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Purpose: The goal of this study was to compare the difference in binocular visual function for high and low-moderate myopes before and after femtosecond laser-assisted in situ keratomileusis (FS-LASIK)., Methods: Thirty-three subjects (17 males and 16 females) were divided into two groups according to their preoperative refractive errors in spherical equivalent (SE): low-moderate myopia group (SE ≥-6.00 D) and high myopia group (SE <-6.00 D). The binocular visual function including accommodative amplitude (AA), accommodative facility (AF), positive and negative relative accommodation (PRA and NRA, respectively), horizontal phoria measurement, positive and negative fusion vergence, accommodative-convergence over accommodation (AC/A) ratio, and stereopsis were assessed with the best-corrected vision before patients received FS-LASIK and 7 and 30 days after the surgery. Repeated measures analysis of variance (ANOVA) was applied to study the change in binocular visual function., Results: The AF values in both groups were significantly reduced after 7 days of FS-LASIK (baseline vs. day 7 (mean): high myopia group: 7.85 vs. 5.62 cpm, repeated ANOVA, P = 0.01; low-moderate myopia group: 5.95 vs. 4.40 cpm, repeated ANOVA, P = 0.04). This change returned to the baseline level 30 days after the operation. In addition, the horizontal phoria values in both groups were significantly reduced for both distant (P = 0.019 and P = 0.001, respectively) and near (P = 0.003 and P = 0.049, respectively) 7 days after the operation, but they rebound to preoperative state after 30 days., Conclusion: A transient change in binocular visual function was noticed after 7 days of FS-LASIK operation, which could cause symptoms of asthenopia. Our data showed all the binocular visual functions returned to baseline level after 30 days of operation., Competing Interests: None

Background: MOXIe was a two-part study evaluating the safety and efficacy of omaveloxolone in patients with Friedreich's ataxia, a rare, progressive neurological disease with no proven therapy. MOXIe part 2, a randomized double-blind placebo-controlled trial, showed omaveloxolone significantly improved modified Friedreich's Ataxia Rating Scale (mFARS) scores relative to placebo. Patients who completed part 1 or 2 were eligible to receive omaveloxolone in an open-label extension study., Objective: The delayed-start study compared mFARS scores at the end of MOXIe part 2 with those at 72 weeks in the open-label extension period (up to 144 weeks) for patients initially randomized to omaveloxolone versus those initially randomized to placebo., Methods: We performed a noninferiority test to compare the difference between treatment groups (placebo to omaveloxolone versus omaveloxolone to omaveloxolone) using a single mixed model repeated measures (MMRM) model. In addition, slopes of the change in mFARS scores were compared between both groups in the open-label extension., Results: The noninferiority testing demonstrated that the difference in mFARS between omaveloxolone and placebo observed at the end of placebo-controlled MOXIe part 2 (-2.17 ± 1.09 points) was preserved after 72 weeks in the extension (-2.91 ± 1.44 points). In addition, patients previously randomized to omaveloxolone in MOXIe part 2 continued to show no worsening in mFARS relative to their extension baseline through 144 weeks., Conclusions: These results support the positive results of MOXIe part 2 and indicate a persistent benefit of omaveloxolone treatment on disease course in Friedreich's ataxia. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Background and Objectives: Alport syndrome is an inherited disease characterized by progressive loss of kidney function. We aimed to evaluate the safety and efficacy of bardoxolone methyl in patients with Alport syndrome., Design, Setting, Participants, & Measurements: We randomly assigned patients with Alport syndrome, ages 12-70 years and eGFR 30-90 ml/min per 1.73 m 2 , to bardoxolone methyl ( n =77) or placebo ( n =80). Primary efficacy end points were change from baseline in eGFR at weeks 48 and 100. Key secondary efficacy end points were change from baseline in eGFR at weeks 52 and 104, after an intended 4 weeks off treatment. Safety was assessed by monitoring for adverse events and change from baseline in vital signs, 12-lead electrocardiograms, laboratory measurements (including, but not limited to, aminotransferases, urinary albumin-creatinine ratio, magnesium, and B-type natriuretic peptide), and body weight., Results: Patients randomized to bardoxolone methyl experienced preservation in eGFR relative to placebo at 48 and 100 weeks (between-group differences: 9.2 [97.5% confidence interval, 5.1 to 13.4; P <0.001] and 7.4 [95% confidence interval, 3.1 to 11.7; P =0.0008] ml/min per 1.73 m 2 , respectively). After a 4-week off-treatment period, corresponding mean differences in eGFR were 5.4 (97.5% confidence interval, 1.8 to 9.1; P <0.001) and 4.4 (95% confidence interval, 0.7 to 8.1; P =0.02) ml/min per 1.73 m 2 at 52 and 104 weeks, respectively. In a post hoc analysis with no imputation of missing eGFR data, the difference at week 104 was not statistically significant (1.5 [95% confidence interval, -1.9 to 4.9] ml/min per 1.73 m 2 ). Discontinuations from treatment were more frequent among patients randomized to bardoxolone methyl; most discontinuations were due to protocol-specified criteria being met for increases in serum transaminases. Serious adverse events were more frequent among patients randomized to placebo. Three patients in each group developed kidney failure., Conclusions: In adolescent and adult patients with Alport syndrome receiving standard of care, treatment with bardoxolone methyl resulted in preservation in eGFR relative to placebo after a 2-year study period; off-treatment results using all available data were not significantly different., Clinical Trial Registry Name and Registration Number: A Phase 2/3 Trial of the Efficacy and Safety of Bardoxolone Methyl in Patients with Alport Syndrome - CARDINAL (CARDINAL), NCT03019185., (Copyright © 2022 by the American Society of Nephrology.)

