The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2.
Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.


Loss of Magel2 impairs the development of hypothalamic Anorexigenic circuits.
Prader-Willi syndrome (PWS) is a genetic disorder characterized by a variety of physiological and behavioral dysregulations, including hyperphagia, a condition that can lead to life-threatening obesity. Feeding behavior is a highly complex process with multiple feedback loops that involve both peripheral and central systems. The arcuate nucleus of the hypothalamus (ARH) is critical for the regulation of homeostatic processes including feeding, and this nucleus develops during neonatal life under of the influence of both environmental and genetic factors. Although much attention has focused on the metabolic and behavioral outcomes of PWS, an understanding of its effects on the development of hypothalamic circuits remains elusive. Here, we show that mice lacking Magel2, one of the genes responsible for the etiology of PWS, display an abnormal development of ARH axonal projections. Notably, the density of anorexigenic α-melanocyte-stimulating hormone axons was reduced in adult Magel2-null mice, while the density of orexigenic agouti-related peptide fibers in the mutant mice appeared identical to that in control mice. On the basis of previous findings showing a pivotal role for metabolic hormones in hypothalamic development, we also measured leptin and ghrelin levels in Magel2-null and control neonates and found that mutant mice have normal leptin and ghrelin levels. In vitro experiments show that Magel2 directly promotes axon growth. Together, these findings suggest that a loss of Magel2 leads to the disruption of hypothalamic feeding circuits, an effect that appears to be independent of the neurodevelopmental effects of leptin and ghrelin and likely involves a direct neurotrophic effect of Magel2.


Intratumoral HPV16-Specific T Cells Constitute a Type I-Oriented Tumor Microenvironment to Improve Survival in HPV16-Driven Oropharyngeal Cancer.
Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome.Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database.Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I-oriented tumor microenvironment, including high numbers of activated CD161+ T cells, CD103+ tissue-resident T cells, dendritic cells (DC), and DC-like macrophages.Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634-47. ©2017 AACRSee related commentary by Laban and Hoffmann, p. 505.


Magnesium sulfate for aneurysmal subarachnoid hemorrhage: the end of the road or more trials?
Delayed cerebral ischemia (DCI) is a feared complication and an important cause of poor outcome after aneurysmal subarachnoid hemorrhage (SAH). In the current study, Wong and colleagues performed a systematic review and meta-analysis of randomized controlled trials that investigated the efficacy of magnesium sulfate in patients with aneurysmal SAH. Outcome measures were DCI, cerebral infarction, and functional outcome 3 and 6 months after SAH. Magnesium sulfate decreased the rate of cerebral infarction, but not of DCI or poor functional outcome. Regarding outcome, a beneficial effect of magnesium sulfate on outcome can not be ruled out because of sample size limitations. Even if this meta-analysis had shown an effect on outcome, the question remains which treatment protocol should be applied in daily practice, since the administration of magnesium sulfate differed between most included studies. The present meta-analysis also underlines the importance of defining clinically relevant endpoints in SAH trials. Clinical deterioration due to DCI is more subject to inter-observer bias compared to cerebral infarction, which represents the ultimate outcome of the ischemic event. The Magnesium in Aneurysmal Subarachnoid Hemorrhage-II (MASH-II: ISRCTN68742385) phase III clinical trial nears completion. It aims to include 1,200 patients, and its results are urgently awaited.


Bovine vaginal antibody responses to immunoaffinity-purified surface antigen of Tritrichomonas foetus.
Bovine trichomoniasis is a prevalent sexually transmitted disease of cattle caused by the protozoan Tritrichomonas foetus. Currently, diagnosis is most often made by culture. In order to provide a faster immunodiagnostic approach, a specific enzyme-linked immunosorbent assay (ELISA) was investigated. A protective surface antigen (TF1.17 antigen) of T. foetus was immunoaffinity purified and used in an ELISA to detect antibodies in vaginal mucus from heifers inoculated with T. foetus. In preliminary studies, antibodies of the immunoglobulin A (IgA) isotype were detected in mucus from all experimentally infected heifers which were tested at 6 weeks postinoculation, whereas IgG1 and IgG2 were not. In addition, IgA responses detected in postinoculation samples were all greater than those detected in preinoculation samples, unlike those detected by a whole-cell antigen ELISA. For these two reasons, IgA antibodies appeared to be useful diagnostically. Further investigation of IgA antibodies used vaginal mucus collected weekly from heifers inoculated intravaginally with 10(2), 10(4), or 10(6) T. foetus organisms. Heifers with positive cultures for T. foetus had similar IgA responses to TF1.17 antigen over the 10 weeks of infection regardless of the initial inoculum dose. This indicates that if the dose is sufficient to establish infection, the magnitude and duration of the immune response are no longer dependent on dose. All of the infected animals receiving all dosages responded with high absorbance values in the IgA anti-TF1.17 antigen ELISA by 6 weeks postinoculation, and all absorbance values remained high at 10 weeks. To determine the duration of the IgA response, four other heifers inoculated with 7 x 10(6) T. foetus organisms were studied through 24 weeks postinoculation. IgA antibody responses to TF1.17 antigen were still high at 24 weeks, even though some heifers cleared the infection several weeks earlier. These results indicate that experimentally infected heifers produced detectable long-lasting IgA responses to TF1.17 antigen in vaginal mucus, which may be useful in a herd test for the diagnosis of bovine trichomoniasis.


Changes in metabolic syndrome in American and Korean youth, 1997-2008.
Metabolic syndrome (MetSyn) in children and adolescence is increasing worldwide; however, its pattern may be different between Asians and Americans. We compare the prevalence and patterns of MetSyn between American and Korean children and adolescents between roughly 1998 and 2007. Data from the American and Korean versions of the NHANES (NHANES and KNHANES) were used for this study. The main outcome is prevalence and pattern of MetSyn among participants separately in each country. In each survey, stratified multistage probability sampling designs and weighting adjustments were conducted to represent the entire population. The revised National Cholesterol Education Program criteria were used to define MetSyn. Totals of 934, 1781, and 1690 Americans aged 12 to 19 participated in NHANES 1988-1994, NHANES 1999-2002, and NHANES 2003-2006, respectively; and 1225, 976, 705, and 456 Koreans aged 12 to 19 have participated in KNHANES 1998, 2001, 2005, and 2007. The age-adjusted prevalence of MetSyn in American NHANES decreased from 7.3% to 6.7% and 6.5%, whereas in Korean NHANES there was an increase from 4.0% to 5.9%, 6.6%, and 7.8% in each country's respective study. Increases in dyslipidemia and abdominal obesity contributed to the increased prevalence in Korea, whereas in the United States, decreases in low high-density lipoprotein cholesterolemia and high blood pressure contributed to a decreased prevalence. Considering different phenotype changes, different approaches should be conducted at the national level to reduce the burden and consequences of MetSyn between Korea and the United States.


ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes.
The quantity of T-lymphocytes reaching the draining lymph nodes from tumors is likely important to mount effective distant responses and for the establishment of long term systemic memory. Looking into mechanisms behind lymphocyte egress, we directed our attention to leukocyte adhesion mechanisms inside tumors. Here we demonstrate that activated T-cells form intra-tumor aggregates in a LFA-1-ICAM-1-dependent fashion in mouse models of melanoma and breast cancer. We also provide evidence of the presence of T-cell clusters in primary human melanoma. Disruption of LFA-1-ICAM-1 interactions, and thereby T-cell clustering, enhances the arrival of activated CD8+ T-cells to tumor draining lymph nodes in both transplanted and spontaneous cancer models. Interestingly, upon ICAM-1 blockade, the expression of the chemotactic receptor CCR7 augments in tumor infiltrating lymphocytes and in in-vitro de-clustered T cells, as well as their ability to transmigrate across lymphatic endothelial cells. We propose that ICAM-1-mediated homotypic T-lymphocyte aggregation may serve as a tumor-mediated immune retention mechanism entrapping activated CD8+ T cells in the tumor microenvironment. Modulation of T-cell adhesion may be of use to improve the transit of activated lymphocytes toward the lymph nodes and their subsequent recirculation.


Identification of monoclinic phase in CAD/CAM zirconia FPD frameworks.
The aim of this study was to identify the presence of monoclinic zirconia phase (m-ZrO2) in 5 computeraided design/computer-assisted manufacture zirconia systems composed of yttria-stabilized tetragonal zirconia polycrystals (Y-TZP). Three-unit fixedpartial dentures were prepared from Cercon (CR); Lava (LW); Zenotec Zr Bridge (WD); In-Ceram YZ (YZ); and IPS e-max ZirCAD (ZC), all milled in a pre-sintered stage and then fully sintered according to the manufacturers' instructions. Raman spectroscopy was used to identify and map the distribution of the m-ZrO2 phase at cervical crown margins, pontic and connector regions. Three sets of data were obtained from each material (n = 2) and the percentage volume (%Vm) of the m-ZrO2 phase was calculated per region. Statistical analysis was performed by two-way analysis of variance and Tukey test (a = 0.05). The m-ZrO2 phase was detected in all the specimens, with the highest intensity located at the crown margins. WD showed the lowest %Vm content (0- 3.14%), followed by LW (10.26-12.39%), CR (11.72-13.19%), ZC (11.13-14.10%) and YZ (12.15-14.99%). No statistically significant difference was found among LW, CR, ZC, YZ per region. Within each material group, significant differences were found between margin-pontic/connector (WD, YZ), margin-connector (CR, ZC) and margin-pontic (LW). The Y-TZP destabilizing m-ZrO2 phase was identified in all the fully sintered frameworks tested, with the highest %Vm located at the margins. The extent to which the presence of this phase may be implicated with zirconia low temperature degradation or porcelain to zirconia bonding is unknown.


