a DEAE-Sepharose fast-flow column. In total four eluents of water (MFP-1), 0.1 M NaCl (MFP-2), 0.2 M NaCl (MFP-3) and 0.3 M NaCl (MFP-4) were fractionated. Arabinose, galactose, glucose, xylose and mannose were the main components present in MFP-1, while MFP-2 and MFP-3 were composed of arabinose and galactose, and MFP-4 of arabinose, galactose and glucose. High-performance gel permeation chromatography (HP-GPC) analysis indicated a narrower molecular weight distribution in the MFP-1 fraction. Scanning electron microscopy (SEM) exhibited a smooth surface for MFP-1 and MFP-3 fractions, whereas MFP-2 and MFP-4 have pore openings and flocculent fibers on a smooth surface. Tertiary structure analyses indicated that none of the fractions had a triple-helical conformation. On the whole, MFP-3 and MFP-4 showed better antioxidant activity and inhibitory effects on α-amylase and α-glucosidase compared to MFP-1 and MFP-2. These results show significant potential for the antioxidant and hypoglycemic activities of MFP-3 and MFP-4 indicating the need for their further exploration as potential antidiabetic agents.


Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity.
Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using β-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.


Tamoxifen impairs both longitudinal and cortical bone growth in young male rats.
Tamoxifen (Tam) has been used experimentally to treat boys with gynecomastia and girls with McCune-Albright syndrome. This drug was recently shown to inhibit the growth of cultured fetal rat metatarsal bones and thus might also affect bone growth in vivo. Four-week-old Sprague-Dawley rats were gavaged daily with vehicle alone (peanut oil), Tam (40 mg/kg/d; 1 or 4 wk), or estradiol (40 microg/kg/d; 4 wk). Five of the 10 rats in each group were killed after 4 wk and the other five after 14 wk of recovery. Bone growth was followed by repeat DXA scans, whereas other bone parameters and spine length were evaluated by pQCT and X-ray at the time of death. Four-week Tam treatment significantly decreased body weight, nose-anus distance, spinal and tibial bone lengths, trabecular BMD, cortical periosteal circumference, and bone strength and also reduced serum IGF-I levels (424 +/- 54 versus 606 +/- 53 ng/ml in control; p < 0.05). Analysis of the tibial growth plate of treated rats showed elevated chondrocyte proliferation (BrdU) and apoptosis (TUNEL), as well as decreases in the number of hypertrophic chondrocytes and in the size of terminal hypertrophic chondrocytes. Despite a complete catch-up of body weight after 14 wk of recovery, the tibia was still shorter (p < 0.001) and its cortical region was smaller. We conclude that, when administered at a clinically relevant dose, Tam causes persistent retardation of longitudinal and cortical radial bone growth in young male rats. Our findings suggest that this inhibition results from local effects on the growth plate cartilage and systemic suppression of IGF-I production. Based on these rat data, we believe that Tam, if given to growing individuals, might compromise cortical bone growth, bone strength, and adult height.


Modulation of canonical transient receptor potential channel 1 in the proliferation of oligodendrocyte precursor cells by the golli products of the myelin basic protein gene.
Golli proteins, products of the myelin basic protein gene, function as a new type of modulator of intracellular Ca(2+) levels in oligodendrocyte progenitor cells (OPCs). Because of this, they affect a number of Ca(2+)-dependent functions, such as OPC migration and process extension. To examine further the Ca(2+) channels regulated by golli, we studied the store-operated Ca(2+) channels (SOCCs) in OPCs and acute brain slice preparations from golli knock-out and golli-overexpressing mice. Our results showed that pharmacologically induced Ca(2+) release from intracellular stores evoked a significant extracellular Ca(2+) entry after store depletion in OPCs. They also indicated that, under these pharmacological conditions, golli promoted activation of Ca(2+) influx by SOCCs in cultured OPCs as well as in tissue slices. The canonical transient receptor potential family of Ca(2+) channels (TRPCs) has been postulated to be SOCC subunits in oligodendrocytes. Using a small interfering RNA knockdown approach, we provided direct evidence that TRPC1 is involved in store-operated Ca(2+) influx in OPCs and that it is modulated by golli. Furthermore, our data indicated that golli is probably associated with TRPC1 at OPC processes. Additionally, we found that TRPC1 expression is essential for the effects of golli on OPC proliferation. In summary, our data indicate a key role for golli proteins in the regulation of TRPC-mediated Ca(2+) influx, a finding that has profound consequences for the regulation of multiple biological processes in OPCs. More important, we have shown that extracellular Ca(2+) uptake through TRPC1 is an essential component in the mechanism of OPC proliferation.


