Search

Background: We aimed to develop a tool for predicting HNF1B mutations in children with congenital abnormalities of the kidneys and urinary tract (CAKUT)., Methods: The clinical and laboratory data from 234 children and young adults with known HNF1B mutation status were collected and analyzed retrospectively. All subjects were randomly divided into a training (70%) and a validation set (30%). A random forest model was constructed to predict HNF1B mutations. The recursive feature elimination algorithm was used for feature selection for the model, and receiver operating characteristic curve statistics was used to verify its predictive effect., Results: A total of 213 patients were analyzed, including HNF1B-positive (mut + , n = 109) and HNF1B-negative (mut - , n = 104) subjects. The majority of patients had mild chronic kidney disease. Kidney phenotype was similar between groups, but bilateral kidney anomalies were more frequent in the mut + group. Hypomagnesemia and hypermagnesuria were the most common abnormalities in mut + patients and were highly selective of HNF1B. Hypomagnesemia based on age-appropriate norms had a better discriminatory value than the age-independent cutoff of 0.7 mmol/l. Pancreatic anomalies were almost exclusively found in mut + patients. No subjects had hypokalemia; the mean serum potassium level was lower in the HNF1B cohort. The abovementioned, discriminative parameters were selected for the model, which showed a good performance (area under the curve: 0.85; sensitivity of 93.67%, specificity of 73.57%). A corresponding calculator was developed for use and validation., Conclusions: This study developed a simple tool for predicting HNF1B mutations in children and young adults with CAKUT., (© 2024. The Author(s).)

Background and Aims: Pediatric acute liver failure (PALF) is a life-threatening condition. In Europe, the main causes are viral infections (12%-16%) and inherited metabolic diseases (14%-28%). Yet, in up to 50% of cases the underlying etiology remains elusive, challenging clinical management, including liver transplantation. We systematically studied indeterminate PALF cases referred for genetic evaluation by whole-exome sequencing (WES), and analyzed phenotypic and biochemical markers, and the diagnostic yield of WES in this condition., Approach and Results: With this international, multicenter observational study, patients (0-18 y) with indeterminate PALF were analyzed by WES. Data on the clinical and biochemical phenotype were retrieved and systematically analyzed., Results: In total, 260 indeterminate PALF patients from 19 countries were recruited between 2011 and 2022, of whom 59 had recurrent PALF. WES established a genetic diagnosis in 37% of cases (97/260). Diagnostic yield was highest in children with PALF in the first year of life (41%), and in children with recurrent acute liver failure (64%). Thirty-six distinct disease genes were identified. Defects in NBAS (n=20), MPV17 (n=8), and DGUOK (n=7) were the most frequent findings. When categorizing, the most frequent were mitochondrial diseases (45%), disorders of vesicular trafficking (28%), and cytosolic aminoacyl-tRNA synthetase deficiencies (10%). One-third of patients had a fatal outcome. Fifty-six patients received liver transplantation., Conclusions: This study elucidates a large contribution of genetic causes in PALF of indeterminate origin with an increasing spectrum of disease entities. The high proportion of diagnosed cases and potential treatment implications argue for exome or in future rapid genome sequencing in PALF diagnostics., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The primary hyperoxalurias (PH1-3) are rare inherited disorders of the glyoxylate metabolism characterized by endogenous overproduction of oxalate. As oxalate cannot be metabolized by humans, oxalate deposits may affect various organs, primarily the kidneys, bones, heart, and eyes. Vision loss induced by severe retinal deposits is commonly seen in infantile PH1; less frequently and milder retinal alterations are found in non-infantile PH1. Retinal disease has not systematically been investigated in patients with PH2 and PH3., Methods: A comprehensive ophthalmic examination was performed in 19 genetically confirmed PH2 (n = 7) and PH3 (n = 12) patients (median age 11 years, range 3-59)., Results: Median best corrected visual acuity was 20/20. In 18 patients, no retinal oxalate deposits were found. A 30-year-old male with PH2 on maintenance hemodialysis with plasma oxalate (Pox) elevation (> 100 µmol/l; normal < 7.4) demonstrated bilateral drusen-like, hyperreflective deposits which were interpreted as crystallized oxalate. Two siblings of consanguineous parents with PH2 presented with retinal degeneration and vision loss; exome-wide analysis identified a second monogenic disease, NR2E3-associated retinal dystrophy., Conclusions: Retinal disease manifestation in PH2 and PH3 is rare but mild changes can occur at least in PH2-associated kidney failure. Decline in kidney function associated with elevated plasma oxalate levels could increase the risk of systemic oxalosis. Deep phenotyping combined with genomic profiling is vital to differentiate extrarenal disease in multisystem disorders such as PH from independent inherited (retinal) disease. A higher resolution version of the Graphical abstract is available as Supplementary information., (© 2022. The Author(s).)

Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: The physiological number and distribution of mast cells (MCs) in the pediatric gastrointestinal (GI) tract is not well defined and reference values of normality are missing. To define a physiological and disease defining cut-off, a systematic histological exploration of MC distribution from the esophagus to the rectum in healthy as well as in patients with gastrointestinal food allergies (GFA) was performed., Methods: Nine pediatric subjects that exhibited unremarkable histopathological evaluations or underwent endoscopy for surveillance reasons after a previous polypectomy of single colonic juvenile polyps served as reference cohort. In all of these subjects, a chronic inflammatory disease (eg, inflammatory bowel disease, celiac disease) or allergy was excluded. In addition, a group of 15 patients with gastrointestinal complaints suspected to be caused by a GFA were investigated. Immunohistochemistry was performed from all biopsies using CD117 (c-Kit) as a reliable marker to identify MCs in the lamina propria., Results: There were distinct differences of MC counts in all parts of the pediatric GI tract. The highest counts of MCs in both symptomatic patients and control cohort, were found in the duodenum, terminal ileum, cecum and ascending colon. The lowest counts were found in the esophagus. Significant disparities between GFA and healthy subjects were found in the gastric corpus (22.1 ± 4.0/ high power field [HPF] vs 32.0 ± 10.1/HPF; P = 0.034) and ascending colon (44.8 ± 10.4/HPF vs 60.4 ± 24.3/HPF; P = 0.047)., Conclusions: Mucosal MC counts in the pediatric GI tract are higher than previously reported, with a considerable overlap between healthy and GFA patients. These results provide detailed information on distribution and numbers of MCs in pediatric allergic patients while allowing estimates of physiological values in childhood for the first time. With regard to diagnostic procedures in GFA further laboratory parameters have to be integrated., Competing Interests: Conflicts of Interest: C. Ehrsam, A. Hoerning, C. Geppert, M. Raithel, R. Rieker, and A. Hartmann have no conflict of interest to declare., (Copyright © 2021 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPβ, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPβ is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Background: The role of B cells in inflammatory bowel disease (IBD) is ambiguous, as B cells may have both pathogenic and protective functions in IBD. We studied B cell subsets before and after initiation of an anti-tumor necrosis factor alpha (anti-TNFα) therapy in pediatric IBD. The aim of the study was to examine the behavior of B cells in pediatric IBD patients undergoing an anti-TNFα therapy and, more specifically, to clarify their association with a successful or an unsuccessful infliximab (IFX) treatment., Methods: A total of N = 42 pediatric IBD patients (Crohn disease, n = 30; ulcerative colitis, n = 12) for whom an anti-TNFα therapy with and without a concomitant azathioprine (AZA) medication was administered were recruited. Fourteen healthy age-matched children served as control patients. Blood samples were collected before initiation of the anti-TNFα therapy, before the fourth infusion at the end of the induction phase, and after 6 and 12 months under therapy maintenance. Flow cytometry (CD20, CD27, CD38, CD138) and intracellular staining (interleukin 10 [IL10], TNFα, granzyme B) were performed. Responders to successful IFX therapy were classified exhibiting a fecal calprotectin level of below 100 µg/g or achieving levels of <10% of the baseline value at initiation than at the end of the 12-month follow-up period., Results: Before initiation of anti-TNFα therapy, flow cytometry revealed increased percentages of naïve B cells whereas transitional B cells were reduced compared with those in the healthy control patients. The IL10-producing B cells of both ulcerative colitis and Crohn disease patients were reduced at the initiation of IFX therapy, whereas TNFα-producing transitional CD24hiCD38hi B cells in ulcerative colitis patients were increased compared with those in healthy control patients. After 12 months of therapy, we detected a significant increase of IL10-producing transitional B cells in responding patients.The IFX trough levels in the responding patients showed a significant increase until 6 months after IFX initiation, attaining mean values of 9.9 µg/mL, whereas the IFX dosage was significantly lower than that in the nonresponding patients. The IFX trough levels in AZA-treated patients reached earlier therapeutic levels than in patients without AZA comedication, whereas during the course of the IFX therapy, comedication with AZA had no significant effect on the outcome., Conclusions: Attaining a normalization of IL10 production among CD24hiCD38hi B cells after 12 months of therapy may represent additional information about the reconstitution of a patient's immune system in responding patients. The achievement of an IFX trough level of ~10 µg/mL at 6 months of treatment is associated with a successful anti-TNFα therapy. In addition, AZA comedication supports an earlier achievement of therapeutic IFX trough levels., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.

