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Introduction The HIV epidemic in Europe is characterized by a disproportionate number of infections among migrants. Although foreign citizens only made up 7% of the population of the European Union [...], : Introduction: Migrants are overrepresented in the European HIV epidemic. We aimed to understand the barriers and facilitators to HIV testing and current treatment and healthcare needs of migrants living with HIV in Europe. Methods: A cross?sectional study was conducted in 57 HIV clinics in nine countries (Belgium, Germany, Greece, Italy, The Netherlands, Portugal, Spain, Switzerland and United Kingdom), July 2013 to July 2015. HIV?positive patients were eligible for inclusion if they were as follows: 18 years or older; foreign?born residents and diagnosed within five years of recruitment. Questionnaires were completed electronically in one of 15 languages and linked to clinical records. Primary outcomes were access to primary care and previous negative HIV test. Data were analysed using random effects logistic regression. Outcomes of interest are presented for women, heterosexual men and gay/bisexual men. Results: A total of 2093 respondents (658 women, 446 heterosexual men and 989 gay/bisexual men) were included. The prevalence of a previous negative HIV test was 46.7%, 43.4% and 82.0% for women, heterosexual and gay/bisexual men respectively. In multivariable analysis previous testing was positively associated with: receipt of post?migration antenatal care among women, permanent residency among heterosexual men and identifying as gay rather than bisexual among gay/bisexual men. Access to primary care was found to be high (>83%) in all groups and was strongly associated with country of residence. Late diagnosis was common for women and heterosexual men (60.8% and 67.1%, respectively) despite utilization of health services prior to diagnosis. Across all groups almost three?quarters of people on antiretrovirals had an HIV viral load Conclusions: Migrants access healthcare in Europe and while many migrants had previously tested for HIV, that they went on to test positive at a later date suggests that opportunities for HIV prevention are being missed. Expansion of testing beyond sexual health and antenatal settings is still required and testing opportunities should be linked with combination prevention measures such as access to PrEP and treatment as prevention.

BACKGROUND: Depression often coexists with a number of disease states, and patients with a diagnosis of depression often receive multiple medications. Thus, it is desirable to avoid coadministration of agents that have a potential for drug interactions in these patients. Although escitalopram and its metabolites are weak to negligible inhibitors of the cytochrome P450 (CYP) 3A4 isozyme and are therefore unlikely to affect plasma concentrations of ritonavir (a CYP3A4 substrate and prototype CYP3A4 inhibitor), ritonavir may potentially affect plasma concentrations of escitalopram, as CYP3A4 is partially responsible for conversion of escitalopram to its major metabolite, S-demethylcitalopram (S-DCT). OBJECTIVES: The aim of this study was to investigate the potential for pharmacokinetic interaction between escitalopram and ritonavir after concomitant administration of a single dose of each in healthy young subjects. METHODS: In this single-center, randomized, open-label, 3-way crossover study, subjects received each of the following: a single dose of escitalopram 20 mg, a single dose of ritonavir 600 mg, and single doses of both escitalopram 20 mg and ritonavir 600 mg. Blood was collected and plasma was analyzed for the pharmacokinetic parameters (maximum plasma concentration [C(max)], time to C(max) [T(max)], area under the plasma concentration-time curve, plasma elimination half-life, oral clearance, and apparent volume of distribution) of escitalopram, S-DCT, and ritonavir. RESULTS: Of 21 subjects (11 men, 10 women; mean [SD] age, 28.4 [4.4] years) who were enrolled, 18 completed the study. After concomitant administration of escitalopram and ritonavir, no statistically significant differences were noted in the pharmacokinetics of escitalopram, with the exception of apparent volume of distribution, which was reduced by approximately 10% (P < 0.001). The pharmacokinetics of S-DCT were unaffected by coadministration of ritonavir, with the exception of T(max), which was increased in the presence of ritonavir. The pharmacokinetic parameters of ritonavir were also unaffected by coadministration of escitalopram. CONCLUSION: In general, no pharmacokinetic interaction was observed between escitalopram and ritonavir in the present study. [ABSTRACT FROM AUTHOR]

Halogenated estrogens are formed during chlorine-based wastewater disinfection and have been detected in wastewater treatment plant effluent; however, very little is known about their susceptibility to biodegradation in natural waters. To better understand the biodegradation of free and halogenated estrogens in a large river under environmentally relevant conditions, we measured estrogen kinetics in aerobic microcosms containing water and sediment from the Willamette River (OR, USA) at two concentrations (50 and 1250 ng L -1 ). Control microcosms were used to characterize losses due to sorption and other abiotic processes, and microbial dynamics were monitored using 16S rRNA gene sequencing and ATP. We found that estrogen biodegradation occurred on timescales of hours to days and that in river water spiked at 50 ng L -1 half-lives were significantly shorter for 17β-estradiol ( t 1/2,bio = 42 ± 3 h) compared to its monobromo ( t 1/2,bio = 49 ± 5 h), dibromo ( t 1/2,bio = 88 ± 12 h), and dichloro ( t 1/2,bio = 98 ± 16 h) forms. Biodegradation was also faster in microcosms with high initial estrogen concentrations as well as those containing sediment. Free and halogenated estrone were important transformation products in both abiotic and biotic microcosms. Taken together, our findings suggest that biodegradation is a key process for removing free estrogens from surface waters but likely plays a much smaller role for the more highly photolabile halogenated forms.

Competing Interests: Declaration of competing interest None.

Background: The use of pharmaceutical pictograms to enhance patients' understanding of drug regimens has been proven effective in many countries. There are two reference systems for pictograms generally used in pharmacy: the United States Pharmacopeia (USP) and International Pharmacy Federation (FIP). This study aimed to evaluate the effectiveness of USP and FIP pictograms among adults in the Philippines by identifying how many pictograms would pass the American National Standards Institute (ANSI) criterion of 85% comprehension, and to describe the factors affecting pictograms' comprehension., Methods: A descriptive cross-sectional research using a face-to-face interview was performed to evaluate 108 pictograms in 52 Filipino adults enrolled through quota sampling. Descriptive statistics, Mann-Whitney U test (Wilcoxon rank-sum test), univariate linear regression, and multiple linear regression were used to statistically analyze the data collected., Results: Only 17 (16 USP and 1 FIP) out of the 108 pictograms (15.74%) passed the ANSI criterion. The median score of Filipinos was 71 out of 108 pictograms (Interquartile range: 10-96). The multivariate model (R 2  = 0.5645, F (4,47) = 15.23) suggested that the score was lower by 5.85 points if the user was female, 21.58 points lower if the participant was below Grade 12 education level, and 1.20 points lower if the patient was greater than 46 years old. Education level was identified as the significant predictor (p-value < 0.0000*, power = 99.98%). The participant with greater than Grade 12 has a higher comprehension score of rank-sum 952.5 (Expected = 689) compared to only 425.5 (Expected = 689)., Conclusions: Since only 17 pictograms passed as stand-alone tool for patient information material, the researchers recommend the use of verbal and written instructions to complement pictograms to enhance comprehension. Furthermore, the government should consider the inclusion of health pictograms in basic health education., (© 2022. The Author(s).)