A proposed metric for assessing the measurement quality of individual microarrays.
High-density microarray technology is increasingly applied to study gene expression levels on a large scale. Microarray experiments rely on several critical steps that may introduce error and uncertainty in analyses. These steps include mRNA sample extraction, amplification and labeling, hybridization, and scanning. In some cases this may be manifested as systematic spatial variation on the surface of microarray in which expression measurements within an individual array may vary as a function of geographic position on the array surface. We hypothesized that an index of the degree of spatiality of gene expression measurements associated with their physical geographic locations on an array could indicate the summary of the physical reliability of the microarray. We introduced a novel way to formulate this index using a statistical analysis tool. Our approach regressed gene expression intensity measurements on a polynomial response surface of the microarray's Cartesian coordinates. We demonstrated this method using a fixed model and presented results from real and simulated datasets. We demonstrated the potential of such a quantitative metric for assessing the reliability of individual arrays. Moreover, we showed that this procedure can be incorporated into laboratory practice as a means to set quality control specifications and as a tool to determine whether an array has sufficient quality to be retained in terms of spatial correlation of gene expression measurements.


Post-Activation Potentiation: Is there an Optimal Training Volume and Intensity to Induce Improvements in Vertical Jump Ability in Highly-Trained Subjects?
The aim of this study was to compare the acute effects of performing half squats (HSs) with different loading intensities (1, 3, and 5 repetitions maximum [RM], and 60% 1RM) and a different number of sets (1, 2, and 3) on the countermovement jump (CMJ) performance of 18 highly-trained male subjects. Participants were submitted to four experimental conditions (1RM, 3RM, 5RM, and 60% 1RM) in randomized order. The CMJ was assessed before and after each set. Differences in CMJ performance between the distinct experimental conditions and individual responses in CMJ performance induced by the different protocols were analyzed via the magnitude-based inference method. Overall, significant improvements were detected in individual CMJ heights after each activation protocol. It can be concluded that the use of 1 to 3 sets of HSs performed at moderate-to-high loads may be an effective strategy to improve jump performance in highly-trained subjects. Nonetheless, despite the high efficiency of the protocols tested here, coaches and researchers are strongly encouraged to perform individualized assessments within the proposed range of loads and sets, to find optimal and tailored post-activation potentiation protocols.


Adding statistical regularity results in a global slowdown in visual search.
Current statistical learning theories predict that embedding implicit regularities within a task should further improve online performance, beyond general practice. We challenged this assumption by contrasting performance in a visual search task containing either a consistent-mapping (regularity) condition, a random-mapping condition, or both conditions, mixed. Surprisingly, performance in a random visual search, without any regularity, was better than performance in a mixed design search that contained a beneficial regularity. This result was replicated using different stimuli and different regularities, suggesting that mixing consistent and random conditions leads to an overall slowing down of performance. Relying on the predictive-processing framework, we suggest that this global detrimental effect depends on the validity of the regularity: when its predictive value is low, as it is in the case of a mixed design, reliance on all prior information is reduced, resulting in a general slowdown. Our results suggest that our cognitive system does not maximize speed, but rather continues to gather and implement statistical information at the expense of a possible slowdown in performance.


Anti-Inflammatory and Apoptotic Signaling Effect of Fucoxanthin on Benzo(A)Pyrene-Induced Lung Cancer in Mice.
Fucoxanthin, a potent carotenoid present in various natural sources especially from seaweeds; it exhibits several biological effects like anti-neoplastic, anti-mutagenic, anti-diabetic, anti-obesity and anti-inflammatory actions. Fucoxanthin role in chemoprevention of lung cancer in mouse model induced using benzo(a)pyrene [B(a)P] has been presented here. Oral administration of fucoxanthin with and without B(a)P were studied, the results from our study shows that fucoxanthin significantly decreased tumor progression in mice exposed to B(a)P, the obtained data were correlated with increased antioxidant, apoptosis and decreased tumour marker and anti-apoptotic molecules. With respect to apoptosis, fucoxanthin treated animals shows increased apoptosis compared to tumor induced mice by increased expression of caspase 9 and 3 and decreased expression of anti-apoptotic Bcl2 protein. Finally, histopathological and immuno histochemical analysis also revealed that fucoxanthin shows potent anticancer agent by bringing back the damaged tissue treated with B(a)P and also decreases the expression of PCNA in cancer induced mice. The anticancer effect of fucoxanthin may be attributed by several independent mechanisms which play a important roles in the prevention of cancer development, there is also substantial evidences to show that fucoxanthin acts indirectly by increasing the antioxidant capacity of affected tissue and prepared to cope up with oxidative stress which is proved in our study. Thus from our study it is clearly established that fucoxanthin act as a persuasive anticancer drug against lung cancer.