Serum glycine is associated with regional body fat and insulin resistance in functionally-limited older adults.
Metabolic profiling may provide insight into biologic mechanisms related to age-related increases in regional adiposity and insulin resistance. The objectives of the current study were to characterize the association between mid-thigh intermuscular and subcutaneous adipose tissue (IMAT, SCAT, respectively) and, abdominal adiposity with the serum metabolite profile, to identify significant metabolites as further associated with the homeostasis model assessment of insulin resistance (HOMA-IR), and, to develop a HOMA-IR associated metabolite predictor set representative of regional adiposity, in 73 functionally-limited (short physical performance battery ≤10; SPPB) older adults (age range, 70-85 y). Fasting levels of 181 total metabolites, including amino acids, fatty acids and acylcarnitines were measured with use of an untargeted mass spectrometry-based metabolomic approach. Multivariable-adjusted linear regression was used in all analyses. Thirty-two, seven and one metabolite(s) were found to be associated with IMAT, abdominal adiposity and, SCAT, respectively, including the amino acid glycine, which was positively associated with SCAT and, negatively associated with both IMAT and abdominal adiposity. Glycine and four metabolites found to be significantly associated with regional adiposity were additionally associated with HOMA-IR. Separate stepwise regression models identified glycine as a HOMA-IR associated marker of both IMAT (model R(2) = 0.51, p<0.0001) and abdominal adiposity (model R(2) = 0.41, p<0.0001). Our findings for a positive association between glycine with SCAT but, a negative association between glycine with IMAT and abdominal adiposity supports the hypothesis that SCAT metabolic processes are different from that found in other fat depots. In addition, because of the significant associations found between glycine with HOMA-IR, IMAT, SCAT and abdominal adiposity, our results suggest glycine as a serum biomarker of both insulin sensitivity and regional fat mass in functionally-limited older adults.


Indicators for monitoring screening programs with primary HPV test.
following scientific evidence produced in numerous studies, as well as national and international guidelines, organized cervical cancer screening programs in Italy have gradually introduced the HPV test as primary screening test, replacing cytology. As public health interventions, screening programs must ensure equity, improvement in quality of life, and adequate information for the population involved with regards to benefits and possible risks; therefore, it is essential for quality to be constantly checked at every phase of the project.The Italian Cervical Screening Group (Gruppo Italiano per lo Screening Cervicale, GISCi) has written a handbook for the calculation and interpretation of cervical screening program monitoring indicators that take into account the new protocol based on primary HPV test with cytology triage. based on the European guidelines and Italian recommendations on primary HPVbased screening, the working group, which includes professionals from all the fields involved in cervical screening, identified the essential points needed to monitor the screening process, the accuracy of individual tests, and early outcomes, defining a specific indicator for each aspect. The indicators were grouped as follows: baseline indicators, indicators for test repeat after one year, cumulative indicators, and waiting times. For every indicator, the source of data, calculation formula, any standards or critical thresholds, and interpretation were defined. The standards are based on the results of NTCC trials or Italian pilot studies. the main indicators proposed for the organization are the following: number of invitations, compliance with first invitation, with one-year test repeat and with colposcopy; for test and process accuracy, a cohort approach was utilised, where indicators are based on women who must be followed for at least one year, so as to integrate the results obtained after the first HPV test with the outcome of the test's repetition after one year; the proposed indicators are positive HPV test, positive triage cytology, and positive predictive value of the referral to colposcopy at first and test repeat, and detection rate at first and test repeat. monitoring the new screening protocol, with cytology triage and one-year test repeat, is more complex, as it imposes a higher number of indicators and requires a cohort approach, which increases the delay before data are obtained.


Addressing Global Data Sharing Challenges.
This issue of the Journal of Empirical Research on Human Research Ethics highlights the ethical issues that arise when researchers conducting projects in low- and middle-income countries seek to share the data they produce. Although sharing data is considered a best practice, the barriers to doing so are considerable and there is a need for guidance and examples. To that end, the authors of this article reviewed the articles in this special issue to identify challenges common to the five countries and to offer some practical advice to assist researchers in navigating this "uncharted territory," as some termed it. Concerns around informed consent, data management, data dissemination, and validation of research contributions were cited frequently as particularly challenging areas, so the authors focused on these four topics with the goal of providing specific resources to consult as well as examples of successful projects attempting to solve many of the problems raised.


Prevalence, Effect on Functional Outcome, and Treatment of Sleep-Disordered Breathing in Patients With Subacute Stroke.
We aimed to elucidate the prevalence of sleep-disordered breathing (SDB), effect of SDB severity on the functional outcome, and feasibility of continuous positive airway pressure (CPAP) therapy in patients with subacute stroke. We recruited 433 consecutive patients (mean age: 66.5 years, 271 men) admitted to our rehabilitation wards for subacute stroke (8 to 90 days after onset) from August 2011-November 2013, who had undergone at least one successful sleep study within 4 weeks after admission to the wards. We investigated the prevalence of SDB, defined as a respiratory event index (REI) ≥ 5 events/h; the relationship between SDB severity and the functional outcome at discharge; and the number of patients receiving and adhering to CPAP therapy. REIs ≥ 5 and ≥ 15 events/h were observed for 87.3% (n = 378) and 46.4% (n = 201) of patients, respectively. The Functional Independence Measure score at discharge was significantly lower for patients with REI ≥ 15 events/h than for those with REI < 15 events/h. However, REI was not an independent factor for functional outcome after adjustment for potential confounders, irrespective of stroke types. CPAP therapy was administered to 41 patients (9.5%). During the mean follow-up period of 21.6 months, 20 patients (48.8%) dropped out from the CPAP therapy. Among the 23 patients who continued CPAP therapy until discharge, 17 (74%) continued its use throughout the follow-up period or discontinued therapy because of improvement. SDB prevalence was high in patients with subacute stroke at admission. However, SDB severity was not significantly related to functional outcome at discharge. Although the overall adherence was not good, relatively good adherence to CPAP therapy after discharge was observed when CPAP was successfully introduced during hospitalization.


Burnout among Healthcare Providers of COVID-19; a Systematic Review of Epidemiology and Recommendations.
In the current systematic review, we intended to systematically review the epidemiology of burnout and the strategies and recommendations to prevent or reduce it among healthcare providers (HCPs) of COVID-19 wards, so that policymakers can make more appropriate decisions. MEDLINE (accessed from PubMed), Science Direct, and Scopus electronic databases were systematically searched in English from December 01, 2019 to August 15, 2020, using MESH terms and related keywords. After reading the title and the abstract, unrelated studies were excluded. The full texts of the studies were evaluated by authors, independently, and the quality of the studies was determined. Then, the data were extracted and reported. 12 studies were included. Five studies investigated the risks factors associated with burnout; none could establish a causal relationship because of their methodology. No study examined any intervention to prevent or reduce burnout, and the provided recommendations were based on the authors' experiences and opinions. None of the studies followed up the participants, and all assessments were done according to the participants' self-reporting and declaration. Assessing burnout in the HCPs working in the frontline wards was performed in four studies; others evaluated burnout among all HCPs working in the regular and frontline wards. Paying attention to the mental health issues, reducing the workload of HCPs through adjusting their work shifts, reducing job-related stressors, and creating a healthy work environment may prevent or reduce the burnout.


Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini.
The mechanisms responsible for intrapancreatic digestive enzyme activation as well as the relationship between that activation and cell injury during pancreatitis are not understood. We have employed an in vitro system in which freshly prepared pancreatic acini are exposed to a supramaximally stimulating concentration of the CCK analog caerulein to explore these issues. We find that in vitro trypsinogen activation depends on the continued presence of Ca2+ in the suspending medium and that it is half-maximal in the presence of 0.3 mM Ca2+. Caerulein-induced trypsinogen activation can be halted by removal of Ca2+ from the suspending medium or by chelation of intracellular Ca2+. Increasing intracellular Ca2+ with either ionomycin or thapsigargin does not induce trypsinogen activation. We have monitored cell injury by measuring the leakage of lactate dehydrogenase (LDH) from acini and by quantitating intercalation of propidium iodide (PI) into DNA. Leakage of LDH and intercalation of PI in response to supramaximal stimulation with caerulein can be detected only after caerulein-induced trypsinogen activation has already occurred, and these indications of cell injury can be prevented by addition of a cell-permeant protease inhibitor. Our findings indicate that caerulein-induced intra-acinar cell activation of trypsinogen depends on a rise in intracellular Ca2+, which reflects entry of Ca2+ from the suspending medium. Intra-acinar cell activation of trypsinogen is an early as well as a critical event in pancreatitis. The subsequent cell injury in this model is mediated by activated proteases.