Background: Diseases of the heart and vascular system are the leading cause of mortality in the Philippines. Hypertension, the most important modifiable risk factor, has a prevalence rate of 28% and a control rate of 20%. Despite the proven efficacy of pharmacologic treatment, medication adherence is reported to be as low as 66%. While there are publications that reported factors that affect adherence in Filipinos, there are no existing research that evaluated them systematically. This review is conducted to present and synthesize findings of published literatures., Methods: Databases-PubMed, Scopus, Wiley Online library, Science Direct, JSTOR, Web of Science, SAGE journals, and Cochrane-were used to search for articles published from 2000 to 2020 that studied medication adherence in adult Filipino hypertensive population. Out of the initial 1514 articles, 15 articles met the criteria and were included in the analysis. The evidence from the included studies was summarized and discussed in a narrative review using the World Health Organization framework for adherence to long-term therapies as the framework., Result: The factors that were positively associated with adherence were health care system-related factors: good patient-health provider relationship, accessibility of health services, use of specialty clinics and programs for hypertension, and health insurance. The factors found to be negatively associated with adherence are (1) social economic factors: younger age, single civil status, low educational attainment, and unemployment; (2) patient-related factors: low in health literacy and awareness, knowledge on hypertension, attitude towards hypertension, self-efficacy, and social support; (3) therapy-related factors: inconsistent drug regimen schedule, use of Thiazide and complementary and alternative medicines; (4) condition-related factors: low illness perception, and absence of comorbidities., Conclusions: Findings should be interpreted with caution because of methodological limitations. Despite this, given that health systems related factors are modifiable, they can be the focus of interventions and future researches to increase medication adherence. Clinicians may also want to screen their Filipino hypertensive patients for factors that are associated to low adherence in order to provide a tailored advice. Longitudinal research studies with heterogeneous samples of hypertensive Filipinos are imperative so that targeted interventions can be developed for the population., (© 2021. The Author(s).)

Two transplant recipients (1 kidney and 1 hematopoietic stem cell) received maribavir (MBV) after cytomegalovirus (CMV) infection clinically resistant to standard therapy. Both patients achieved CMV DNA clearance within 30 and 18 days; however, the UL97 C480F variant emerged, causing recurrent CMV infection after a cumulative 2 months of MBV and 15 or 4 weeks of ganciclovir treatment, respectively. C480F was not detected under ganciclovir before MBV treatment. Recombinant phenotyping showed that C480F conferred the highest level of MBV resistance and ganciclovir cross-resistance, with impaired viral growth. Clinical follow-up and genotypic and phenotypic studies are essential for the assessment and optimization of patients with suspected MBV resistance., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Simplified antiretroviral therapy (ART) regimens are desirable for people with HIV. We investigated the efficacy and safety of switching from triple ART to dual dolutegravir plus lamivudine therapy., Methods: DOLAM is a phase 4, randomised, open-label, non-inferiority trial, done at six HIV clinics in Catalonia, Spain. Adults with HIV-1 receiving a triple ART regimen, aged 18 years or older, with virological suppression, a CD4 nadir of at least 200 cells per μL, who were HBsAg-negative, and without previous viral failure or resistance mutations to study drugs were eligible. Participants underwent computer-generated randomisation, stratified by the class of the third drug, and were assigned (1:1) to switch to oral dolutegravir 50 mg and lamivudine 300 mg once daily or to continue triple ART for 48 weeks. The primary endpoint was the proportion of people with an HIV RNA value of at least 50 copies per mL at week 48 (US Food and Drug Administration snapshot algorithm, 8% non-inferiority margin). Both the primary and safety outcomes were evaluated in the intention-to-treat exposed population. The study is completed and was registered with EudraCT 201500027435., Findings: Between July 7, 2015, and Oct 31, 2018, 265 participants were randomly assigned to switch to dolutegravir plus lamivudine (n=131) or to maintain triple ART (n=134) and all received at least one dose. Nine (7%) participants in the dual therapy group and ten (7%) in the triple therapy group were excluded before 48 weeks, mostly due to treatment discontinuations or virological failure. Participants were predominantly male (116 [87%] of 134 in the triple ART group and 111 [85%] of 131 in the dolutegravir plus lamivudine group). The difference in the proportion of participants with HIV RNA values of at least 50 copies per mL at 48 weeks between the dual therapy group (three [2%] of 131) and triple therapy group (two [1%] of 134) was 0·8 percentage points (95% CI -3·3 to 5·2), showing non-inferiority of dolutegravir plus lamivudine dual therapy compared with triple ART. 73 (56%) of 131 participants allocated to dual therapy had 150 adverse effects, compared with 78 (58%) of 134 participants allocated to triple therapy who also had 150 adverse events (p=0·68). Drug discontinuation due to adverse effects occurred in four people in the triple therapy group and three people in the dual therapy group., Interpretation: Our findings show the efficacy and safety of dolutegravir plus lamivudine as a simplified therapy switch option for selected people with HIV with virological suppression on triple ART., Funding: Instituto de Salud Carlos III, Red de Investigación en Sida, and ViiV Healthcare., Competing Interests: Declaration of interests PD reports grants and personal fees from Gilead Sciences, ViiV Healthcare, and Janssen & Cilag, and personal fees from Merck, Sharp & Dohme and Theratechnologies, outside the submitted work. JT reports personal fees from Gilead Sciences, ViiV Healthcare, Merck, Sharp & Dohme, and Janssen, outside the submitted work. ER reports personal fees from Merck, Sharp & Dohme, Gilead Sciences, Janssen, and ViiV Healthcare, outside the submitted work. DP reports research grants and honoraria for advisory boards and conferences from Viiv Healthcare, Pfizer, Bristol Myers Squibb, Gilead Sciences, Janssen, and Merck, Sharp & Dohme, outside the submitted work. RP reports grants from ViiV Healthcare during the conduct of the study, and grants and personal fees from Merck, Sharp & Dohme and Gilead Sciences, outside the submitted work. JMG is currently a full employee at ViiV Healthcare, reports grants from ViiV Healthcare during the conduct of the study, and personal fees from ViiV Healthcare, outside the submitted work. JLB reports grants, personal fees, and non-financial support from ViiV Healthcare and Janssen, and personal fees and non-financial support from Merck, Sharp & Dohme, Gilead Sciences, and Theratechnologies, outside the submitted work. EM reports grants from Instituto de Salud Carlos III and ViiV Healthcare during the conduct of the study, grants and personal fees from Merck, Sharp & Dohme and ViiV Healthcare, and personal fees from Gilead Sciences and Janssen, outside the submitted work. All other authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Several proteins and peptides in saliva were shown to stimulate gingival wound repair, but the role of salivary metabolites in this process remains unexplored. In vitro gingival re-epithelialization kinetics were determined using unstimulated saliva samples from healthy individuals collected during an experimental gingivitis study. Elastic net regression with stability selection identified a specific metabolite signature in a training dataset that was associated with the observed re-epithelialization kinetics and enabled its prediction for all saliva samples obtained in the clinical study. This signature encompassed ten metabolites, including plasmalogens, diacylglycerol and amino acid derivatives, which reflect enhanced host-microbe interactions. This association is in agreement with the positive correlation of the metabolite signature with the individual's gingival bleeding index. Remarkably, intra-individual signature-variation over time was associated with elevated risk for gingivitis development. Unravelling how these metabolites stimulate wound repair could provide novel avenues towards therapeutic approaches in patients with impaired wound healing capacity.