Tri-modal microscopy with multiphoton and optical coherence microscopy/tomography for multi-scale and multi-contrast imaging.
Multi-scale multimodal microscopy is a very useful technique by providing multiple imaging contrasts with adjustable field of views and spatial resolutions. Here, we present a tri-modal microscope combining multiphoton microscopy (MPM), optical coherence microscopy (OCM) and optical coherence tomography (OCT) for subsurface visualization of biological tissues. The advantages of the tri-modal system are demonstrated on various biological samples. It enables the visualization of multiple intrinsic contrasts including scattering, two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). It also enables a rapid scanning over a large tissue area and a high resolution zoom-in for cellular-level structures on regions of interest. The tri-modal microscope can be important for label-free imaging to obtain a sufficient set of parameters for reliable sample analysis.


Binding of benzo[a]pyrene 7,8-diol-9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity.
The formation, stereostructure, and cellular reactions of the 7,8-diol-9,10-epoxide metabolites of the carcinogen benzo[a]pyrene have been examined after topical application of benzo[a]pyrene to the skin of mice. In this known target tissue, polymer adducts from diastereomeric diol epoxides, (+)-(7S, 8R, 9R, 10R) and (+)-(7R, 8S, 9R, 10R), were formed stereospecifically from their corresponding 7,8-dihydrodiols. Both diol epoxides bind with proteins, RNA, and DNA in vivo. For the nucleic acids, binding occurs preferentially at the 2-amino group of guanine in cellular RNA and DNA in vivo. Methods for establishing the structure of the cellular adducts as well as the possible biological implications of their formation are discussed.


Gender equity in treatment for cardiac heart disease in Portugal.
Equity in health care delivery is one of the objectives of the Portuguese health care system. To date, research on this issue has mainly focused on income-related equity. This is the first study to shed light on gender equity, using a large data base that includes all patients admitted with cardiac heart disease at Portuguese NHS hospitals over the 2000-2006 period (259,519 discharges from 57 hospitals). In this paper we compare the use of catheterization and revascularization between men and women, controlling for age, comorbidities and hospital characteristics. Our findings show that women receive notably less catheterization and revascularization, with no significant change in this pattern over the 2000-2006 period. In addition, we observe that (i) gender differences disfavouring women are higher prior to detection of acute disease than after; (ii) women are significantly more likely to die during hospitalization despite equal treatment; (iii) gender differences against women are higher for non-elective admissions, and women are more often admitted through emergency units. These additional findings suggest that gender differences in detection, referral and treatment at early stages of the disease are likely to play a crucial role. They could possibly explain part of the higher gender differences before acute disease has been detected; they also lead women to be treated later, to be more frequently admitted through emergency units and to experience worse outcomes. However, alternative explanations cannot be discarded. The higher women's in-patient mortality may also signal gender differences in recovery from treatment, and the higher gap among emergency admissions could point to women's lower willingness to be treated. Further investigation should help to disentangle the precise role of each of these causal factors.


