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Abstract: Improved long-term survival rates after allogeneic hematopoietic cell transplantation (alloHCT) make family planning for young adult cancer survivors an important topic. However, treatment-related infertility risk poses challenges. To assess pregnancy and birth rates in a contemporary cohort, we conducted a national multicenter study using data from the German Transplant Registry, focusing on adult women aged 18 to 40 years who underwent alloHCT between 2003 and 2018. Of 2654 women who underwent transplantation, 50 women experienced 74 pregnancies, occurring at a median of 4.7 years after transplant. Fifty-seven of these resulted in live births (77%). The annual first birth rate among HCT recipients was 0.45%, which is >6 times lower than in the general population. The probability of a live birth 10 years after HCT was 3.4%. Factors associated with an increased likelihood of pregnancy were younger age at alloHCT, nonmalignant transplant indications, no total body irradiation or a cumulative dose of <8 Gy, and nonmyeloablative/reduced-intensity conditioning. Notably, 72% of pregnancies occurred spontaneously, with assisted reproductive technologies used in the remaining cases. Preterm delivery and low birth weight were more common than in the general population. This study represents the largest data set reporting pregnancies in a cohort of adult female alloHCT recipients. Our findings underscore a meaningful chance of pregnancy in alloHCT recipients. Assisted reproductive technologies techniques are important and funding should be made available. However, the potential for spontaneous pregnancies should not be underestimated, and patients should be informed of the possibility of unexpected pregnancy despite reduced fertility. Further research is warranted to understand the impact of conditioning decisions on fertility preservation., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Constructed wetlands (CWs) offer an efficient alternative technology for removing emerging organic contaminants (EOCs) from wastewater. Optimizing CW performance requires understanding the impact of CW configuration on EOC removal and microbial community dynamics. This study investigated EOC removal and microbial communities in horizontal subsurface flow (HSSF) CWs over a 26-month operational period. Comparison between tuff-filled and gravel-filled CWs highlighted the superior EOC removal in tuff-filled CWs during extended operation, likely caused by the larger surface area of the tuff substrate fostering microbial growth, sorption, and biodegradation. Removal of partially positively charged EOCs, like atenolol (29-98 %) and fexofenadine (21-87 %), remained constant in the different CWs, and was mainly attributed to sorption. In contrast, removal rates for polar non-sorbing compounds, including diclofenac (3-64 %), acyclovir (9-85 %), and artificial sweeteners acesulfame (5-60 %) and saccharin (1-48 %), seemed to increase over time due to enhanced biodegradation. The presence of vegetation and different planting methods (single vs. mixed plantation) had a limited impact, underscoring the dominance of substrate type in the CW performance. Microbial community analysis identified two stages: a startup phase (1-7 months) and a maturation phase (19-26 months). During this transition, highly diverse communities dominated by specific species in the early stages gave way to more evenly distributed and relatively stable communities. Proteobacteria and Bacteroidetes remained dominant throughout. Alphaproteobacteria, Acidobacteria, Planctomycetes, Salinimicrobium, and Sphingomonas were enriched during the maturation phase, potentially serving as bioindicators for EOC removal. In conclusion, this study emphasizes the pivotal role of substrate type and maturation in the removal of EOCs in HSSF CW, considering the complex interplay with EOC physicochemical properties. Insights into microbial community dynamics underscore the importance of taxonomic and functional diversity in assessing CW effectiveness. This knowledge aids in optimizing HSSF CWs for sustainable wastewater treatment, EOC removal, and ecological risk assessment, ultimately contributing to environmental protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Aim: Evaluating the clinical survival and quality parameters of class-II restorations using 3M™ Filtek TM Bulk Fill Posterior Restorative compared to 3M™ Filtek TM Supreme XTE Universal Restorative over a period of five years., Materials and Methods: A longitudinal, randomized, prospective split-mouth study with 60 patients (29 female, 31 male; mean age 44 y; range 20-77 y) and a total of 120 load-bearing class II restorations (TEST: n=60 Filtek Bulk Fill Posterior Restorative; CONTROL: n=60 Filtek Supreme XTE Universal Restorative) was conducted. Clinical evaluation was performed by blinded evaluators according to FDI criteria. Kaplan-Meier method was used for survival analysis and an intergroup comparison (Mann-Whitney-U-Test) was carried out. A basic significance level of 0.05 was corrected by the Bonferroni method to account for multiple testing (significance after correction: p<0.00067)., Results: The mean overall survival of restorations was 92% after 56.98±1.51 months in the TEST group (95 CI= 54.02;59.94) and 92% after 57.25±1.46 months (95 CI= 54.39; 60.12) in the CONTROL group (log-rank p=0.995). In total, four failures occurred in both TEST and CONTROL group during the observation period (mean annual failure rate: 1.6%). The most common reasons for failure were chipping-fractures, debonding, cracked-tooth-syndrome and recurrent decay. With regard to the FDI criteria, no significant differences between TEST and CONTROL material occurred for any of the evaluated variables. In the TEST group two restorations had to be repaired and two had to be replaced, in the CONTROL group four restorations had to be replaced., Conclusion: Both materials showed acceptable clinical performance and survival during the 5-year observation period., Clinical Significance: The use of a nanofilled bulk-fill composite proved to be an aesthetically, functionally and biologically satisfactory alternative in posterior dentition., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Introduction: Duplication of the urethra is a rare congenital anomaly in females which, if left unidentified, might cause refractory urinary incontinence. This video demonstrates its identification and treatment., Patient & Methods: A 5-year-old female was referred for primary refractory diurnal incontinence without any dry moment since birth. Initial clinical examination showed permanent urine leakage from an opening cranial to the clitoris. Ultrasound did not show any bladder or kidney abnormalities. A diagnostic cystoscopy under anesthesia revealed an accessory meatus located cranially to the orthotopic meatus and to the clitoris., Results: Lying in dorsal decubitus under general anesthesia, catheters were inserted in respectively the orthotopic and accessory meatus. The accessory meatus and urethra were dissected away into the bladder dome from a circumferential skin incision. Defects were closed and bladder drainage per urethram was left in situ for 10 days. P ostoperative recovery was uncomplicated. Complete resolution of the incontinence was immediately achieved at catheter removal. Daytime and night-time continence are observed with 3 years follow-up., Conclusion: Urethral duplication is a rare finding in females but should be excluded during the workup of primary incontinence. In girls with isolated urethral duplication, resection of the accessory urethra is the best surgical option., (Copyright © 2022 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

The disposal of untreated sanitary sewage in the soil has several consequences for human health and leads to environmental risks; thus, it is necessary investigating, monitoring and remediating the affected sites. The aims of the current study are to evaluate ecotoxicological effects on Eisenia andrei earthworms exposed to soil subjected to sources of sanitary sewage discharge and to investigate whether prevention values established by the Brazilian legislation for soil quality, associated with the incidence of chemical substances in it, are satisfactory enough to assure the necessary quality for different organisms. Earthworms' behavior, reproduction, acetylcholinesterase activity, catalase, superoxide dismutase and malondialdehyde levels were evaluated. The reproduction and behavior of earthworms exposed to sanitary sewage were adversely affected. Increased superoxide dismutase and catalase activity acted as antioxidant defense mechanism. Significantly increased lipid peroxidation levels and acetylcholinesterase activity inhibition have indicated lipid peroxidation in cell membrane and neurotransmission changes, respectively. Results have confirmed that sanitary sewage induced oxidative stress in earthworms. In addition, based on biochemical data analysis, the integrated biomarker response (IBR) has evidenced different toxicity levels in earthworms between the investigated points. Finally, results have indicated that effluents released into the soil, without proper treatment, lead to contaminant accumulation due to soil saturation and it can hinder different processes and biological development taking place in the soil. In addition, the current study has shown that physical-chemical analyses alone are not enough to assess soil quality, since it is also requires adopting an ecotoxicological approach. Brazilian legislation focused on soil quality must be revised and new guiding values must be proposed., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor β-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications., (© 2021 by The American Society of Hematology.)

Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) is frequently associated with impaired oral intake and malnutrition, which potentially increases morbidity and mortality. Therefore, nutrition is one of the major challenges in the post-transplant period., Methods: To document the current clinical approach in nutritional treatment, we designed a questionnaire concerning the current practice in nutrition after alloHSCT and distributed it to German speaking centers performing alloHSCT in Germany, Austria and Switzerland between November 2018 and March 2020. Twenty-eight (39%) of 72 contacted centers completed the survey, 23 from Germany, two from Austria and three from Switzerland, representing 50% of alloHSCT activity within the participating countries in 2018., Results: All centers reported having nutritional guidelines for patients undergoing alloHSCT, whereby 86% (n = 24) provided a low-microbial diet during the neutropenic phase. The criteria to start parenteral nutrition (PN) directly after alloHSCT seemed to be consistent, 75% (n = 21) of the corresponding centers started PN if the oral nutritional intake or the bodyweight dropped below a certain limit. In the setting of intestinal graft-versus-host disease (GvHD) the current practice appeared to be more heterogenous. About 64% (n = 18) of the centers followed a special diet, added food stepwise modulated by GvHD symptoms, while only four centers regularly stopped oral intake completely (intestinal GvHD grade >1). Half of the centers (54%, n = 15) applied a lactose-free diet, followed by 43% (n = 12) which provided fat- and 18% (n = 5) gluten-free food in patients with intestinal GvHD. Supplementation of micronutrients in acute intestinal GvHD patients was performed by 54% (n = 15) of the centers, whereas vitamin D (89%, n = 25) and vitamin B 12 (68%, n = 19) was added regularly independently of the presence of GvHD. Only 5 (18%) participating centers ever observed a food-associated infection during hospitalization, whereas food-associated infections were reported to occur more often in the outpatient setting (64%, n = 18)., Conclusion: The survey documented a general consensus about the need for nutritional guidelines for patients undergoing alloHSCT. However, the nutritional treatment in clinical practice (i.e. lactose-, gluten- or fat-free in intestinal GvHD) as well as the use of food supplements was very heterogeneous. In line with current general recommendations the centers seemed to focus on safe food handling practice rather than providing a strict neutropenic diet. More high-quality data are required to provide evidence-based nutrition to patients during and after alloHSCT., Competing Interests: Conflict of interest R.T. received a research grant from Bayer AG, travel support from Falk-Pharma and serves as consultant for Bayer AG. D.W. received honoraria from Falk-Pharma. R.T., H.G., A.L., J.H., A.B., A.S., J.A., P.J., J.M., I.H., S.K., E.M. W-D., C.S., G.B. and D.W. did not receive payment or services from a third party for any aspect of the submitted work and have nothing further to declare., (Copyright © 2021 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

This study aimed to investigate the influence of cropping method and substrate type on the fate and the removal of bacterial and antibiotic resistance genes (ARGs) indicators from primary wastewater by constructed wetlands (CWs) during startup and maturation stages. Four small-scale CWs differing in their plantation pattern (monoculture vs. polyculture) and substrate type were constructed and operated under field conditions. While for bacteria, the greatest impact of the cropping method and substrate type on removal was during the startup stage rather than the maturation stage, for ARGs, such impact was significant at both stages. During startup, the removal efficiencies of heterotrophic bacteria, fecal coliforms, E. coli, 16S rRNA genes and lacZ increased with the operation time. At maturation, the removal efficiencies were constant and were within the range of 89.2-99.4%, 93.7-98.9%, 89-98.8%, 94.1-99.6% and 92.9-98.7%, respectively. The removal efficiencies of intl1, tetM, intl1, sul1, ermB and total ARGs were also increased with the operation time. However, they were ARG type and configuration-dependent; at maturation they ranged between 50.7%-89.4%, 85.9%-97%, 49.6%-92.9%, 58.2%-96.7% and 79.9-94.3%, respectively. The tuff-filled serially planted CW was also the only one capable of removing these genes at similar high efficiency. Metagenomic analysis showed that none of the ARGs was among the most common ARGs in water and biofilm samples; rather most ARGs belonged to bacterial efflux transporter superfamilies. Although ARGs were removed, they were still detected in substrate biofilm and their relative concentrations were increased in the effluents. While the removal of both bacteria and ARGs was higher during summer compared to winter, the season had no effect on the removal pattern of ARGs. Hence, combination of the serial plantation with substrate having high surface area is a potential strategy that can be used to improve the performance of CWs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Phalloplasty for penile insufficiency in biological men differs from trans-men by incorporating native tissue. The study objective was to report surgical and functional outcomes of phalloplasty in biological men., Patients & Methods: Phalloplasty was performed with a radial free forearm (RFFA) or pedicled anterolateral thigh (ALT) flap. The tube-within-tube technique was used for urethroplasty. Complications <30 d postoperative were categorized according to Clavien-Dindo., Results: 30 patients, median (IQR) age of 21 (18-30)y, were included. 17 patients (57%) had penile insufficiency due to exstrophy or hypospadias surgeries, 7 patients (23%) due to sexual development disorders. In 16 patients RFFA was used. 19 patients needed urethroplasty, the rest catheterized through stoma. Median follow-up was 33 (14-80)mo. Within 30 d postoperative, 1 patients (3.3%) had Clavien-Dindo II and 6 patients (20%) Clavien-Dindo III complications. On long-term, 10 patients (33%) developed fistula, 6 needing urethroplasty. 7 patients (23%) had urethral strictures, all needing surgical intervention. All patients but one (98%) had erogenous sensitivity in the neo-phallus. All urethroplasty patients reported antegrade ejaculation. 16 patients (84%) voided through the urethra. 21 patients (70%) had an erectile device implanted., Conclusion: RFFA and ALT result in good erogenous sensitivity but fistulas and strictures are frequent., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Background: In Fontan patients, attrition of ventricular function is well recognized, but early detection of ventricular dysfunction is difficult. The aim of this study is to longitudinally assess ventricular strain in Fontan patients using a new method for cardiac magnetic resonance (CMR) feature tracking, and to investigate the relationship between ventricular strain and cardiac systolic function., Methods and Results: In this prospective, standardized follow-up study in 51 Fontan patients, age ≥ 10 years, CMR and concomitant clinical assessment was done at the start of the study and after 2 years. CMR feature tracking was done combining the dominant and hypoplastic ventricles. Global longitudinal strain (GLS) (-17.3% versus -15.9%, P = 0.041) and global circumferential strain (GCS) (-17.7 versus -16.1, P = 0.047) decreased over 2 years' time. Ejection fraction (EF) (57%), cardiac index (CI) (2.7 l/min/m 2 ) and NYHA functional class (97% in class I/II) were preserved. The strain values of the combined dominant and hypoplastic ventricles were significantly worse compared to those of the dominant ventricle only (GLS -16.8 (-19.5 to -14.0) versus -18.8 (-21.3 to -15.3) respectively, P = 0.001, GCS -18.3 (-22.1 to -14.8) versus -22.5 (-26.3 to -19.4) respectively, P < 0.001)., Conclusions: This study showed a decrease in cardiac strain over 2 years in Fontan patients without clinical signs of Fontan failure, where EF, CI and clinical status were still preserved. Cardiac strain might be a sensitive early indicator of systolic ventricular decline. Furthermore, combined strain of the hypoplastic and dominant ventricles seems a more accurate representation of cardiac strain in functionally univentricular hearts., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Competing Interests: Conflict-of-interest disclosure: D.W. received honoraria from Novartis, Falk Pharma, and Mallinckrodt.