Human milk oligosaccharides (HMO) and prebiotic oligosaccharides are proposed to confer several health benefits to the infant. They shape the microbiota, have anti-inflammatory properties, and support epithelial barrier functioning. However, in order to select the best oligosaccharides for inclusion in infant formulas, there is a need to increase our understanding of the specific effects of HMO and prebiotics on the host immune system. Therefore, we investigated the effects of the HMO sialyllactose (SL), and galactooligosaccharides (GOS) on epithelial barrier functioning, microbiota composition, and SCFA production. The effect of GOS and SL on epithelial barrier functioning and microbiota composition was investigated using in vitro models. Epithelial barrier function was investigated by transcriptome analysis of fully polarized Caco-2 cells exposed for 6 h to SL or GOS. In addition, epithelial cell growth, alkaline phosphatase production, and re-epithelization was studied. Further, we investigated the effect of SL and GOS on microbiota composition and SCFA production using in vitro fecal batch cultures. Transcriptome analysis showed that SL and GOS both induced pathways that regulate cell cycle control. This gene-expression profile translated to a phenotype of halted proliferation and included the induction of alkaline phosphatase activity, a marker of epithelial cell differentiation. SL and GOS also promoted re-epithelialization in an in vitro epithelial wound repair assay. SL and GOS did show distinct modulation of microbiota composition, promoting the outgrowth of Bacteroides and bifidobacteria, respectively, which resulted in distinct changes in SCFA production profiles. Our results show that SL and GOS can both modulate epithelial barrier function by inducing differentiation and epithelial wound repair, but differentially promote the growth of specific genera in the microbiota, which is associated with differential changes in SCFA profiles.

Aim: To determine if there are ethnic differences in the prevalence of antiphospholipid syndrome (APS), clinical presentation and autoantibody profile between Roma and Caucasian patients with systemic lupus erythematosus (SLE)., Method: A cross-sectional study was conducted including data from Roma and Caucasian SLE patients consecutively attending six hospitals in Spain. Socio-demographic characteristics, prevalence of APS, clinical and analytical features of SLE and APS were compared between ethnic groups., Results: Data from 52 Roma and 98 Caucasian SLE patients were included. Roma SLE patients had a higher risk (odds ratio 2.56, 95% CI 1.02-6.39) and prevalence of APS (28.8% vs. 13.3%, P = 0.027). Furthermore, Roma SLE patients had a statistically significant higher prevalence of abortions (23.5% vs. 10.2%, P = 0.049). In relation to other APS diagnostic criteria, Roma SLE patients had a non-statistically significant higher prevalence of fetal deaths (14.3% vs. 5.1%, P = 0.106) and thrombotic events (21.1% vs. 12.2%, P = 0.160). In relation to SLE clinical features, Roma patients had a significantly higher prevalence of arthritis (75% vs. 57.1%, P = 0.034) and non-significant higher prevalence of serositis (44.2% vs. 29.6%, P = 0.104), discoid lesions (11.5% vs. 5.1%, P = 0.191), oral ulcers (46.1% vs. 34.7%, P = 0.218) and livedo reticularis (21.1% vs. 15.3%, P = 0.374). No statistically significant differences were found in the Systemic Lupus International Collaborating Clinics Damage Index or the autoimmune serological profile., Conclusion: Prevalence and risk of APS were significantly higher in Roma SLE patients. Furthermore, Roma patients had a significantly higher prevalence of abortions and a non-significant higher prevalence of fetal deaths and thrombotic events., (© 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Background: Scientific and professional development opportunities for early career scientists in low- and middle- income countries (LMICs) are limited and not consistent. There is a disproportionately low number of biomedical and clinical researchers in LMIC's relative to their high burden of disease, a disparity that is aggravated by emigration of up to 70% of scientists from their countries of birth for education and employment elsewhere. To help address this need, a novel University-accredited, immersive fellowship program was established by a large public-academic-private network. We sought to describe the program and summarize progress and lessons learned over its first 7-years., Methods: Hallmarks of the program are a structured learning curriculum and bespoke research activities tailored to the needs of each fellow. Research projects expose the scientists to state-of-the-art methodologies and leading experts in their fields while also ensuring that learnings are implementable within their home infrastructure. Fellows run seminars on drug discovery and development that reinforce themes of scientific leadership and teamwork together with practical modules on addressing healthcare challenges within their local systems. Industry mentors achieve mutual learning to better understand healthcare needs in traditionally underserved settings. We evaluated the impact of the program through an online survey of participants and by assessing research output., Results: More than 140 scientists and clinicians from 25 countries participated over the 7-year period. Evaluation revealed strong evidence of knowledge and skills transfer, and beneficial self-reported impact on fellow's research output and career trajectories. Examples of program impact included completion of post-graduate qualifications; establishment and implementation of good laboratory- and clinical- practice mechanisms; and becoming lead investigators in local programs. There was a high retention of fellows in their home countries (> 75%) and an enduring professional network among the fellows and their mentors., Conclusions: Our experience demonstrates an example for how multi-sectoral partners can contribute to scientific and professional development of researchers in LMICs and supports the idea that capacity-building efforts should be tailored to the specific needs of beneficiaries to be maximally effective. Lessons learned may be applied to the design and conduct of other programs to strengthen science ecosystems in LMICs.