The systemic inflammation-based Glasgow Prognostic Score as a powerful prognostic factor in patients with upper tract urothelial carcinoma.
The combination of C-reactive protein and albumin, the Glasgow Prognostic Score (GPS), had independent prognostic value in patients with varying cancers, except for upper tract urothelial carcinoma (UTUC). The aim of this study was to describe the relationship between GPS and survival in patients with UTUC after adjustment for other prognostic factors. We queried 2 UTUC databases. Retrospective clinical series on patients with localized UTUC managed by nephroureterectomy with bladder cuff, for whom data from the Yamaguchi Uro-Oncology Group and Osaka Medical College registry, including age, presence of bladder cancer, pT stage, lymphovascular invasion, C-reactive protein (CRP) and albumin, were analyzed. The GPS was constructed by combining CRP and albumin. Cancer specific survival (CSS) and overall survival (OS) and relative excess risk of death were estimated by GPS categories after adjusting for gender, age, ECOG performance status (PS), grade, and lymphovascular invasion (LVI). Seven hundred and twenty four UTUC patients were identified. Our final cohort included 574 patients; of these, 29.2% died during a maximum follow up of 16.7 years. The estimated mean 10-year CSS of patients with GPS of scre-0, -1, and -2 was 99.5, 95.1, and 75.9 months, respectively. Patients with GPS of score-2 had poorest 10-year estimated mean OS of 67.6 months (57.2-77.9). Raised GPS also had a significant association with excess risk of cancer death at 10 years (GPS 2: Relative Excess Risk = 1.74, 95% CI 1.20-2.54) after adjusting for gender, patients' age, ECOG PS, and tumor focality. C-index of GPS both for CSS and OS were superior to patients' age and tumor focality, and comparable to grade. The GPS is an independent prognostic factor for CSS and OS after surgery with curative intent for localized UTUC. It significantly increases the accuracy of established prognostic factors. The GPS may provide a meaningful adjunct for patient counseling and clinical trial design.


In vitro chemopreventive effects of plant polysaccharides (Aloe barbadensis miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor).
A plant polysaccharide, Aloe gel extract, was reported to have an inhibitory effect on benzo[a]pyrene (B[a]P)-DNA adduct formation in vitro and in vivo. Hence, chemopreventive effects of plant polysaccharides [Aloe barbadensis Miller (APS), Lentinus edodes (LPS), Ganoderma lucidum (GPS) and Coriolus versicolor (CPS)] were compared using in vitro short-term screening methods associated with both initiation and promotion processes in carcinogenesis. In B[a]P-DNA adduct formation, APS (180 micrograms/ml) was the most effective in inhibition of B[a]P binding to DNA in mouse liver cells. Oxidative DNA damage (by 8-hydroxydeoxyguanosine) was significantly decreased by APS (180 micrograms/ml) and CPS (180 micrograms/ml). In induction of glutathione S-transferase activity, GPS was found to be the most effective among plant polysaccharides. In screening anti-tumor promoting effects, APS (180 micrograms/ml) significantly inhibited phorbol myristic acetate (PMA)-induced ornithine decarboxylase activity in Balb/3T3 cells. In addition, APS significantly inhibited PMA-induced tyrosine kinase activity in human leukemic cells. APS and CPS significantly inhibited superoxide anion formation. These results suggest that some plant polysaccharides produced both anti-genotoxic and anti-tumor promoting activities in in vitro models and, therefore, might be considered as potential agents for cancer chemoprevention.


The form of NY-ESO-1 antigen has an impact on the clinical efficacy of anti-tumor vaccination.
Anti-tumor vaccination is being evaluated as a prophylactic and therapeutic strategy against cancer growth, dissemination (spreading) or recurrence. Although a large number of studies investigate in detail the identity of antigens to be used for efficacious immune intervention, there have been few studies investigating the optimal form for antigen to be used in the vaccine. Here we show in a mouse H-2(d) MHC background and for NY-ESO-1 that genetic (plasmid DNA) but not full length protein vaccine is capable of inducing a protective prophylactic anti-tumor cytotoxic T-cell immune response in vivo. Peptide vaccination using nominal MHC class I epitope adjuvanted with a Toll Like Receptor agonist such as stabilized RNA can also provide some anti-tumor protection. Our results highlight the idea that when evaluating the clinical efficacy of a cancer vaccine, not only the identity of the antigen but also the format of the vaccine is of the utmost importance.