Background: The total cost of treating the 3 million Americans chronically infected with hepatitis C virus (HCV) represents a substantial affordability challenge requiring treatment prioritization. This study compares the health and economic outcomes of alternative treatment prioritization schedules., Methods: We developed a multiyear HCV treatment budget allocation model to evaluate the tradeoffs of 7 prioritization strategies. We used optimization to identify the priority schedule that maximizes population net monetary benefit (NMB). We compared prioritization schedules in terms of the number of individuals treated, the number of individuals who progress to end-stage liver disease (ESLD), and population total quality-adjusted life years (QALYs). We applied the model to the population of treatment-naive patients with a total annual HCV treatment budget of US$8.6 billion., Results: First-come, first-served (FCFS) treats the fewest people with advanced fibrosis, prevents the fewest cases of ESLD, and gains the fewest QALYs. A schedule developed from optimizing population NMB prioritizes treatment in the first year to patients with moderate to severe fibrosis who are younger than 65 years, followed by older individuals with moderate to severe fibrosis. While this strategy yields the greatest population QALYs, prioritization by disease severity alone prevents more cases of ESLD. Sensitivity analysis indicated that the differences between prioritization schedules are greater when the budget is smaller. A 10% annual treatment price reduction enabled treatment 1 year sooner to several patient subgroups, specifically older patients and those with less severe liver fibrosis., Conclusion: In the absence of a sufficient budget to treat all patients, explicit prioritization targeting younger people with more severe disease first provides the greatest health benefits. We provide our spreadsheet model so that decision makers can compare health tradeoffs of different budget levels and various prioritization strategies with inputs tailored to their population.

Background: Patients with diabetes can be affected by several comorbidities that require immediate action when occurring as they may otherwise cause fatal or consequential damage. For this reason, patients must closely monitor their metabolism and inject insulin when necessary. The documentation of glucose values and other relevant measurements is often still on paper in a diabetes diary., Objective: The goal of this work is to develop and implement a novel mobile health system for the secure collection of relevant data referring to a person's metabolis and to digitize the diabetes diary to enable continuous monitoring for both patients and treating physicians. One specific subgoal is to enable data transmission of health parameters to secure data storage., Methods: The process of implementing the system consists of (1) requirements analysis with patients and physicians to identify patient needs and specify relevant functionalities, (2) design and development of the app and the data transmission, and (3) usability study., Results: We developed and implemented the mobile app GlucoMan to support data collection pertaining to a person's metabolism. An automated transfer of measured values from a glucometer was implemented. Medication and nutrition data could be entered using product barcodes. Relevant background knowledge such as information on carbohydrates was collected from existing databases. The recorded data was transmitted using international interoperability standards to the MIDATA.coop storage platform. The usability study revealed some design issues that needs to be solved, but in principle, the study results show that the app is easy to use and provides useful features., Conclusions: Data collection on a patient's metabolism can be supported with a multifunctional app such as GlucoMan. Besides monitoring, continuous data can be documented and made available to the treating physician. GlucoMan allows patients to monitor disease-relevant parameters and decide who accesses their health data. In this way, patients are empowered not only to manage diabetes but also manage their health data., (©Kaspar S Schmocker, Fabian S Zwahlen, Kerstin Denecke. Originally published in JMIR Diabetes (http://diabetes.jmir.org), 26.02.2018.)

To address the impact of cellular origin on acute myeloid leukemia (AML), we generated an inducible transgenic mouse model for MLL-AF9-driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSC) in vitro resulted in dispersed clonogenic growth and expression of genes involved in migration and invasion. In vivo, 20% LT-HSC-derived AML were particularly aggressive with extensive tissue infiltration, chemoresistance, and expressed genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulator ZEB1 significantly reduced leukemic blast invasion. By classifying mouse and human leukemias according to Evi1/EVI1 and Erg/ERG expression, reflecting aggressiveness and cell of origin, and performing comparative transcriptomics, we identified several EMT-related genes that were significantly associated with poor overall survival of AML patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation.