Sticking with the Pointy End? Molecular Configuration of Chloro Boron-Subphthalocyanine on Cu(111).
In this combined low-temperature scanning tunneling microscopy (STM) and density functional theory (DFT) study, we investigate self-assembly of the dipolar nonplanar organic semiconductor chloro boron-subphthalocyanine (ClB-SubPc) on Cu(111). We observe multiple distinct adsorption configurations and demonstrate that these can only be understood by taking surface-catalyzed dechlorination into account. A detailed investigation of possible adsorption configurations and the comparison of experimental and computational STM images demonstrates that the configurations correspond to "Cl-up" molecules with the B-Cl moiety pointing toward the vacuum side of the interface, and dechlorinated molecules. In contrast to the standard interpretation of adsorption of nonplanar molecules in the phthalocyanine family, we find no evidence for "Cl-down" molecules where the B-Cl moiety would be pointing toward the Cu surface. We show computationally that such a configuration is unstable and thus is highly unlikely to occur for ClB-SubPc on Cu(111). Using these assignments, we discuss the different self-assembly motifs in the submonolayer coverage regime. The combination of DFT and STM is essential to gain a full atomistic understanding of the surface-molecule interactions, and our findings imply that phthalocyanines may undergo surface-catalyzed reactions hitherto not considered. Our results also indicate that care has to be taken when analyzing possible adsorption configurations of polar members of the phthalocyanine family, especially when they are adsorbed on comparably reactive surfaces like Cu(111).


Antimicrobial potentials of different solvent extracted samples from Physalis ixocarpa.
The present study investigates the antimicrobial activities of different solvent extracted samples isolated from different parts of Physalis ixocarpa through disc diffusion assay using three different concentrations. Statistical analysis of the data revealed that different parts of the plant showed varying degree of inhibition against different bacteria at different concentrations. Different solvent extracted samples from the calyx showed inhibitory activity against most of the bacteria under study. Extracts from leaf and fruit samples showed activity against S. aureus and K. pneumoniae and extracts from the stem tissues were effective to control the growth of E. coli and K. pneumoniae. Crude methanolic extract from the stem and n-butanol extracted samples from fruit exhibited strong inhibitory activity against Klebsiella pneumoniae at highest concentrations. Antifungal activity was observed only in crude methanol extract from the leaf against Rhizopus stolinifer, Aspergillus niger and Penicillium chrysogenum.


Diarrhoeal disease and subsequent risk of death in infants and children residing in low-income and middle-income countries: analysis of the GEMS case-control study and 12-month GEMS-1A follow-on study.
The Global Enteric Multicenter Study (GEMS) was a 3-year case-control study that measured the burden, aetiology, and consequences of moderate-to-severe diarrhoea (MSD) in children aged 0-59 months. GEMS-1A, a 12-month follow-on study, comprised two parallel case-control studies, one assessing MSD and the other less-severe diarrhoea (LSD). In this report, we analyse the risk of death with each diarrhoea type and the specific pathogens associated with fatal outcomes. GEMS was a prospective, age-stratified, matched case-control study done at seven sites in Africa and Asia. Children aged 0-59 months with MSD seeking care at sentinel health centres were recruited along with one to three randomly selected matched community control children without diarrhoea. In the 12-month GEMS-1A follow-on study, children with LSD and matched controls, in addition to children with MSD and matched controls, were recruited at six of the seven sites; only cases of MSD and controls were enrolled at the seventh site. We compared risk of death during the period between enrolment and one follow-up household visit done about 60 days later (range 50-90 days) in children with MSD and LSD and in their respective controls. Approximately 50 pathogens were detected using, as appropriate, classic bacteriology, immunoassays, gel-based PCR and reverse transcriptase PCR, and quantitative real-time PCR (qPCR). Specimens from a subset of GEMS cases and controls were also tested by a TaqMan Array Card that compartmentalised probe-based qPCR for 32 enteropathogens. 223 (2·0%) of 11 108 children with MSD and 43 (0·3%) of 16 369 matched controls died between study enrolment and the follow-up visit at about 60 days (hazard ratio [HR] 8·16, 95% CI 5·69-11·68, p<0·0001). 12 (0·4%) of 2962 children with LSD and seven (0·2%) of 4074 matched controls died during the follow-up period (HR 2·78, 95% CI 0·95-8·11, p=0·061). Risk of death was lower in children with dysenteric MSD than in children with non-dysenteric MSD (HR 0·20, 95% CI 0·05-0·87, p=0·032), and lower in children with LSD than in those with non-dysenteric MSD (HR 0·29, 0·14-0·59, p=0·0006). In children younger than 24 months with MSD, infection with typical enteropathogenic Escherichia coli, enterotoxigenic E coli encoding heat-stable toxin, enteroaggregative E coli, Shigella spp (non-dysentery cases), Aeromonas spp, Cryptosporidium spp, and Entamoeba histolytica increased risk of death. Of 61 deaths in children aged 12-59 months with non-dysenteric MSD, 31 occurred among 942 children qPCR-positive for Shigella spp and 30 deaths occurred in 1384 qPCR-negative children (HR 2·2, 95% CI 1·2-3·9, p=0·0090), showing that Shigella was strongly associated with increased risk of death. Risk of death is increased following MSD and, to a lesser extent, LSD. Considering there are approximately three times more cases of LSD than MSD in the population, more deaths are expected among children with LSD than in those with MSD. Because the major attributable LSD-associated and MSD-associated pathogens are the same, implementing vaccines and rapid diagnosis and treatment interventions against these major pathogens are rational investments. Bill & Melinda Gates Foundation.


Oridonin induces G2/M cell cycle arrest and apoptosis through MAPK and p53 signaling pathways in HepG2 cells.
Oridonin, the main active constituent of Isodon rubescen, has antihepatocarcinoma activity in experimental and clinical settings. The aims of the study were to explore the anticancer effect of oridonin in HepG2 cells and to investigate the underlying mechanisms. Results showed that oridonin treatment for 24 or 48 h resulted in a marked decrease in cell viability time- and dose-dependently. IC50 values were determined as 38.86 microM and 24.90 microM for 24-h and 48-h treatments, respectively. Flow cytometric analysis showed that a 24-h treatment of 40 microM oridonin induced G2/M cell cycle arrest and apoptosis. Typical apoptotic nucleus alterations were observed with fluorescence microscope after DAPI staining. Immunoblot analysis demonstrated that oridonin treatment increased expression levels of p-JNK, p-p38, p-p53 and p21, elevated the level of cyclin B1/p-Cdc2 (Tyr15) complex, and inhibited the expression of p-ERK. Moreover, oridonin treatment activated caspase-9 and caspase-3. In conclusion, oridonin induced G2/M cell cycle arrest and apoptosis in HepG2 cells through MAPK and p53 pathways, which advances our understanding on the molecular mechanisms of oridonin in hepatocarcinoma management.


Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.
CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.


Coherent anti-Stokes Raman scattering in a microcavity.
We study tight focus coherent anti-Stokes Raman scattering (CARS) emission in a microcavity where the active medium is squeezed between two independent planar mirrors. We show strong modifications in the CARS forward and backward far-field radiation patterns. For low-order cavities, we demonstrate that most of the emitted power can be concentrated into a direction perpendicular to the mirrors.


Neurologic complications after sublaminar wiring. An experimental study in lambs.
To study possible neurologic complications, five lambs were operated on by sublaminar wiring at the thoracolumbar spine. Monitoring of the central motor pathway was carried out by percutaneous electrostimulation. A comparative anatomic study was designed to compare the magnitude of the spinal canal and the area occupied by the spinal cord at the low thoracic and lumbar level in 15 lamb, 8 pig, and 8 human spines. The following parameters were measured by slide caliper: the anteroposterior diameter of the spinal canal and the spinal cord, the length and thickness of the laminas, and the distance between each consecutive lamina. All five operated lambs showed major neurologic deficits after surgery. The results of the anatomic study suggest that there exists in humans a sufficiently ample "safety zone" that permits wire insertion without risking injury to neurologic structures. Such a "safety zone" is nonexistent in lambs and pigs unless an extensive laminectomy is performed to decrease the depth of wire penetration.