Arsenic (As) groundwater contamination is common yet spatially heterogeneous within most environments. It is therefore necessary to measure As concentrations to determine whether a water source is safe to drink. Measurement of As in the field involves using a test strip that changes color in the presence of As. These tests are relatively inexpensive, but results are subjective and provide binned categorical data rather than exact determinations of As concentration. The goal of this work was to determine if photos of field kit test strips taken on mobile phone cameras could be used to extract more precise, continuous As concentrations. As concentrations for 376 wells sampled from Araihazar, Bangladesh were analyzed using ICP-MS, field kit and the new mobile phone photo method. Results from the field and lab indicate that normalized RGB color data extracted from images were able to accurately predict As concentrations as measured by ICP-MS, achieving detection limits of 9.2μg/L, and 21.9μg/L for the lab and field respectively. Data analysis is most consistent in the laboratory, but can successfully be carried out offline following image analysis, or on the mobile phone using basic image analysis software. The accuracy of the field method was limited by variability in image saturation, and variation in the illumination spectrum (lighting) and camera response. This work indicates that mobile phone cameras can be used as an analytical tool for quantitative measures of As and could change how water samples are analyzed in the field more widely, and that modest improvements in the consistency of photographic image collection and processing could yield measurements that are both accurate and precise., (Copyright © 2016. Published by Elsevier B.V.)

Chronic graft-versus-host disease (cGVHD) remains one of the most significant long-term complications after allogeneic blood and marrow transplantation. Diagnostic biomarkers for cGVHD are needed for early diagnosis and may guide identification of prognostic markers. No cGVHD biomarker has yet been validated for use in clinical practice. We evaluated both previously known markers and performed discovery-based analysis for cGVHD biomarkers in a 2 independent test sets (total of 36 cases ≤1 month from diagnosis and 31 time-matched controls with no cGVHD). On the basis of these results, 11 markers were selected and evaluated in 2 independent replication cohorts (total of 134 cGVHD cases and 154 controls). cGVHD cases and controls were evaluated for several clinical covariates, and their impact on biomarkers was identified by univariate analysis. The 2 replications sets were relatively disparate in the biomarkers they replicated. Only sBAFF and, most consistently, CXCL10 were identified as significant in both replication sets. Other markers identified as significant in only 1 replication set included intercellular adhesion molecule 1 (ICAM-1), anti-LG3, aminopeptidase N, CXCL9, endothelin-1, and gelsolin. Multivariate analysis found that all covariates evaluated affected interpretation of the biomarkers. CXCL10 had an increased significance in combination with anti-LG3 and CXCL9, or inversely with CXCR3(+)CD56(bright) natural killer (NK) cells. There was significant heterogeneity of cGVHD biomarkers in a large comprehensive evaluation of cGVHD biomarkers impacted by several covariates. Only CXCL10 strongly correlated in both replication sets. Future analyses for plasma cGVHD biomarkers will need to be performed on very large patient groups with consideration of multiple covariates., (© 2016 by The American Society of Hematology.)

Background: Patients with a Fontan circulation tend to develop liver fibrosis, liver cirrhosis and even hepatocellular carcinoma. The aim of this study is to use the magnetic resonance technique diffusing-weighted imaging (DWI) for detecting liver fibrosis/cirrhosis in Fontan patients and to establish whether DWI results are associated with functional aspects of the Fontan circulation., Methods: In a cross-sectional study, 59 Fontan patients were evaluated by liver DWI. The association between apparent diffusion coefficients (ADC) and patient characteristics, laboratory measurements and functional aspects of the Fontan circulation (NYHA class, maximum oxygen uptake during exercise and cardiac index) was assessed., Results: Liver ADC values were low (0.82×10(-3)±0.11×10(-3) mm2/s) compared with literature values for healthy volunteers and correlated negatively with calculated liver fibrosis/cirrhosis scores (Fib-4 score, p=0.019; AST/ALT ratio, p=0.009) and gamma-glutamyl transferase (p=0.001). Furthermore, ADC values correlated negatively with follow-up duration (p<0.001) and positively with cardiac index (p=0.019). No correlation between ADC values and exercise tests was found. In multivariable analysis, the ADC values were independently correlated with follow-up duration after Fontan completion., Conclusions: The results of the current study suggest that progressive liver damage due to chronic congestion and potential hypoperfusion is reflected in the liver ADC values in Fontan patients. This study highlights that liver damage in the context of the Fontan circulation might be far more common than previously thought, and that the implementation of liver assessment in the routine follow-up of Fontan patients is recommendable., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Indole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation., (© 2015 by The American Society of Hematology.)