Background: The large global burden of rheumatic heart disease (RHD) has come to light in recent years following robust epidemiologic studies. As an operational research component of a broad program aimed at primary and secondary prevention of RHD, we sought to determine the current prevalence of RHD in the country's capital, Lusaka, using a modern imaging-based screening methodology. In addition, we wished to evaluate the practicality of training local radiographers in echocardiography screening methods., Methods: Echocardiography was conducted on a random sample of students in 15 schools utilizing a previously validated, abbreviated screening protocol. Through a task-shifting scheme, and in the spirit of capacity-building to enhance local diagnostic and research skills, general radiographers based at Lusaka University Teaching Hospital (UTH) were newly trained to use portable echocardiography devices. Students deemed as screen-positive were referred for comprehensive echocardiography and clinical examination at UTH. Cardiac abnormalities were classified according to standard World Heart Federation criteria., Results: Of 1102 students that were consented and screened, 53 students were referred for confirmatory echocardiography. Three students had definite RHD, 10 had borderline RHD, 29 were normal, and 11 students were lost to follow-up. The rates of definite, borderline, and total RHD were 2.7 per 1000, 9.1 per 1000, and 11.8 per 1000, respectively. Anterior mitral valve leaflet thickening and chordal thickening were the most common morphological defects. The pairwise kappa test showed fair agreement between the local radiographers and an echocardiographer quality assurance specialist., Conclusion: The prevalence of asymptomatic RHD in urban communities in Zambia is within the range of results reported in other sub-Saharan African countries using the WHF criteria. Task-shifting local radiographers to conduct echocardiography was feasible. The results of this study will be used to inform ongoing efforts in Zambia to control and eventually eliminate RHD., Trial Registration: The study was registered on clinicaltrials.gov ( #NCT02661763 ).

Background: In vitro scratch assays have been widely used to study the influence of bioactive substances on the processes of cell migration and proliferation that are involved in re-epithelialization. The development of high-throughput microscopy and image analysis has enabled scratch assays to become compatible with high-throughput research. However, effective processing and in-depth analysis of such high-throughput image datasets are far from trivial and require integration of multiple image processing and data extraction software tools., Findings: We developed and implemented a kinetic re-epithelialization analysis pipeline (KREAP) in Galaxy. The KREAP toolbox incorporates freely available image analysis tools and automatically performs image segmentation and feature extraction of each image series, followed by automatic quantification of cells inside and outside the scratched area over time. The enumeration of infiltrating cells over time is modeled to extract three biologically relevant parameters that describe re-epithelialization kinetics. The output of the tools is organized, displayed, and saved in the Galaxy environment for future reference., Conclusions: The KREAP toolbox in Galaxy provides an open-source, easy-to-use, web-based platform for reproducible image processing and data analysis of high-throughput scratch assays. The KREAP toolbox could assist a broad scientific community in the discovery of compounds that are able to modulate re-epithelialization kinetics.

Gingival re-epithelialization represents an essential phase of oral wound healing in which epithelial integrity is re-establish. We developed an automated high-throughput re-epithelialization kinetic model, using the gingival epithelial cell line Ca9-22. The model was employed to screen 39 lactic acid bacteria, predominantly including oral isolates, for their capacity to accelerate gingival re-epithelialization. This screen identified several strains of Streptococcus salivarius that stimulated re-epithelialization. Further analysis revealed that S. salivarius strain MS-oral-D6 significantly promoted re-epithelialization through a secreted proteinaceous compound and subsequent experiments identified a secreted serine protease as the most likely candidate to be involved in re-epithelialization stimulation. The identification of bacteria or their products that stimulate gingival wound repair may inspire novel strategies for the maintenance of oral health.

Objective: To evaluate tissue effect of J-Plasma® (Bovie Medical Corporation, Clearwater, Florida) in porcine liver, kidney, muscle, ovarian, and uterine tissue blocks., Design: Prospective study utilizing porcine tissue blocks to evaluate the thermal spread of J-Plasma® device on liver, kidney, muscle, ovarian, and uterine tissue at various power settings, gas flow, and exposure times., Materials and Methods: J-Plasma® helium was used in porcine liver, kidney, and muscle tissue at 20%, 50%, and 100% power, and 1 L/min, 3 L/min, and 5 L/min gas flow at one, five, and 10-second intervals. J-Plasma® was then used in ovarian and uterine tissue at maximum power and gas flow settings in intervals of one, five, 10, and 30 seconds. Histologic evaluation of each tissue was then performed to measure thermal spread., Results: Regardless of tissue type, increased power setting, gas flow rate, and exposure time correlated with greater depth of thermal spread in liver, kidney, and muscle tissue. J-Plasma® did not exceed 2 mm thermal spread on liver, kidney, muscle, ovarian, and uterine tissue, even at a maximum setting of 100% power and 5 L/min gas flow after five seconds. Prolonged exposure to J-Plasma® of up to 30 seconds resulted in increased length and width of thermal spread of up to 12 mm, but did not result in significantly increased depth at 2.84 mm., Conclusions: The J-Plasma® helium device has minimal lateral and depth of thermal spread in a variety of tissue types and can likely be used for a multitude of gynecologic surgical procedures. However, further studies are needed to demonstrate device safety in a clinical setting.

