[Analysis of the new health module of the National Socioeconomic Characterization Survey 2000].
The National Socioeconomic Characterization Survey (CASEN) was modified in the year 2000, to include an assessment of the inequalities in the access to health services. To analyze the health issues of year 2000 survey database. During 2000, 38338 urban and 26698 rural dwellings were surveyed, totaling 240000 people analyzing ascription to public or private health services and the need demand and use of these services. A higher risk population (lower income, higher age and women) is ascribed to public health services. Sixty five percent of the population self perceives their health as good, this figure decreases along with age and women have a worse self perception than men. In the 30 days prior to the survey, 13% of the population had a health related event; this figure was higher among women and the lower income quintiles. Expressed health demand was higher among women and lower income quintiles. A multivariate analysis identified an age below 14 years, pertaining to a minority ethnic group, ascription to private health services, residing in rural areas, pertaining to the lower income quintile and male sex, as factors associated to a lack access to health care. In the adjusted model, pertaining to the National Health Fund (a public system) is a protective factor to receive health services. The significant inequalities in the access to health care should be corrected with the new Health Reform. The new module incorporated to the National Socioeconomic Characterization Survey, is useful to assess the access to health care in Chile.


Comparative sequence analyses of a new mammalian reovirus genome and the mammalian reovirus S1 genes from six new serotype 2 human isolates.
We previously described isolation of a potentially new mammalian reovirus, designated BYD1, which can cause clinical symptoms similar to that of severe acute respiratory syndrome (SARS) in guinea pigs and macaques, from throat swabs of one SARS patient of Beijing, in 2003. For this study, we determined the genome sequences of BYD1 and the S1 gene sequences of other five mammalian reovirus isolates (BLD, JP, and BYL were isolated from different SARS patients during the outbreak, 302I and 302II were isolated from fecal specimens of two children of Beijing in 1982) to allow molecular comparison with other previously reported mammalian reoviruses (MRVs). Comparative analyses of the BYD1 genome with those of three prototype mammalian reovirus strains demonstrated that BYD1 is a novel reassortant virus, with its S1 gene segment coming from a previously unidentified serotype 2 isolate and other nine segments coming from ancestors of homologous T1L and T3D segments, which supports the hypothesis that mammalian reovirus gene segments reassort in nature. Further analyses of the S1 segments of the six isolates showed that all the isolates are novel serotype 2 MRVs based on their S1 gene sequences, which are markedly different from those of all previously reported, and the S1 genes of the four new isolates share more than 99% identity with each other, proving that they diverged from a common ancestor most recently, and the S1 genes of the four new isolates share about 65% identity with those of the two strains isolated in 1982.


Removal of periocular veins by sclerotherapy.
Prominent periocular veins, especially of the lower eyelid, are not uncommon and patients often seek their removal. Sclerotherapy is a procedure that has been successfully used to permanently remove varicose and telangiectatic veins of the lower extremity and less frequently at other sites. Although it has been successfully used to remove dilated facial veins, it is seldom performed and often not recommended in the periocular region for fear of complications occurring in adjacent structures. The purpose of this study was to determine whether sclerotherapy could safely and effectively eradicate prominent periocular veins. Noncomparative case series. Fifty adult female patients with prominent periocular veins in the lower eyelid were treated unilaterally. Sclerotherapy was performed with a 0.75% solution of sodium tetradecyl sulfate. All patients were followed for at least 12 months after treatment. Complete clinical disappearance of the treated vein was the criterion for success. All 50 patients were successfully treated with uneventful resorption of their ectatic periocular veins. No patient required a second treatment and there was no evidence of treatment failure at 12 months. No new veins developed at the treated sites and no patient experienced any ophthalmologic or neurologic side effects or complications. Sclerotherapy appears to be a safe and effective means of permanently eradicating periocular veins.


A critique of international indicators of sexual risk behaviour.
To investigate whether the indicators of sexual risk behaviour, defined by UNAIDS for use among members of general populations, have been found as risk factors, to examine how information on sexual behaviour is collected and summarised in order to calculate the indicators, and to look for possible sources of error in the data and in interpretation of those indicators. The literature on risk factors for HIV infection was reviewed. Indicators were calculated for countries where data were available for two or more points in time. Indicators of sexual behaviour describe behaviours that are relevant to HIV risk and that are amenable to change. These behaviours do not correspond closely to the individual risk factors for HIV infection that have been identified in observational studies. Although potential errors of both measurement and interpretation exist, most of the indicators currently defined can fulfil their purpose, providing they are used with caution. Many of the indicators should not be interpreted in isolation but need supporting information to make sense of trends or differences between groups. Much of this information is provided by other indicators. The source of the data used to calculate the indicator is potentially important and should always be provided with indicator estimates. Some estimate of the accuracy of the estimate, either by means of confidence intervals or the number of respondents, should be given.


Thyroid hormone treatment decreases hepatic glucose production and renal reabsorption of glucose in alloxan-induced diabetic Wistar rats.
The thyroid hormone (TH) plays an important role in glucose metabolism. Recently, we showed that the TH improves glycemia control by decreasing cytokines expression in the adipose tissue and skeletal muscle of alloxan-induced diabetic rats, which were also shown to present primary hypothyroidism. In this context, this study aims to investigate whether the chronic treatment of diabetic rats with T3 could affect other tissues that are involved in the control of glucose homeostasis, as the liver and kidney. Adult Male Wistar rats were divided into nondiabetic, diabetic, and diabetic treated with T3 (1.5 μg/100 g BW for 4 weeks). Diabetes was induced by alloxan monohydrate (150 mg/kg, BW, i.p.). Animals showing fasting blood glucose levels greater than 250 mg/dL were selected for the study. After treatment, we measured the blood glucose, serum T3, T4, TSH, and insulin concentration, hepatic glucose production by liver perfusion, liver PEPCK, GAPDH, and pAKT expression, as well as urine glucose concentration and renal expression of SGLT2 and GLUT2. T3 reduced blood glucose, hepatic glucose production, liver PEPCK, GAPDH, and pAKT content and the renal expression of SGLT2 and increased glycosuria. Results suggest that the decreased hepatic glucose output and increased glucose excretion induced by T3 treatment are important mechanisms that contribute to reduce serum concentration of glucose, accounting for the improvement of glucose homeostasis control in diabetic rats.


Inhibition of matrix metalloproteinases-2 and -9 prevents cognitive impairment induced by pneumococcal meningitis in Wistar rats.
Pneumococcal meningitis is a relevant clinical disease characterized by an intense inflammatory reaction into the subarachnoid and ventricular spaces, leading to blood-brain barrier breakdown, hearing loss, and cognitive impairment. Matrix metalloproteinases (MMPs) are capable of degrading components of the basal laminin, thus contributing to BBB damage and neuronal injury. In the present study, we evaluated the effects of MMP-2, MMP-9, and MMP-2/9 inhibitors on BBB integrity, learning, and memory in Wistar rats subjected to pneumococcal meningitis. The animals underwent a magna cistern tap and received either 10 µL sterile saline as a placebo or an equivalent volume of a Streptococcus pneumoniae suspension at a concentration of 5 × 10(9)cfu/mL. The rats were randomized into different groups that received adjuvant treatment with MMP-2, MMP-9 or MMP-2/9 inhibitors. The BBB integrity was evaluated, and the animals were habituated to open-field and object recognition tasks 10 days after meningitis induction. Adjuvant treatments with inhibitors of MMP-2 or MMP-2/9 prevented BBB breakdown in the hippocampus, and treatments with inhibitors of MMP-2, MMP-9 or MMP-2/9 prevented BBB breakdown in the cortex. Ten days after meningitis induction, the animals that received adjuvant treatment with the inhibitor of MMP-2/9 demonstrated that animals habituated to the open-field task faster and enhanced memory during short-term and long-term retention test sessions in the object recognition task. Further investigation is necessary to provide support for MMP inhibitors as an alternative treatment for bacterial meningitis; however, these findings suggest that the meningitis model could be a good research tool for studying the biological mechanisms involved in the behavioral alterations associated with pneumococcal meningitis.


Motesanib diphosphate in progressive differentiated thyroid cancer.
The expression of vascular endothelial growth factor (VEGF) is characteristic of differentiated thyroid cancer and is associated with aggressive tumor behavior and a poor clinical outcome. Motesanib diphosphate (AMG 706) is a novel oral inhibitor of VEGF receptors, platelet-derived growth-factor receptor, and KIT. In an open-label, single-group, phase 2 study, we treated 93 patients who had progressive, locally advanced or metastatic, radioiodine-resistant differentiated thyroid cancer with 125 mg of motesanib diphosphate, administered orally once daily. The primary end point was an objective response as assessed by an independent radiographic review. Additional end points included the duration of the response, progression-free survival, safety, and changes in serum thyroglobulin concentration. Of the 93 patients, 57 (61%) had papillary thyroid carcinoma. The objective response rate was 14%. Stable disease was achieved in 67% of the patients, and stable disease was maintained for 24 weeks or longer in 35%; 8% had progressive disease as the best response. The Kaplan-Meier estimate of the median duration of the response was 32 weeks (the lower limit of the 95% confidence interval [CI] was 24; the upper limit could not be estimated because of an insufficient number of events); the estimate of median progression-free survival was 40 weeks (95% CI, 32 to 50). Among the 75 patients in whom thyroglobulin analysis was performed, 81% had decreased serum thyroglobulin concentrations during treatment, as compared with baseline levels. The most common treatment-related adverse events were diarrhea (in 59% of the patients), hypertension (56%), fatigue (46%), and weight loss (40%). Motesanib diphosphate can induce partial responses in patients with advanced or metastatic differentiated thyroid cancer that is progressive. (ClinicalTrials.gov number, NCT00121628.)