Objectives: Restorative rehabilitation of missing single teeth in the posterior dentition is achieved primarily by dental implants or fixed dental prostheses. Here we introduce an uncommon, minimally invasive treatment option in order to close single tooth gaps., Methods: The purpose of this study was to evaluate the clinical quality and survival of 45 direct composite buildups in the posterior dentition. A detailed clinical examination was performed, and restorations were rated qualitatively using the modified USPHS/FDI criteria., Results: The mean extension of the direct composite buildups was 2.96 (1.12)mm per tooth. Interdental closures from both adjacent teeth had a significantly higher extension of the direct composite buildups than one-sided interdental closures (p=0.03). Patients with a history of prior tooth loss showed a significantly greater extension of direct composite buildups than patients without prior tooth loss (p=0.006). During the follow-up period, no restoration showed unfavorable events. The overall survival rate for the direct composite buildups was 100% after median follow-up of 78.96 months. The clinical quality rating indicated that most of the restorations (>94%) displayed excellent or good quality. No periodontal destruction, signs of gingival inflammation or increased plaque accumulation was observed., Conclusions: The posterior direct composite buildups evaluated in this study showed promising clinical survival data and excellent quality parameters after a median observation time of 6.5 years., Clinical Significance: If a minimally or non-invasive treatment approach is indicated, posterior direct composite buildups provide a successful treatment alternative for the closure of single-tooth gaps to implant-supported or fixed dental prostheses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The Fontan circulation is a palliation for patients with a functionally univentricular heart. It is characterized by gradual attrition over time. An increase in pulmonary vascular resistance could be a key factor in the long-term failure of the Fontan circulation. In this study we aimed to identify pulmonary vascular remodeling in patients with a Fontan circulation., Methods: Pulmonary vascular histomorphometric analysis and immunohistochemistry were performed in lung tissue obtained at autopsy from 12 Fontan patients. These patients had died either peri-operatively (Group A: death during or <15 days after Fontan completion; n = 5) or in mid to long-term follow-up (Group B: death >5 years after Fontan completion; n = 7). Two age-matched control groups (n = 10 and n = 14, respectively) were included., Results: Intra-acinar pulmonary vessels in the Fontan Group B patients showed decreased medial thickness (p = 0.028) compared with age-matched controls, whereas intimal thickness was increased (p = 0.002). Intimal thickness in the Fontan Group B patients correlated with age at death (r = 0.964, p < 0.001) and with the length of time that the Fontan circulation had been in place (r = 0.714, p = 0.036). Immunohistochemistry revealed a reduction of vascular smooth muscles cells in the medial layer of the intra-acinar pulmonary vessels. The eccentric intimal thickening was composed of mainly acellular fibrosis with collagen deposition., Conclusions: We observed a unique pattern of adverse pulmonary vascular remodeling in patients with a long-standing Fontan circulation who had died during follow-up. This remodeling pattern may play a major role in long-term attrition of the Fontan circulation., (Copyright © 2015 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Objectives: Adhesive resin composite technology enables dentists to add composite material to tooth surfaces to close gaps and reshape tooth form without cavity preparation. This option creates new possibilities for minimally invasive dentistry as the tooth shape, position and colour can be altered without loss of tooth substance. However, evidence-based data on direct composite buildups are rare., Methods: The purpose of this study was to evaluate the outcome and to document the occurrence of unfavourable events and clinical findings of 176 direct composite buildups that had been performed in the Department of Conservative Dentistry, University Hospital Heidelberg, Germany, between 2002 and 2008. Outcome was categorised as failure (F), survival (SR) or success (S). Restorations still in place and without prior failure at follow-up were qualitatively rated using modified USPHS/FDI criteria., Results: During the follow-up period, 30 restorations were found to have had unfavourable events or clinical findings. All restorations were repaired, and they remained in situ (SR). No complete loss (F) was recorded. The overall survival rate was 84.6% after 60 months (95%, confidence interval [CI]: 78.5 and 90.6). Clinical quality was rated excellent or good for most (>90%) of the restorations examined., Conclusions: The direct composite buildups observed in this study were found to have promising clinical outcome and good quality parameters after 5 years. If a noninvasive or minimally invasive treatment approach is indicated, direct composite buildups provide an acceptable treatment alternative for the aesthetic correction and reshaping of anterior teeth., Clinical Significance: The application of direct composite buildups in clinical cases in which a non- or minimally invasive treatment approach is indicated provides an excellent treatment alternative for gold-standard treatment options like laboratory-made crowns and porcelain veneers. Restorations in the present investigation showed a functional survival rate of 100%, an overall survival rate of 84.6% after 5 years., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The granulocyte colony-stimulating factor (G-CSF) is utilized to reduce neutropenic complications in patients receiving cancer chemotherapy. This study represents a systematic review and evidence summary of the impact of G-CSF support on chemotherapy dose intensity and overall mortality., Materials and Methods: All randomized controlled trials (RCTs) comparing chemotherapy with or without G-CSF support and reporting all-cause mortality with at least 2 years of follow-up were sought. Dual-blind data abstraction of disease, treatment, patient and outcome study results with conflict resolution by third party was carried out., Results: The search revealed 61 randomized comparisons of chemotherapy with or without initial G-CSF support. Death was reported in 4251 patients randomized to G-CSFs and in 5188 controls. Relative risk (RR) with G-CSF support for all-cause mortality was 0.93 (95% confidence interval: 0.90-0.96; P < 0.001). RR for mortality varied by intended chemotherapy dose and schedule: same dose and schedule (RR = 0.96; P = 0.060), dose dense (RR = 0.89; P < 0.001), dose escalation (RR = 0.92; P = 0.019) and drug substitution or addition (RR = 0.94; P = 0.003). Greater RR reduction was observed among studies with longer follow-up (P = 0.02), where treatment was for curative intent (RR = 0.91; P < 0.001), and where survival was the primary outcome (RR = 0.91; P < 0.001)., Conclusions: All-cause mortality is reduced in patients receiving chemotherapy with primary G-CSF support. The greatest impact was observed in RCTs in patients receiving dose-dense schedules.

The Wickerhamomyces ciferrii strain NRRL Y-1031 F-60-10A is a well-known producer of tetraacetylphytosphingosine (TAPS) and used for the biotechnological production of sphingolipids and ceramides. It was our aim to gain new biological insights into the sphingolipid metabolism by employing a dual platform mass spectrometry strategy. The first step comprised metabolic (15)N-labeling in combination with label-free proteomics using high resolution LTQ Orbitrap mass spectrometry. Then selected reaction monitoring tandem mass spectrometry served for the targeted quantification of sphingoid base biosynthesis enzymes. The non-producer strain NRRL Y-1031-27 served as a reference strain. In total, 1697 proteins were identified, and 123 enzymes of main metabolic pathways were observed as differentially expressed. Major findings were: 1) the likely presence of higher carbon flux in NRRL Y-1031 F-60-10A, resulting in higher availability of pyruvate and acetyl-CoA; 2) indications of oleaginous yeast-like behavior in NRRL Y-1031 F-60-10A; and 3) approx. 2-fold higher abundance of eight sphingolipid biosynthesis enzymes in NRRL Y-1031 F-60-10A. Taken together, in NRRL Y-1031 F-60-10A, the levels of glycolytic enzymes apparently gear carbon flux towards higher acetyl-CoA synthesis rates, while simultaneously reducing protein biosynthesis in the process. Thereby, C2 building blocks for acyl-moieties and downstream sphingoid base acetylation are provided to maintain TAPS production., Biological Significance: First quantitative proteome study for a phytosphingosine-producing yeast., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The authors conducted a literature review to determine how dentofacial esthetics can be evaluated in restorative dentistry and which quantifiable clinical parameters can be used for this assessment of dentofacial esthetics., Types of Studies Reviewed: The authors selected 35 studies that focused on assessment strategies for dental professionals. The primary inclusion criteria were intraoral and extraoral esthetic assessment methods and indexes or rating scales evaluating esthetics in restorative dentistry., Results: The studies' protocols and assessment methods were heterogeneous. The authors grouped the studies into six categories according to topic: golden proportion, soft-tissue measurement, smile and smile line assessment, orofacial indexes and scales, incisor proportion and angulation, and facial esthetics. These categories included various esthetic parameters, including the smile line, lip line, incisal offset, location of dental and facial midline, incisor angulations and width to height ratios of the maxillary anterior teeth, gingival contour, and root coverage and papilla height. These parameters should be considered when providing dental treatment in the anterior area, as they allow for quantification and objective judgment., Clinical Implications: The findings of this review might increase interest in a comprehensive dental esthetic index that allows for objective quantification and intrastudy and interstudy comparison of dental treatment outcomes.