Roles of Arabidopsis patatin-related phospholipases a in root development are related to auxin responses and phosphate deficiency.
Phospholipase A enzymes cleave phospho- and galactolipids to generate free fatty acids and lysolipids that function in animal and plant hormone signaling. Here, we describe three Arabidopsis patatin-related phospholipase A (pPLA) genes AtPLAIVA, AtPLAIVB, and AtPLAIVC and their corresponding proteins. Loss-of-function mutants reveal roles for these pPLAs in roots during normal development and under phosphate deprivation. AtPLAIVA is expressed strongly and exclusively in roots and AtplaIVA-null mutants have reduced lateral root development, characteristic of an impaired auxin response. By contrast, AtPLAIVB is expressed weakly in roots, cotyledons, and leaves but is transcriptionally induced by auxin, although AtplaIVB mutants develop normally. AtPLAIVC is expressed in the floral gynaecium and is induced by abscisic acid (ABA) or phosphate deficiency in roots. While an AtplaIVC-1 loss-of-function mutant displays ABA responsiveness, it exhibits an impaired response to phosphate deficiency during root development. Recombinant AtPLA proteins hydrolyze preferentially galactolipids and, less efficiently, phospholipids, although these enzymes are not localized in chloroplasts. We find that AtPLAIVA and AtPLAIVB are phosphorylated by calcium-dependent protein kinases in vitro and this enhances their activities on phosphatidylcholine but not on phosphatidylglycerol. Taken together, the data reveal novel functions of pPLAs in root development with individual roles at the interface between phosphate deficiency and auxin signaling.


Simulations of the folding of a globular protein.
Dynamic Monte Carlo simulations of the folding of a globular protein, apoplastocyanin, have been undertaken in the context of a new lattice model of proteins that includes both side chains and a-carbon backbone atoms and that can approximate native conformations at the level of 2 angstroms (root mean square) or better. Starting from random-coil unfolded states, the model apoplastocyanin was folded to a native conformation that is topologically similar to the real protein. The present simulations used a marginal propensity for local secondary structure consistent with but by no means enforcing the native conformation and a full hydrophobicity scale in which any nonbonded pair of side chains could interact. These molecules folded through a punctuated on-site mechanism of assembly where folding initiated at or near one of the turns ultimately found in the native conformation. Thus these simulations represent a partial solution to the globular-protein folding problem.


TGFB1 genetic polymorphisms and coronary heart disease risk: a meta-analysis.
Genetic variations in TGFB1 gene have been studied in relation to coronary heart disease (CHD) risk, but the results were inconsistent. We performed a systematic review of published studies on the potential role of TGFB1 genetic variation in CHD risk. Articles that reported the association of TGFB1 genetic variants with CHD as primary outcome were searched via Medline and HuGE Navigator through July 2011. The reference lists from included articles were also reviewed. Data were available from 4 studies involving 1777 cases and 7172 controls for rs1800468, 7 studies involving 5935 cases and 10677 controls for rs1800469, 7 studies involving 6634 cases and 9620 controls for rs1982073, 5 studies involving 5452 cases and 9999 controls for rs1800471, and 4 studies involving 5143 cases and 4229 controls for rs1800472. The pooled odds ratios (ORs) for CHD among minor T allele carriers of rs1800469, minor C allele carriers of rs1982073, and minor C allele carriers of rs1800471 versus homozygous major allele carriers was 1.14 (95% confidence interval [CI]: 1.05-1.24), 1.18 (95% CI: 1.04-1.35), and 1.16 (95% CI: 1.02-1.32), respectively. No substantial heterogeneity for ORs was detected among the included Caucasian populations for all SNPs. However, for rs1800471, the statistical significance disappeared after adjusting for potential publication bias. No significant association was found between rs1800468 and rs1800472 variants and CHD risk. Minor allele carriers of two genetic variants (rs1800469 and rs1982073) in TGFB1 have a 15% increased risk of CHD.


Serodiagnosis of Lyme borreliosis using detection of different immunoglobulin (sub)classes by enzyme-linked immunosorbent assay and Western blotting.
To improve the performance of enzyme-linked immunosorbent assays for the serodiagnosis of Lyme borreliosis, the prevalence of several immunoglobulin classes and subclasses against various antigens of Borrelia burgdorferi was investigated by Western blotting. The sera of 40 early Lyme borreliosis patients (ELB), 27 late Lyme borreliosis patients (LLB), 62 healthy controls and 140 non-Lyme borreliosis patients were used. Detection of IgG1 versus total IgG was found to be more sensitive in detecting Borrelia burgdorferi antigens, especially flagellin (41 kD) protein, but did not improve the performance of Western blotting. The use of IgG1 detection showed an increase in sensitivity and specificity for the early Lyme borreliosis patient group compared to the standard IgG and IgM detection method by enzyme immunoassays using purified Borrelia burgdorferi flagellum. However, in an enzyme immunoassay using a total sonicate, sensitivity in detecting early Lyme borreliosis and late Lyme borreliosis with IgG1 remained lower compared to the detection of early Lyme borreliosis by IgM antibodies and late Lyme borreliosis by total IgG antibodies.