PARP-1 transcriptional activity is regulated by sumoylation upon heat shock.
Heat shock and other environmental stresses rapidly induce transcriptional responses subject to regulation by a variety of post-translational modifications. Among these, poly(ADP-ribosyl)ation and sumoylation have received growing attention. Here we show that the SUMO E3 ligase PIASy interacts with the poly(ADP-ribose) polymerase PARP-1, and that PIASy mediates heat shock-induced poly-sumoylation of PARP-1. Furthermore, PIASy, and hence sumoylation, appears indispensable for full activation of the inducible HSP70.1 gene. Chromatin immunoprecipitation experiments show that PIASy, SUMO and the SUMO-conjugating enzyme Ubc9 are rapidly recruited to the HSP70.1 promoter upon heat shock, and that they are subsequently released with kinetics similar to PARP-1. Finally, we provide evidence that the SUMO-targeted ubiquitin ligase RNF4 mediates heat-shock-inducible ubiquitination of PARP-1, regulates the stability of PARP-1, and, like PIASy, is a positive regulator of HSP70.1 gene activity. These results, thus, point to a novel mechanism for regulating PARP-1 transcription function, and suggest crosstalk between sumoylation and RNF4-mediated ubiquitination in regulating gene expression in response to heat shock.


Microfluidic in situ mechanical testing of photopolymerized gels.
Gels are a functional template for micro-particle fabrication and microbiology experiments. The control and knowledge of their mechanical properties is critical in a number of applications, but no simple in situ method exists to determine these properties. We propose a novel microfluidic based method that directly measures the mechanical properties of the gel upon its fabrication. We measure the deformation of a gel beam under a controlled flow forcing, which gives us a direct access to the Young's modulus of the material itself. We then use this method to determine the mechanical properties of poly(ethylene glycol) diacrylate (PEGDA) under various experimental conditions. The mechanical properties of the gel can be highly tuned, yielding two order of magnitude in the Young's modulus. The method can be easily implemented to allow for an in situ direct measurement and control of Young's moduli under various experimental conditions.


Accuracy of sentinel lymph node biopsy for patients with T2 and T3 breast cancers.
Although numerous studies have demonstrated that sentinel lymph node (SLN) biopsy can accurately determine the axillary nodal status for early breast cancer some studies have suggested that SLN biopsy may be less reliable for tumors >2 cm in size. This analysis was performed to determine whether tumor size affects the accuracy of SLN biopsy. The University of Louisville Breast Cancer Sentinel Lymph Node Study is a prospective multi-institutional study involving 226 surgeons. The study was approved by the Institutional Review Board of each institution, and informed consent was obtained from all patients. Patients with clinical stage T1-2 N0 breast cancer were eligible for the study. Some patients with T3 tumors were included because they were clinically staged as T2 but on final pathology were found to have tumors >5 cm. This analysis includes 2148 patients who were enrolled from August 1997 through October 2000. All patients underwent SLN biopsy using a combination of radioactive colloid and blue dye injection followed by completion Level I/II axillary dissection. Statistical comparison was performed by chi-square analysis. The SLN identification rate, false negative rate, and overall accuracy of SLN biopsy were not significantly different among tumor stages T1, T2, and T3. We conclude that SLN biopsy is no less accurate for T2-3 breast cancers compared with T1 tumors.


Reliable identification of the auditory thalamus using multi-modal structural analyses.
The medial geniculate body (MGB) of the thalamus is a key component of the auditory system. It is involved in relaying and transforming auditory information to the cortex and in top-down modulation of processing in the midbrain, brainstem, and ear. Functional imaging investigations of this region in humans, however, have been limited by the difficulty of distinguishing MGB from other thalamic nuclei. Here, we introduce two methods for reliably delineating MGB anatomically in individuals based on conventional and diffusion MRI data. The first uses high-resolution proton density weighted scanning optimized for subcortical grey-white contrast. The second uses diffusion-weighted imaging and probabilistic tractography to automatically segment the medial and lateral geniculate nuclei from surrounding structures based on their distinctive patterns of connectivity to the rest of the brain. Both methods produce highly replicable results that are consistent with published atlases. Importantly, both methods rely on commonly available imaging sequences and standard hardware, a significant advantage over previously described approaches. In addition to providing useful approaches for identifying the MGB and LGN in vivo, our study offers further validation of diffusion tractography for the parcellation of grey matter regions on the basis of their connectivity patterns.


Analysis of TP53 mutation status in human cancer cell lines: a reassessment.
Tumor-derived cell lines play an important role in the investigation of tumor biology and genetics. Across a wide array of studies, they have been tools of choice for the discovery of important genes involved in cancer and for the analysis of the cellular pathways that are impaired by diverse oncogenic events. They are also invaluable for screening novel anticancer drugs. The TP53 protein is a major component of multiple pathways that regulate cellular response to various types of stress. Therefore, TP53 status affects the phenotype of tumor cell lines profoundly and must be carefully ascertained for any experimental project. In the present review, we use the 2014 release of the UMD TP53 database to show that TP53 status is still controversial for numerous cell lines, including some widely used lines from the NCI-60 panel. Our analysis clearly confirms that, despite numerous warnings, the misidentification of cell lines is still present as a silent and neglected issue, and that extreme care must be taken when determining the status of p53, because errors may lead to disastrous experimental interpretations. A novel compendium gathering the TP53 status of 2,500 cell lines has been made available (http://p53.fr). A stand-alone application can be used to browse the database and extract pertinent information on cell lines and associated TP53 mutations. It will be updated regularly to minimize any scientific issues associated with the use of misidentified cell lines (http://p53.fr).


Early animal evolution: emerging views from comparative biology and geology.
The Cambrian appearance of fossils representing diverse phyla has long inspired hypotheses about possible genetic or environmental catalysts of early animal evolution. Only recently, however, have data begun to emerge that can resolve the sequence of genetic and morphological innovations, environmental events, and ecological interactions that collectively shaped Cambrian evolution. Assembly of the modern genetic tool kit for development and the initial divergence of major animal clades occurred during the Proterozoic Eon. Crown group morphologies diversified in the Cambrian through changes in the genetic regulatory networks that organize animal ontogeny. Cambrian radiation may have been triggered by environmental perturbation near the Proterozoic-Cambrian boundary and subsequently amplified by ecological interactions within reorganized ecosystems.


Alterations of DNA-dependent DNA polymerase activities in the immature quail oviduct in response to estrogen stimulation.
Administration of diethylstilbestrol, an estrogen analogue, to immature female quails causes an increase of extractable DNA-dependent DNA polymerase activities from the oviduct. At least two forms of polymerases have been determined, a high molecular weight polymerase (210,000 daltons) and a low molecular weight polymerase (34,000 daltons) calculated from column chromatography Sephadex G-200. During the primary hormone stimulation the amount of extractable enzyme reaches a maximum on the fifth day after daily injections of the hormone. In the period of withdrawal the activities decrease and reach values similar to those determined in the unstimulated oviducts. During secondary stimulation the polymerase activities increase again the first day; subsequently the values decrease drastically. The alterations in enzyme activity correlate with the DNA synthesis in the oviduct, as measured by analytical determination of the DNA content.


Genome-wide discovery and verification of novel structured RNAs in Plasmodium falciparum.
We undertook a genome-wide search for novel noncoding RNAs (ncRNA) in the malaria parasite Plasmodium falciparum. We used the RNAz program to predict structures in the noncoding regions of the P. falciparum 3D7 genome that were conserved with at least one of seven other Plasmodium spp. genome sequences. By using Northern blot analysis for 76 high-scoring predictions and microarray analysis for the majority of candidates, we have verified the expression of 33 novel ncRNA transcripts including four members of a ncRNA family in the asexual blood stage. These transcripts represent novel structured ncRNAs in P. falciparum and are not represented in any RNA databases. We provide supporting evidence for purifying selection acting on the experimentally verified ncRNAs by comparing the nucleotide substitutions in the predicted ncRNA candidate structures in P. falciparum with the closely related chimp malaria parasite P. reichenowi. The high confirmation rate within a single parasite life cycle stage suggests that many more of the predictions may be expressed in other stages of the organism's life cycle.


Statistical representation of high-dimensional deformation fields with application to statistically constrained 3D warping.
This paper proposes a 3D statistical model aiming at effectively capturing statistics of high-dimensional deformation fields and then uses this prior knowledge to constrain 3D image warping. The conventional statistical shape model methods, such as the active shape model (ASM), have been very successful in modeling shape variability. However, their accuracy and effectiveness typically drop dramatically in high-dimensionality problems involving relatively small training datasets, which is customary in 3D and 4D medical imaging applications. The proposed statistical model of deformation (SMD) uses wavelet-based decompositions coupled with PCA in each wavelet band, in order to more accurately estimate the pdf of high-dimensional deformation fields, when a relatively small number of training samples are available. SMD is further used as statistical prior to regularize the deformation field in an SMD-constrained deformable registration framework. As a result, more robust registration results are obtained relative to using generic smoothness constraints on deformation fields, such as Laplacian-based regularization. In experiments, we first illustrate the performance of SMD in representing the variability of deformation fields and then evaluate the performance of the SMD-constrained registration, via comparing a hierarchical volumetric image registration algorithm, HAMMER, with its SMD-constrained version, referred to as SMD+HAMMER. This SMD-constrained deformable registration framework can potentially incorporate various registration algorithms to improve robustness and stability via statistical shape constraints.