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

Objectives: Irregular tooth shape and position in the anterior maxilla and mandible are major aesthetic problems for patients. In recent years such conditions have increasingly been treated minimally or even non-invasively by recontouring teeth with direct composite resin buildups. Although clinical experience with this treatment option is promising, evidence-based data about longevity are limited., Methods: The authors evaluated survival and quality of 327 composite buildups that were placed in 101 patients in the Department of Conservative Dentistry, University of Heidelberg, between 2002 and 2008. Follow-up intervals and failures were recorded. Quality was assessed by grading restorations (modified USPHS/Ryge critera) still in situ without adverse event during the follow-up period., Results: After a mean follow-up interval of 27.8 months, 284 restorations were in situ and had no event. Forty-two restorations were in situ but had events that were mostly minor fractures of the composite resin. One restoration had been lost. Analysis of the time from insertion to any event or end of follow-up yielded an estimated 5-year survival rate of 79.2% (95% CI, 70.5-87.9). Quality was assessed by grading in situ restorations without event. More than 90% of graded restorations were categorized as "clinically excellent" (1) or "clinically good" (2). A correlation between those rated "clinically sufficient" or worse (≥3) and the follow-up period was observed., Conclusions: Direct composite buildups are aesthetic, functional, and biologically sound treatment options for recontouring teeth and closing diastemas with clinically promising survival rates. Therefore, tooth shape correction can be recommended in cases in which minimally invasive or non-invasive procedures on healthy teeth are indicated., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Allogeneic stem cell transplantation (ASCT) after reduced-intensity conditioning has become a reasonable treatment option for patients with advanced myelofibrosis. The role of characteristic molecular genetic abnormalities, such as JAK2V617F on outcome of ASCT, is not yet elucidated. In 139 of 162 myelofibrosis patients with known JAK2V617F mutation status who received ASCT after reduced-intensity conditioning, the impact of JAK2 genotype, JAK2V617F allele burden, and clearance of mutation after ASCT was evaluated. Overall survival was significantly reduced in multivariate analysis in patients harboring JAK2 wild-type (hazard ratio = 2.14, P = .01) compared with JAK2 mutated patients. No significant influence on outcome was noted for the mutated allele burden analyzed either as continuous variable or after dividing into quartiles. Achievement of JAK2V617F negativity after ASCT was significantly associated with a decreased incidence of relapse (hazard ratio = 0.22, P = .04). In a landmark analysis, patients who cleared JAK2 mutation level in peripheral blood 6 months after ASCT had a significant lower risk of relapse (5% vs 35%, P = .03). We conclude that JAK2V617F-mutated status, but not allele frequency, resulted in an improved survival and rapid clearance after allografting reduces the risk of relapse.

Introduction: The use of mineral trioxide aggregate (MTA) might improve the prognosis of teeth after pulp exposure. The treatment outcome of teeth after direct pulp capping, either with mineral trioxide aggregate (MTA) or calcium hydroxide (controls), was investigated, taking into account possible confounding factors., Methods: One hundred forty-nine patients treated between 2001 and 2006 who received direct pulp capping treatment in 167 teeth met the inclusion criteria. Treatment was performed by supervised undergraduate students (72%) and dentists (28%). Assessment of clinical and radiographic outcomes was performed by calibrated examiners 12-80 months after treatment (median, 27 months)., Results: One hundred eight patients (122 treated teeth) were available for follow-up (72.5% recall rate). A successful outcome was recorded for 78% of teeth (54 of 69) in the MTA group and for 60% of teeth (32 of 53) in the the calcium hydroxide group. The univariate analysis (generalized estimation equations model [GEE model] showed a significant difference in the success rate (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.05-5.32; P = .04). In the multiple analysis (GEE model), the OR is marginally inside the nonsignificant range (OR, 0.43; 95% CI, 0.19-1.02; P = .05) when conspicuous confounding factors are stabilized (univariate analysis). Multiple analysis showed that teeth that were permanently restored >or=2 days after capping had a significantly worse prognosis in both groups (OR, 0.24; 95% CI, 0.09-0.66; P = .01)., Conclusions: MTA appears to be more effective than calcium hydroxide for maintaining long-term pulp vitality after direct pulp capping. The immediate and definitive restoration of teeth after direct pulp capping should always be aimed for., (Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: The purpose of this study was to evaluate both reliability and validity of the assessment of the shape of the mandibular condyle in panoramic images of the TMJ., Study Design: Forty subjects were included and were examined according to the Research Diagnostic Criteria for Temporomandibular Disorders. Panoramic radiographs (PRs) and magnetic resonance images (MRIs) were completed for all subjects. Both MRIs and PRs were rated by raters blinded to the clinical diagnosis. Kappa statistics were used to compare the results of the raters of the PRs. Additionally, the specificity and the sensitivity of the PRs were calculated for 2 scenarios: one with MRI and the other with clinical findings as the gold standard., Results: The sensitivity was 0.94 (specificity = 0.45) for the assumption that MRI is the gold standard and 0.86 (specificity = 0.49) for the assumption that the clinical examination is the gold standard. For reliability, the results for kappa ranged from 0.06 to 0.327., Conclusion: It can be concluded that PRs are not a reliable method of accurately judging the shape of the mandibular condyle.

To assess the role of NOD2/CARD15 variants on the long-term outcome of allogeneic stem cell transplantation in a genetically homogeneous group, we extended our previous study (cohort I, n = 78) and typed DNA for NOD2/CARD15 single nucleotide polymorphisms (SNPs) from an additional 225 recipients and their HLA-identical sibling donors (cohort II) treated at four other European centers. Results of genotyping were compared with clinical outcome. The strong association of NOD2/CARD15 variants with transplantation-related mortality (TRM) was confirmed in univariate and multivariate analysis; TRM increased from 20% in cohort I/22% in cohort II in recipient/donor pairs without any NOD2/CARD15 variants to 47% in cohort I/32% in cohort II in the presence of one variant in either donor or recipient and further to 57% in cohort I/74% in cohort II in the presence of 2 or more variants (P < .002 in both cohorts). NOD2/CARD15 SNPs were not associated with relapse rate but had a strong impact on overall survival. In an analysis of center effects, the type of gastrointestinal decontamination was the only factor interfering with the prognostic significance of NOD2/CARD15 SNPs. Our data further support an interaction between gastrointestinal defense mechanisms, activation of the innate immune system, and specific transplant-related complications.

CD4+CD25+ regulatory T cells (Tregs) control immune responses to self- and foreign antigens and play a pivotal role in autoimmune diseases, infectious and noninfectious inflammation, and graft rejection. Since recent experimental studies have indicated that Tregs were able to ameliorate graft-versus-host disease (GvHD), we analyzed the number of infiltrating Tregs in the intestinal mucosa as one site of GvH reactivity using immunoenzymatic labeling to enumerate FOXP3+ T cells in 95 intestinal biopsies from 49 allografted patients in comparison with healthy controls and patients with infectious inflammation. While patients with cytomegalovirus (CMV)-colitis or diverticulitis showed a concomitant increase of CD8+ effectors and Tregs, acute and chronic GvHD were characterized by the complete lack of a counter-regulation indicated by a FOXP3+/CD8+ T-cell ratio identical to healthy controls. In contrast, specimens without histologic signs of GvHD demonstrated increased numbers of FOXP3+ per CD8+ T cells, indicating that the potential for Treg expansion is principally maintained in allografted patients. Our findings provide evidence that GvHD is associated with an insufficient up-regulation of Tregs in intestinal GvHD lesions. The determination of FOXP3+/CD8+ ratio can be a helpful tool to discriminate GvHD from infectious inflammation after allogeneic stem cell transplantation.