Objective: Friedreich ataxia (FA) is a progressive genetic neurodegenerative disorder with no approved treatment. Omaveloxolone, an Nrf2 activator, improves mitochondrial function, restores redox balance, and reduces inflammation in models of FA. We investigated the safety and efficacy of omaveloxolone in patients with FA., Methods: We conducted an international, double-blind, randomized, placebo-controlled, parallel-group, registrational phase 2 trial at 11 institutions in the United States, Europe, and Australia (NCT02255435, EudraCT2015-002762-23). Eligible patients, 16 to 40 years of age with genetically confirmed FA and baseline modified Friedreich's Ataxia Rating Scale (mFARS) scores between 20 and 80, were randomized 1:1 to placebo or 150mg per day of omaveloxolone. The primary outcome was change from baseline in the mFARS score in those treated with omaveloxolone compared with those on placebo at 48 weeks., Results: One hundred fifty-five patients were screened, and 103 were randomly assigned to receive omaveloxolone (n = 51) or placebo (n = 52), with 40 omaveloxolone patients and 42 placebo patients analyzed in the full analysis set. Changes from baseline in mFARS scores in omaveloxolone (-1.55 ± 0.69) and placebo (0.85 ± 0.64) patients showed a difference between treatment groups of -2.40 ± 0.96 (p = 0.014). Transient reversible increases in aminotransferase levels were observed with omaveloxolone without increases in total bilirubin or other signs of liver injury. Headache, nausea, and fatigue were also more common among patients receiving omaveloxolone., Interpretation: In the MOXIe trial, omaveloxolone significantly improved neurological function compared to placebo and was generally safe and well tolerated. It represents a potential therapeutic agent in FA. ANN NEUROL 2021;89:212-225., (© 2020 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

In a multinational placebo-controlled phase III clinical trial in 2,185 patients with type 2 diabetes mellitus and stage 4 chronic kidney disease, treatment with the Nrf2 activator bardoxolone methyl increased estimated glomerular filtration rate, a measure of kidney function, but also resulted in increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase. These increases in liver enzyme level(s) were maximal after 4 weeks of treatment and reversible, trending back toward baseline through week 48. Total bilirubin concentrations did not increase, and no cases met Hy's Law criteria, although two subjects had ALT concentrations that exceeded 10 × the upper limit of the population reference range leading to discontinuation of treatment. Animal and cell culture experiments suggested that the increases in ALT and AST induced by bardoxolone methyl may be related to its pharmacological activity. Bardoxolone methyl significantly induced the mRNA expression of ALT and AST isoforms in cultured cells. Expression of ALT and AST isoforms in liver and kidney also positively correlated with Nrf2 status in mice. Overall, these data suggest that the increases in ALT and AST observed clinically were, at least in part, related to the pharmacological induction of aminotransferases via Nrf2 activation, rather than to any intrinsic form of hepatotoxicity., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Introduction: Alport syndrome is a rare genetic disorder that affects as many as 60,000 persons in the USA and a total of 103,000 persons (<5 per 10,000) in the European Union [1, 2]. It is the second most common inherited cause of kidney failure and is characterized by progressive loss of kidney function that often leads to end-stage kidney disease. Currently, there are no approved disease-specific agents for therapeutic use. We designed a phase 3 study (CARDINAL; NCT03019185) to evaluate the safety, tolerability, and efficacy of bardoxolone methyl in patients with Alport syndrome., Methods: The CARDINAL phase 3 study is an international, multicenter, double-blind, placebo-controlled, randomized registrational trial. Eligible patients were of ages 12-70 years with confirmed genetic or histologic diagnosis of Alport syndrome, eGFR 30-90 mL/min/1.73 m2, and urinary albumin to creatinine ratio (UACR) ≤3,500 mg/g. Patients with B-type natriuretic peptide values >200 pg/mL at baseline or with significant cardiovascular histories were excluded. Patients were randomized 1:1 to bardoxolone methyl or placebo, with stratification by baseline UACR., Results: A total of 371 patients were screened, and 157 patients were randomly assigned to receive bardoxolone methyl (n = 77) or placebo (n = 80). The average age at screening was 39.2 years, and 23 (15%) were <18 years of age. Of the randomized population, 146 (93%) had confirmed genetic diagnosis of Alport syndrome, and 62% of patients had X-linked mode of inheritance. Mean baseline eGFR was 62.7 mL/min/1.73 m2, and the geometric mean UACR was 141.0 mg/g. The average annual rate of eGFR decline prior to enrollment in the study was -4.9 mL/min/1.73 m2 despite 78% of the patient population receiving ACE inhibitor (ACEi) or ARB therapy., Discussion/conclusion: CARDINAL is one of the largest interventional, randomized controlled trials in Alport syndrome conducted to date. Despite the use of ACEi or ARB, patients were experiencing significant loss of kidney function prior to study entry., (© 2021 The Author(s). Published by S. Karger AG, Basel.)

Bardoxolone methyl attenuates inflammation by inducing nuclear factor erythroid-derived 2-related factor 2 and suppressing nuclear factor κB. The Bardoxolone Methyl Evaluation in Patients With Chronic Kidney Disease and Type 2 Diabetes (BEACON) trial was a phase 3 placebo-controlled, randomized, double-blind, parallel-group, international, multicenter trial in 2185 patients with type 2 diabetes mellitus and stage 4 chronic kidney disease. BEACON was terminated because of safety concerns, largely related to a significant increase in early heart failure events in patients randomized to bardoxolone methyl. Bardoxolone methyl resulted in increased estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio. Herein, we present post hoc analyses characterizing the relation between the urine albumin-to-creatinine ratio and eGFR. The urine albumin-to-creatinine ratio and eGFR were assessed every four weeks through Week 12, followed by assessments every eight weeks thereafter, and 4 weeks after the last dose of bardoxolone methyl was administered. The initial increases in urine albumin-to-creatinine ratio observed in patients randomized to bardoxolone methyl were attenuated after six months. Multivariable regression analysis identified baseline eGFR and eGFR over time as the dominant factors associated with change in the urine albumin-to-creatinine ratio. Relative to placebo, bardoxolone methyl resulted in a significant decrease in albuminuria when indexed to eGFR (least-squared means: -0.035 [95% confidence interval -0.031 to -0.039]). Thus, among patients with type 2 diabetes mellitus and stage 4 chronic kidney disease treated with bardoxolone methyl, changes in albuminuria are directly related to changes in eGFR, challenging the conventional construct that increases in albuminuria universally reflect kidney injury and denote harm., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Treatment with bardoxolone methyl (Bard) in a multinational phase 3 trial, Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes (BEACON), resulted in increases in estimated glomerular filtration rate with concurrent reductions in serum magnesium. We analyzed data from several trials to characterize reductions in magnesium with Bard., Methods: BEACON randomized patients (n = 2,185) with type 2 diabetes (T2DM) and stage 4 chronic kidney disease (CKD) 1:1 to receive Bard (20 mg) or placebo once daily. In a separate open-label study, magnesium levels from 24-hour urine and sublingual epithelial cell samples were analyzed in patients with stage 3b-4 CKD and T2DM administered 20 mg Bard for 56 consecutive days., Results: BEACON patients randomized to Bard experienced significant reductions in serum magnesium from baseline relative to patients randomized to placebo (-0.17 mEq/L, 95% CI -0.18 to -0.60 mEq/L; p < 0.001). A separate study showed intracellular and urinary magnesium levels were unchanged with Bard treatment., Conclusions: Bard treatment results in significant decreases in serum magnesium that are not associated with changes in intracellular and urinary magnesium levels, indicating that magnesium decreases are not due to renal magnesium wasting or total body magnesium depletion. Importantly, the decreases in serum magnesium with Bard are not associated with adverse effects on QT interval., (The Author(s). Published by S. Karger AG, Basel.)