Physical Activity Guidelines Compliance and Its Relationship With Preventative Health Behaviors and Risky Health Behaviors.
People who are physically active enjoy a multitude of health benefits across their lifespan compared with people who are not physically active. However, little research has sought to determine whether those who meet the physical activity (PA) guidelines also engage in other healthy behaviors. The purpose of this study was to compare healthy behaviors of people who met the PA guidelines set forth by the U.S. Department of Health and Human Services to those who did not meet the guidelines. This was a cross-sectional study using data from the Behavioral Risk Factor Surveillance System survey conducted in 2017. Descriptive statistical analyses were performed using chi-square tests. Odds and adjusted odds ratios were calculated using multiple logistic regressions. Those who met the PA guidelines were more likely to get a flu shot, have a medical checkup, take human immunodeficiency virus tests, wear seatbelts, and binge drink more frequently, compared with those who did not meet the guidelines. This group is also less likely to be smokers and be overweight or obese in comparison to their inactive counterparts. The positive association between PA and other healthy behaviors represents synergistic health activities, with healthy behaviors supporting others.


Soil Functional Zone Management: A Vehicle for Enhancing Production and Soil Ecosystem Services in Row-Crop Agroecosystems.
There is increasing global demand for food, bioenergy feedstocks and a wide variety of bio-based products. In response, agriculture has advanced production, but is increasingly depleting soil regulating and supporting ecosystem services. New production systems have emerged, such as no-tillage, that can enhance soil services but may limit yields. Moving forward, agricultural systems must reduce trade-offs between production and soil services. Soil functional zone management (SFZM) is a novel strategy for developing sustainable production systems that attempts to integrate the benefits of conventional, intensive agriculture, and no-tillage. SFZM creates distinct functional zones within crop row and inter-row spaces. By incorporating decimeter-scale spatial and temporal heterogeneity, SFZM attempts to foster greater soil biodiversity and integrate complementary soil processes at the sub-field level. Such integration maximizes soil services by creating zones of 'active turnover', optimized for crop growth and yield (provisioning services); and adjacent zones of 'soil building', that promote soil structure development, carbon storage, and moisture regulation (regulating and supporting services). These zones allow SFZM to secure existing agricultural productivity while avoiding or minimizing trade-offs with soil ecosystem services. Moreover, the specific properties of SFZM may enable sustainable increases in provisioning services via temporal intensification (expanding the portion of the year during which harvestable crops are grown). We present a conceptual model of 'virtuous cycles', illustrating how increases in crop yields within SFZM systems could create self-reinforcing feedback processes with desirable effects, including mitigation of trade-offs between yield maximization and soil ecosystem services. Through the creation of functionally distinct but interacting zones, SFZM may provide a vehicle for optimizing the delivery of multiple goods and services in agricultural systems, allowing sustainable temporal intensification while protecting and enhancing soil functioning.


Safety, adherence and acceptability of intermittent tenofovir/emtricitabine as HIV pre-exposure prophylaxis (PrEP) among HIV-uninfected Ugandan volunteers living in HIV-serodiscordant relationships: a randomized, clinical trial.
Efficacy of oral pre-exposure prophylaxis (PrEP) in prevention of HIV acquisition has been evaluated using a daily regimen. However, adherence to long term daily medication is rarely perfect. Intermittent regimen may be a feasible alternative. Preclinical studies have demonstrated effectiveness of intermittent PrEP in SHIV prevention among animals. However, little is known about intermittent PrEP regimens. Seventy two HIV-uninfected volunteers in HIV serodiscordant couple relationships in Uganda were randomly assigned to receive daily oral Tenofovir/Emtricitabine (TDF/FTC-Truvada) or placebo, or intermittent (Monday, Friday and within 2 hours after sex, not to exceed one dose per day) oral TDF/FTC or placebo in a 2:1:2:1 ratio. Volunteers and study staff were blinded to drug assignment, but not to regimen assignment. Volunteers were followed for 4 months after randomization, with monthly clinical and laboratory safety assessments and comprehensive HIV risk reduction services. Adherence was monitored using medication event monitoring system (MEMS) and self-report. Sexual activity data were collected via daily short text message (SMS) and self-report. HIV-specific immune responses were assessed by IFN-γ ELISPOT. Both daily and intermittent oral TDF/FTC regimens were well tolerated. Median MEMS adherence rates were 98% (IQR: 93-100) for daily PrEP regimen, 91% (IQR: 73-97) for fixed intermittent dosing and 45% (IQR: 20-63) for post-coital dosing. SMS response rate was 74%, but increased to 80% after excluding server outages; results may have been affected by the novelty of this measure. The majority of volunteers expressed willingness with no particular preference for either regimen. Both daily and intermittent oral PrEP dosing regimens were safe. Adherence was high for daily and fixed intermittent dosing; post-coital dosing was associated with poor adherence. Fixed intermittent PrEP regimens may be feasible especially if a minimum effective drug concentration correlating with HIV prevention can be achieved with this dosing. Clinicaltrials.gov number NCT00931346.


Arterial hyperoxia and in-hospital mortality after resuscitation from cardiac arrest.
Hyperoxia has recently been reported as an independent risk factor for mortality in patients resuscitated from cardiac arrest. We examined the independent relationship between hyperoxia and outcomes in such patients. We divided patients resuscitated from nontraumatic cardiac arrest from 125 intensive care units (ICUs) into three groups according to worst PaO2 level or alveolar-arterial O2 gradient in the first 24 hours after admission. We defined 'hyperoxia' as PaO2 of 300 mmHg or greater, 'hypoxia/poor O2 transfer' as either PaO2 < 60 mmHg or ratio of PaO2 to fraction of inspired oxygen (FiO2 ) < 300, 'normoxia' as any value between hypoxia and hyperoxia and 'isolated hypoxemia' as PaO2 < 60 mmHg regardless of FiO2. Mortality at hospital discharge was the main outcome measure. Of 12,108 total patients, 1,285 (10.6%) had hyperoxia, 8,904 (73.5%) had hypoxia/poor O2 transfer, 1,919 (15.9%) had normoxia and 1,168 (9.7%) had isolated hypoxemia (PaO2 < 60 mmHg). The hyperoxia group had higher mortality (754 (59%) of 1,285 patients; 95% confidence interval (95% CI), 56% to 61%) than the normoxia group (911 (47%) of 1,919 patients; 95% CI, 45% to 50%) with a proportional difference of 11% (95% CI, 8% to 15%), but not higher than the hypoxia group (5,303 (60%) of 8,904 patients; 95% CI, 59% to 61%). In a multivariable model controlling for some potential confounders, including illness severity, hyperoxia had an odds ratio for hospital death of 1.2 (95% CI, 1.1 to 1.6). However, once we applied Cox proportional hazards modelling of survival, sensitivity analyses using deciles of hypoxemia, time period matching and hyperoxia defined as PaO2 > 400 mmHg, hyperoxia had no independent association with mortality. Importantly, after adjustment for FiO2 and the relevant covariates, PaO2 was no longer predictive of hospital mortality (P = 0.21). Among patients admitted to the ICU after cardiac arrest, hyperoxia did not have a robust or consistently reproducible association with mortality. We urge caution in implementing policies of deliberate decreases in FiO2 in these patients.


The GiSAS study: rationale and design of a pragmatic randomized controlled trial on aripiprazole, olanzapine and haloperidol in the long-term treatment of schizophrenia.
Given the controversy about the comparative efficacy of first- compared with second-generation antipsychotics in the treatment of schizophrenia, more large-scale evidence is needed to guide clinicians in their prescriptions. Most randomized controlled trials (RCTs) were conducted in centers of excellence on highly selected samples, poorly representative of real-world patients, and often suffered conflicts of interest as they were sponsored by drug companies. The primary aim of the present study is to compare the effectiveness of haloperidol, olanzapine and aripiprazole in a representative sample of schizophrenia patients. The GiSAS trial is an open-label, independent, pragmatic RCT in Italian community-based public psychiatric services. At least 260 patients meeting the DSM-IV criteria for schizophrenia will be randomly allocated to one of the study drugs and followed up for one year. A two-year observational phase will follow. The primary outcome for tolerability will be the onset of metabolic syndrome. The primary endpoint for effectiveness will be discontinuation of antipsychotic monotherapy. Secondary measures include global functioning, time to discontinuation due to side-effects, change of lipid profile, extrapyramidal symptoms and other adverse effects. In the last four years, the GiSAS study group has been working to implement this multicenter RCT. The trial mechanism is now fully functional and working. As of end of February 2011, 260 subjects were randomized by 54 study investigators in 33 out of 43 participating centers.


Hospital responses to the leapfrog group in local markets.
The Leapfrog (LF) initiative, directed at improving patient safety in hospitals, may be the most ambitious, coordinated attempt to date on the part of large employers to shape the delivery of health care in America. This article assesses the role of market conditions and other factors in influencing hospital responses to LF activities at the community level. Community characteristics were found to be important in explaining hospital participation in a LF safety standards survey at the study sites. However, characteristics of the individual hospitals, and of the LF goals themselves, were more important in explaining the relatively limited progress by hospitals across all sites in achieving those goals over a 5-year period.