Aim: The aim of this retrospective study was to investigate whether radical (segmental resection) or conservative (marginal) resection of mandibular bone influenced patients, survival., Patients and Methods: A series of 136 patients selected for partial mandibular resection for the treatment of squamous cell carcinomas of the lower oral cavity was evaluated retrospectively. Fifty-four patients underwent marginal and 82 cases segmental resection of the mandible. The mean follow-up periods were 91.1+/-30.1 months and 91.5+/-26.2 months, respectively. Data was extracted from the patient records and transferred into a database for statistical evaluation. Results Mean survival was 63.0+/-35.3 months for marginal and 53.1+/-32.3 months for segmental resection. The overall survival rate was analysed according to Kaplan-Meier and the test of significance (Log Rank) which yielded no statistically significant difference in the survival rate between both groups (p=0.1119). Numbers of recurrences, second primaries, metastases and/or postoperative complications were similar in both groups, i.e. no statistically significant differences were observed (ANOVA, Post-hoc Scheffé test). Conclusion In cases with an indication for bone resection, marginal resection may achieve satisfactory control and is as effective as segmental resection.

New conditioning regimens are being explored to reduce toxicity and enable allogeneic bone marrow transplantation in patients not eligible for conventional transplantation. We have investigated treosulfan, an alkylating agent, with the aim of developing an efficient and reliable but less-toxic conditioning regimen. A series of 30 patients who were not eligible for standard conditioning therapy received transplants from HLA-matched related (n = 14) or unrelated (n = 16) donors after administration of treosulfan 10 g/m2 intravenously daily for 3 days and fludarabine 30 mg/m2 intravenously daily for 5 days. Patients receiving grafts from unrelated donors also were given rabbit antithymocyte globulin 10 mg/kg intravenously daily for 3 days. All patients achieved prompt neutrophil and platelet recovery. Extramedullary toxicity was generally mild with Common Toxicity Criteria (CTC) grade 3 or 4 attributable to the conditioning seen only with transaminases. Complete donor chimerism was achieved by 90% of the patients. Acute graft-versus-host disease (GVHD) grade III or IV developed in 14% of the patients and chronic GVHD in 39%. An estimated overall survival rate of 73% and an event-free survival rate of 49% have been reached after a median of 22 months (range, 7.4-33.4 months). In summary, the combination of treosulfan and fludarabine is a safe and efficient conditioning regimen.

Objective: To sequence and characterize the expression of the prothrombinase Fgl-2 in the Sprague-Dawley rat., Methods: Reverse-transcriptase polymerase chain reaction was performed on RNA from spontaneously cycling adult pregnant Sprague-Dawley rats by using specific Fgl-2 primers. The resulting amplicon was also used to screen a rat spleen bacteriophage library and to probe a Northern blot of various tissues. The rat Fgl-2 amino acid sequence was compared with the known sequences in mouse and human., Results: Fgl-2-specific amplicon bands were observed in the rat brain, kidney, liver, ovary, spleen, and gestational day 22 and postpartum uterus. The rat Fgl-2 cDNA and amino acid sequence were found to be homologous with those of human (86% and 74%, respectively) and mouse (91% and 87%, respectively). Northern blotting demonstrated two different-sized transcripts (1.3 and 3.4 kb), and expression was observed in the cervix, heart, liver, ovary, and nongestational and gestational day 22 myometrium., Conclusion: Thrombin is classically generated from the cleavage of the proenzyme prothrombin by activated factors V and X. In tissues thrombin appears to be generated by a novel prothrombinase Fgl-2 (fibrinogen-like protein) whose activity is stimulated by proinflammatory mediators. Fgl-2 provides the mechanistic coupling between proinflammatory cytokines and the generation of active thrombin independent of the coagulation cascade. Our studies confirmed the expression of Fgl-2 mRNA in several rat tissues, including the pregnant uterus, where it could play a key role in the initiation of parturition especially in response to local or systemic infection.

Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.

We have designed a method that permits to isolate highly purified Golgi vesicles deprived of endoplasmic reticulum (ER), main contaminant of Golgi fractions. To this end, we prepared a rabbit polyclonal antibody against the cytosolic N-terminal oligopeptide of the enzyme heparan glucosaminyl N-deacetylase/N-sulphotransferase (HSST), a specific marker for Golgi apparatus. The Golgi localization of HSST was confirmed by indirect immunofluorescence microscopy. The antibody binding to Golgi vesicles was demonstrated by immunoelectronmicroscopy and allowed the immunopurification by magnetic sorting. Golgi vesicles subjected to purification by magnetic sorting showed the presence of HSST and p28, which is an integral membrane protein on the cis-Golgi also used as a specific Golgi marker. The purified material was devoid of calreticulin, a specific ER marker. This purification method will allow to improve studies requiring highly purified Golgi membranes such as identification of specific receptors and the electrophysiological characterization of Golgi membrane ion channels, which have been jeopardized up to now by ER membrane contamination.

C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface., (Copyright 2000 Academic Press.)

N(omega)propyl-l-arginine (NPA) and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea (TFMPITU) inhibit selectively the neuronal nitric oxide (NO) synthase (nNOS) isoform. In the presence of Ca(2+) and calmodulin (CaM), NPA and TFMPITU produce a time- and concentration-dependent suppression of nNOS catalyzed NO formation. This suppression of activity occurs by a first order kinetic process as revealed from linear Kitz-Wilson plots but does not depend on catalytic turnover since it occurs in the absence of NADPH. Following full suppression of NO synthetic activity by either NPA or TFMPITU, NO synthesis can be restored slowly by excess arginine or by dilution, indicating that the effects of these agents are reversible. This behavior is consistent with a dissociation of NPA and TFMPITU from nNOS slowed by a conformational transition produced by Ca(2+) CaM-binding. NPA and TFMPITU bind to nNOS rapidly producing a heme-substrate interaction as revealed by difference spectrophotometry. At physiological conditions (100 microM extracellular arginine), NPA and TFMPITU inhibit Ca(2+)-dependent NO formation by GH(3) pituitary cells with IC(50) values of 19 and 47 microM, respectively, but require millimolar concentrations to inhibit NO formation by cytokine-induced RAW 264.7 murine macrophages. The inhibition of NO formation by these agents in GH(3) cells is rapidly reversible and not due to suppression of cellular arginine uptake., (Copyright 2000 Academic Press.)

Purpose: To correlate the X inactivation pattern, as determined by one or more molecular assays, with phenotype in individuals with structurally abnormal X chromosomes., Methods: We utilized methylation analysis of androgen receptor (AR) and Fragile X (FMR1) genes and expression studies of an XIST polymorphism to assess X inactivation patterns of 28 females with structurally abnormal X chromosomes. Individuals were placed in one of three categories: (1) completely nonrandom inactivation of one X chromosome, (2) preferential or skewed inactivation of one X chromosome, or (3) random inactivation of either X chromosome., Results: In 19 of 21 cases with complete (>97%) skewing of X inactivation, the phenotype was either normal, consistent with a single gene disorder, or consistent with classical Turner syndrome; two cases with completely nonrandom X inactivation had unexplained mental retardation phenotypes. In contrast, six of seven cases that did not exhibit completely nonrandom X inactivation were phenotypically abnormal. Carriers of two balanced translocations, two duplicated Xs, one deleted X, and one 45,X/46,X,r(X) presented with mental retardation and/or multiple congenital anomalies., Conclusion: In patients with random or skewed X inactivation, the abnormal phenotype was hypothesized to be due to functional nullisomy or disomy of X-linked genes. Based on these results, we propose that X inactivation studies should be performed on all women with structurally abnormal X chromosomes. This should aid in the understanding of abnormal phenotypes in liveborn individuals with abnormal X chromosomes and may help to predict phenotypes for prenatally detected cases in the future.