Purpose: The purpose of this paper is to introduce a novel trans-illuminating culdotomy and uterine manipulator device., Materials and Methods: The study was a prospective, non-randomized, non-blinded observational clinical study involving 50 female patients undergoing total laparoscopic hysterectomy (TLH) or laparoscopic supracervical hysterectomy (LSH) for benign indications. The surgeries were performed from March through May 2012 at two institutions. The primary study objectives were to demonstrate the safety and adequate clinical performance of the uterine manipulator device and to illustrate its potential widespread future use in minimally invasive gynecologic procedures., Results: Average patient age was 45.1 years and, of the 50 patients, 33 had undergone previous intra-abdominal surgery. There were no reports of adverse events, difficulty with placement of the instrument, multiple attempts at placement, or difficulty with uterine manipulation. There was only one device-related uterine perforation, and pneumoperitoneum was maintained in all cases during culdotomy. Vaginal tissue left on subjects was less than 5mm. Overall, there were no ureteral injuries, there were two reported incidental cystotomies, and average blood loss was 99.0cc. Postoperative courses were normal for all patients, with only two reported postoperative complications: a possible vaginal cuff abscess and a 2cm vaginal mucosal cuff separation., Conclusions: The McCarus-Volker ForniSee® (LSI Solutions, Inc., Victor, New York) is a novel trans-illuminating culdotomy device and uterine manipulator that is safe, efficient, functional, and easy to use. Trans-illumination additionally delineates and enhances identification of critical anatomic planes, such as the vesicovaginal junction and cervicovaginal junction.

A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.

Packaging is associated with a high environmental impact. This is also the case in the food industry despite packaging being necessary for maintaining food quality, safety assurance and preventing food waste. The aim of the present study was to identify improvements in food packaging solutions able to minimize environmental externalities while maximizing the economic sustainability. To this end, the life cycle assessment (LCA) methodology was applied to evaluate the environmental performance of new packaging solutions. The environmental impact of packaging and food losses and the balance between the two were examined in relation to a cheesecake that is normally packaged in low density polyethylene film and has a limited shelf life due to microbial growth. A shelf life extension was sought via application of the well-established modified atmosphere packaging (MAP) technique. Samples for MAP (N 2 /CO 2 : 70/30) were placed inside multilayer gas barrier trays, which were then wrapped with a multilayer gas and water barrier film (i.e. AerPack packaging); control batches were packaged in gas barrier recycled polyethylene terephthalate (XrPet) trays and wrapped with a XrPet film. Samples were then stored at 20°C and inspected at regular intervals for chemical-physical, microbiological and sensory parameters. Results show that the new packaging solution could considerably extend the shelf life of cheesecakes, thereby reducing food waste and decreasing the overall environmental impact. Moreover, the new packaging allows one to minimize transport costs and to generate economies of scale in manufacturing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell., Competing Interests: A patent pertaining to the use of strains expressing lactococcal pili was filed by TIFN. The affiliations to TIFN and NIZO food research do not alter our adherence to PLOS ONE policies on sharing data and materials.

This study sought to describe the characteristics of deaths with leprosy as the underlying cause recorded in the Mortality Information System (SIM) and compare these characteristics with the groups of cases where the cause of death was reported, or failed to be reported, in the National Case Registration Database (Sinan). Deaths with leprosy as the underlying cause occurring in Brazil in the 2004-2009 period, and cases of leprosy from 1975 to 2010 were included. The probabilistic bases of SIM and Sinan were compared. Of the 1,463 deaths from leprosy recorded in SIM, 44.2% were not recorded in Sinan. Of the total number of deaths, the majority were men (72.5%), aged 60 or older (56.6%), occurring in hospitals (65.3%) and with due care (45.8%). Of the 820 deaths identified in Sinan, 92% were patients with multibacillary disease, 45.2% were discharged as cured by Sinan and 38.9% died. Deaths due to leprosy were found on SIM that were not notified to Sinan. The data base linkage enabled identification of ancillary records and inconsistencies between the systems.

Introduction: This article presents the results of a research whose objective was to verify the prevalence of the perception reports regarding quality of life of library attendees in the public libraries in the Brazilian capital Federal District (FD) and the surrounding region and to analyse the factors related to dissatisfaction., Methods: An epidemiological transversal study was conducted in 592 individuals aged above 12 years old through the application of the WHOQOL-BREF/WHO questionnaire., Results: Higher frequencies of dissatisfaction were observed among women with ages above 25, with lower personal income and lower educational level. Dissatisfaction regarding the physical domain was more prevalent in the surrounding region than in the FD. Under the psychological domain, dissatisfaction predominated in people in the FD. Negative feelings, concentration difficulties and dissatisfaction regarding personal safety were referred by more than 25% of participants in both regions. Regarding the environment domain, lack of money and of leisure opportunities were the main complaints. In spite of these findings, interviewees referred being very satisfied with their health and quality of life., Conclusions: The results can be a sign that the quality of life in the study region is in alert level. A careful look at these data is needed to identify alternatives to change this situation, with effective actions for Health Promotion and development strategies for the study area. A planning and an intervention in the area of health education in public libraries is recommended, since these are very important social loci, that can be engaged in health promotion and disease prevention actions in the communities.

ACT-280778 is a novel nondihydropyridine dual L/T-type calcium channel blocker. Two clinical studies (AC-067-101 and AC-067-102) were conducted to characterize its safety, tolerability, and pharmacokinetics in healthy male subjects after oral administration of single and multiple doses. Both trials were single-center, randomized, double-blind, placebo-controlled, adaptive design, ascending-dose studies, in which ACT-280778 was administrated as single doses of 2, 5, 15, or 40 mg, or as once-daily doses of 5 or 15 mg for 7 days. Single and multiple doses up to and including 15 mg were well tolerated, and no serious or severe adverse event was reported in either study. A single dose of 40 mg was associated with abnormal electrocardiogram findings resulting in the discontinuation of further treatment at this dose or higher doses. ACT-280778 was rapidly absorbed, and larger than dose-proportional increases of the maximum plasma concentration and area under the plasma concentration-time curve were observed. Food intake delayed the time to maximum plasma concentration and doubled exposure. Urinary excretion of unchanged ACT-280778 was negligible, and accumulation at steady state was modest. Overall, pharmacokinetic and tolerability profiles of ACT-280778 observed in these 2 studies warranted further evaluation of ACT-280778 in a proof-of-concept study in patients with hypertension.