Background: Bardoxolone methyl has been shown to increase eGFR in several clinical trials, including a phase 3 trial in patients with type 2 diabetes and stage 4 CKD (BEACON), which was terminated early due to an increase in heart failure events in bardoxolone methyl-treated patients. A separate, "thorough QT" study was conducted in parallel with BEACON to evaluate the cardiovascular safety of bardoxolone methyl in healthy subjects., Methods: Subjects in the "thorough QT" study were randomized to receive bardoxolone methyl 20 mg (therapeutic dose) or 80 mg (supratherapeutic dose), placebo, or moxifloxacin (400 mg; an active comparator). ECG results and supine blood pressure measurements were analyzed. The effects of bardoxolone methyl on QT interval changes from baseline were quantified compared to the effect of placebo by calculating mean, time-matched, placebo-corrected, baseline-adjusted QTcF values (ΔΔQTcF) after 6 days of daily administration of bardoxolone methyl., Results: The study was halted early due to emerging safety information from the BEACON trial; however, 142/179 patients received all doses of the study drug and completed the study. For both bardoxolone methyl-treated groups (20 and 80 mg), the upper limits of the 2-sided 90% confidence interval for ΔΔQTcF were less than the significance limit (10 ms) at all time points. Changes in blood pressure were similar in all treatment groups, and no serious adverse events were reported., Conclusions: In healthy subjects, treatment with 20 or 80 mg bardoxolone methyl did not affect the QTcF interval., (The Author(s). Published by S. Karger AG, Basel.)

Aims: Obesity is associated with progression of chronic kidney disease (CKD). Treatment with bardoxolone methyl in a multinational phase 3 trial, Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes (BEACON), resulted in increases in estimated glomerular filtration rate (eGFR) with concurrent reductions in body weight. We performed post-hoc analyses to further characterize reductions in body weight with bardoxolone methyl., Methods: Eligible patients with type 2 diabetes (T2DM) and CKD stage 4 (eGFR 15 to <30 mL/min/1.73 m 2 ) were randomized 1:1 to receive once-daily oral dose of bardoxolone methyl (20 mg) or placebo., Results: BEACON enrolled 2185 patients. Patients randomized to bardoxolone methyl experienced significant reductions in body weight from baseline relative to patients randomized to placebo (-5.7 kg; 95% CI: -6.0 to -5.3 kg; p < 0.001). In patients randomized to bardoxolone methyl, rate and magnitude of body weight loss were proportional to baseline BMI. Bardoxolone methyl resulted in significant reductions in waist circumference and improved glycemic control., Conclusions: Bardoxolone methyl resulted in significant weight loss in a generally obese patient population with T2DM and stage 4 CKD, with the magnitude and rate dependent on baseline BMI., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Increases in measured inulin clearance, measured creatinine clearance, and estimated glomerular filtration rate (eGFR) have been observed with bardoxolone methyl in 7 studies enrolling approximately 2,600 patients with type 2 diabetes (T2D) and chronic kidney disease (CKD). The largest of these studies was Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes (BEACON), a multinational, randomized, double-blind, placebo-controlled phase 3 trial which enrolled patients with T2D and CKD stage 4. The BEACON trial was terminated after preliminary analyses showed that patients randomized to bardoxolone methyl experienced significantly higher rates of heart failure events. We performed post-hoc analyses to characterize changes in kidney function induced by bardoxolone methyl., Methods: Patients in -BEACON (n = 2,185) were randomized 1: 1 to receive once-daily bardoxolone methyl (20 mg) or placebo. We compared the effects of bardoxolone methyl and placebo on a post-hoc composite renal endpoint consisting of ≥30% decline from baseline in eGFR, eGFR <15 mL/min/1.73 m2, and end-stage renal disease (ESRD) events (provision of dialysis or kidney transplantation)., Results: Consistent with prior studies, patients randomized to bardoxolone methyl experienced mean increases in eGFR that were sustained through study week 48. Moreover, increases in eGFR from baseline were sustained 4 weeks after cessation of treatment. Patients randomized to bardoxolone methyl were significantly less likely to experience the composite renal endpoint (hazards ratio 0.48 [95% CI 0.36-0.64]; p < 0.0001)., Conclusions: Bardoxolone methyl preserves kidney function and may delay the onset of ESRD in patients with T2D and stage 4 CKD., (© 2018 The Author(s) Published by S. Karger AG, Basel.)

Purpose: People in Hong Kong generally live in a densely populated area and their homes are smaller compared with most other cities worldwide. Interestingly, East Asian cities with high population densities seem to have higher myopia prevalence, but the association between them has not been established. This study investigated whether the crowded habitat in Hong Kong is associated with refractive error among children., Methods: In total, 1075 subjects [Mean age (S.D.): 9.95 years (0.97), 586 boys] were recruited. Information such as demographics, living environment, parental education and ocular status were collected using parental questionnaires. The ocular axial length and refractive status of all subjects were measured by qualified personnel., Results: Ocular axial length was found to be significantly longer among those living in districts with a higher population density (F 2,1072  = 6.15, p = 0.002) and those living in a smaller home (F 2,1072  = 3.16, p = 0.04). Axial lengths were the same among different types of housing (F 3,1071  = 1.24, p = 0.29). Non-cycloplegic autorefraction suggested a more negative refractive error in those living in districts with a higher population density (F 2,1072  = 7.88, p < 0.001) and those living in a smaller home (F 2,1072  = 4.25, p = 0.02). After adjustment for other confounding covariates, the population density and home size also significantly predicted axial length and non-cycloplegic refractive error in the multiple linear regression model, while axial length and refractive error had no relationship with types of housing., Conclusions: Axial length in children and childhood refractive error were associated with high population density and small home size. A constricted living space may be an environmental threat for myopia development in children., (© 2017 The Authors Ophthalmic & Physiological Optics © 2017 The College of Optometrists.)