Parasitism in Gambusia affinis: Fitness Effects in an Incipient Matrotroph.
Gambusia affinis (western mosquitofish) serves as a host for a variety of larval and adult parasites. Gambusia affinis is also an incipient matrotroph, exhibiting adjustments in post-fertilization provisioning to some offspring within a brood using recently acquired resources. Nutrient transfer to embryos is expected to limit the loss of embryo mass during development resulting in larger offspring. Since larger offspring are more likely to survive, maternal contributions are expected to increase fitness. The presence of parasites, particularly intestinal helminths, potentially reduces body condition and resources available for developing offspring, thereby reducing host fitness. The effects of parasitism on the fitness of G. affinis were investigated in the present study. Fish were collected from 3 sites monthly from June 2015 through August 2016. All helminth parasites were collected during necropsy and identified. Brood size and embryo developmental stage were recorded for each female fish. Additionally, 10 ova/embryos of each developmental stage from each female fish collected from May through August 2016 were haphazardly selected and individually weighed. From 429 female mosquitofish, 5,072 helminths were collected. Brood size varied among collection sites and was positively influenced by maternal body condition, the number of daylight hours, water temperature, and the intensity of both plerocercoid and adult Schyzocotyle acheilognathi. However, brood size was negatively related to the intensity of Neoechinorhynchus cylindratus cystacanth and an increasing number of days between collection and dissection. Embryo weight increased with the presence of either Camallanidae or Contracaecum multipapulatum, embryo developmental stage, and relative host density. These results indicate that some parasitic helminth species negatively affect the fitness of G. affinis, while some positively affect fitness, and that effect can vary with intensity.


Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis.
We recently developed a direct fluorescence ratio assay (Zhai, Y., and G.G. Borisy. 1994. J. Cell Sci. 107:881-890) to quantify microtubule (MT) polymer in order to determine if net MT depolymerization occurred upon anaphase onset as the spindle was disassembled. Our results showed no net decrease in polymer, indicating that the disassembly of kinetochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level. We now examine the reverse process, the interphase-mitosis transition. Specifically, we quantitated both the level of MT polymer and the dynamics of MTs during the G2/M transition using the fluorescence ratio assay and a fluorescence photoactivation approach, respectively. Prophase cells before nuclear envelope breakdown (NEB) had high levels of MT polymer (62%) similar to that previously reported for random interphase populations (68%). However, prophase cells just after NEB had significantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 47%; metaphase, 56%). The abrupt reorganization of MTs at NEB was corroborated by anti-tubulin immunofluorescence staining using a variety of fixation protocols. Sensitivity to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turnover (pre-NEB) increased post-NEB and then became slower later in mitosis. Our results indicate that the interphase-mitosis (G2/M) transition of the MT array does not occur by a simple redistribution of tubulin at constant polymer level as the mitosis-interphase (M/G1) transition. Rather, an abrupt decrease in MT polymer level and increase in MT dynamics occurs tightly correlated with NEB. A subsequent increase in MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understanding spindle morphogenesis. They indicate that changes in MT dynamics may cause the steady-state MT polymer level in mitotic cells to be lower than in interphase. We propose that tension exerted on the kMTs may lead to their lengthening and thereby lead to an increase in the MT polymer level as chromosomes attach to the spindle.


Considerations surrounding reconstruction after resection of musculoskeletal sarcomas.
The defects left by resection of bone and soft-tissue sarcomas often require reconstructive surgery to provide adequate wound coverage, preserve limb function, and optimize cosmetic results. Immediate reconstruction should always be considered after resection with a negative margin, and should be attempted whenever possible. The choice of reconstructive method and tissue flap depends on multiple factors, including body site, donor site morbidity, functional requirements, size of the vascular pedicle, and aesthetics. Preoperative planning before the resection should anticipate the defect size and resulting functional and cosmetic deficits; the success of such planning depends on a collaborative approach between the teams performing the primary resection and the reconstruction. Vigilant postoperative care and flap monitoring is key to avoiding flap or graft failure, hematoma, infection, and other reconstruction-related complications.


The effects of age, gender, hopelessness, and exposure to violence on sleep disorder symptoms and daytime sleepiness among adolescents in impoverished neighborhoods.
Although sleep problems are associated with negative outcomes among adolescents, studies have not focused on sleep disorder symptoms among adolescents living in impoverished neighborhoods and how sleep problems may be related to two factors common in those environments: hopelessness and exposure to violence. This study used data from the longitudinal Mobile Youth Survey (MYS; N = 11,838, 49% female, 93% African-American) to examine trajectories of sleep problems by age (10-18 years) among impoverished adolescents as a function of gender, feelings of hopelessness, and exposure to violence. The results indicate that sleep problems associated with traumatic stress decline with age, with four notable distinctions. First, the steepest decline occurs during the early adolescent years. Second, the rate of decline is steeper for males than for females. Third, exposure to violence impedes the rate of decline for all adolescents, but more dramatically for females than for males. Fourth, the rate of decline is smallest for adolescents with feelings of hopelessness who also had been exposed to violence. To explore the generalizability of these results to other types of sleep disorders, we analyzed cross-sectional data collected from a subsample of 14- and 15-year-old MYS participants (N = 263, 49% female, 100% African-American) who completed a sleep symptoms questionnaire. Four results from the cross-sectional analysis extend the findings of the longitudinal analysis. First, the cross-sectional results showed that symptoms of apnea, insomnia, nightmares, and restless legs syndrome or periodic limb movement disorder (RLS/PLMD), as well as daytime sleepiness, increase as a function of hopelessness. Second, symptoms of insomnia, RLS, and nightmares, as well as daytime sleepiness, increase as function of exposure to violence. Third, symptoms of insomnia and RLS/PLMD are greater under conditions of combined hopelessness and exposure to violence than for either condition alone. Fourth, symptoms of RLS/PLMD are worst for females who have been exposed to violence and experience hopelessness. Overall, the findings suggest that hopelessness and exposure to violence have negative independent and multiplicative effects on adolescent sleep, particularly for females. Understanding the causal factors associated with inadequate sleep in impoverished adolescents is important for three reasons. First, sleep is an important aspect of adolescent development. Second, inadequate sleep has severe consequences for adolescent morbidity, mortality, and overall quality of life. Third, impoverished adolescents are at the most severe risk for poor outcomes, and improvement in their sleep may produce large gains.


Electrophysiologic properties and nature of ventrolateral thalamic nucleus neurons reactive to converging inputs of paleo- and neocerebellar origin.
Acute experiments were carried out in chloralose-anesthetized, curarized, and artificially ventilated cats to investigate the consistency and functional characteristics of converging cerebellar nuclear (fastigial, interpositus, dentate nuclei) inputs to single neurons of the ventrolateral thalamic nucleus, and the nature (thalamocortical relay or interneuronal) of its convergent neuronal population responsive to stimulation of the cerebellar nuclei. From unitary analysis of both extra- and juxtacellular recordings, we conclude that convergence of paleo- and neocerebellar impulses on thalamic ventrolateral neurons occurs widely, and that both excitatory (mono- and polysynaptic) and inhibitory (di- and polysynaptic) effects are induced on the same neurons by cerebellar nuclei stimulation; these effects concern about 40% of the thalamic ventrolateral neuronal population tested using the extracellular recording technique and about 75% of the ventrolateral nucleus neurons tested using the juxtacellular recording; activation of neocerebellar nuclear outputs generates mainly excitatory and excitatory-inhibitory response patterns, whereas activation of the paleocerebellar nuclear outputs evokes predominantly inhibitory and excitatory-inhibitory response patterns; the convergent paleo- neocerebellar neurons are found commonly in the more anterior ventrolateral nucleus stereotaxic planes; and from the results obtained by stimulation of the somatomotor cortical areas, a large proportion of the ventrolateral nucleus convergent units consists of thalamocortical relay cells. The results have implications for a possible integration between posture and voluntary movements at the level of the thalamic relay nucleus of the cerebellar-cortical pathway, and an interaction between central feedback and peripheral feedback in the programming, execution, and correction of voluntary movements.


Clinical and microbiological features of infection in alcoholic hepatitis: an international cohort study.
Previous studies have described the clinical impact of infection in alcoholic hepatitis (AH) but none have comprehensively explored the aetiopathogenesis of infection in this setting. We examined the causes, consequences and treatment of infection in a cohort of patients with AH. We undertook a retrospective cohort study of patients with AH admitted between 2009 and 2014 to seven centres in Europe and the USA. Clinical and microbiological data were extracted from medical records. Survival was analysed with Kaplan-Meier analysis and Cox proportional hazards analysis to control the data for competing factors. Propensity score matching was used to examine the efficacy of prophylactic antibiotics administered in the absence of infection. We identified 404 patients with AH. Of these, 199 (49%) showed clinical or culture evidence of infection. Gut commensal bacteria, particularly Escherichia coli and Enterobacter species, were most commonly isolated in culture. Fungal infection was rarely seen. Cultured organisms and antibiotic resistance differed markedly between centres. Infection was an independent risk factor for death (hazard ratio for death at 90 days 2.33, 95% confidence interval 1.63-3.35, p < 0.001). Initiation of antibiotic therapy on admission in the absence of infection did not reduce mortality or alter the incidence of subsequent infections. Corticosteroid use increased the incidence of infection but this did not impact on survival. In this large real-world cohort of patients with AH, infection was common and was associated with reduced short-term survival. Gram-negative, gut commensal bacteria were the predominant infective organisms, consistent with increased translocation of gut bacteria in AH; however, the characteristics of infection differ between centres. Infection should be actively sought and treated, but we saw no benefits of prophylactic antibiotics.