What Is Known: Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension (PAH). Since bosentan is frequently used to treat pediatric PAH patients, a pediatric formulation was developed., Aim: To evaluate the pharmacokinetic properties of bosentan and its active metabolite, Ro 48-5033, of the quadrisected, dispersible pediatric vs. the adult tablet after single-dose administration to healthy subjects. Secondary objectives of the study were to compare the pharmacokinetics of two inactive metabolites and the safety of both formulations., Materials and Methods: In this open-label, two-way crossover study, subjects (20 - 43 years) were randomized to receive single oral doses of 62.5 mg of bosentan as 1 adult tablet and 64 mg as 2 pediatric tablets of 32 mg. Blood samples were drawn over a 48-hour period to measure bosentan and its metabolites., Results: 16 subjects were enrolled and completed the study. Following treatment with the pediatric formulation, values for Cmax and AUC0-∞ of bosentan were lower than with the adult formulation with geometric mean ratios (90% confidence interval) of 0.82 (0.65, 1.04) and 0.87 (0.78, 0.97), respectively. Similar results were obtained for the primary active metabolite Ro 48-5033. Both treatments were well tolerated., What Is New and Conclusion: Although the 90% confidence intervals of the geometric mean ratios of Cmax and AUC0-∞ were not entirely within the conventional bioequivalence range (0.80 - 1.25), no clinically relevant effect of formulation on the pharmacokinetics of bosentan and Ro 48-5033 was detected. Both formulations were well tolerated.

Background: Epoprostenol sodium for injection is approved for the treatment of severe cases of primary pulmonary arterial hypertension. Currently, there are 3 approved formulations of this drug containing the same active ingredient (epoprostenol sodium) but differing with regard to excipients. When compared with epoprostenol sodium formulated with glycine-mannitol (epoprostenol GM), 2 new formulations of epoprostenol sodium, one formulated with arginine-mannitol (epoprostenol AM) and one formulated with arginine-sucrose (epoprostenol AS), have improved stability after reconstitution and dilution. The biocomparability of epoprostenol AM and epoprostenol GM, with regard to pharmacokinetic (PK), pharmacodynamic (PD), safety, and tolerability profiles, has been shown previously., Objective: This study compared PK, PD, safety, and tolerability profiles of the 3 different formulations of epoprostenol sodium for injection., Methods: This was a prospective, single-center, open-label, 2-period, 2-treatment, randomized, crossover, ascending dose study in 2 parts. Twenty healthy men in part 1 and 20 different individuals in part 2 received epoprostenol AM and epoprostenol AS and epoprostenol GM and epoprostenol AS, respectively, in a crossover fashion, as sequential IV infusions of 2, 4, 6, and 8 ng/kg/min for 2 hours each. In each part, the PK profile of epoprostenol was characterized via analysis of the concentration-time profiles of its 2 primary metabolites: 6-keto-prostacyclin F1α and 6,15-diketo-13,14-dihydro-prostacyclin F1α. The effect of the formulations was assessed using the 90% CI of the geometric mean ratio calculated for the exposure PK parameters. The PD variables cardiac output, cardiac index, and heart rate were assessed using echocardiography. Adverse events were recorded through the study., Results: The plasma concentration versus time curves of epoprostenol AM and epoprostenol AS in part 1 and epoprostenol GM and epoprostenol AS in part 2 were similar in shape and almost superimposable. For each study part, the 90% CIs of ratios of geometric means for AUC0-∞ of the assessed epoprostenol formulations were within the range for bioequivalence (0.8-1.25). The increases in cardiac output, cardiac index, and heart rate resulting from infusion with epoprostenol sodium were comparable between all formulations, with maximum values attained after 8 hours. Almost all study participants reported at least one treatment-emergent adverse event, the most common being headache, which was reported in 80% to 85% of study participants., Conclusions: Overall, the PK, PD, safety, and tolerability profiles of the 3 formulations of epoprostenol sodium for injection are comparable and meet the criteria of bioequivalence. Australian New Zealand Clinical Trials Registry identifier: ACTRN12612001086853., (Copyright © 2013 Elsevier HS Journals, Inc. All rights reserved.)

Aim: The aim of the study was to report the first thorough characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of epoprostenol in an integrated manner., Method: Twenty healthy male subjects received two formulations of i.v. epoprostenol, in a crossover design, in sequential infusions of 2, 4, 6 and 8 ng kg(-1)  min(-1) for 2 h each. A sensitive assay was developed which allowed accurate PK characterization of epoprostenol via analysis of the concentration-time profiles of its two primary metabolites, 6-keto-prostacyclin F(1α) and 6,15-diketo-13,14-dihydro-prostacyclin F(1α) . PD parameters included cardiac output (CO), cardiac index (CIn) and heart rate (HR)., Results: The pharmacokinetics of epoprostenol deviated slightly from dose-proportionality, probably due to a food effect. After infusion of the two formulations of epoprostenol, the t(1/2) values expressed as geometric mean (95% confidence interval) were 0.25 h (0.14, 0.46) and 0.22 h (0.13, 0.38) for 6-keto-prostacyclin F(1α) , and 0.32 h (0.22, 0.45) and 0.34 h (0.26, 0.46) for 6,15-diketo-13,14-dihydro-prostacyclin F(1α) . A single compartment infusion model with first order elimination adequately described the PK of 6-keto-prostacyclin F(1α) . This model also characterized the food effect. Stepwise infusions with epoprostenol resulted in a progressive increase in CO, CIn and HR., Conclusion: Of the two metabolites analyzed, the appearance of 6-keto-prostacyclin F(1α) in plasma was more closely associated with the haemodynamic effects of i.v. epoprostenol. PK and PD profiles showed that CIn relates proportionally and linearly to the plasma concentrations of 6-keto-prostacyclin F(1α) . These results suggest that 6-keto-prostacyclin F(1α) is a suitable surrogate marker of plasma concentrations of epoprostenol., (© 2012 Actelion Pharmaceuticals Ltd.. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.)