Adeno-associated virus (AAV) vectors have emerged as a safe and efficient gene therapy platform. One complication is that a significant amount of empty particles have always been generated as impurities during AAV vector production. However, the effects of such particles on AAV vector performance remain unclear. Here we systemically evaluated the biological properties of three types of "empty" AAV particles: syngeneic pseudo-vectors with partial AAV genomes derived from DNA of the corresponding full particles, allogeneic pseudo-vectors with partial genomes different from the corresponding full particles, and null pseudo-vectors with no DNA inside the capsids. The syngeneic particles in excess increased the corresponding full AAV vector transgene expression both in vivo and in vitro. However, such effects were not observed with null or allogeneic particles. The observed differences among these pseudo-AAV particles may be ascribed to the syngeneic pseudo-vector DNA facilitating the complementary DNA synthesis of the corresponding full AAV particles. Our study suggests that the DNA content in the pseudo-vectors plays a key role in dictating their effects on AAV transduction. The effects of residual "empty" particles should be adequately assessed when comparing AAV vector performance. The syngeneic AAV pseudo-vectors may be used to enhance the efficacy of gene therapy.

Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed, heavier particles found in rAAV preparations have traditionally been ignored due to their reported low in vitro transduction efficiency. In this study, the biological properties of regular and high-density rAAV serotype 8 vectors, rAAV RD and rAAV HD , were systemically compared. Results demonstrated that both rAAV RD and rAAV HD exhibited similar DNA packaging profiles, while rAAV HD capsids contained fewer VP1 and VP2 proteins, indicating that the rAAV HD particles contained a higher DNA/protein ratio than that of rAAV RD particles. Dynamic light scattering and transmission electron microscopy data revealed that the diameter of rAAV HD was smaller than that of rAAV RD . In vitro, rAAV HD was two- to fourfold less efficient in transduction compared with rAAV RD . However, the transduction performance of rAAV HD and rAAV RD was similar in vivo. No significant difference in neutralizing antibody formation against rAAV RD and rAAV HD was observed, suggesting that the surface epitopes of rAAV RD and rAAV HD are congruent. In summary, the results of this study demonstrate that rAAV RD and rAAV HD are equally competent for in vivo transduction, despite their difference in vitro. Therefore, the use of rAAV HD vectors in human gene therapy should be further evaluated., Competing Interests: Author Disclosure No competing financial interests exist.

The canine is the most important large animal model for testing novel hemophilia A (HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, different biological properties between cFVIII and human FVIII (hFVIII) indicated that the development of novel HA treatment may require careful characterization of non-human FVIII. To investigate whether the data obtained using cFVIII can translate to HA treatment in human, we analyzed the differential biological properties of canine heavy chain (cHC) and light chain (cLC) by comparing with human heavy chain (hHC) and light chain (hLC). The secretion of cHC was 5-30-fold higher than hHC, with or without light chains (LCs). cHC+hLC group exhibited ~18-fold increase in coagulation activity compared with hHC+hLC delivery by recombinant adeno-associated viral vectors. Unlike hHC, the secretion of cHC was independent of LCs. cLC improves the specific activity of FVIII by two- to threefold compared with hLC. Moreover, the cLC, but not cHC, contributes to the higher stability of cFVIII. Our results suggested that the cFVIII expression results in the canine model should be interpreted with caution as the cHC secreted more efficiently than hHC and cLC exhibited a more active and stable phenotype than hLC.

Monte Carlo (MC) codes are increasingly spreading in the hadrontherapy community due to their detailed description of radiation transport and interaction with matter. The suitability of a MC code for application to hadrontherapy demands accurate and reliable physical models capable of handling all components of the expected radiation field. This becomes extremely important for correctly performing not only physical but also biologically based dose calculations, especially in cases where ions heavier than protons are involved. In addition, accurate prediction of emerging secondary radiation is of utmost importance in innovative areas of research aiming at in vivo treatment verification. This contribution will address the recent developments of the FLUKA MC code and its practical applications in this field. Refinements of the FLUKA nuclear models in the therapeutic energy interval lead to an improved description of the mixed radiation field as shown in the presented benchmarks against experimental data with both (4)He and (12)C ion beams. Accurate description of ionization energy losses and of particle scattering and interactions lead to the excellent agreement of calculated depth-dose profiles with those measured at leading European hadron therapy centers, both with proton and ion beams. In order to support the application of FLUKA in hospital-based environments, Flair, the FLUKA graphical interface, has been enhanced with the capability of translating CT DICOM images into voxel-based computational phantoms in a fast and well-structured way. The interface is capable of importing also radiotherapy treatment data described in DICOM RT standard. In addition, the interface is equipped with an intuitive PET scanner geometry generator and automatic recording of coincidence events. Clinically, similar cases will be presented both in terms of absorbed dose and biological dose calculations describing the various available features.