Interaction of caldesmon with endoplasmic reticulum membrane: effects on the mobility of phospholipids in the membrane and on the phosphatidylserine base-exchange reaction.
We have previously demonstrated by tryptophan fluorescence the interaction of caldesmon with anionic phospholipid vesicles [Czurylo, Zborowski and Dabrowska (1993) Biochem. J. 291, 403-408]. In the present work we investigated the interaction of caldesmon with natural-membrane (rat liver endoplasmic reticulum) phospholipids by co-sedimentation assay. The results indicate that 1 mol of caldesmon binds approx. 170 mol of membrane phospholipids with a binding affinity constant of 7.3 x 10(6) M-1. The caldesmon-membrane phospholipid complex dissociates with increasing salt concentration and in the presence of Ca2+/calmodulin. As indicated by EPR measurements of membrane lipids labelled with 5-doxyl stearate and TEMPO-phosphatidylethanolamine, binding of caldesmon results in an increase in mobility of the acyl chains (in the region of carbon 5) and a decrease in polar headgroup mobility of phospholipids. Interaction of caldesmon with phospholipids is accompanied by inhibition of phosphatidylethanolamine synthesis via a phospholipid base-exchange reaction, with phosphatidylserine as substrate. This shows that, of the endoplasmic reticulum membrane phospholipids, the main target of caldesmon is phosphatidylserine.


Using mitochondrial activity to select for potent human hematopoietic stem cells.
Hematopoietic cell transplantation is a critical curative approach for many blood disorders. However, obtaining grafts with sufficient numbers of hematopoietic stem cells (HSCs) that maintain long-term engraftment remains challenging; this is due partly to metabolic modulations that restrict the potency of HSCs outside of their native environment. To address this, we focused on mitochondria. We found that human HSCs are heterogeneous in their mitochondrial activity as measured by mitochondrial membrane potential (MMP) even within the highly purified CD34+CD38-CD45RA-CD90+CD49f+ HSC population. We further found that the most potent HSCs exhibit the lowest mitochondrial activity in the population. We showed that the frequency of long-term culture initiating cells in MMP-low is significantly greater than in MMP-high CD34+CD38-CD45RA-CD90+ (CD90+) HSCs. Notably, these 2 populations were distinct in their long-term repopulating capacity when transplanted into immunodeficient mice. The level of chimerism 7 months posttransplantation was >50-fold higher in the blood of MMP-low relative to MMP-high CD90+ HSC recipients. Although more than 90% of both HSC subsets were in G0, MMP-low CD90+ HSCs exhibited delayed cell-cycle priming profile relative to MMP-high HSCs. These functional differences were associated with distinct mitochondrial morphology; MMP-low in contrast to MMP-high HSCs contained fragmented mitochondria. Our findings suggest that the lowest MMP level selects for the most potent, likely dormant, stem cells within the highly purified HSC population. These results identify a new approach for isolating highly potent human HSCs for further clinical applications. They also implicate mitochondria in the intrinsic regulation of human HSC quiescence and potency.


Relationship between files that bind at the apical foramen and foramen openings in maxillary central incisors--a SEM study.
Considering that instrumentation of the apical foramen has been suggested for root canal infection control, this study analyzed the relationship between the files that bind at the apical foramen and the foraminal openings in 50 maxillary central incisors. After preparation of the pulp chamber, access to the canal was obtained with #1 and 2 LA Axxess and K-files with tip cut were inserted up to the apical foramen until binding was felt. The files were fixed with methyl cyanoacrylate and the tooth-file sets were cross-sectioned 10 mm short of the apex. Scanning electron microscopic analysis was carried out and files and foraminal areas were measured using Image Tool software. Statistically significant difference (p<0.0001) was found between files and the apical foraminal areas. The mean foraminal area was 3.8 times larger than the mean file area. The results of this study suggest that it would require 4 files of greater size beyond the one that bound to the foramen in order to allow a better relationship between files and apical openings of maxillary central incisors.


Role of tyrosine kinase inhibitors in lung cancer.
Protein tyrosine kinases are enzymes which catalyze the phosphorylation of tyrosine residues and activate a downstream cascade of cellular signalling pathways which regulate cell proliferation, differentiation and apoptosis and a wide variety of cellular functions. Clinical developments over the past decade have identified several novel therapeutic agents which inhibit tyrosine kinase activity, either by direct receptor inhibition or indirect inhibition of tyrosine kinase controlled pathways. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKI), such as gefitinib and erlotinib have been studied extensively in patients with refractory non-small cell lung cancer (NSCLC). Early studies with gefitinib showed undoubted clinical activity but failed to show a survival benefit, whereas studies with erlotinib showed a small but statistically significant benefit in overall survival. Subsequent studies explored the possibility of synergistic activity between targeted agents (gefitinib or erlotinib) and conventional chemotherapy drugs reporting disappointing results. Clinical trial results with gefitinib and erlotinib, either as monotherapy or in combination with chemotherapy, have failed to match the encouraging results noted in the pre-clinical setting. It is now increasingly recognised that clinical exploration of molecular targeted agents may not conform well to traditional phase I/II/III drug trial designs. Therapeutic responses may be limited to a small subpopulation of patients, therefore diluting the overall therapeutic effect. Hypothesising a genetic basis for the heterogeneity in trial results, biomarkers (such as EGFR gene mutation analysis, EGFR protein expression, and increased EGFR gene copy number) have been studied with a view to identifying a target population most likely to benefit from these drugs. Future clinical trials with targeted agents need to be carefully designed to incorporate correlative translational research elements that will allow selection of appropriate treatment strategies for individual patients. For assessment of phase III trial results in advanced disease, progression free survival may serve as a more appropriate end-point than response rate in an adequately designed trial in the appropriately selected population, although there should be no substitute for the overall survival and quality of life end points. The role of EFGR TKI in NSCLC will be discussed in detail and data from these studies will be used to illustrate the challenges in designing clinical trials and interpreting outcomes.


Imaging of metastatic lymph nodes by X-ray phase-contrast micro-tomography.
Invasive cancer causes a change in density in the affected tissue, which can be visualized by x-ray phase-contrast tomography. However, the diagnostic value of this method has so far not been investigated in detail. Therefore, the purpose of this study was, in a blinded manner, to investigate whether malignancy could be revealed by non-invasive x-ray phase-contrast tomography in lymph nodes from breast cancer patients. Seventeen formalin-fixed paraffin-embedded lymph nodes from 10 female patients (age range 37-83 years) diagnosed with invasive ductal carcinomas were analyzed by X-ray phase-contrast tomography. Ten lymph nodes had metastatic deposits and 7 were benign. The phase-contrast images were analyzed according to standards for conventional CT images looking for characteristics usually only visible by pathological examinations. Histopathology was used as reference. The result of this study was that the diagnostic sensitivity of the image analysis for detecting malignancy was 100% and the specificity was 87%. The positive predictive value was 91% for detecting malignancy and the negative predictive value was 100%. We conclude that x-ray phase-contrast imaging can accurately detect density variations to obtain information regarding lymph node involvement previously inaccessible with standard absorption x-ray imaging.


Treatment of Displaced Midshaft Clavicle Fractures: Figure-of-Eight Harness Versus Anterior Plate Osteosynthesis: A Randomized Controlled Trial.
Most midshaft clavicle fractures affect the economically active population, which is negatively impacted by transient limb impairment during the treatment. There is still debate about the advantages and disadvantages of surgical treatment for these fractures. In this prospective randomized controlled trial, 117 patients were allocated to 1 of 2 groups: nonsurgical treatment with a figure-of-eight harness or surgical treatment with anteroinferior plate osteosynthesis. The primary outcome was upper-limb limitation measured with the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire at 6 months. Other outcomes included pain, radiographic findings, satisfaction with the cosmetic result, complications, and time to return to previous work and activities. Participants were assessed at 6 weeks, 6 months, and 1 year after the intervention. No difference between the 2 groups was detected in the DASH score at any time point (p = 0.398, 0.403, and 0.877 at 6 weeks, 6 months, and 1 year, respectively), pain levels measured with a visual analogue scale (VAS), time to return to previous activities, or dissatisfaction with the cosmetic result. Seven patients (14.9%) developed nonunion after nonsurgical treatment, a nonunion rate that was significantly higher than that in the surgical group, in which all fractures had healed (p = 0.004). The patients in the nonsurgical group had radiographic evidence of greater clavicle shortening (p < 0.001) and more of the patients in that group answered "yes" when asked if their clavicle felt short (p < 0.001) and if they felt bone prominence (p < 0.001). More patients answered "yes" when asked if they felt paresthesia in the surgical group (7; 13.7%) than in the nonsurgical group (1; 2.1%) (p = 0.036). This study did not demonstrate a difference in limb function between patients who underwent surgical treatment and those nonsurgically treated for a dislocated midshaft clavicle fracture. Meanwhile, surgical treatment decreased the likelihood of nonunion. Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.