Purpose: To evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of escalating single oral doses of ACT-077825, a novel orally active renin inhibitor, in healthy male subjects., Methods: In this single-center, double-blind, placebo- and active-controlled (with enalapril) randomized study, 70 subjects received a single dose of ACT-077825 (1-1,000 mg), placebo, or enalapril 20 mg under fasted conditions. The main pharmacokinetic endpoints were area under the plasma ACT-077825 concentration-time curve from time zero to infinity and the terminal half-life (t(1/2)). The pharmacodynamic endpoints included immunoactive active renin (iAR) plasma concentrations and plasma renin activity (PRA). Standard laboratory and safety data were collected., Results: Of the few adverse events reported, diarrhea and headache were the most frequent. The pharmacokinetics of ACT-077825 were dose-proportional in the dose range 100 to 1,000 mg. Terminal t(1/2), best characterized following a dose of 1,000 mg, was 41.6 h and t(max) 4-5 h post-dose. ACT-077825 dose-dependently increased iAR and decreased PRA, effects that were associated with a decrease in blood pressure at 1,000 mg, similar to following treatment with enalapril., Conclusion: The results provide evidence that ACT-077825, with a pharmacokinetic profile consistent with a once-a-day dosing regimen, may represent an effective antihypertensive agent and pave the way toward a multiple-ascending dose study.

Objective: Open-label trials with the selective serotonin reuptake inhibitor citalopram suggest that this agent is effective and safe for the treatment of depressive symptoms in children and adolescents. The current study investigated the efficacy and safety of citalopram compared with placebo in the treatment of pediatric patients with major depression., Method: An 8-week, randomized, double-blind, placebo-controlled study compared the safety and efficacy of citalopram with placebo in the treatment of children (ages 7-11) and adolescents (ages 12-17) with major depressive disorder. Diagnosis was established with the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version. Patients (N=174) were treated initially with placebo or 20 mg/day of citalopram, with an option to increase the dose to 40 mg/day at week 4 if clinically indicated. The primary outcome measure was score on the Children's Depression Rating Scale-Revised; the response criterion was defined as a score of < or =28., Results: The overall mean citalopram dose was approximately 24 mg/day. Mean Children's Depression Rating Scale-Revised scores decreased significantly more from baseline in the citalopram treatment group than in the placebo treatment group, beginning at week 1 and continuing at every observation point to the end of the study (effect size=2.9). The difference in response rate at week 8 between placebo (24%) and citalopram (36%) also was statistically significant. Citalopram treatment was well tolerated. Rates of discontinuation due to adverse events were comparable in the placebo and citalopram groups (5.9% versus 5.6%, respectively). Rhinitis, nausea, and abdominal pain were the only adverse events to occur with a frequency exceeding 10% in either treatment group., Conclusions: In this population of children and adolescents, treatment with citalopram reduced depressive symptoms to a significantly greater extent than placebo treatment and was well tolerated.

Background: Citalopram tablets fulfill most dosing needs in the treatment of depression, but some patients may have difficulty swallowing tablets and thus may be less likely to comply with their medication regimen. A liquid formulation of citalopram could be beneficial for such patients., Objective: This study was undertaken to compare the pharmacokinetic profiles of oral solution and tablet formulations of citalopram in healthy volunteers., Methods: In this open-label, single-dose, randomized, crossover, bioequivalence study, healthy volunteers alternately received one 60-mg dose of citalopram as an oral solution (10 mg/5 mL) and one 60-mg dose as a tablet. Doses were separated by a 14-day interval., Results: Of 24 subjects enrolled (mean age 27 years), 24 (16 men and 8 women) received the citalopram oral solution and 23 (15 men and 8 women) received the tablet; 1 subject discontinued before receiving the tablet. Citalopram was rapidly absorbed, with peak plasma concentrations occurring at approximately 4 hours with both formulations. The rate and extent of absorption were similar between the 2 formulations, and no statistically significant differences were observed in half-life or oral clearance between formulations. Similarly, the pharmacokinetic profile for demethylcitalopram (the major metabolite of citalopram) did not differ between the 2 formulations. Both formulations were well tolerated, with no serious adverse events reported., Conclusion: The oral solution and tablet formulations of citalopram 60 mg were determined to be bioequivalent in this population.

Familial ligand-defective apolipoprotein B (apoB) is a group of disorders caused by mutations in the apoB gene. In this report the R3531C mutation is characterized further using a monoclonal antibody MB19/dynamic laser light scattering technique to measure ratios of Cys(3531) to normal low density lipoprotein (LDL) particles. All six subjects studied showed a preferential accumulation of particles carrying the defective apoB allotype. We determined binding properties of LDL from R3531C heterozygotes by measurement of high-affinity binding to LDL receptors on fibroblasts and its ability promote growth of U937 cells. LDL from R3531C heterozygotes, compared to normal LDL, had 49.3% of the binding affinity and was 74% as effective in a U937 cell proliferation assay. To identify new probands, we screened 2570 subjects for the R3531C mutation. Nine probands were found with 15 affected relatives. Of the seven haplotypes we uncovered, two were novel, while five were identical to one initially reported as associated with Cys3531. Three silent mutations were detected also: T3540T, N3542N and T3552T. Analysis of lipid profiles of R3531C families showed, as with the R3500Q mutation, variable expression of the phenotype, modulated by environmental and other genetic factors. Both mutations tend to produce lower plasma levels of LDL in affected subjects than do defects of the LDL receptor (familial hypercholesterolemia, FH). This study shows that the Cys(3531) LDL particles are not only defective at binding to the LDL receptor, as determined by two separate methods, but that in all cases they accumulate preferentially compared to the normal allotype.-Pullinger, C. R., D. Gaffney, M. M. Gutierrez, M. J. Malloy, V. N. Schumaker, C. J. Packard, and J. P. Kane. Apolipoprotein B R3531C mutation: characteristics of 24 subjects from 9 kindreds. .