Tyrosine phosphorylation of Grb2-associated proteins correlates with phospholipase C gamma 1 activation in T cells.
Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.


Evaluation of Selected Cucurbitaceous Vegetables for Resistance to Zucchini yellow mosaic virus.
Zucchini yellow mosaic virus (ZYMV) causes considerable losses of cucurbitaceous vegetables grown nearly all over the world; indeed, the commonly planted cultivars are highly susceptible to ZYMV. In all, 3 cultivars of American and 8 of European summer squash (Cucurbita pepo), and 6 Japanese and 21 European cucumber lines (Cucumis sativus), including both slicing and pickling species, were selected for the evaluation of their resistance to the most virulent Czech strain, ZYMV-H (GenBank accession number DQ144054). Butternut squash (Cucurbita moschata) 'Menina 15', Chinese slicing cucumber 'Taichung Mou Gua-1' (TMG-1), and watermelon (Citrullus lanatus) accession PI 595203 were included in the experiment, because they were reported to be resistant to ZYMV. The tested plants were mechanically inoculated by ZYMV-H and their resistance was assessed through a comparison of the relative virus protein concentrations and visual symptoms. Butternut squash Menina 15, Chinese slicing cucumber TMG-1, Japanese slicing cucumber breeds 'G22' and 'A192-18', and watermelon PI 595203 were evaluated as immune: the virus concentration in their leaves was zero, as verified by polymerase chain reaction. American summer squash 'Cougar' and Japanese slicing cucumber breeds 'A202-18', 'R10', and 'S93-18' were clearly resistant, because the virus multiplied at a low rate in these plants. The remaining tested cultivars were tolerant or susceptible to ZYMV.


"Being all alone makes me sad": loneliness in older adults with depressive symptoms.
ABSTRACT Background: The consequences of co-occurring persistent loneliness and late life depression are yet unknown. The aim of this study was to get a deeper insight into the mental health consequences of loneliness in older persons with depressive symptoms and their perspectives of emotional distress by using a mixed-methods study design. Methods: Two hundred and forty nine community-dwelling older persons with depressive symptoms according to the Patient Health Questionnaire-9 (≥6) were included. A validated cut-off score on the Loneliness Scale was used to distinguish lonely elders from elders who were not lonely. Quantitative and qualitative data were used to examine differences in mental health and perspectives on emotional distress between lonely and not lonely older persons with depressive symptoms. Results: Loneliness was highly prevalent among older persons with depressive symptoms (87.8%). Lonely people suffered from worse mental ill-health (e.g., more severe depressive symptoms, more often a depressive disorder and a lower quality of life) compared to not lonely individuals. Depressive symptoms were regarded as a logical consequence of loneliness. Lonely people perceived little command over their situation: causes of loneliness were attributed externally to perceived deficits in their social networks and they mainly expressed the need to be listened to. Conclusion: Our findings underline the importance of paying considerable attention to (severe) loneliness in older adults with depressive symptoms given its high prevalence and serious mental health consequences. Future studies should look into whether addressing loneliness when discussing depressive symptoms in clinical practice may provide an opportunity to better adjust to older persons' depression perceptions and might therefore improve care utilization.


Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules.
We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.


miR-9 is a tumor suppressor in pediatric AML with t(8;21).
MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of acute myeloid leukemia (AML) with t(8;21), inv(16) or mixed lineage leukemia (MLL) rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor-suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor-suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner.


The effect of a PP2A inhibitor on the nuclear receptor corepressor pathway in glioma.
Nuclear receptor corepressor (N-CoR) forms a complex that maintains neural stem cells in an undifferentiated state through transcriptional repression. Recently, it has been shown that N-CoR is overexpressed in glioblastoma multiforme (GBM) tumor stem cells and has a putative role in maintaining these cells in an undifferentiated immortal state. To determine the effects of disruption of N-CoR complex function by serine/threonine protein phosphatase 2A (PP2A) inhibition on GBM tumor cell differentiation and proliferation, the authors developed and investigated a competitive small molecule inhibitor (LB1) of PP2A in GBM. The authors investigated the effects of LB1 on GBM proliferation and molecular differentiation pathways using in vitro and in vivo studies. The LB1 inhibited PP2A, leading to increased levels of phosphorylated Akt kinase and decreased NCoR expression, as well as dose-dependent antiproliferative activity in cultured U87 and U251 malignant glioma cells (dose range 1-10 microM). Systemic LB1 treatment (1.5 mg/kg/day for 21 days) had significant tumor antiproliferative effects in mice harboring U87 glioma xenografts (73% mean reduction in tumor volume compared with controls; p < 0.001). Moreover, a reduction in PP2A expression and activity after LB1 treatment in vivo correlated with increased Akt phosphorylation, reduced nuclear N-CoR expression and N-CoR cytoplasmic translocation, and increased accumulation of acetylated core histones, which coincided with the appearance of glial fibrillary acidic protein-expressing tumor cells. These findings indicate that PP2A inhibition effectively disrupts N-CoR complex function/expression and leads to cytoplasmic translocation of N-CoR with subsequent tumor cell differentiation and/or death. Therapeutic paradigms that target N-CoR function in the cancer stem cell component of malignant gliomas may have treatment utility.


Implicit and explicit memory in young, old, and demented adults.
Previous study of scopolamine and memory (Grober et al., 1989) showed that young adults given moderate or high doses of scopolamine maintained maximum cued recall in spite of a dose-dependent decrement in free recall when memory was assessed by cued selective reminding (CSR), a procedure which circumvents inattention and induces semantic processing. Intact recall by CSR indicates either that scopolamine impairs memory indirectly through effects on attention and information processing or that it impairs explicit memory but not implicit memory. In the present study which was done to determine if CSR reflects explicit or implicit memory, a free association test was used to estimate implicit memory after CSR was administered; explicit memory was estimated with a final trial of cued recall. Data from young, nondemented, and demented adults indicate that CSR reflects explicit memory supporting the interpretation of the previous study that scopolamine does not produce direct impairment of explicit memory.


Bacteriophage predation promotes serovar diversification in Listeria monocytogenes.
Listeria monocytogenes is a bacterial pathogen classified into distinct serovars (SVs) based on somatic and flagellar antigens. To correlate phenotype with genetic variation, we analyzed the wall teichoic acid (WTA) glycosylation genes of SV 1/2, 3 and 7 strains, which differ in decoration of the ribitol-phosphate backbone with N-acetylglucosamine (GlcNAc) and/or rhamnose. Inactivation of lmo1080 or the dTDP-l-rhamnose biosynthesis genes rmlACBD (lmo1081-1084) resulted in loss of rhamnose, whereas disruption of lmo1079 led to GlcNAc deficiency. We found that all SV 3 and 7 strains actually originate from a SV 1/2 background, as a result of small mutations in WTA rhamnosylation and/or GlcNAcylation genes. Genetic complementation of different SV 3 and 7 isolates using intact alleles fully restored a characteristic SV 1/2 WTA carbohydrate pattern, including antisera reactions and phage adsorption. Intriguingly, phage-resistant L. monocytogenes EGDe (SV 1/2a) isolates featured the same glycosylation gene mutations and were serotyped as SV 3 or 7 respectively. Again, genetic complementation restored both carbohydrate antigens and phage susceptibility. Taken together, our data demonstrate that L. monocytogenes SV 3 and 7 originate from point mutations in glycosylation genes, and we show that phage predation represents a major driving force for serovar diversification and evolution of L. monocytogenes.


Dietary patterns in six european populations: results from EURALIM, a collaborative European data harmonization and information campaign.
To determine and describe the extent to which European dietary data collected in disparate surveys can be meaningfully compared. Seven independent population-based surveys from six European countries were initially included. Differences in study designs and methodological approaches were examined. Risk factor data for 31,289 adults aged 40-59 y were harmonized and pooled in a common, centralized database. Direct comparisons of dietary measures across studies were not deemed appropriate due to methodological heterogeneity. Nonetheless, comparisons of intra-population contrasts by gender across sites were considered valid. Women consumed fruit and vegetables more often than men. Age-standardized gender differences in the prevalence of low fruit and vegetable consumption ranged from 7 to 18% and 5 to 15%, respectively. Data on energy intake showed good agreement across study populations. The proportion of total energy from macronutrients was similar for women and men. Gender differences for relative intakes of saturated fatty acids (percentage energy) were small and only in France were they significant. Dietary fibre density was significantly higher in women than in men. Overall, the participating Southern European populations from Italy and Spain exhibited more healthful food composition patterns. Contrasts in dietary patterns by gender across populations may provide the basis for health promotion campaigns. The most favourable patterns observed may serve as attainable goals for other populations. An international risk factor surveillance programme based upon locally run, good quality studies has the potential to provide the needed data. European Community (DG V), project 96CVVF3-446-0; Swiss Federal Office for Education and Science, OFES 96.0089.