[Non-Necrotizing Acute Dermo-Hypodermal Infections: Erysipela and Infectious Cellulitis].
Non-necrotizing acute dermo-hypodermal infections are infectious processes that include erysipela and infectious cellulitis, and are mainly caused by group A β-haemolytic streptococcus. The lower limbs are affected in more than 80% of cases and the risk factors are disruption of cutaneous barrier, lymphoedema and obesity. Diagnosis is clinical and in a typical setting we observe an acute inflammatory plaque with fever, lymphangitis, adenopathy and leucocytosis. Bacteriology is usually not helpful because of low sensitivity or delayed positivity. In case of atypical presentations, erysipela must be distinguished from necrotizing fasciitis and acute vein thrombosis. Flucloxacillin and cefradine remain the first line of treatment. Recurrence is the main complication, so correct treatment of the risk factors is crucial.


Construct-A-Vis: Exploring the Free-Form Visualization Processes of Children.
Building data analysis skills is part of modern elementary school curricula. Recent research has explored how to facilitate children's understanding of visual data representations through completion exercises which highlight links between concrete and abstract mappings. This approach scaffolds visualization activities by presenting a target visualization to children. But how can we engage children in more free-form visual data mapping exercises that are driven by their own mapping ideas? How can we scaffold a creative exploration of visualization techniques and mapping possibilities? We present Construct-A-Vis, a tablet-based tool designed to explore the feasibility of free-form and constructive visualization activities with elementary school children. Construct-A-Vis provides adjustable levels of scaffolding visual mapping processes. It can be used by children individually or as part of collaborative activities. Findings from a study with elementary school children using Construct-A-Vis individually and in pairs highlight the potential of this free-form constructive approach, as visible in children's diverse visualization outcomes and their critical engagement with the data and mapping processes. Based on our study findings we contribute insights into the design of free-form visualization tools for children, including the role of tool-based scaffolding mechanisms and shared interactions to guide visualization activities with children.


Impact of navigators on completion of steps in the kidney transplant process: a randomized, controlled trial.
Many patients with ESRD, particularly minorities and women, face barriers in completing the steps required to obtain a transplant. These eight sequential steps are as follows: medical suitability, interest in transplant, referral to a transplant center, first visit to center, transplant workup, successful candidate, waiting list or identify living donor, and receive transplant. This study sought to determine the effect of navigators on completion of steps. Cluster randomized, controlled trial at 23 Ohio hemodialysis facilities. One hundred sixty-seven patients were recruited between January 2009 and August 2009 and were followed for up to 24 months or until study end in February 2011. Trained kidney transplant recipients met monthly with intervention participants (n=92), determined their step in the transplant process, and provided tailored information and assistance in completing the step. Control participants (n=75) continued to receive usual care. The primary outcome was the number of transplant process steps completed. Starting step did not significantly differ between the two groups. By the end of the trial, intervention participants completed more than twice as many steps as control participants (3.5 versus 1.6 steps; difference, 1.9 steps; 95% confidence interval, 1.3-2.5 steps). The effect of the intervention on step completion was similar across race and sex subgroups. Use of trained transplant recipients as navigators resulted in increased completion of transplant process steps.


Mass burden and estimated flux of heavy metals in Pakistan coast: sedimentary pollution and eco-toxicological concerns.
Heavy-metal contamination in coastal areas poses a serious threat to aquatic life and public health due to their high toxicity and bio-accumulation potential. In the present study, levels of different heavy metals (Cu, Cd, Cr, Ni, Co, Pb, Zn, and Mn), their spatial distribution, geochemical status, and enrichment indices (Cu, Cd, Cr, Ni, Co, Pb, Zn) were investigated in the sediment samples from 18 coastal sites of Pakistan. The analyses of coastal sediments indicated the presence of heavy metals in order such as Cr > Zn > Cu > Pb > Ni > Mn > Co > Cd. Geo-accumulation index (I geo), enrichment factor (EF), and contamination factor (CF) showed diverse range in heavy-metal enrichment site by site. Pollution load index (PLI) has shown that average pollution load along the entire coastal belt was not significant. Based on the mean effect range medium quotient, coastal sediments of Pakistan had 21% probability of toxicity. The estimated sedimentary load of selected heavy metals was recorded in the range of 0.3-44.7 g/cm(2)/year, while the depositional flux was in the range of 0.07-43.5 t/year. Heavy-metal inventories of 9.8 × 10(2)-3.8 × 10(5) t were estimated in the coastal sediments of Pakistan. The enrichment and contamination factors (EF and CF) suggested significant influence of anthropogenic and industrial activities along the coastal belt of Pakistan.


Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.
Contrary to the traditional view that microtubules pull chromosomes polewards during the anaphase stage of meiotic and mitotic cell divisions, new evidence suggests that the chromosome movements are driven by a motor located at the kinetochore. The process of chromosome segregation involves proper arrangement of kinetochores for spindle attachment, followed by spindle attachment and chromosome movement. Mechanisms in Drosophila for chromosome segregation in meiosis differ in males and females, implying the action of different gene products in the two sexes. A product encoded at the claret locus in Drosophila is required for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. Here we show that the predicted amino-acid sequence of this product is related to the heavy chain of kinesin. The conserved region corresponds to the kinesin motor domain and includes the ATP-binding site and a region that can bind microtubules. A second region contains a leucine repeat motif which may mediate protein-subunit interactions necessary for attachment of chromosomes to the spindle. The mutant phenotype of chromosome nondisjunction and loss, and its similarity to the kinesin ATP-binding domain, suggest that the product encoded at claret not only stabilizes chromosome attachments to the spindle, but may also be a motor that drives chromosome segregation in female meiosis.


Carvacrol Ameliorates Ligation-Induced Periodontitis in Rats.
This study aims to evaluate the ameliorative effect of carvacrol, an anti-inflammatory monoterpenoid phenol and a major component of Plectranthus amboinicus, on periodontal damage in an experimental rat model of periodontitis. Forty Sprague-Dawley rats were divided into ligation (Lig), non-ligation (n-Lig), and two ligation plus carvacrol (Lig+C) groups. Carvacrol (17.5 or 35.0 mg/kg body weight/day) was administered intragastrically from 1 day before ligation. On day 8, dental alveolar bone loss and gingival inflammation in periodontal specimens were examined by dental radiography, microcomputed tomography, and histology. Expressions of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase messenger (m)RNAs, and levels of matrix metalloproteinase (MMP)-2 and MMP-9 in gingiva were examined by reverse transcription-polymerase chain reaction and zymography. Dental radiography revealed periodontal bone-supporting ratios in Lig and Lig+C groups were lower than the n-Lig group, with Lig group ratios being lowest. Compared with the n-Lig group, the cemento-enamel junction-bone distance was significantly longer in Lig and Lig+C groups, with Lig+C groups showing shorter distances regardless of radiographic methods used. Histology and histometry showed less inflammatory area and stronger connective tissue attachment in Lig+C groups than in the Lig group. Cytokine/mediator mRNA expression and MMP-9 levels were reduced in the Lig+C groups. Carvacrol reduced tissue damage and bone loss caused by ligation-induced periodontitis. The present results indicate that carvacrol might reduce tissue destruction by downregulating expression of proinflammatory mediators and MMP-9.


Microbial toxicity of isopropyl myristate used for sterility testing of petrolatum-based ophthalmic ointments.
The toxicity of isopropyl myristate (IPM) to microorganisms varies from lot to lot and between manufacturers. The toxicity is not directly proportional to the number and level of IPM homologues present nor to the small quantities of fatty acids, alcohols, aldehydes, or ketones found in both filter- and heat-sterilized IPM. The toxicity of IPM, both filter- and heat-sterilized, has no correlation with the usual physical and chemical characteristics of IPM but shows excellent correlation (correlation coefficient, r = 0.88) with the pH of a water extract of IPM. The toxic compounds can be removed and the toxicity of both filter- and heat-sterilized IPM can be reduced by basic alumina treatment. These findings may indicate that trace amounts of acidic catalysts remaining after production of IPM are responsible for the toxic effect.


Efficient Bayesian approach for multilocus association mapping including gene-gene interactions.
since the introduction of large-scale genotyping methods that can be utilized in genome-wide association (GWA) studies for deciphering complex diseases, statistical genetics has been posed with a tremendous challenge of how to most appropriately analyze such data. A plethora of advanced model-based methods for genetic mapping of traits has been available for more than 10 years in animal and plant breeding. However, most such methods are computationally intractable in the context of genome-wide studies. Therefore, it is hardly surprising that GWA analyses have in practice been dominated by simple statistical tests concerned with a single marker locus at a time, while the more advanced approaches have appeared only relatively recently in the biomedical and statistical literature. we introduce a novel Bayesian modeling framework for association mapping which enables the detection of multiple loci and their interactions that influence a dichotomous phenotype of interest. The method is shown to perform well in a simulation study when compared to widely used standard alternatives and its computational complexity is typically considerably smaller than that of a maximum likelihood based approach. We also discuss in detail the sensitivity of the Bayesian inferences with respect to the choice of prior distributions in the GWA context. our results show that the Bayesian model averaging approach which explicitly considers gene-gene interactions may improve the detection of disease associated genetic markers in two respects: first, by providing better estimates of the locations of the causal loci; second, by reducing the number of false positives. The benefits are most apparent when the interacting genes exhibit no main effects. However, our findings also illustrate that such an approach is somewhat sensitive to the prior distribution assigned on the model structure.