Background: There is currently limited information about ethnic differences in myopia prevalence within mainland China, especially in rural or semi-rural areas. We examined the prevalence of refractive errors, visual impairment and spectacle coverage in school children of varying ethnicity in Turpan, Xinjiang province., Methods: A community eye care service was provided for five schools. Presenting monocular distance and near visual acuity (VA), and ocular alignment were assessed. Retinoscopy and cycloplegic subjective refraction were performed for participants with presenting visual impairment (distance VA worse than 0.3 logMAR; Snellen 6/12 or 20/40) or abnormal binocular vision. Questionnaires administered prior to the eye examinations were used to collect information regarding personal lifestyle and parental myopia., Results: A total of 646 out of 690 (94%) subjects aged four to 19 years (11.9 ± 2.6; mean ± S.D.) completed the eye examination. Three hundred and eighty-two (59%) of participants were of Uyghur ethnicity, followed by Han, 176 (27%) and Hui, 74 (12%). The mean age of Uyghur, Han and Hui students was 12.3 ± 2.7, 11.4 ± 2.6 and 11.4 ± 2.3 years respectively, in which the Uyghur students were significantly older than the Han and Hui students (F(3,631) = 5.58 p < 0.001). In total, 170 (27%) and 85 (13%) subjects failed the screening examination for one eye or both eyes, respectively. The prevalence of presenting visual impairment was not significantly different among the ethnic groups (p = 0.26). After cycloplegic refraction, most subjects' VA (98%) improved to better than 0.3 logMAR (Snellen 6/12 or 20/40). The prevalence of "clinically-significant myopia" (≤-0.50 dioptres) was 27%, 18% and 13% in Han, Hui and Uyghur children, respectively (p < 0.001). In contrast, Uyghur students had the highest prevalence of astigmatism (Uyghur 12%, Han 5%, Hui 4%). The overall spectacle coverage was 36%, while spectacle coverage among ethnic groups were similar (Han, 41%; Uyghur, 32%; Hui, 41%; χ(2) = 2.23, df = 2, p = 0.33)., Conclusion: The prevalence of clinically significant myopia varied markedly with ethnicity in school children sampled from a semi-rural region of mainland China (Han > Hui > Uyghur). As reported previously, uncorrected/under-corrected refractive error was the main cause of presenting visual impairment., (© 2015 The Authors Ophthalmic & Physiological Optics © 2015 The College of Optometrists.)

The electrical response of the retina was examined as a function of retinal region, using stimuli of various spatial frequencies in the first experiment. In the second experiment, the regional response of the retina to defocus at high and low spatial frequencies was investigated. Twenty three subjects were recruited for global flash multifocal electroretinogram (mfERG) in experiment 1. Black and white gratings (printed on plastic transparent sheets) of four spatial frequencies (SF), 0.24, 1.2, 2.4 and 4.8 cycle per degree were presented in front of the mfERG stimulation. The amplitudes and implicit times of the direct (DC) and induced (IC) components of mfERG responses were pooled into six concentric rings for analysis. There was low amplitude DC at low SF, which increased with increasing SF, and which decreased with increasing eccentricity. The IC was high in amplitude at all SF and reduced in amplitude with increasing eccentricity. Our findings suggested that outer and inner retina had different characteristics in processing spatial details. In experiment 2, Twenty-three young adults were recruited for mfERG measurement. The retinal electrical responses for low (0.24cpd) and high (4.8cpd) SF under fully corrected conditions of short-term negative defocus (-2D) and short term positive defocus (+2D) conditions were measured. There was a sign-dependent response to defocus in the DC response, mainly in peripheral regions. The sign dependent response at low SF was more obvious than that at high SF, and was located more peripherally. The IC response showed no clear trends for either defocus condition. The human retina seems to have a decoding system for optical defocus, which was tuned for low spatial frequency, and was located in the retinal near periphery.

Background: A phase 3 randomized clinical trial was designed to test whether bardoxolone methyl, a nuclear factor erythroid-2-related factor 2 (Nrf2) activator, slows progression to end-stage renal disease in patients with stage 4 chronic kidney disease and type 2 diabetes mellitus. The trial was terminated because of an increase in heart failure in the bardoxolone methyl group; many of the events were clinically associated with fluid retention., Methods and Results: We randomized 2,185 patients with type 2 diabetes mellitus (T2DM) and stage 4 chronic kidney disease (CKD) (estimated glomerular filtration rate 15 to <30 mL min(-1) 1.73 m(-2)) to once-daily bardoxolone methyl (20 mg) or placebo. We used classification and regression tree analysis to identify baseline factors predictive of heart failure or fluid overload events. Elevated baseline B-type natriuretic peptide and previous hospitalization for heart failure were identified as predictors of heart failure events; bardoxolone methyl increased the risk of heart failure by 60% in patients with these risk factors. For patients without these baseline characteristics, the risk for heart failure events among bardoxolone methyl- and placebo-treated patients was similar (2%). The same risk factors were also identified as predictors of fluid overload and appeared to be related to other serious adverse events., Conclusions: Bardoxolone methyl contributed to events related to heart failure and/or fluid overload in a subpopulation of susceptible patients with an increased risk for heart failure at baseline. Careful selection of participants and vigilant monitoring of the study drug will be required in any future trials of bardoxolone methyl to mitigate the risk of heart failure and other serious adverse events., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Severe genetic bottleneck occurs during HIV-1 sexual transmission whereby most infections are initiated by a single transmitted/founder (T/F) virus. Similar observations had been made in nonhuman primates exposed mucosally to SIV/SHIV. We previously reported variable clinical outcome in rhesus macaques inoculated intravaginally (ivg) with a high dose of R5 SHIVSF162P3N. Given the potential contributions of viral diversity to HIV-1 persistence and AIDS pathogenesis and recombination between retroviral genomes increases the genetic diversity, we tested the hypothesis that transmission of multiple variants contributes to heightened levels of virus replication and faster disease progression in the SHIVSF162P3N ivg-infected monkeys., Results: We found that the differences in viral replication and disease progression between the transiently viremic (TV; n = 2), chronically-infected (CP; n = 8) and rapid progressor (RP; n = 4) ivg-infected macaques cannot be explained by which variant in the inoculum was infecting the animal. Rather, transmission of a single variant was observed in both TV rhesus, with 1-2 T/F viruses found in the CPs and 2-4 in all four RP macaques. Moreover, the genetic relatedness of the T/F viruses in the CP monkeys with multivariant transmission was greater than that seen in the RPs. Biological characterization of a subset of T/F envelopes from chronic and rapid progressors revealed differences in their ability to mediate entry into monocyte-derived macrophages, with enhanced macrophage tropism observed in the former as compared to the latter., Conclusion: Our study supports the tenet that sequence diversity of the infecting virus contributes to higher steady-state levels of HIV-1 virus replication and faster disease progression and highlights the role of macrophage tropism in HIV-1 transmission and persistence.