Environmental enrichment, training, and habitat characteristics of common bottlenose dolphins (Tursiops truncatus) and Indo-Pacific bottlenose dolphins (Tursiops aduncus).
In recent decades, animal welfare science has evolved to utilize a multidisciplinary approach to assess the welfare of animals in accredited zoos and aquariums. Science-based animal welfare assessments have become an essential component of management programs and widespread application is expected by animal care professionals. Management practices for bottlenose dolphins in accredited facilities incorporate several programs that potentially impact animal welfare including environmental enrichment and animal training. Additionally, habitat characteristics, such as the dimensions of the habitat, have been proposed to affect welfare. While accredited facilities are required to meet high standards of care, habitat characteristics and management practices are not standardized across locations. Knowledge and subsequent application of these practices and habitat characteristics can enhance our understanding of factors associated with positive welfare. As part of a larger study of dolphin welfare titled "Towards understanding the welfare of cetaceans in zoos and aquariums" (colloquially called the Cetacean Welfare Study), survey data were collected from 86 bottlenose dolphins in 40 habitats at 38 facilities in seven countries. The major aims of this paper are to provide general descriptive information regarding dolphin management in accredited zoos and aquariums and to provide supplemental context to the other research published from the Cetacean Welfare Study data set. This paper provides a review of current habitat characteristics and management practices at those 38 accredited zoos and aquariums. These data enabled the identification and quantification of how cetacean management practices differed between participating facilities accredited by the Alliance for Marine Mammal Parks and Aquariums and the Association of Zoos & Aquariums. Variables were selected based on their potential association with welfare including the physical habitat, environmental enrichment, and training programs. The variables were also used for subsequent research in this collection of related papers to investigate important connections between potential indicators of welfare and habitat characteristics, environmental enrichment, and training programs.


Endometriosis: a high-risk population for major chronic diseases?
Despite an estimated prevalence of 10% in women, the etiology of endometriosis remains poorly understood. Over recent decades, endometriosis has been associated with risk of several chronic diseases, such as cancer, autoimmune diseases, asthma/atopic diseases and cardiovascular diseases. A deeper understanding of these associations is needed as they may provide new leads into the causes or consequences of endometriosis. This review summarizes the available epidemiological findings on the associations between endometriosis and other chronic diseases and discusses hypotheses for underlying mechanisms, potential sources of bias and methodological complexities. We performed a comprehensive search of the PubMed/Medline and ISI Web of Knowledge databases for all studies reporting on the associations between endometriosis and other diseases published in English through to May 2014, using numerous search terms. We additionally examined the reference lists of all identified papers to capture any additional articles that were not identified through computer searches. We identified 21 studies on the associations between endometriosis and ovarian cancer, 14 for breast cancer, 8 for endometrial cancer, 4 for cervical cancer, 12 for cutaneous melanoma and 3 for non-Hodgkin's lymphoma, as well as 9 on the links between endometriosis and autoimmune diseases, 6 on the links with asthma and atopic diseases, and 4 on the links with cardiovascular diseases. Endometriosis patients were reported to be at higher risk of ovarian and breast cancers, cutaneous melanoma, asthma, and some autoimmune, cardiovascular and atopic diseases, and at decreased risk of cervical cancer. Increasing evidence suggests that endometriosis patients are at higher risk of several chronic diseases. Although the underlying mechanisms are not yet understood, the available data to date suggest that endometriosis is not harmless with respects to women's long-term health. If these relationships are confirmed, these findings may have important implications in screening practices and in the management and care of endometriosis patients.


Synthesis, structures and electrochemical and photophysical properties of anilido-benzoxazole boron difluoride (ABB) complexes.
A new series of four-ring-fused π-conjugated anilido-benzoxazole boron difluoride (ABB) dyes were synthesized by employing an unsymmetrical bidentate ligand under a mild reaction condition. X-ray structural analysis demonstrated that the four-ring-fused π-conjugated skeleton is nearly coplanar, and almost orthogonal to the side anilido phenyl group with dihedral angles of 74-86°. The synthesized complexes exhibit very bright luminescence in solution (Φf = 0.45-0.96 in CH2Cl2) and in the solid-state (Φf = 0.07-0.37). These complexes show a larger Stokes shift (56-128 nm) than the well-known boron dipyrromethene dyes (8-12 nm, in most cases). The role of molecular packing patterns elucidated by the assistance of their X-ray crystal structures rationalizes the solid-state fluorescence. One of the tested compounds displayed aggregation induced emission (AIE). First-principle-based quantum-chemical studies were carried out on complexes . Time-dependent DFT (TD-DFT) calculations support the experimental results. The participation of the anilido phenyl moiety and the fluorine atoms was found to be negligible in the LUMO orbitals.


Incorporating diverse data and realistic complexity into demographic estimation procedures for sea otters.
Reliable information on historical and current population dynamics is central to understanding patterns of growth and decline in animal populations. We developed a maximum likelihood-based analysis to estimate spatial and temporal trends in age/sex-specific survival rates for the threatened southern sea otter (Enhydra lutris nereis), using annual population censuses and the age structure of salvaged carcass collections. We evaluated a wide range of possible spatial and temporal effects and used model averaging to incorporate model uncertainty into the resulting estimates of key vital rates and their variances. We compared these results to current demographic parameters estimated in a telemetry-based study conducted between 2001 and 2004. These results show that survival has decreased substantially from the early 1990s to the present and is generally lowest in the north-central portion of the population's range. The greatest temporal decrease in survival was for adult females, and variation in the survival of this age/sex class is primarily responsible for regulating population growth and driving population trends. Our results can be used to focus future research on southern sea otters by highlighting the life history stages and mortality factors most relevant to conservation. More broadly, we have illustrated how the powerful and relatively straightforward tools of information-theoretic-based model fitting can be used to sort through and parameterize quite complex demographic modeling frameworks.


Differential Phosphorylation of RNA Polymerase III and the Initiation Factor TFIIIB in Saccharomyces cerevisiae.
The production of ribosomes and tRNAs for protein synthesis has a high energetic cost and is under tight transcriptional control to ensure that the level of RNA synthesis is balanced with nutrient availability and the prevailing environmental conditions. In the RNA polymerase (pol) III system in yeast, nutrients and stress affect transcription through a bifurcated signaling pathway in which protein kinase A (PKA) and TORC1 activity directly or indirectly, through downstream kinases, alter the phosphorylation state and function of the Maf1 repressor and Rpc53, a TFIIF-like subunit of the polymerase. However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components. To address this issue, we systematically examined all 17 subunits of pol III along with the three subunits of the initiation factor TFIIIB for evidence of differential phosphorylation in response to inhibition of TORC1. A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated. Bdp1 is phosphorylated on four major sites during exponential growth and the protein is variably dephosphorylated under conditions that inhibit tRNA gene transcription. PKA, the TORC1-regulated kinase Sch9 and protein kinase CK2 are all implicated in the phosphorylation of Bdp1. Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression. The new findings suggest an integrated regulatory model for signaling events controlling pol III transcription.


Low-loss, flat-topped and spectrally uniform silicon-nanowire-based 5th-order CROW fabricated by ArF-immersion lithography process on a 300-mm SOI wafer.
We report superior spectral characteristics of silicon-nanowire-based 5th-order coupled resonator optical waveguides (CROW) fabricated by 193-nm ArF-immersion lithography process on a 300-mm silicon-on-insulator wafer. We theoretically analyze spectral characteristics, considering random phase errors caused by micro fabrication process. It will be experimentally demonstrated that the fabricated devices exhibit a low excess loss of 0.4 ± 0.2 dB, a high out-of-band rejection ratio of >40dB, and a wide flatband width of ~2 nm. Furthermore, we evaluate manufacturing tolerances for intra-dies and inter-dies, comparing with the cases for 248-nm KrF-dry lithography process. It will be shown that the 193-nm ArF-immersion lithography process can provide much less excess phase errors of Si-nanowire waveguides, thus enabling to give better filter spectral characteristics. Finally, spectral superiorities will be reconfirmed by measuring 25 Gbps modulated signals launched into the fabricated device. Clear eye diagrams are observed when the wavelengths of modulated signals are stayed within almost passband of the 5th-order CROW.


Epileptic Focus Localization Using Discrete Wavelet Transform Based on Interictal Intracranial EEG.
Over the past decade, with the development of machine learning, discrete wavelet transform (DWT) has been widely used in computer-aided epileptic electroencephalography (EEG) signal analysis as a powerful time-frequency tool. But some important problems have not yet been benefitted from DWT, including epileptic focus localization, a key task in epilepsy diagnosis and treatment. Additionally, the parameters and settings for DWT are chosen empirically or arbitrarily in previous work. In this work, we propose a framework to use DWT and support vector machine (SVM) for epileptic focus localization problem based on EEG. To provide a guideline in selecting the best settings for DWT, we decompose the EEG segments in seven commonly used wavelet families to their maximum theoretical levels. The wavelet and its level of decomposition providing the highest accuracy in each wavelet family are then used in a grid search for obtaining the optimal frequency bands and wavelet coefficient features. Our approach achieves promising performance on two widely-recognized intrancranial EEG datasets that are also seizure-free, with an accuracy of 83.07% on the Bern-Barcelona dataset and an accuracy of 88.00% on the UBonn dataset. Compared with existing DWT-based approaches in epileptic EEG analysis, the proposed approach leads to more accurate and robust results. A guideline for DWT parameter setting is provided at the end of the paper.