Survey Results of the Expert Meeting on Laparoscopic Living Donor Hepatectomy and Literature Review.
Because laparoscopic donor surgery has been successful in pediatric living donor liver transplantation, its application is expanding to right hepatectomy. However, there is no consensus on the indications for laparoscopic donor hepatectomy or on the details of the surgical technique. To evaluate the current status of laparoscopic donor hepatectomy and to summarize the expert opinion on it. Before the expert meeting on September 8, 2016, in Seoul, Korea, a survey was undertaken from expert liver surgeons from around the world. Fifteen of 17 (88.2%) surgeons responded to the survey. The selection criteria for laparoscopic donor surgery are stricter than for open surgery in terms of the anatomy, remnant liver volume, and recipient's condition. There is no consensus on the instruments or equipment used. A literature review of laparoscopic donor hepatectomy showed that the use of this method is increasing and the short-term outcomes are similar to those of open surgery. This survey and literature review show that laparoscopic donor hepatectomy is performed by experienced surgeons in selected cases, and that its incidence is increasing worldwide.


A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies.
Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.


[Exploration on academic thought of Zhang Zhongjing on processing of Chinese herbal medicine].
For exploration on the academic thought on processing of Chinese herbal medicine the basic theory and methods, textual research of the scientific of Zhang Zhongiing on processing of Chinese herbal medicine was summarized, and the historical significance on academic thought of Zhang Zhongjing on processing of Chinese herbal medicine was analyzed. Regarded Zhang Zhongjing is the ancestor of processing of Chinese herbal medicine to lay the theoretical foundation on processing of Chinese herbal medicine for later.


[Preliminary study on serotyping of Klebsiella pneumoniae and its clinical significance].
This paper reports the results of 32 type-specific klebsiella antisera preparations made in our laboratory, through immunizing New Zealand rabbits with standard klebsiella strains. The significant titres of these antisera ranged from 1:8-1:256. Using above mentioned a antisera, 282 isolates of klebsiella mainly isolated from sputa were typed with the method of coagglutination test. The typeable rate of total isolates was 69.1% (195/282). The most common serotypes were K1 and K33, representing 13.1 and 9.6% of total isolates respectively. Of 207 isolates mainly from sputa, 144 isolates (69.6%) were typeable, Serotypes K1 and K33 were also most common. It suggested that serotypes K1 and K33 were probably the most common types in infections of respiratory and urinary tracts in Shanghai. The relationship between serotypes of klebsiella pneumoniae and pathogenicity was discussed.


Regulation of proliferation, cell fate specification and differentiation by the homeodomain proteins Prox1, Six3, and Chx10 in the developing retina.
The coordination of proliferation and cell fate specification is of particular importance in the developing central nervous system, because different classes of neurons are generated at different stages of development. As a consequence, if too many or too few multipotent progenitor cells exit the cell cycle during the early stages of development, then a predictable shift in the ratio of neuronal cell types would occur. Such a perturbation could prove devastating for normal neural function. In the past several years, we have made numerous advances toward understanding the roles of cell cycle proteins in the development of the retina, a specialized region of the central nervous system. More recently, researchers have identified and characterized a number of homeodomain transcription factors that regulate the proliferation of retinal progenitor cells and the specification of cell fate. In this review, I will present recent findings about the homeodomain protein Prox1 and compare them with studies of two other homeodomain proteins that are important regulators of retinal development, Chx10 and Six3. Together, the research on the genes that encode these three proteins raises several new questions about the mechanisms underlying the coordination of proliferation and cell fate specification in the developing central nervous system.


Transcriptome profile of yeast reveals the essential role of PMA2 and uncharacterized gene YBR056W-A (MNC1) in adaptation to toxic manganese concentration.
Adaptation of S. cerevisiae to toxic concentrations of manganese provides a physiological model of heavy metal homeostasis. Transcriptome analysis of adapted yeast cells reveals upregulation of cell wall and plasma membrane proteins including membrane transporters. The gene expression in adapted cells differs from that of cells under short-term toxic metal stress. Among the most significantly upregulated genes are PMA2, encoding an ortholog of Pma1 H+-ATPase of the plasma membrane, and YBR056W-A, encoding a putative membrane protein Mnc1 that belongs to the CYSTM family and presumably chelates manganese at the cell surface. We demonstrate that these genes are essential for the adaptation to toxic manganese concentration and propose an extended scheme of manganese detoxification in yeast.


A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.
Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner.


miRGate: a curated database of human, mouse and rat miRNA-mRNA targets.
MicroRNAs (miRNAs) are small non-coding elements involved in the post-transcriptional down-regulation of gene expression through base pairing with messenger RNAs (mRNAs). Through this mechanism, several miRNA-mRNA pairs have been described as critical in the regulation of multiple cellular processes, including early embryonic development and pathological conditions. Many of these pairs (such as miR-15 b/BCL2 in apoptosis or BART-6/BCL6 in diffuse large B-cell lymphomas) were experimentally discovered and/or computationally predicted. Available tools for target prediction are usually based on sequence matching, thermodynamics and conservation, among other approaches. Nevertheless, the main issue on miRNA-mRNA pair prediction is the little overlapping results among different prediction methods, or even with experimentally validated pairs lists, despite the fact that all rely on similar principles. To circumvent this problem, we have developed miRGate, a database containing novel computational predicted miRNA-mRNA pairs that are calculated using well-established algorithms. In addition, it includes an updated and complete dataset of sequences for both miRNA and mRNAs 3'-Untranslated region from human (including human viruses), mouse and rat, as well as experimentally validated data from four well-known databases. The underlying methodology of miRGate has been successfully applied to independent datasets providing predictions that were convincingly validated by functional assays. miRGate is an open resource available at http://mirgate.bioinfo.cnio.es. For programmatic access, we have provided a representational state transfer web service application programming interface that allows accessing the database at http://mirgate.bioinfo.cnio.es/API/ Database URL: http://mirgate.bioinfo.cnio.es


[Ecological risk and methods for its assessment].
The authors prove that signals of ecologic influence on public health could be detected only by statistic analysis of relatively rare diseases. Serious restrictions therefore should cover use of some health parameters in ecologic ranking. The article discusses controversies of legal basis for ecologic ranking of territories.


Extrapyramidal motor signs in degenerative ataxias.
Extrapyramidal motor signs (EPS) are well-known symptoms of degenerative ataxia. However, little is known about frequency and appearance of EPS in subtypes of ataxia. We characterized 311 patients with ataxia clinically and genetically. Course of the disease and EPS were investigated according to a standardized protocol. Diagnostic and prognostic impact of EPS in subtypes of ataxia was analyzed by Kaplan-Meier plots. Extrapyramidal motor signs occurred in all forms of ataxia, but frequency and type of EPS varied between genetically and clinically defined subtypes. Postural tremor in hereditary ataxias was typical for spinocerebellar ataxia type 2 (SCA2). Dystonia was generally rare in ataxias, but, if present, suggested SCA3. We observed a parkinsonian variant of SCA3 in which parkinsonism was present in the beginning of the disease and responded well to levodopa therapy, leading to diagnostic confusion. Parkinsonism in SCA3 was independent of CAG repeat length but ran in families, suggesting modifying genes. In idiopathic sporadic cerebellar ataxia (ISCA), EPS are more frequent in late-onset than in early-onset forms. In 50% of ISCA patients with parkinsonism, the diagnosis of multiple system atrophy remained questionable because of normal autonomic function. Extrapyramidal motor signs can help to predict the genetic subtype of ataxia. Extrapyramidal motor signs were more frequent in genetic subtypes in which basal ganglia affection has been demonstrated by postmortem studies. However, no type of EPS was specific for an underlying mutation. In ISCA, EPS are an adverse prognostic factor. Parkinsonism is especially associated with a more rapid course of the disease. Arch Neurol. 2000;57:1495-1500


Prediction of recurrence and prognosis in patients with hepatocellular carcinoma after resection by use of CLIP score.
The survival time of patients with hepatocellular carcinoma (HCC) after resection is hard to predict. Both residual liver function and tumor extension factors should be considered. A new scoring system has recently been proposed by the Cancer of the Liver Italian Program (CLIP). CLIP score was confirmed to be one of the best ways to stage patients with HCC. To our knowledge, however, the literature concerning the correlation between CLIP score and prognosis for patients with HCC after resection was not published. The aim of this study is to evaluate the recurrence and prognostic value of CLIP score for the patients with HCC after resection. A retrospective survey was carried out in 174 patients undergoing resection of HCC from January 1986 to June 1998. Six patients who died in the hospital after operation and 11 patients with the recurrence of the disease were excluded at 1 month after hepatectomy. By the end of June 2001, 4 patients were lost and 153 patients with curative resection have been followed up for at least three years. Among 153 patients, 115 developed intrahepatic recurrence and 10 developed extrahepatic recurrence, whereas the other 28 remained free of recurrence. Recurrences were classified into early (< or =3 year) and late (>3 year) recurrence. The CLIP score included the parameters involved in the Child-Pugh stage (0-2), plus macroscopic tumor morphology (0-2), AFP levels (0-1), and the presence or absence of portal thrombosis (0-1). By contrast, portal vein thrombosis was defined as the presence of tumor emboli within vascular channel analyzed by microscopic examination in this study. Risk factors for recurrence and prognostic factors for survival in each group were analyzed by the chi-square test, the Kaplan-Meier estimation and the COX proportional hazards model respectively. The 1-, 3-, 5-, 7-,and 10-year disease-free survival rates after curative resection of HCC were 57.2%, 28.3%, 23.5%, 18.8%, and 17.8%, respectively. Median survival time was 28, 10, 4, and 5 mo for CLIP score 0, 1, 2, 3, and 4 to 5, respectively. Early and late recurrence developed in 109 patients and 16 patients respectively. By the chi-square test, tumor size, microsatellite, venous invasion, tumor type (uninodular, multinodular, massive), tumor extension (< or = or >50% of liver parenchyma replaced by tumor), TNM stage, CLIP score, and resection margin were the risk factors for early recurrence, whereas CLIP score and Child-Pugh stage were significant risk factors for late recurrence. In univariate survival analysis, Child-Pugh stages, resection margin, tumor size, microsatellite, venous invasion, tumor type, tumor extension, TNM stages, and CLIP score were associated with prognosis. The multivariate analysis by COX proportional hazards model showed that the independent predictive factors of survival were resection margins and TNM stages. CLIP score has displayed a unique superiority in predicting the tumor early and late recurrence and prognosis in the patients with HCC after resection.