Background: Bardoxolone methyl, an Nrf2-activating and nuclear factor-κB-inhibiting semisynthetic oleanane triterpenoid compound, was evaluated in a phase 3 trial (BEACON) in patients with type 2 diabetes mellitus (T2DM) and stage 4 chronic kidney disease (CKD). The trial was terminated because of an increase in heart failure events in the bardoxolone methyl group, many of which appeared related to fluid retention. Thus, additional analyses were conducted to explain these serious adverse events., Methods: Patients (n = 2,185) were randomized to receive once-daily bardoxolone methyl (20 mg) or placebo. Twenty-four-hour urine collections were analyzed in a subset of the BEACON population and from a separate, open-label pharmacology study in patients with stage 3b/4 CKD and T2DM administered 20 mg bardoxolone methyl once daily for 56 consecutive days., Results: Bardoxolone-methyl-treated patients in the BEACON substudy had a clinically meaningful reduction in urine volume and sodium excretion at week 4 relative to baseline (p < 0.05), and a separate study revealed that decreased sodium excretion and urine output occurred in some patients with stage 4 CKD but not those with stage 3b CKD. The clinical phenotype of fluid overload and heart failure in BEACON was similar to that observed with endothelin receptor antagonists in advanced CKD patients, and preclinical data demonstrate that bardoxolone methyl modifies endothelin signaling., Conclusions: The totality of the evidence suggests that through modulation of the endothelin pathway, bardoxolone methyl may pharmacologically promote acute sodium and volume retention and increase blood pressure in patients with more advanced CKD.

Lycium Barbarum Polysaccharides (LBP) are the active components of Wolfberry (a traditional Chinese medicine) which has long been used for improving visual function. This study aims to investigate localized changes of retinal function in a partial optic nerve transection (PONT) model, and effects of LBP on visual function. The multifocal electroretinograms (mfERG) were obtained from 30 eyes of 30 Sprague-Dawley rats. The rats were divided into 6 groups (five treatment groups and one control group). Starting from the first day of the experiment, the rats in the (PONT+LBP) group and the (LBP) group were dosed with LBP; rats in the (PONT+PBS (phosphate buffered saline)) group and the (PBS) group were dosed with PBS via nasogastric tube every day until euthanized. The dorsal part of the optic nerve was transected in the (PONT), (PONT+LBP) and (PONT+PBS) groups at the end of week 1 (day 7 after LBP or PBS feeding began). The mfERG was measured at three time points: week 2, week 3 and week 5. Significant reduction of P1 and PhNR amplitudes of the mfERG were observed in all retinal regions a week after PONT. Feeding with LBP prior to PONT preserved retinal function. All mfERG responses returned to the normal range in the superior retina, which corresponds to the transected dorsal region of the optic nerve, while most of the inferior retinal responses were significantly increased at week 4 after PONT. The ventral part of the retina had secondary degeneration which was not only limited to the ganglion cell layer, but is a widespread effect affecting the outer retina. LBP altered the functional reduction caused by PONT by regulating the signal from the outer retina.

Monte Carlo simulations play a crucial role for in-vivo treatment monitoring based on PET and prompt gamma imaging in proton and carbon-ion therapies. The accuracy of the nuclear fragmentation models implemented in these codes might affect the quality of the treatment verification. In this paper, we investigate the nuclear models implemented in GATE/Geant4 and FLUKA by comparing the angular and energy distributions of secondary particles exiting a homogeneous target of PMMA. Comparison results were restricted to fragmentation of (16)O and (12)C. Despite the very simple target and set-up, substantial discrepancies were observed between the two codes. For instance, the number of high energy (>1 MeV) prompt gammas exiting the target was about twice as large with GATE/Geant4 than with FLUKA both for proton and carbon ion beams. Such differences were not observed for the predicted annihilation photon production yields, for which ratios of 1.09 and 1.20 were obtained between GATE and FLUKA for the proton beam and the carbon ion beam, respectively. For neutrons and protons, discrepancies from 14% (exiting protons-carbon ion beam) to 57% (exiting neutrons-proton beam) have been identified in production yields as well as in the energy spectra for neutrons.

Background: HIV-1 subtype B and subtype F are prevalent in the AIDS epidemic of Brazil. Recombinations between these subtypes have generated at least four BF circulating recombinant forms (CRFs). CRF28&#95;BF and CRF29&#95;BF are among the first two BF recombinants being identified in Brazil and they contributed significantly to the epidemic. However, the evolution and demographic histories of the CRFs are unclear., Methodology/principal Findings: A collection of gag and pol sequences sampled within Brazil was screened for CRF28&#95;BF-like and CRF29&#95;BF-like recombination patterns. A Bayesian coalescent framework was employed to delineate the phylogenetic, divergence time and population dynamics of the virus having CRF28&#95;BF-like and CRF29&#95;BF-like genotype. These recombinants were phylogenetically related to each other and formed a well-supported monophyletic clade dated to 1988-1989. The effective number of infections by these recombinants grew exponentially over a five-year period after their emergence, but then decreased toward the present following a logistic model of population growth. The demographic pattern of both recombinants closely resembles those previously reported for CRF31&#95;BC., Conclusions: We revealed that HIV-1 recombinants of the CRF28&#95;BF/CRF29&#95;BF clade are still circulating in the Brazilian population. These recombinants did not exhibit a strong founder effect and showed a decreasing prevalence in the AIDS epidemic of Brazil. Our data suggested that multiple URFs may also play a role in shaping the epidemic of recombinant BF HIV-1 in the region.

The presence of iNOS and nitrotyrosine in cutaneous melanomas has been correlated with poor survival rates of patients, suggesting that NO plays a role in the tumor pathophysiology. However, the concentrations of NO that melanoma cells are exposed to in vivo have been unknown. To provide cell kinetic data for use in predicting those concentrations, synthesis and consumption of NO was examined in A375 melanoma cells. Nitric oxide synthesis was undetectable. The rate of intracellular NO consumption was determined by continuous monitoring of NO concentrations following injection of NO solutions in a closed chamber. After correcting for autoxidation and consumption from media-generated O(2)(-), the rate constant obtained for cellular consumption was 7.1±1.1 s(-1). This information was combined with previous data on macrophage NO kinetics to develop a mathematical model to predict NO levels in cutaneous melanomas. Synthesis of NO by macrophages in the stroma was found to give a maximum concentration at the tumor periphery of 0.2 μM. Because of the high rates of cellular consumption, the elevation in NO concentration is predicted to be very localized, approximately 90% of the concentration decay occurring within 30 μm of the tumor edge. High NO concentrations at the periphery of a melanoma may contribute to metastasis by stimulating cell proliferation, inhibiting apoptosis, or acting as a lymphangiogenic factor., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1) sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants., Results: HIV-1 lacking SL1 (NLΔSL1) did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K) on day 18 postinfection and C1907T in the SP1 domain (P10L) on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1., Conclusions: HIV-1 evolved to overcome a deletion in SL1 and restored infectivity by acquiring compensatory mutations in the N-terminal matrix or SP1 domain of Gag. These data shed light on the functions of the N-terminal matrix and SP1 domains and suggest that both regions may have a role in Gag interactions with spliced viral RNA.