N(G)-Amino-l-arginine, N(5)-(1-iminoethyl)-l-ornithine, N(6)-(1-iminoethyl)-l-lysine, and aminoguanidine were studied for the mechanisms by which they produce suicidal inactivation of the neuronal nitric oxide synthase isoform (nNOS). All of the inactivators that were amino acid structural analogs targeted the heme residue at the nNOS active site and led to its destruction as evidenced by the time- and concentration-dependent loss of the nNOS heme fluorescence, which reflects the disruption of the protoporphyrin-conjugated structure. The loss of heme was exclusively associated with the dimeric population of the nNOS. This inactivator-mediated loss of the nNOS heme never reached more than 60%, suggesting that only half of the dimeric heme is involved in catalytic activation of mechanism-based inactivators studied. Aminoguanidine-induced nNOS inactivation produced covalent modification of the nNOS protein chain with a stoichiometry of 0.8 mol of aminoguanidine per mole of the nNOS monomer. Specific covalent modification by aminoguanidine was exclusively associated with the oxygenase domain of the nNOS. The mechanisms by which N(6)-(1-iminoethyl)-l-lysine and aminoguanidine inactivate the nNOS and iNOS do not differ between the isoforms. The selectivity of these inactivators toward the iNOS isoform is a reflection of their much lower partition ratios, which were determined to be 0.16 +/- 0. 1 for N(6)-(1-iminoethyl)-l-lysine and 12 +/- 1.5 for aminoguanidine in case of the iNOS isoform while the same inactivators produced the partition ratios of 17 +/- 2 and 206 +/- 4, respectively, for the nNOS isoform.

Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60 x 10(6) and 90 x 10(6) g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples.

Nitric oxide formation by GH3 pituitary cells is stimulated by depolarizing concentrations of K+ and by the L-channel Ca2+ agonist Bay kappa 8644 in an additive manner that depends on extracellular Ca2+. Ca(2+)-dependent NO formation at 100 microM arginine was inhibited 50% over a 30-min period by 5 microM NG-amino-L-arginine, 30 microM N6-iminoethyl-L-ornithine (NIO) and 520 microM N5-iminoethyl-L-lysine (NIL) but required concentrations of aminoguanidine (AG) greater than 3 mM. As measured at 100 microM extracellular arginine, intracellular neuronal nitric oxide synthase (nNOS) was inactivated 50% over a 30-min period by 150 microM NG-amino-L-arginine and 1500 microM NIO, but required concentrations of NIL or AG greater than 5 mM. The inactivation of nNOS by these agents occurred only under conditions that mobilized extracellular Ca2+ and was inhibited by increased extracellular arginine. Thus these agents inhibit cellular Ca(2+)-dependent NO formation at concentrations far lower than those required to inactivate the cellular nNOS. Inhibition of NO formation by these agents was not attributable to effects on cellular arginine uptake. In contrast diphenyliodonium chloride produced a rapid and complete inactivation of cellular NO formation and nNOS activity. These inactivations produced by diphenyliodonium chloride occurred with identical kinetics but displayed no requirement for Ca2+. These data support the assertion that neuronal NO synthase is refractory to mechanism-based inactivation in GH3 pituitary cells.

As measured at 100 microM extracellular arginine, aminoguanidine produced a time- and concentration-dependent inactivation of nitric oxide (NO) synthesis by cytokine-induced RAW cells. Inactivation obeyed first-order kinetics and occurred at a maximal rate of 0.22 min(-1) with a half-maximal inactivation rate observed at a concentration of 670 microM aminoguanidine (K(I) value). Inactivation of NO synthetic activity in the presence of N(G)-methyl-L-arginine similarly followed first-order kinetics with a maximal inactivation rate of 0.07 min(-1) and a K(I) value of 170 microM. Inactivation of NO synthetic activity in the presence of diphenyliodonium chloride occurred with a maximal inactivation rate of 0.24 min(-1) with a K(I) value of 14 microM. Diphenyliodonium chloride also produced a first-order rate of inactivation of cytokine-inducible nitric oxide synthase (iNOS) activity affinity purified from cytokine-induced RAW cells with a maximal inactivation rate of its cytochrome c reductase activity of 0.24 min(-1) with a K(I) value of 18 microM. Cytokine-induced RAW cells were treated with aminoguanidine, N(G)-methyl-L-arginine, and diphenyliodonium chloride at concentrations and for a time sufficient to completely inactivate NO synthesis by the cells and were allowed to recover in drug-free medium. Despite the presence of cycloheximide, NO synthetic rate recovered from 70 to 90% of its pretreatment activity over 4 h in cells exposed to either aminoguanidine or N(G)-methyl-L-arginine but did not recover from exposure to diphenyliodonium chloride. Analysis by sucrose density gradient centrifugation of the cytochrome c reductase and citrulline-forming activities in extracts of cells recovered from aminoguanidine treatment revealed that recovery was accompanied by a diminished population of iNOS monomers with an increased population of iNOS dimers. This observation is consistent with the hypothesis that for the mechanism-based inactivator aminoguanidine, functional dimers can be assembled from "drug-undamaged" monomers during the recovery period.

Protoporphyrin IX inhibits citrulline formation by all three nitric oxide synthase isoforms in a manner reversible by dilution. Zinc protoporphyrin IX, by contrast, produces a time- and concentration-dependent inactivation of all three nitric oxide synthase isoforms, not reversible by dilution. The inhibition of citrulline formation by protoporphyrin IX occurs with IC50 values of 0.8, 4, and 5 microM for the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition by N-methyl-protoporphyrin IX occurs at IC50 values of 6, 5, and 8 microM for the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition of nitric oxide synthase by protoporphyrin IX is a multisite, positively cooperative inhibition that exhibits a Hill coefficient of 2.3 for the iNOS isoform. Protoporphyrin IX reduces the maximal velocity of citrulline formation for both the iNOS and nNOS isoforms without altering the K(m) for the arginine substrate or the EC50 value for the tetrahydrobiopterin cofactor. Protoporphyrin IX inhibits the arginine-independent NADPH oxidase activity of nNOS with an IC50 value of 1 microM but has no effect on cytochrome c reductase activity at concentrations as high as 30 microM. At concentrations of 10 and 20 microM, protoporphyrin IX inhibits NO formation by cytokine-induced murine RAW 264.7 cells; however, these inhibitions are accompanied by significant cellular cytotoxicity. Coproporphyrins I and III, uroporphyrins I and III, and porphobilinogen, intermediates in the biosynthesis of heme that accumulate in hepatic porphyrias, are ineffective as inhibitors of the nitric oxide synthase isoforms. Since protoporphyrin IX is the immediate biosynthetic precursor of heme that accumulates in hepatic protoporphyria, iron deficiency anemia, and lead poisoning, protoporphyrin IX inhibition of nitric oxide synthase may contribute to the pathophysiology of these conditions.