The effect of blueberry interventions on cognitive performance and mood: A systematic review of randomized controlled trials.
Blueberries are rich in polyphenols that may be beneficial to cognitive performance and mood. The aim of this systematic review was to evaluate randomized controlled trials investigating the effects of blueberries and blueberry products on measures of cognition and mood. In total, eleven articles (that included 12 studies) were identified using freeze-dried blueberries (n = 9 studies), whole blueberries (n = 2) and blueberry concentrate (n = 1). These studies were conducted in children (n = 5), young adults (n = 1), and older people with either no known cognitive impairment (n = 4) or indicated cognitive impairment (n = 2). Eight studies reported blueberry consumption or supplementation at various doses and time lengths to improve measures of cognitive performance, particularly short- and long-term memory and spatial memory. For mood, one study reported significant between-group improvements in positive affect from blueberry products, whereas four studies reported no improvement. Low risk of bias were observed across all studies. Based on the current evidence, blueberries may improve some measures of cognitive performance. However, considerable differences in study design, dosages, and anthocyanin content hinder between-study comparison. The use of standardized blueberry interventions, consideration of placebo formulations, and consistently reported cognitive performance tools are recommended in future trials. PROSPERO registration no. CRD42018100888.


Increased mindfulness is related to improved stress and mood following participation in a mindfulness-based stress reduction program in individuals with cancer.
Mindfulness-based stress reduction (MBSR) has demonstrated efficacy for alleviating cancer-related distress. Although theorized to be the means by which people improve, it is yet to be determined whether outcomes are related to the development or enhancement of mindfulness among participants. This study examined the effect of participation in an MBSR program on levels of mindfulness in a heterogeneous sample of individuals with cancer, and if these changes were related to improvements in stress and mood outcomes. In all, 268 individuals with cancer completed self-report assessments of stress and mood disturbances before and after participation in an 8-week MBSR program. Of these, 177 participants completed the Mindful Attention Awareness Scale and 91 participants completed the Five Facet Mindfulness Questionnaire, at both time points. Levels of mindfulness on both measures increased significantly over the course of the program. These were accompanied by significant reductions in mood disturbance (55%) and symptoms of stress (29%). Increases in mindfulness accounted for a significant percentage of the reductions in mood disturbance (21%) and symptoms of stress (14%). Being aware of the present moment and refraining from judging inner experience were the 2 most important mindfulness skills for improvements of psychological functioning among cancer patients. These results add to a growing literature measuring the impact of mindfulness and its relationship to improved psychological health. Moreover, specific mindfulness skills may be important in supporting these improvements.


Diabetes mellitus is independently associated with an increased risk of mortality in patients with clear cell renal cell carcinoma.
Conflicting data exist on the interaction of diabetes mellitus with outcomes in patients with renal cell carcinoma. We evaluated the association of diabetes mellitus with survival in patients with clear cell renal cell carcinoma treated with nephrectomy. We reviewed the records of 1,964 patients treated surgically for sporadic, unilateral, M0 clear cell renal cell carcinoma between 1990 and 2008. One pathologist re-reviewed all specimens to confirm clear cell renal cell carcinoma. We matched 257 patients with diabetes 1:2 to referent patients without diabetes according to clinicopathological and surgical features. Cancer specific and overall survival was estimated using the Kaplan-Meier method. Cox models were used to evaluate associations with outcomes. A total of 257 patients (13%) had diabetes mellitus. They were significantly older and more likely to be obese, and had higher Charlson scores, renal impairment and smoking rates, and worse performance status at surgery (p <0.001). Pathological features were similar between the groups. Median postoperative followup was 8.7 years. Five-year cancer specific survival was similar in patients with and without diabetes (82% vs 86%, p = 0.1) while 5-year overall survival was significantly worse in those with diabetes (65% vs 74%, p <0.001). On multivariable analysis diabetes mellitus independently predicted cancer specific mortality (HR 1.55, 95% CI 1.08-2.21, p = 0.02) and all-cause mortality (HR 1.32, 95% CI 1.06-1.64, p = 0.01). Our results suggest that diabetes mellitus is independently associated with decreased cancer specific and overall survival in patients with surgically treated clear cell renal cell carcinoma.