Relative effects of juvenile and adult environmental factors on mate attraction and recognition in the cricket, Allonemobius socius.
Finding a mate is a fundamental aspect of sexual reproduction. To this end, specific-mate recognition systems (SMRS) have evolved that facilitate copulation between producers of the mating signal and their opposite-sex responders. Environmental variation, however, may compromise the efficiency with which SMRS operate. In this study, the degree to which seasonal climate experienced during juvenile and adult life-cycle stages affects the SMRS of a cricket, Allonemobius socius (Scudder) (Orthoptera: Gryllidae) was assessed. Results from two-choice behavioral trials suggest that adult ambient temperature, along with population and family origins, mediate variation in male mating call, and to a lesser extent directional response of females for those calls. Restricted maximum-likelihood estimates of heritability for male mating call components and for female response to mating call appeared statistically nonsignificant. However, appreciable "maternal genetic effects" suggest that maternal egg provisioning and other indirect maternal determinants of the embryonic environment significantly contributed to variation in male mating call and female response to mating calls. Thus, environmental factors can generate substantial variation in A. socius mating call, and, more importantly, their marginal effect on female responses to either fast-chirp or long-chirp mating calls suggest negative fitness consequences to males producing alternative types of calls. Future studies of sexual selection and SMRS evolution, particularly those focused on hybrid zone dynamics, should take explicit account of the loose concordance between signal producers and responders suggested by the current findings.


COX-1 Inhibitors: Beyond Structure Toward Therapy.
Biosynthesis of prostaglandins from arachidonic acid (AA) is catalyzed by cyclooxygenase (COX), which exists as COX-1 and COX-2. AA is in turn released from the cell membrane upon neopathological stimuli. COX inhibitors interfere in this catalytic and disease onset process. The recent prominent discovery involvements of COX-1 are mainly in cancer and inflammation. Five classes of COX-1 inhibitors are known up to now and this classification is based on chemical features of both synthetic compounds and substances from natural sources. Physicochemical interactions identification between such molecules and COX-1 active site was achieved through X-ray, mutagenesis experiments, specific assays and docking investigations, as well as through a pharmacometric predictive model building. All these insights allowed the design of new highly selective COX-1 inhibitors to be tested into those disease models in which COX-1 is involved. Particularly, COX-1 is expressed at high levels in the early to advanced stages of human epithelial ovarian cancer, and it also seems to play a pivotal role in cancer progression. The refinement of COX-1 selective inhibitor structure has progressed to the stage that some of the inhibitors described in this review could be considered as promising active principle ingredients of drugs and hence part of specific therapeutic protocols. This review aims to outline achievements, in the last 5 years, dealing with the identification of highly selective synthetic and from plant extracts COX-1 inhibitors and their theranostic use in neuroinflammation and ovarian cancer. Their gastrotoxic effect is also discussed.


Observations on the leech Placobdella ornata feeding from bony tissues of turtles.
The leech Placobdella ornata was observed feeding from the blood sinuses of the plastron and carapace bones of Chelydra serpentina and Chrysemys picta. Evidence of successful feeding included blood upwelling from the point of attachment and gastric ceca of the leeches freshly filled with blood after removal. There was an apparent preference for the sulci between scales of the shell.


Flood risk reduction in the northern coast of Central Java Province, Indonesia: An application of stakeholder's analysis.
The northern coast of Central Java Province, Indonesia, is considered as the critical area of flooding. The area always suffers from flooding because of heavy rainfall and/or sea level rise. Flooding brings a lot of consequences, including people's suffering, property damage and property loss. A number of efforts have been carried out to manage flood problems, yet the achievement is incomplete without stakeholder involvement. Stakeholders have a very important role in flood risk reduction. A common understanding on the existence and role of stakeholders is important in achieving community resilience. The aim of this article was to analyse interest and cooperation among stakeholders in flood risk reduction. In-depth interviews were conducted to identify and analyse interest and influence of stakeholders in relation to flood risk reduction. MIC-MAC (Cross-impact Matrix Multiplication Applied to Classification) and MACTOR (Matrix of Alliance and Conflict: Tactics, Objectives, and Recommendations) methods were employed to analyse collected data. The results of this study indicated that restoration and humanity are the key objectives with major influence in lowering flood risk. The objectives are critical for the success of flood risk reduction efforts. The Disaster Management Agency is identified as the main stakeholder with the most important role in reducing flood risk in Central Java Province.


Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture.
To characterize phenotypic and antigenic changes in isolated Müller cells during proliferation in extended culture. Müller cells were isolated from porcine retina by sequential papain and DNase digestion, trituration, and density gradient centrifugation. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and delta smooth muscle actin (alpha SMA). Continuously proliferating cells established in culture were examined for changes in the expression of these antigens. Primary cultures of purified Müller cells, incubated under routine culture conditions, were proliferative and lost immunodetectable CRALBP within 2 weeks. The expression of CA-II also diminished with time, but at an apparently lower rate than that of CRALBP. Loss of GFAP expression was even more gradual and was complete by passage 5. De novo expression of alpha SMA was detectable in some cells within 12 days in culture and by all cells by passage 5. During this period, vimentin expression remained qualitatively unchanged. Isolated porcine Muller cells in culture undergo a phenotypic dedifferentiation to a fibroblast-like cell, which includes loss of detectable CRALBP, CA-II, and GFAP, and they acquire expression of the myoid marker alpha SMA.


Is preoperative chronic kidney disease status associated with oncologic outcomes in upper urinary tract urothelial carcinoma? A multicenter propensity score-matched analysis.
The aim of this study was to determine the effect of preoperative chronic kidney disease (CKD) on the prognosis of patients with upper urinary tract urothelial carcinoma (UTUC) who had undergone radical nephroureterectomy (RNU). The median follow-up period was 31.1 months (interquartile range: 16.2-55.7 months). Among the study patients, 224 patients in the non-CKD group were selected via propensity score matching. The median recurrence-free, cancer-specific, and overall survival were significantly shorter for patients with preoperative CKD than for non-CKD patients (p = 0.001, p = 0.001, and p = 0.001, respectively). According to multivariable Cox regression analysis, preoperative CKD was related to worse recurrence-free (hazard ratio [HR]: 1.81, 95% confidence interval [CI]: 1.15-2.86, p = 0.011), cancer-specific (HR: 2.44, 95% CI: 1.44-4.14, p = 0.001), and overall survival (HR: 1.66, 95% CI: 1.15-2.40, p = 0.007). A total of 566 patients who underwent RNU at 6 institutions from 2004 to 2014 were retrospectively reviewed. Of these patients, 342 had an estimated glomerular filtration rate (eGFR) ≥ 60 ml/min/1.73 m2 (non-CKD group) and 224 patients had an eGFR <60 ml/min/1.73 m2 (CKD group). To adjust for potential baseline confounders, 224 patients in the non-CKD group were selected by propensity matching. Clinicopathological variables and survival rates were compared between the 2 groups. Preoperative CKD appears to be an important independent prognostic factor for oncologic outcomes in patients with UTUC.


Liver nonparenchymal cells are stimulated to provide interleukin 6 for induction of the hepatic acute-phase response in endotoxemia but not in remote localized inflammation.
It has been postulated that Kupffer cells provide signals that regulate hepatocyte responses in sepsis and inflammation. Although in vitro data support such a hypothesis, to our knowledge, no in vivo evidence has been reported. We injected rats with lipopolysaccharide intraperitoneally to simulate sepsis or turpentine intramuscularly to mimic localized inflammation. Both treatments are known to induce the hepatic acute-phase response. Liver nonparenchymal cells and hepatocytes were isolated and placed in culture. Hepatocyte fibrinogen synthesis was measured as an indication of interleukin 6 exposure, while nonparenchymal interleukin 6 production was measured directly. Both lipopolysaccharide and turpentine stimulated a sharp increase in hepatocyte fibrinogen synthesis (turpentine greater than lipopolysaccharide). However, only lipopolysaccharide injection was associated with increased nonparenchymal cell interleukin 6 synthesis. Increased circulating levels of interleukin 6 could be found only after lipopolysaccharide injection. In addition, tumor necrosis factor synthesis was enhanced by lipopolysaccharide but not turpentine. Our data show that nonparenchymal cells are stimulated to provide the interleukin 6 signal to hepatocytes in endotoxemia but not in remote localized inflammation, even though both treatments stimulate the hepatic acute-phase response. Our findings support paracrine functions for liver sinusoidal cells in certain septic states.


Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations.
A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.