Quantification and biodistribution of iron oxide nanoparticles in the primary clearance organs of mice using T1 contrast for heating.
To use contrast based on longitudinal relaxation times (T1 ) or rates (R1 ) to quantify the biodistribution of iron oxide nanoparticles (IONPs), which are of interest for hyperthermia therapy, cell targeting, and drug delivery, within primary clearance organs. Mesoporous silica-coated IONPs (msIONPs) were intravenously injected into 15 naïve mice. Imaging and mapping of the longitudinal relaxation rate constant at 24 h or 1 week postinjection were performed with an echoless pulse sequence (SWIFT). Alternating magnetic field heating measurements were also performed on ex vivo tissues. Signal enhancement from positive T1 contrast caused by IONPs was observed and quantified in vivo in liver, spleen, and kidney at concentrations up to 3.2 mg Fe/(g tissue wt.) (61 mM Fe). In most cases, each organ had a linear correlation between the R1 and the tissue iron concentration despite variations in intra-organ distribution, degradation, and IONP surface charge. Linear correlation between R1 and volumetric SAR in hyperthermia therapy was observed. The linear dependence between R1 and tissue iron concentration in major organs allows quantitative monitoring of IONP biodistribution in a dosage range relevant to magnetic hyperthermia applications, which falls into the concentration gap between CT and conventional MRI techniques. Magn Reson Med 78:702-712, 2017. © 2016 International Society for Magnetic Resonance in Medicine.


Tenascin supports lymphocyte rolling.
Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.


State-specific trends in lung cancer incidence and smoking--United States, 1999-2008.
Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death in the United States. Most deaths from lung cancer are caused by cigarette smoking and exposure to secondhand smoke. Large variations in lung cancer, smoking behavior, and tobacco control programs and policies have been observed among states. Effective tobacco control policies can decrease smoking prevalence, ultimately leading to decreases in lung cancer. To assess lung cancer incidence by state, CDC analyzed data from the National Program of Cancer Registries (NPCR) and the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program for the period 1999-2008. To assess smoking behavior by state, data from the Behavioral Risk Factor Surveillance System (BRFSS) for the period 1994-2009 were analyzed. This report summarizes the results of these analyses. From 1999 to 2008, decreases in lung cancer incidence were observed among men in 35 states and among women in six states. Regionally, the lowest rates and most rapid rate of decline in lung cancer were concentrated among states in the West, correlating with low smoking prevalence and high ratios of former smokers to ever smokers. Further reductions in smoking prevalence are critical to continue the decline in lung cancer incidence.


Protein phosphatases decrease their activity during capacitation: a new requirement for this event.
There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.


Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).
In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.


The control of calcium release in heart muscle.
The control of calcium release from intracellular stores (the sarcoplasmic reticulum) in cardiac muscle was examined with the use of a confocal microscope and voltage clamp techniques. Depolarization evoked graded calcium release by altering the extent of spatial and temporal summation of elementary calcium release events called "calcium sparks." These evoked sparks were triggered by local L-type calcium channel currents in a stochastic manner, were similar at different potentials, and resembled spontaneous calcium sparks. Once triggered, the calcium release from the sarcoplasmic reticulum during a calcium spark was independent of the duration of the triggering calcium influx. These results were used to develop a unifying model for cardiac excitation-contraction coupling that explains the large (but paradoxically stable) amplification of the trigger calcium influx by a combination of digital and analog behavior.


Mitochondrial reprogramming via ATP5H loss promotes multimodal cancer therapy resistance.
The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1α stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer.


Inhibition of TNF-alpha processing and TACE-mediated ectodomain shedding by ethanol.
Alcohol (EtOH) is a well-documented immunosuppressant. Acute EtOH-induced immunosuppression is partially due to suppression of tumor necrosis factor alpha (TNF-alpha) secretion. We investigated the mechanism of acute EtOH-induced TNF-alpha suppression in two monocytic cell lines, Mono Mac 6 and DRM. EtOH inhibited TNF-alpha secretion in a dose-dependent manner. However, TNF-alpha transcription was not affected by EtOH. Enzyme-linked immunosorbent assay and confocal microscopy showed that EtOH treatment increased cell-associated TNF-alpha. Ectodomain shedding of TNF-alpha from the cell surface is mediated by TNF-alpha converting enzyme (TACE). In contrast with TNF-alpha, EtOH did not inhibit interleukin-8 (IL-8) secretion, which does not require shedding. Furthermore, TNF p75 receptor shedding, a biomarker for TACE activity, was inhibited by EtOH in both cell lines. EtOH also inhibited TNF p75 receptor shedding in TACE-reconstituted fibroblasts, suggesting that EtOH inhibits the shedding process. These data show that acute EtOH exposure can posttranscriptionally suppress TNF-alpha production, resulting in specific defects in immune defense.


[Erectile dysfunction: a marker for myocardial perfusion impairment?].
To study the correlation between erectile dysfunction (ED) and myocardial perfusion impairment in men with suspected or diagnosed coronary artery disease (CAD). In this prospective study a self-administered IIEF-5 questionnaire was answered by 287 patients that underwent myocardial perfusion imaging under both resting and stress condition with technetium-99m sestamibi, through gated SPECT nuclear scintigraphy technique, before and after physical or pharmacological stress. Some degree of erectile dysfunction (group ED+) was found in 137 (47.8%) patients and in these, age was significantly higher (60.60+/-9.84 vs 50.67+/-9.94 - p<0.001)) than in those without erectile dysfunction (group ED-). In the ED+ group, it was observed a higher prevalence of hypertension, diabetes, myocardial infarction (MI) and percutaneous coronary angioplasty (PCTA). Regardless of the age factor, ED+ patients also presented higher occurrence of myocardial perfusion impairment (necrosis and/or ischemia) and left ventricular systolic wall motion and thickening abnormalities. Patients with ED have higher estimated probability of presenting segmental myocardial perfusion and functional contraction impairment and, therefore, CAD, independent of the aging factor. The authors conclude that ED may be considered not only a marker for CAD but also a condition related to the occurrence of major coronary events such as MI and PCTA.


Using low-moisture molasses-based blocks to supplement Ca salts of soybean oil to forage-fed beef cows.
This experiment compared plasma fatty acid (FA) profile of forage-fed beef cows receiving a molasses-based supplement enriched with Ca salts of soybean oil [CSSO; 24.7% of dry matter (DM)] via a self-fed low-moisture block (LMB) or hand-fed granular concentrate daily (CONC). Thirty-six nonlactating, nonpregnant, multiparous beef cows were blocked by age (three blocks), ranked within blocks by body weight (BW) and body condition score (BCS), and allocated to 1 of three drylot pens (27 × 10 m) per block. Nine pens with four cows each were enrolled in a replicated 3 × 2 Latin square design with two periods of 42 d, and a 21-d washout interval. On day 0, pens within each block were randomly assigned to receive one of the three treatments, in a manner that pens did not receive the same treatment in both periods (total n = 6 pens per treatment). Cows received hay (Cynodon dactylon), water, and a mineral-vitamin mix for ad libitum consumption during the study. Hay intake was recorded daily from days 0 to 42, and LMB intake was recorded from days 14 to 42 to allow cows to adapt to supplement with minimal interference from days 0 to 13. The CONC was offered at 0.420 kg/cow daily (DM basis) from days 0 to 13 and then adjusted (days 14 to 42) to match LMB intake. Cow BW and BCS were recorded, and blood samples were collected on days 0, 14, 28, and 42. Average LMB intake during the initial 13 d was 0.846 ± 0.107 kg/cow daily (DM basis). Supplement DM intake did not differ (P = 0.39) between LMB and CONC cows from days 14 to 42 as designed (0.570 vs. 0.583 kg/d, respectively; SEM = 0.011), despite a greater variation in daily intake of LMB vs. CONC (treatment × day interaction; P < 0.01). No treatments effects were noted (P ≥ 0.40) for hay intake, BCS, and BW. Treatment × day interactions were detected (P ≤ 0.01) for plasma concentrations of ω-6 polyunsaturated FA and total FA. On day 0, plasma FA profile did not differ (P ≥ 0.20) between treatments. From days 14 to 42, plasma concentrations of linoleic acid, ω-6 polyunsaturated FA, and total FA were greater (P < 0.01) in CONC and LMB vs. NOSUPP cows. Plasma concentrations of these FA were also greater (P ≤ 0.03) in LMB vs. CONC cows on day 14, but did not differ (P ≥ 0.35) on days 28 and 42. These results indicate that CSSO inclusion into LMB resulted in similar incorporation of ω-6 polyunsaturated and total FA in the circulation compared with CONC offered at the same daily rate. Hence, the use of self-fed LMB appears to be a valid strategy to provide CSSO to forage-fed beef cattle with reduced labor needs.