High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B/B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly ( approximately 30%) less frequently in B/F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had complex patterns, and the crossover junctions were distributed throughout the pol gene. In contrast, selected B/F recombinants had limited recombination patterns and restricted crossover junction distribution. These results provide evidence for the evolved coadapted sites in variants from different subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses.

Knowledge of the rates at which macrophages and epithelial cells synthesize NO is critical for predicting the concentrations of NO and other reactive nitrogen species in colonic crypts during inflammation, and elucidating the linkage between inflammatory bowel disease, NO, and cancer. Macrophage-like RAW264.7 cells, primary bone marrow-derived macrophages (BMDM), and HCT116 colonic epithelial cells were subjected to simulated inflammatory conditions, and rates of formation and consumption were determined for NO, O(2), and O(2)(-). Production rates of NO were determined in either of two ways: continuous monitoring of NO concentrations in a closed chamber with corrections for autoxidation, or NO(2)(-) accumulation measurements in an open system with corrections for diffusional losses of NO. The results obtained using the two methods were in excellent agreement. Rates of NO synthesis (2.3 +/- 0.6 pmol s(-1) 10(6) cells(-1)), NO consumption (1.3 +/- 0.3 s(-1)), and O(2) consumption (59 +/- 17 pmol s(-1) 10(6) cells(-1) when NO is negligible) for activated BMDM were indistinguishable from those of activated RAW264.7 cells. NO production rates calculated from NO(2)(-) accumulation data for HCT116 cells infected with Helicobacter cinaedi (3.9 +/- 0.1 pmol s(-1) 10(6) cells(-1)) were somewhat greater than those of RAW264.7 macrophages infected under similar conditions (2.6 +/- 0.1 pmol s(-1) 10(6) cells(-1)). Thus, RAW264.7 cells have NO kinetics nearly identical to those of primary macrophages, and stimulated epithelial cells are capable of synthesizing NO at rates comparable to those of macrophages. Using these cellular kinetic parameters, simulations of NO diffusion and reaction in a colonic crypt during inflammation predict maximum NO concentrations of about 0.2 microM at the base of a crypt.

The collision type central to BNCT is (10)B(n, alpha)(7)Li, however, other types of nuclear reactions also take place in the patient. In addition to the major elements (H, C, N, O), minor elements such as Na, Mg, P, S, Cl, K, Ca and Fe present in body tissues also interact in neutron collisions. Detailed accounting of the above not only provides a better understanding of radiation transport in the human body during BNCT, but such knowledge affects the design of the facility, as well as treatment planning, imaging and verification for a given BNCT agent. Of the methods of investigation currently available, only Monte Carlo simulation could provide the detailed accounting and breakdown of the quantities required. We report Monte Carlo simulation of an anthropomorphic voxel phantom, the VIP-Man and show how these quantities change with different (10)B concentrations in the tumour, the blood and the remaining tissues. The (10)B biodistribution has been chosen to be the variable of interest, since it is not accurately known, is frequently approximated and is a crucial quantity upon which dose calculations are based.

We investigated Geant4 Monte Carlo simulation of protons interactions in (18)O and (14)N. With proton incident energies of 10 and 30 MeV, we compared the different Geant4 models available for proton inelastic collisions: the low-energy parameterisation, Bertini cascade and precompound models. The models produced different (1) combinations of secondary particles and (2) final states of secondary particles. The non-convergence suggests that the hadronic models, yet to be benchmarked for this energy range, are not ready for production-mode problem-solving.

The generation of genetic diversity is a fundamental characteristic of HIV-1 replication, allowing the virus to successfully evade the immune response and antiviral therapies. Although mutations are the first step towards diversity, mixing of the mutations through the process of recombination increases the variation and allows for the faster establishment of advantageous strains within the viral population. Therefore, studying recombination of HIV-1 provides insights into not only the mechanisms of HIV-1 replication but also into the potential for spread of antiviral drug resistance mutations within and across viral subtypes. This chapter describes, in detail, a highly sensitive recombination assay designed to measure the frequency of recombination between two viruses. This assay allows us to investigate the requirements, mechanisms, and final products of recombination. Additionally, software-based phylogenetic tools are described in this chapter, which allow for the identification of specific recombination events within patient samples or viral progeny from the recombination assay.

Nitric oxide production in the colon has been linked to inflammatory bowel disease (IBD) and increased risk for colon cancer. However, measurements of NO concentration in the inflamed colon have not been available and it is not known what NO levels are pathophysiological. A computational model, based on anatomical length scales and rates of NO production measured in cell cultures, was used to predict spatially varying NO concentrations within a colonic crypt under inflammatory conditions. A variety of scenarios were considered, including different spatial distributions of macrophages and a range of possible macrophage and epithelial synthesis rates for NO. Activated macrophages arranged as a monolayer at the base of the crypt elicited maximum NO concentrations of approximately 0.3 microM. The epithelial contribution to NO synthesis was calculated to be negligible. Assuming a uniform macrophage layer, NO synthesis rates greater than 20 microM/s, or more than three times that measured in vitro, would be necessary to achieve maximum NO concentrations of 1 microM in the crypt. Thus, unless NO synthesis rates in macrophages and/or epithelial cells greatly exceed those measured in cell cultures, NO concentrations will remain submicromolar in the crypt during inflammation. Additionally, the results were used to predict the range of NO concentrations (<0.3 microM) and cumulative NO dose (560 microM min) experienced by a given epithelial cell migrating from the base to the top of the crypt. These estimates of NO concentrations in inflamed crypts should facilitate efforts to elucidate the molecular biological linkage between NO exposure and carcinogenesis in IBD.