Joint action of a chemical carcinogen and a neoplastic virus to induce cancer in rabbits; results of exposing epidermal cells to a carcinogenic hydrocarbon at time of infection with the Shope papilloma virus.
Areas of rabbit skin previously rendered hyperplastic with turpentine were scarified, inoculated with the Shope papilloma virus, and covered with a dressing that contained 20-methylcholanthrene (MC) or 9:10-dimethyl-1:2-benzanthracene (9:10). The dressing was left on until healing had been well completed, a matter of 5 to 7 days. The papillomas which subsequently arose often appeared later, were fewer, and remained less vigorous than those due to the action of virus alone, but throughout several months they appeared to differ from these in no other ways. Then, more or less abruptly, the large majority became carcinomatous, frequently at several situations, whereas with few exceptions the control growths underwent no such alteration. The cancers were of the sorts ordinarily deriving, by secondary change, from epidermal cells infected with the virus. Collateral data have made plain that the hydrocarbons acted in their carcinogenic capacity to bring on the cancers. Indeed in control tests 9: 10 sometimes conferred latent neoplastic potentialities on uninoculated epidermis exposed to it while healing after scarification, a fact disclosed months later by painting these expanses with chloroform until hyperplasia occurred. Under the promoting influence of this agent papillomas formed which had the distinctive morphology of those induced by the chemical carcinogens. So strong and enduring were the effects of MC and 9:10 as to cause cancers to arise from many virus papillomas which were retrogressing after months of proliferation, that is to say under circumstances ordinarily unfavorable to malignant change. The facts justify the conclusion that the virus and the hydrocarbons acted jointly and in their carcinogenic capacities.


Patient outcomes associated with post-tuberculosis lung damage in Malawi: a prospective cohort study.
Post-tuberculosis lung damage (PTLD) is a recognised consequence of pulmonary TB (pTB). However, little is known about its prevalence, patterns and associated outcomes, especially in sub-Saharan Africa and HIV-positive adults. Adult (≥15 years) survivors of a first episode of pTB in Blantyre, Malawi, completed the St George's Respiratory Questionnaire, 6-minute walk test, spirometry and high-resolution CT (HRCT) chest imaging at TB treatment completion. Symptom, spirometry, health seeking, TB-retreatment and mortality data were collected prospectively to 1 year. Risk factors for persistent symptoms, pulmonary function decline and respiratory-related health-seeking were identified through multivariable regression modelling. Between February 2016 and April 2017, 405 participants were recruited. Median age was 35 years (IQR: 28 to 41), 77.3% (313/405) had had microbiologically proven pTB, and 60.3% (244/403) were HIV-positive. At pTB treatment completion, 60.7% (246/405) reported respiratory symptoms, 34.2% (125/365) had abnormal spirometry, 44.2% (170/385) had bronchiectasis ≥1 lobe and 9.4% (36/385) had ≥1 destroyed lobe on HRCT imaging. At 1 year, 30.7% (113/368) reported respiratory symptoms, 19.3% (59/305) and 14.1% (43/305) of patients had experienced declines in FEV1 or FVC of ≥100 mL, 16.3% (62/380) had reported ≥1 acute respiratory event and 12.2% (45/368) had symptoms affecting their ability to work. PTLD is a common and under-recognised consequence of pTB that is disabling for patients and associated with adverse outcomes beyond pTB treatment completion. Increased efforts to prevent PTLD and guidelines for management of established disease are urgently needed. Low-cost clinical interventions to improve patient outcomes must be evaluated.


Inputs of nutrients and fecal bacteria to freshwaters from irrigated agriculture: case studies in Australia and New Zealand.
Increasing demand for global food production is leading to greater use of irrigation to supplement rainfall and enable more intensive use of land. Minimizing adverse impacts of this intensification on surface water and groundwater resources is of critical importance for the achievement of sustainable land use. In this paper we examine the linkages between irrigation runoff and resulting changes in quality of receiving surface waters and groundwaters in Australia and New Zealand. Case studies are used to illustrate impacts under different irrigation techniques (notably flood and sprinkler systems) and land uses, particularly where irrigation has led to intensification of land use. For flood irrigation, changes in surface water contaminant concentrations are directly influenced by the amount of runoff, and the intensity and kind of land use. Mitigation for flood irrigation is best achieved by optimizing irrigation efficiency. For sprinkler irrigation, leaching to groundwater is the main transport path for contaminants, notably nitrate. Mitigation measures for sprinkler irrigation should take into account irrigation efficiency and the proximity of intensive land uses to sensitive waters. Relating contaminant concentrations in receiving groundwaters to their dominant causes is often complicated by uncertainty about the subsurface flow paths and the possible pollutant sources, viz. drainage from irrigated land. This highlights the need for identification of the patterns and dynamics of surface and subsurface waters to identify such sources of contaminants and minimize their impacts on the receiving environments.


"A Great First Step into Research": Stepping Into Research Is an Effective and Sustainable Model for Research Training in Clinical Settings: A Report of 6-Year Outcomes.
Cultivating a dynamic research culture is critical to growing the allied health professions. The Stepping into Research training program was developed in 2008 using small group training and mentoring to train allied health clinicians at a large health service in basic research skills. This paper describes the outcomes 6 years after its implementation, including translation of the program to a second health service. This mixed-methods observational evaluation included (1) quantitative data on research outputs (publications, conference presentations- and enrolment in further research training) and (2) qualitative analysis of the experiences of the first cohort of participants following translation of the program to a second site, with comparison to a previous evaluation conducted at the first site. Of the 55 participants enrolled in the program over 6 years, 49 completed the basic program resulting in 22 academic publications, 21 conference presentations, and 5 PhD enrolments. Qualitative data suggest the first cohort of participants and mentors at both sites experienced similar benefits and challenges. The Stepping into Research program has led to tangible research outcomes, has been sustained over 6 years, and been translated to a second health service. Questions remain about the impact of the program on clinical outcomes and research culture.


In vitro effects of listerial hemolysin on rat brain mitochondria.
Crude hemolysin derived from Listeria monocytogenes, strains 9-125 and 1122-3, reduced phosphate utilization of rat brain mitochondria in a succinate system. Oxidation rates were not altered by addition of hemolysin to a concentration of 42 hemolytic units/ml, with a mitochondrial protein concentration of 2.3 mg/ml. At a mitochondrial protein concentration of 1.8 mg/ml, 42 hemolytic units/ml of hemolysin increased the inhibition of phosphate utilization and also reduced the oxygen uptake. An age differential was apparent, with mitochondria from young rats demonstrating uncoupling at a lower concentration of hemolysin.


Biomechanical evaluation of endosseous implants at early implantation times: a study in dogs.
This study tested the null hypothesis that differences in surgical instrumentation, macrogeometry, and surface treatment imposed by different implant systems do not affect early biomechanical fixation in a canine mandible model. The lower premolars of 6 beagle dogs were extracted and the ridges allowed to heal for 8 weeks. Thirty-six (n = 12 each group) implants were bilaterally placed, remaining for 1 and 3 weeks in vivo. The implant groups were as follows: group 1, Ti-6Al-4V with a dual acid-etched surface with nanometer scale discrete crystalline deposition (Nanotite; Certain Biomet-3i, West Palm Springs, FL); group 2, Ti-6Al-4V with a titanium oxide-blasted fluoride-modified surface chemistry (Osseospeed 4.0 S; Astra Tech, Mölndal, Sweden); group 3: Ti-6Al-4V with a bioceramic microblasted surface (Ossean; Intra-Lock International, Boca Raton, FL). Following euthanasia, implants were torqued to interface failure and histologically evaluated. General linear modeling (ANOVA) at 95% level of significance was performed. Histology showed that interfacial bone remodeling and initial woven bone formation were observed around all implant groups at 1 and 3 weeks. Torque values were significantly affected by time in vivo, implant group, and their interaction (P = .016, P < .001, and P = .001, respectively). Regarding torque values, group 3, group 2, and group 1 ranked highest, intermediate, and lowest, respectively. Early biomechanical fixation at 1 and 3 weeks was affected by surgical instrumentation, macrogeometry, and surface treatment present for one of the implant systems tested. The null hypothesis was rejected.


Carboxylate-Directed Pd-Catalyzed β-C(sp3)-H Arylation of N-Methyl Alanine Derivatives for Diversification of Bioactive Peptides.
This study presents a Pd(II)-catalyzed method for the β-C(sp3)-H arylation of N-Cbz- or N-Fmoc-protected N-methyl alanines, providing ready access to building blocks for N-methylated peptide synthesis. For this transformation, the native carboxylate was exploited as the directing group, attributing its success to the use of a monoprotected amino-pyridine ligand. Its synthetic utility was demonstrated by facile generation of nine analogues of the naturally occurring N-methylated cyclic peptide cycloaspeptide A.


The payer's role in addressing the opioid epidemic: It's more than money.
County, State, and Federal agencies are addressing the public health opioid crisis. Ohio's 51 county-based Alcohol, Drug Addiction and Mental Health Services (ADAMHS) Boards finance and regulate opioid treatment services within their jurisdictions. This three-year comparative trial collaborated with ADAMHS Boards (n = 14) to test the Advancing Recovery Framework, a suite of organizational and system change strategies designed to promote use of buprenorphine for opioid agonist therapy. A multi-level intervention directed payers and treatment agencies to leverage their roles in increasing the use of buprenorphine. Half of the boards partnered with local substance use disorder treatment providers using the partnership strategies recommended by the Advancing Recovery (AR) framework. The comparison boards did not use the partnership strategies. A logistic regression analysis detected increases in the number of patients receiving buprenorphine in both conditions. Buprenorphine use, as a percentage of patients with an opioid use disorder diagnosis, was significantly greater among the boards using the Advancing Recovery strategies during the three-year experimental period (odds ratio (OR) 1.63, 95% CI, 1.50 to 1.76, p < .001) and a one-year maintenance period (OR 2.13, 95% CI, 1.85 to 2.46, p < .001). Boards in both groups provided similar levels of financial support to implement and maintain buprenorphine prescribing. Strategy differences between the study conditions existed in use of a committee that facilitated payer-provider partnering and the ADAMHS boards setting expectations for using buprenorphine. Payer-provider partnerships achieved greater improvements and maximized the effectiveness of funding in increasing access to buprenorphine.