Cannabinoids to treat spinal cord injury.
Spinal cord injury (SCI) is a devastating condition for which there is no standard treatment beyond rehabilitation strategies. In this review, we discuss the current knowledge on the use of cannabinoids to treat this condition. The endocannabinoid system is expressed in the intact spinal cord, and it is dramatically upregulated after lesion. Endogenous activation of this system counteracts secondary damage following SCI, and treatments with endocannabinoids or synthetic cannabinoid receptor agonists promote a better functional outcome in experimental models. The use of cannabinoids in SCI is a new research field and many questions remain open. Here, we discuss caveats and suggest some future directions that may help to understand the role of cannabinoids in SCI and how to take advantage of this system to regain functions after spinal cord damage.


Salmonella bacteriuria in a cat fed a Salmonella-contaminated diet.
A 9-year-old castrated male domestic shorthair cat was evaluated because of hematuria and weight loss after an 8-year history of intermittent signs of feline lower urinary tract disease (FLUTD). A complete diet history revealed that the cat was eating a commercial diet that does not undergo the same processing procedures as most pet foods and so might be at increased risk for bacterial contamination owing to a nonstandard industry cooking procedure. The cat had a history consistent with FLUTD, but bacteriologic culture of the urine revealed Salmonella organisms. Additional analysis revealed Salmonella enterica serotype I:ROUGH-O:g,m,s:- in samples of urine and feces as well as Salmonella enterica serotype Johannesburg and Salmonella enterica serotype Senftenberg in the diet. The cat responded positively to antimicrobial treatment for the Salmonella bacteriuria as well as to dietary and environmental management for the clinical signs associated with FLUTD. Findings in this case highlighted an additional health consequence associated with ingestion of Salmonella-contaminated food. Such contamination is of particular concern with raw meat-based diets or diets that have not undergone standard industry cooking practices. Veterinarians should obtain a diet history for every companion animal during every evaluation to help with diagnosis and optimal treatment.


Cutting edge: A-kinase anchor proteins are involved in maintaining resting T cells in an inactive state.
A-kinase anchor proteins (AKAPs) target protein kinase A (PKA) to different subcellular locations and are thought to play important roles in the cAMP signaling pathway. The aims of this study were to determine whether T cells express AKAPs and, if so, to establish their physiological significance. CD4(+) T cells were found to express eight AKAPs. Disruption of the AKAP-PKA interaction caused high levels of IL-2, IL-4, IL-5, and IFN-gamma production in the absence of stimulation via CD3epsilon and CD28 molecules. Disruption of the AKAP-PKA interaction acted synergistically with suboptimal doses of Ag in boosting proliferative responses of T cells. Finally, disruption of the AKAP-PKA interaction rendered T cells insensitive to cAMP-elevating agents. It was concluded that AKAPs, through their association with PKA, are involved in maintaining T cell homeostasis and in regulating the sensitivity of T cells to incoming cAMP signals.


Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy.
Mitochondrial quality control (MQC) is crucial for regulating CNS homeostasis, and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human postmortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide therapeutic options for the management of DR-associated complications.


Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosa-luteal cells.
Until recently, the primate brain was thought to contain only one form of GnRH known as mammalian GnRH (GnRH-I). The recent cloning of a second form of GnRH (GnRH-II) with characteristics of chicken GnRH-II in the primate brain has prompted a reevaluation of the role of GnRH in reproductive functions. In the present study, we investigated the hormonal regulation of GnRH-II messenger RNA (mRNA) and its functional role in the human granulosa-luteal cells (hGLCs), and we provided novel evidence for differential hormonal regulation of GnRH-II vs. GnRH-I mRNA expression. Human GLCs were treated with various concentrations of GnRH-II, GnRH-II agonist (GnRH-II-a), or GnRH-I agonist (GnRH-I-a; leuprolide) in the absence or presence of FSH or human CG (hCG). The expression levels of GnRH-II, GnRH-I, and GnRH receptor (GnRHR) mRNA were investigated using semiquantitative or competitive RT-PCR. A significant decrease in GnRH-II and GnRHR mRNA levels was observed in cells treated with GnRH-II or GnRH-II-a. In contrast, GnRH-I-a revealed a biphasic effect (up- and down-regulation) of GnRH-I and GnRHR mRNA, suggesting that GnRH-I and GnRH-II may differentially regulate GnRHR and their ligands (GnRH-I and GnRH-II). Treatment with FSH or hCG increased GnRH-II mRNA levels but decreased GnRH-I mRNA levels, further indicating that GnRH-I and GnRH-II mRNA levels are differentially regulated. To investigate the physiological role of GnRH-II, hGLCs were treated with GnRH-II or GnRH-II-a in the presence or absence of hCG, for 24 h, and progesterone secretion was measured by RIA. Both GnRH-II and GnRH-II-a inhibited basal and hCG-stimulated progesterone secretion, effects which were similar to the effects of GnRH-I treatment on ovarian steroidogenesis. Next, hGLCs were treated with various concentrations of GnRH-II, GnRH-II-a, or GnRH-I-a; and the expression levels of FSH receptor and LH receptor were investigated using semiquantitative RT-PCR. A significant down-regulation of FSH receptor and LH receptor was observed in cells treated with GnRH-II, GnRH-II-a, and GnRH-I-a, demonstrating that GnRH-II and GnRH-I may exert their antigonadotropic effect by down-regulating gonadotropin receptors. Interestingly, GnRH-II and GnRH-II-a did not affect basal and hCG-stimulated intracellular cAMP accumulation, suggesting that the antigonadotropic effect of GnRH-II may be independent of modulation of cAMP levels. Taken together, these results suggest that GnRH-II may have biological effects similar to those of GnRH-I but is under differential hormonal regulation in the human ovary.


Apolipoprotein epsilon 3 alleles are associated with indicators of neuronal resilience.
Epilepsy is associated with precocious development of Alzheimer-type neuropathological changes, including appearance of senile plaques, neuronal loss and glial activation. As inheritance of APOE ε4 allele(s) is reported to favor this outcome, we sought to investigate neuronal and glial responses that differ according to APOE genotype. With an eye toward defining ways in which APOE ε3 alleles may foster neuronal well-being in epilepsy and/or APOE ε4 alleles exacerbate neuronal decline, neuronal and glial characteristics were studied in temporal lobectomy specimens from epilepsy patients of either APOE ε4,4 or APOE ε3,3 genotype. Tissue and/or cellular expressions of interleukin-1 alpha (IL-1α), apolipoprotein E (ApoE), amyloid β (Aβ) precursor protein (βAPP), synaptophysin, phosphorylated tau, and Aβ were determined in frozen and paraffin-embedded tissues from 52 APOE ε3,3 and 7 APOE ε4,4 (0.25 to 71 years) epilepsy patients, and 5 neurologically normal patients using Western blot, RT-PCR, and fluorescence immunohistochemistry. Tissue levels of IL-1α were elevated in patients of both APOE ε3,3 and APOE ε4,4 genotypes, and this elevation was apparent as an increase in the number of activated microglia per neuron (APOE ε3,3 vs APOE ε4,4 = 3.7 ± 1.2 vs 1.5 ± 0.4; P < 0.05). This, together with increases in βAPP and ApoE, was associated with apparent neuronal sparing in that APOE ε4,4 genotype was associated with smaller neuron size (APOE ε4,4 vs APOE ε3,3 = 173 ± 27 vs 356 ± 45; P ≤ 0.01) and greater DNA damage (APOE ε4,4 vs APOE ε3,3 = 67 ± 10 vs 39 ± 2; P = 0.01). 3) Aβ plaques were noted at early ages in our epilepsy patients, regardless of APOE genotype (APOE ε4,4 age 10; APOE ε3,3 age 17). Our findings of neuronal and glial events, which correlate with lesser neuronal DNA damage and larger, more robust neurons in epilepsy patients of APOE ε3,3 genotype compared to APOE ε4,4 genotype carriers, are consistent with the idea that the APOE ε3,3 genotype better protects neurons subjected to the hyperexcitability of epilepsy and thus confers less risk of AD (Alzheimer's disease).Please see related article: http://www.biomedcentral.com/1741-7015/10/36.


Synergic interplay of the La motif, RRM1 and the interdomain linker of LARP6 in the recognition of collagen mRNA expands the RNA binding repertoire of the La module.
The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3' UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5' UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.


A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia.
DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.