HIV-1 intersubtype recombinants have an increasingly important role in shaping the AIDS pandemic. We sought to understand the molecular mechanisms that generate intersubtype HIV-1 recombinants. We analyzed recombinants of HIV-1 subtypes B and C, and identified their crossover junctions in the viral genome from the 5' long terminal repeat (LTR) to the end of pol. We identified 56 recombination events in 56 proviruses; the distribution of these events indicated an apparent recombination gradient: there were significantly more crossover junctions in the 3' half than in the 5' half of the region analyzed. HIV-1 subtypes B and C have different dimerization initiation signal (DIS). We hypothesized that the inability of subtype B and C RNAs to form perfect base-pairing of the DIS affects the dimeric RNA structure and causes a decrease in recombination events at the 5' end of the viral genome. To test this hypothesis, we examined recombinants generated from a subtype C virus and a modified subtype B virus containing a subtype C DIS. In the 56 proviruses analyzed, we identified 96 recombination events, which are significantly more frequent than in the B/C recombinants. Furthermore, these crossover junctions were distributed evenly throughout the region analyzed, indicating that the recombination gradient was corrected by matching the DIS. Therefore, base-pairing at the DIS has an important function during HIV-1 reverse transcription, most likely in maintaining nucleic-acid structure in the complex. These findings reveal elements important to retroviral recombination and provide insights into the generation of HIV-1 intersubtype recombinants that are important to the AIDS epidemic.

Sequence differences in the dimerization initiation signal (DIS) affect the rate of recombination between subtype B and subtype C HIV-1. To test the hypothesis that DIS sequences can be used to predict intersubtype recombination potentials, we measured the recombination rate between CRF01&#95;A/E (AE) and B, which contain mismatches in the DIS, and between AE and C, which have an identical DIS. Compared with the intrasubtype recombination rate, the recombination rate between AE and subtype B virus was 9-fold lower, and the rate between AE and subtype C virus was 2-fold lower. Thus, DIS sequences can be used to predict the recombination potential between HIV-1 subtypes. Further analyses revealed that the 2-fold lower recombination rate between AE and C viruses can be restored to the intrasubtype recombination rate by matching a part of the LTR and a portion of the viral genome. Therefore, the lower intersubtype recombination rate between AE and C is not caused by a given region but is a cumulative effect by more than one region.

Genetic recombination increases diversity in HIV-1 populations, thereby allowing variants to escape from host immunity or antiviral therapies. In addition to the currently described nine subtypes of HIV-1, many of the circulating strains are intersubtype recombinants. In this study, we determined the recombination rate between two HIV-1 subtype C viruses and between a subtype B virus and a subtype C virus during a single round of virus replication. Although HIV-1 subtype C recombines at a high rate, similar to that of HIV-1 subtype B, the recombination rate between a subtype B virus and a subtype C virus is much lower than the intrasubtype recombination rate. A 3-nt sequence difference in the dimerization initiation signal (DIS) region between HIV-1 subtypes B and C accounts for most of the reduction of intersubtype recombination. By matching the DIS sequences, the B/C intersub-type recombination rate was elevated 4-fold; by introducing mismatches in the 3-nt sequences, the B/B intrasubtype recombination rate was reduced 4-fold. Further analyses showed that the intermolecular template-switching frequency was unaffected by the sequence identity of the DIS region. These results support the hypothesis that mismatched sequences in the DIS region alter the formation of heterozygous virions, thereby lowering the observable recombination rate. Here, we present the discovery of a major restriction in HIV-1 intersubtype recombination. These results have important implications for virus evolution, the mechanism of HIV-1 RNA packaging, high negative interference in recombination, and the generation of circulating intersubtype recombinants within the infected population.

Incubation of a rabbit endometrial cell line (HRE-H9 cells) with KCl (5-60 mM) for 30 min enhanced IR-GnRH secretion, with 30 and 60 mM producing the greatest stimulatory effect (280 +/- 19% and 298 +/- 49% of control group, respectively). By adding 30 mM KCl into HRE-H9 culture and increasing the incubation time to 90 min, there was a stepwise increase in IR-GnRH secretion. In the third experiment, treatment of HRE-H9 cells with estradiol (E2, 10(-9)-10(-8) M) for 48 h stimulated IR-GnRH secretion (215 +/- 17%, 168 +/- 19%, respectively), whereas P4 treatment did not produce any significant change. Treatment with E2 + P4 at all doses tested (10(-10)-10(-6) M) augmented the secretion of IR-GnRH (140 +/- 16%, 153 +/- 14%, 276 +/- 23%, 259 +/- 26%, 198 +/- 16%, respectively). Increased IR-GnRH secretion by E2 (10(-9) M) and E2 + P4 (10(-8)-10(-7) M) resulted in a reduction in cell content of IR-GnRH (p < 0.05). In conclusion, secretion of IR-GnRH by HRE-H9 cells can be induced by KCl depolarization. Treatment of HRE-H9 cells with E2 and E2 + P4 enhanced their secretion of IR-GnRH. Under such conditions, secreted IR-GnRH appears to be derived primarily from intracellular IR-GnRH pools.

The purpose of this study was to immortalize porcine endometrial cells and to characterize the transformed cells. Primary porcine endometrial cells were transfected with the plasmid vector (pmk16) containing SV40 DNA using a liposome-mediated method. The viral DNA was from a replication-defective, origin-minus, temperature-sensitive mutant strain (A58). One clone, designated PE-1, has been propagated for over 120 passages. PE-1 cells grown at 33C (33C cells) exhibit spindle-shaped morphology; when cultured at 40C (40C cells), they took on a polygonal or spherical shape. Morphology of 40C cells returned to the spindle shape after culture flasks were shifted back to 33C. During a 2-week period, 33C cells propagated approximately 30-fold faster than 40C cells, whereas protein concentration was higher in 40C cells. Southern blot analysis of PE-1 cells demonstrated successful integration of the ts-SV40 DNA sequence into the porcine endometrial cells, possibly at multiple sites. The presence of cytokeratin on PE-1 cell membranes was shown by immunocytochemical studies, suggesting that the PE-1 cell clone was of epithelial origin. Reverse phase (RP)-HPLC analysis of PE-1 cell extract indicated that the majority of immunoreactive beta-endorphin (ir-BEND) eluted with a hydrophobicity similar to that of synthetic BEND and alpha-N-acetylated BEND (Nac-BEND). These results demonstrate that a porcine endometrial cell line has been established, and that this cell line possesses characteristics of temperature sensitivity in cell morphology, growth rate, and protein synthesis.