Monoclonal antibody complexes have proven very useful in the study of low-density lipoproteins (LDLs). Thus, complexes composed of two different monoclonal antibodies, selected from a panel of 11 different antibodies, and LDL have been employed to map apolipoprotein B (apoB) on the surface of the LDL. In this way, apoB was found to surround the LDL as a ribbon with a bow [Chatterton, J. E., et al. (1995) J. Lipid Res. 36, 2027-2037]. Moreover, monoclonal MB19, which recognizes a polymorphic site, has been employed to quantitate the two different allelic forms of apoB found on LDL in human sera, and in this way, we assessed the effect of most of the known common polymorphisms of this protein as well as detected the depletion of the normal allele product in two forms of familial defective apoB-100 [Chatterton, J. E., et al. (1995) Biochemistry 34, 9571-9580; Pullinger, C. R., et al. (1995) J. Clin. Invest. 95, 1225-1234]. In this paper, these studies have been extended by examining by dynamic light scattering and sedimentation velocity techniques the complexes formed with only one antibody, and complexes formed using two antibodies. Our data show that the largest complex formed with a single monoclonal antibody was that of an LDL dimer; no larger, nonspecific complexes were present. With two antibodies, a variety of complexes were seen. Thus, monoclonal antibodies MB47 and 4G3, which bound about 55 degrees apart, formed a very stable dimer. Monoclonal antibodies MB47 and 2D8, which bound 136 degrees apart, formed a very stable tetramer, with four LDLs held together in probably a circular structure with four monoclonal antibodies. Finally, monoclonal antibodies 2D8 and 1D1, which bound 86 degrees apart, probably formed a less stable LDL tetramer, held together by three to four monoclonal antibodies. A rationale for these structures is discussed, as well as the biological relevance of these complexes.

Individuals homozygous for the e2 allele encoding apolipoprotein E exhibit a remnant removal defect and accumulate substantial levels of intestinally derived particles containing apolipoprotein B-48 (apoB-48). Such lipoproteins were isolated from the plasma of E2/E2 individuals, and further purified by affinity chromatography using a polyclonal antibody specific for selective binding and removal of apoB-100-containing lipoproteins. The unbound lipoproteins, termed chylomicron remnants, were particles with average hydrated diameters of 31.2 nm as determined by dynamic light scattering. They contained apoB-48 and ApoE as their only protein components. The number of apoB-48 molecules on each lipoprotein was assessed by counting the number of antibody molecules bound to the surface of the chylomicron remnants, using either a monoclonal antibody specific for a single epitope on apoB-48 or a mixture of two such monoclonal antibodies specific for widely separated epitopes. The results of this analysis seem unambiguous: no more than one apoB-48 resides on the chylomicron remnant. Because apoB appears to be unable to transfer among lipoprotein particles, it may be inferred that nascent chylomicrons also contain a single copy of apoB-48.

Rare mutations in apolipoprotein B (apoB) can cause defective binding of low-density lipoproteins (LDLs) to the LDL receptor, leading to elevated plasma cholesterol levels and premature atherosclerosis. This communication describes a novel approach to study the effects of apoB mutations on LDL metabolism. Monoclonal antibody MB19 identifies a common polymorphism in apoB, an Ile/Thr substitution at residue 71, by binding with a 60-fold higher affinity to apoB(Ile71)-containing LDL. Because each LDL contains a single apoB, a maximum of two LDLs may be bound by the bivalent monoclonal antibody. Thus, at the appropriate concentration, an equivalent amount of MB19 will promote substantial dimer formation of LDL containing the strongly binding apoB(Ile71), but little dimer formation of LDL containing the weakly binding apoB(Thr71). For LDL isolated from heterozygous individuals, the amount of dimer formed, determined by dynamic light scattering, yields an estimate of the allelic ratio of the two forms of LDL. For such individuals, not only the effect of the polymorphism recognized by MB19 but also the effects of other polymorphisms on the LDL allelic ratio can be determined. Examination of six normolipemic MB19 heterozygotes gave percent allelic ratios between 48:52 and 51:49 tight:weak-binding LDL, not significantly different from a 50:50 ratio. These individuals were also heterozygous for six common apoB polymorphisms, allowing calculation of the odds that each of these polymorphisms caused significant alterations in lipid levels. In contrast, the rare mutation at residue 3500 causing defective binding to the LDL receptor and familial defective apoB100 (FDB) resulted in substantial changes (26:74 and 13:87) in LDL allelic ratio in both of two FDB individuals examined.

In this study, Xenopus laevis oocytes injected with poly(A)+ RNA (mRNA) isolated from human kidney were used to express a Na(+)-nucleoside cotransporter. Na(+)-stimulated [3H]thymidine uptake was enhanced 2-3-fold in oocytes injected with 50 ng poly(A)+ RNA and 4-5-fold in oocytes injected with 20 ng of a size-fractionated human renal cortex mRNA fragment (2-3 kb) in comparison with water-injected oocytes. Na(+)-dependent thymidine uptake in oocytes injected with the 2-3 kb mRNA fragment was inhibited significantly by thymidine and guanosine but not by formycin B, consistent with the N4 Na(+)-nucleoside cotransporter. The Km (28 microM) of Na(+)-dependent thymidine uptake in the oocytes injected with the 2-3 kb mRNA fragment was similar to the Km (27 microM) of Na(+)-dependent thymidine uptake obtained in human renal brush border membrane vesicles. These data suggest for the first time that a Na(+)-nucleoside cotransporter from human kidney can be expressed in X. laevis oocytes.

The Na+/nucleoside cotransporter in rabbit choroid plexus differs from Na+/nucleoside cotransporters in other tissues in terms of substrate selectivity and stoichiometry. The overall goal of this study was to further characterize the kinetics of this system (N3). Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP and treated with valinomycin and K+. Na+/thymidine uptake at 30 s in the presence of an inside negative potential difference was significantly greater than in the absence of a potential difference. Na+/thymidine uptake was not significantly affected by replacing chloride with either thiocyanate or sulfate. The Km of Na+/guanosine uptake was 149, 85.2 and 30.5 microM in the presence of a 25, 50 and 100 mM Na+ gradient, respectively, whereas the Vmax was unaffected, suggesting that Na+ binds first to the cotransporter, then, the nucleoside. Therapeutically relevant base-modified nucleoside analogs, 5-fluorouridine, 2-chloroadenosine and 5-iododeoxyuridine, significantly inhibited Na+/thymidine uptake with IC50 values (mean +/- S.E.) of 12.0 +/- 2.3, 21.3 +/- 2.2 and 24.4 +/- 2.1 microM, respectively, whereas nucleoside analogs structurally modified on the ribose ring, 3'-azidothymidine, dideoxyinosine and dideoxycytidine (100 microM) did not. These studies suggest that Na+/nucleoside cotransport in the choroid plexus is electrogenic and is not dependent on chloride. This cotransporter, which is present in choroid plexus but not in renal brush-border membrane vesicles from rabbit, may play a role in the disposition of clinically relevant base-modified nucleoside analogs into and out of the brain.