The effect of chronically occluded coronary artery recanalisation on baroreflex sensitivity and left ventricular systolic function.
Recanalisation of chronic total occlusion (CTO) of a coronary artery can be reflected by improvements in various clinical parameters. Revealing increased parasympathetic activity would constitute an additional argument for performing this procedure. To assess the effect of CTO recanalisation on baroreflex sensitivity (BRS) and left ventricular ejection fraction (LVEF) in stable symptomatic patients with coronary artery disease. BRS (spectral analysis, transfer function, Blackman-Tukey algorithm, 0.03 Hz bandwidth Parzen window) and LVEF (echocardiography, Simpson's method) were analysed in 23 patients: one day (R1) before, one day (R2) after, and three months (R3) after CTO recanalisation. Patients were divided into two groups: those with depressed (≤ 3 ms/mm Hg) or preserved(> 3 ms/mm Hg) BRS. Significant BRS changes were observed in the study group compared to baseline values (p = 0.016). In the patients with a depressed reflex, BRS in R2 was similar to R1 and almost doubled in R3 (p = 0.018). In the patients with a preserved reflex, BRS significantly decreased in R2 (p = 0.024) and returned to the baseline value in R3. The behaviour of LVEF was homogenous in the groups, showing an increase from R1 to R3. The improvement in autonomic nervous system activity after successful CTO recanalisation is reflected by an increase in BRS, and the changes are dependent on the baseline value of the measurement: patients with a depressed BRS before recanalisation present a greater BRS improvement than patients with a preserved BRS. The CTO recanalisation leads to the improvement of LVEF in both groups.


Waist Circumference and Risk of Liver Cancer: A Systematic Review and Meta-Analysis of over 2 Million Cohort Study Participants.
Liver cancer is the sixth most common type of cancer worldwide, and waist circumference (WC) is associated with its risk beyond body mass index (BMI). This dose-response meta-analysis was performed to investigate the association between WC and the risk of incident liver cancer using prospective cohort studies. A comprehensive systematic search was conducted in MEDLINE/PubMed, Web of Science databases, Scopus, and Coch-rane from inception to May 2019. Studies with retrospective or prospective cohort design that reported hazard ratio (HR), risk ratio, or odds ratio, and the corresponding 95% confidence intervals (CI) for liver cancer based on WC categories were included in this meta-analysis. Combined HRs with 95% CIs was estimated by DerSimonian and Laird random-effects models. Associations between WC and liver cancer were reported in 5 articles with 2,547,188 participants. All studies were published between 2013 and 2019. Pooled results showed a strong significant association with minimum heterogeneity between WC and risk of liver cancer (HR 1.59, 95% CI 1.38-1.83, pheterogeneity = 0.42: I2 = 0%). Moreover, a dose-response model indicated a significant positive association between WC and risk of liver cancer (exp(b) = 1.018, p < 0.001). This systematic review and dose-response meta-analysis highlights WC as a significant risk factor related to the incidence of liver cancer.


NAD Metabolome Analysis in Human Cells Using ¹H NMR Spectroscopy.
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.


New Insulin Glargine 300 Units/mL Versus Glargine 100 Units/mL in People With Type 1 Diabetes: A Randomized, Phase 3a, Open-Label Clinical Trial (EDITION 4).
Insulin therapy in type 1 diabetes still provides suboptimal outcomes. Insulin glargine 300 units/mL (Gla-300), with a flatter pharmacodynamic profile compared with insulin glargine 100 units/mL (Gla-100), is an approach to this problem. People with type 1 diabetes, using a mealtime and basal insulin regimen, were randomized open-label to Gla-300 or Gla-100 and to morning or evening injection, continuing the mealtime analog, and followed for 6 months. Participants (n = 549) were a mean age of 47 years and had a mean duration of diabetes of 21 years and BMI of 27.6 kg/m(2). The change in HbA1c (primary end point; baseline 8.1%) was equivalent in the two treatment groups (difference, 0.04% [95% CI -0.10 to 0.19]) (0.4 mmol/mol [-1.1 to 2.1]), and Gla-300 was thus noninferior. Similar results with wider 95% CIs were found for morning and evening injection times and for prebreakfast self-measured plasma glucose (SMPG) overall. Results were also similar for Gla-300 when morning and evening injection time was compared, including overlapping 8-point SMPG profiles. Hypoglycemia did not differ, except for the first 8 weeks of the study, when nocturnal confirmed or severe hypoglycemia was lower with Gla-300 (rate ratio 0.69 [95% CI 0.53-0.91]). Hypoglycemia with Gla-300 did not differ by time of injection. The basal insulin dose was somewhat higher at 6 months for Gla-300. The adverse event profile did not differ and was independent of the Gla-300 time of injection. Weight gain was lower with Gla-300. In long-duration type 1 diabetes, Gla-300 provides similar glucose control to Gla-100, with a lower risk of hypoglycemia after transfer from other insulins, independent of time of injection, and less weight gain.


A Genome-Wide Loss-of-Function Screen Identifies SLC26A2 as a Novel Mediator of TRAIL Resistance.
TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome-wide shRNA screen for genes that regulate TRAIL sensitivity in sublines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including angiotensin II receptor 2, Crk-like protein, T-Box Transcription Factor 2, and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared with normal cells, and in breast cancer, SLC26A2 is associated with a significant decrease in relapse-free survival.Implication: Our results shed light on novel resistance mechanisms that could affect the efficacy of TRAIL agonist therapies and highlight the possibility of using these proteins as biomarkers to identify TRAIL-resistant tumors, or as potential therapeutic targets in combination with TRAIL. Mol Cancer Res; 15(4); 382-94. ©2017 AACR.


Modification of Ganglioside Content of Human Gastric Epithelial Cell Membrane Decreases Helicobacter pylori Adhesion.
In polarized cells, ganglioside location determines ganglioside function. Diet alters ganglioside content and composition in cell membranes. Ganglioside acts as a receptor for Helicobacter pylori. H pylori infects the stomach epithelium and may cause peptic ulcer disease and gastric cancer. The present study used purified gangliosides to modify the ganglioside composition of human gastric epithelial cells in vitro to reduce H pylori adhesion. A human gastric epithelial cell line (NCI-N87) was cultured with a ganglioside mix or with pure ganglioside (GM3 or GD3) at different concentrations (0-30 μg/mL) and ganglioside membrane content of gastric cells was determined after 48 hours. LC/triple quadrupole MS was used to analyse ganglioside concentration. H pylori was inoculated into the culture media of gastric cells previously treated with gangliosides GM3 or GD3 or a combination of GM3 and GD3. GD3 and GM3 content increased in the plasma membrane in a dose-dependent manner. Gastric cells treated with GD3 showed more GM3 content than GD3 (P < 0.01). Ganglioside content was modified in the apical membrane, but GM3 and GD3 were also found in the basolateral membrane after treatments. Gastric cells treated with GM3, GD3 or the combination of GM3:GD3 decreased H pylori adhesion to gastric cells at all ganglioside concentrations tested by 80% compared with untreated gastric cells (P < 0.05). These observations suggest that GD3 and GM3 present in the stomach lumen may be taken up into the apical gastric membrane and decrease H pylori adhesion to the epithelium.


Diastolic filling dynamics in patients with aortic stenosis.
Left ventricular filling dynamics were investigated in 24 patients with aortic stenosis (AS). Biplane cineangiography was performed with simultaneous micromanometry in these 24 patients and in six control subjects. Twelve of the patients with AS had moderate hypertrophy with a left ventricular muscle mass index of less than 180 g/m2 (ASI group) and 12 had severe hypertrophy with an index of 180 g/m2 or more (AS2 group). Filling dynamics were also evaluated postoperatively in eight patients in the AS1 and six patients in the AS2 group. Preoperatively, end-diastolic and end-systolic volume indexes were larger and ejection fraction was lower in the AS2 compared with the control or AS1 group. Percent volume increase during the first half of diastole (%V1) was smaller in the AS1 than in the AS2 group. Peak filling rate in the first half of diastole (PFR 1) was higher in the AS2 than in the control or in AS1 group, while peak filling rate in the second half of diastole (PFR2) was considerably greater in the AS1 group than in the other two groups. The time constant of left ventricular pressure decline, an index of the rate of relaxation, was prolonged in the AS2 group. In contrast, mitral valve opening pressure (MVOP) was significantly higher in this group than in the other two groups. The constant of left ventricular chamber stiffness was slightly but not significantly greater in both AS groups than in the control subjects. After surgery in patients in the AS1 group, preoperatively reduced %V1 had increased and preoperatively enhanced PFR2 had decreased. In patients in the AS2 group, excluding one with a persistent low ejection fraction after surgery, preoperatively enhanced PFR1 decreased in association with a decrease in MVOP. Thus, left ventricular filling dynamics vary in patients with AS depending on the degree of left ventricular hypertrophy and systolic function. In patients with AS and moderate hypertrophy %V1 is slightly reduced but is compensated for by a forceful atrial contraction. In those with severe hypertrophy and systolic dysfunction increased driving pressure allows %V1 to remain within normal limits, despite prolonged left ventricular relaxation and decreased elastic recoil. Both changes in left ventricular filling dynamics tend to normalize after surgery in association with a reduction in left ventricular hypertrophy and/or an improvement of systolic function.