Diaminoguanidine (DAG) and NG-amino-L-arginine each produced a time- and concentration-dependent inactivation of the citrulline-forming activity of all three NOS isoforms. DAG inactivates both the NADPH-oxidase and the citrulline-forming activities of GH3 pituitary nNOS while NG-amino-L-arginine inactivates only its citrulline-forming activity. The inactivation by DAG of GH3 nNOS NADPH-oxidase and citrulline forming activities is stimulated by (6R)-5,6,7,8-tetrahydrobiopterin (BH4) cofactor, follows pseudo-first-order kinetics and is not substrate saturable. DAG-induced inactivation of the citrulline-forming activity for the iNOS and eNOS isoforms displayed maximal inactivation rates of 0.37 and 0.14 min-1 and Ki values of 385 and 670 microM, respectively. At 1 mM DAG and saturating BH4, half-times of inactivation of 0.7, 8, and 2 min were observed for the nNOS, eNOS, and iNOS isoforms, respectively. NG-Amino-L-arginine-induced inactivation of the citrulline-forming activity of the nNOS, iNOS, and eNOS isoforms displayed maximal inactivation rates of 0.35, 0.26, and 0.53 min-1 and Ki values of 0.3, 3, and 2.5 microM, respectively. The inactivation of the NOS activities by both DAG and NG-amino-L-arginine in preincubations required the presence of oxygen and Ca2+, consistent with an inactivation mechanism that requires active metabolism by NOS. Methylguanidine and 1,1-dimethylguanidine exhibited a reversible inhibition pattern in contrast to all three NOS isoforms. Neither agent exhibited significant isoform selectivity.

Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed. To examine the properties of chimeric Fab derived from these monoclonal antibodies, we have developed an expression system which allows the rapid production of milligram quantities of chimeric Fab. Both the chimeric light chain and the chimeric Fd were co-expressed from the same vector, pAPEX-3P. This vector contains the SV40 origin of replication, which allows the rapid production of chimeric Fab in COS cells for preliminary characterization. Additionally, pAPEX-3P contains the Epstein-Barr virus origin of replication and a puromycin selectable marker for maintenance as a stable episome in human cell lines. A production system consisting of transfected 293-EBNA cells cultured in serum free medium followed by protein G-Sepharose chromatography of the conditioned medium was found to be sufficient for the rapid production of purified chimeric Fab. Here we have utilized this expression system to demonstrate that an anti-human C5 chimeric Fab was a potent inhibitor of complement activation in both in vitro activation assays and an ex vivo model of complement-mediated tissue damage.

Aminoguanidine produces a time-dependent inactivation of the citrulline forming activity of all three nitric oxide synthase isoforms that is blocked by arginine. Aminoguanidine inactivates both the NADPH oxidase and citrulline forming activities of GH3 pituitary constitutive nitric oxide synthase (cNOS) but does not alter its cytochrome c reductase activity. GH3 pituitary cells contain an NOS isoform identical physically, kinetically, and immunologically to cerebellar neuronal NOS (Wolff and Datto, Biochemical J. (1992) 285, 201-206). The inactivation of GH3 cNOS NADPH oxidase activity, as measured without added tetrahydrobiopterin cofactor, is saturable, is inhibited by arginine, and follows pseudo-first-order kinetics with an inactivation rate constant of 0.25 min-1 and a Ki value of 0.83 mM aminoguanidine. The inactivation of the citrulline forming activity of GH3 cNOS by aminoguanidine was not saturable by aminoguanidine. Aminoguanidine, at concentrations in the millimolar range, inhibited the citrulline forming activity of endothelial cNOS by an apparently nonsaturable mechanism. Aminoguanidine inactivates the citrulline forming activity of murine macrophage iNOS. The inactivation is saturable and follows pseudo-first-order kinetics with an inactivation rate constant of 0.46 min-1 and a Ki value of 16 microM. The inactivation of the constitutive isoforms of nitric oxide synthase by aminoguanidine required the concurrent presence of Ca2+, calmodulin, NADPH, tetrahydrobiopterin, and oxygen in preincubations and was not reversed either by dilution or dialysis. These observations support the assertion that aminoguanidine is a mechanism-based inactivator of the nitric oxide synthase isoforms and exhibits marked specificity for the inactivation of the inducible isoform.

Citrulline formation by the Ca2+ CaM-dependent nitric oxide synthase of bovine endothelium is inhibited reversibly by 7-nitroindazole, 1-phenylimidazole, and imidazole. As measured at 0.67 microM (6R)-5,6,7,8-tetrahydrobiopterin (BH4), IC50 values of 0.8, 200, and 50 microM were determined for 7-nitroindazole, 1-phenylimidazole, and imidazole, respectively. Increasing concentrations of added BH4 cofactor increased the IC50 values for 7-nitroindazole and 1-phenylimidazole but did not alter the IC50 value for imidazole. 7-nitroindazole inhibited citrulline formation by the endothelial cNOS noncompetitively versus arginine substrate but competitively versus BH4 with a Ki value of 0.8 microM. 1-Phenylimidazole inhibited citrulline formation by the endothelial cNOS competitively versus both arginine substrate and BH4 with a Ki value of 50 microM. Imidazole inhibited citrulline formation competitively versus arginine substrate but noncompetitively versus BH4 with a Ki value of 50 microM. Neither 7-nitroindazole, 1-phenylimidazole, nor imidazole inhibited the cytochrome c reductase activity of endothelial cNOS at concentrations up to 5000-fold higher than their Ki values for inhibition of citrulline formation. By comparison with the previously determined kinetic properties of the other nitric oxide synthase isoforms, these observations establish that 1-phenylimidazole displays marked specificity for inhibiting the inducible nitric oxide synthase isoform and, since 7-nitroindazole has been reported not to elevate blood pressure (McCall et al., 1991, Br. J. Pharmacol. 102, 234-238), fails to confirm the expected insensitivity of the constitutive endothelial nitric oxide synthase to inhibition by 7-nitroindazole.

Citrulline formation by Ca(2+)-calmodulin (CaM)-dependent nitric oxide synthase from bovine brain is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 2.3 mM, 1.15 mM, 40 microM, and 2.5 microM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a Ki value of 0.16 microM and was competitive versus both arginine substrate and (6R)-5,6,7,8-tetrahydrobiopterin cofactor. The NADPH oxidase activity of bovine brain CaM-dependent nitric oxide synthase was inhibited by 7-nitroindazole with an IC50 value of 0.6 microM. Citrulline formation by the interferon-gamma/lipopolysaccharide-inducible nitric oxide synthase of murine macrophages (264.7 cell line) is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 470, 240, 56, and 20 microM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a Ki value of 1.6 microM and was noncompetitive versus arginine substrate but competitive versus (6R)-5,6,7,8-tetrahydrobiopterin cofactor. None of the indazoles tested inhibited the cytochrome c reductase activity of either nitric oxide synthase isoform at concentrations up to 1000-fold higher than their IC50 values for inhibition of citrulline formation. These observations are consistent with the proposal that the indazoles exert their inhibitory actions by interaction with the heme-iron of nitric oxide synthase such that oxygen does not bind.