Purpose: During healthy pregnancy, a distinct but limited invasion of trophoblast cells into the uterus occurs. In contrast, excessive trophoblast invasion is associated with placental choriocarcinoma (CC). Overexpression of the cytoskeletal protein LASP-1 was shown to contribute to cancer aggressiveness. Here, the yet unknown role of LASP-1 in CC cells is analysed., Methods: Expression of LASP-1 in human primary carcinoma was assessed by immunohistochemistry and confirmed in CC-derived cell lines by immunocytochemistry, RT-PCR and Western blot. After down-regulation of LASP-1 expression with specific si-RNA in CC-derived cell lines, migratory and proliferative activities were analysed by matrigel migration assay and WST-8 test., Results: LASP-1 expression was detected in human primary choriocarcinoma and in JEG-3, JAR and BeWo cells. Knock down of LASP-1 resulted in a decreased expression of LASP-1 protein in JEG-3 and JAR cells accompanied by a diminished migration and a decreased proliferative activity of these two cell lines. Knockdown of LASP-1 in BeWo cells failed. In consequence, migratory function and proliferation was unaffected., Conclusion: This is the first study describing LASP-1 expression in CC cells. Detecting an affection of migratory processes after LASP-1 silencing, we propose that LASP-1 could impact on metastasis of CC cells.

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression as well as its potential clinical and functional impact in colon cancer. Based on 30 tumor and 30 adjacent normal tissues we examined ITIH5 mRNA expression and promoter methylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA) platform. In addition, ITIH5 protein expression was evaluated using immunohistochemistry. ITIH5 mRNA expression loss was significantly associated (P<0.001) with hypermethylation of the ITIH5 promoter in primary colon tumors. In addition, treatment of tumor cell lines with demethylating (DAC) and histone acetylating (TSA) agents induced ITIH5 expression. In line, independent TCGA data revealed a significant expression loss of ITIH5, particularly in the MSI-high and CIMP-positive phenotype concordant with an increased ITIH5 hypermethylation in CIMP-positive colon tumors (P<0.001). In proximal, i.e., right-sided tumors, abundant ITIH5 expression was associated with longer overall survival (OS, P = 0.049) and the CIMP-positive (P = 0.032) subgroup. Functionally, ITIH5 re-expression mediated a reduced proliferation in HCT116 and CaCo2 cells. In conclusion, our results indicate that ITIH5 is a novel putative tumor suppressor gene in colon cancer with a potential impact in the CIMP-related pathway. ITIH5 may serve as a novel epigenetic-based diagnostic biomarker with further clinical impact for risk stratification of CIMP-positive colon cancer patients.

Soil salinity is one of the most severe abiotic stress factors threatening agriculture worldwide. Hence, particular interest exists in unraveling mechanisms leading to salt tolerance and improved crop plant performance on saline soils. Barley is considered to be one of the most salinity-tolerant crops, but varying levels of tolerance are well characterized. A proteomic analysis of the roots of two contrasting cultivars (cv. Steptoe and cv. Morex) is presented. Young plants were exposed to a period of 1, 4, 7, or 10 d at 0, 100, or 150 mM NaCl. The root proteome was analyzed based on two-dimensional gel electrophoresis. A number of cultivar-specific and salinity stress-responsive proteins were identified. Mass spectrometry-based identification was successful for 74 proteins, and a hierarchical clustering analysis grouped these into five clusters based on similarity of expression profile. The rank product method was applied to statistically access the early and late responses, and this delivered a number of new candidate proteins underlying salinity tolerance in barley. Among these were some germin-like proteins, some pathogenesis-related proteins, and numerous as-yet uncharacterized proteins. Notably, proteins involved in detoxification pathways and terpenoid biosynthesis were detected as early responsive to salinity and may function as a means of modulating growth-regulating mechanisms and membrane stability via fine tuning of phytohormone and secondary metabolism in the root.

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics., (Copyright © 2013 John Wiley & Sons, Ltd.)

Mass spectrometry has made possible the field of proteomics and become an invaluable tool for identifying and quantifying post-translational modifications of proteins from complex mixtures. Because PTMs are recognized as key factors of biological activity, reliable PTM characterization is essential to understanding the relationship between protein isoform and activity. Protein glycosylations, in particular, can be especially difficult to characterize due to the range of different oligosaccharide entities that may be attached at any particular site. In this month' Special Feature Dr Daniel Kolarich of the Dept. of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces and his collaborators point out that quantitative label-free glycoproteomics can yield information about glycoprotein microand macroheterogeniety if the tools are sufficiently accurate. To understand and characterize the performance capability of MS tools they synthesized a panel of peptides and their glycopeptide derivatives as references and used these to investigate the qualitative and quantitative results from various ionization techniques and mass analyzers., (Copyright © 2013 John Wiley & Sons, Ltd.)

We examined the influence of a single exercise session on quantitative muscle fat fraction MRI measurements. Ten healthy volunteers were scanned on a 3T body scanner before and after a session of bilateral squats until muscular fatigue. Axial in- and opposed phase images were acquired at a fixed distance from the knee joint and fat fractions were calculated using a 2-point Dixon technique as well as muscle cross sectional area at the same position. After the squat session, calculated fat fraction in the quadriceps bilaterally appeared to be significantly decreased, while all but one non-exercised muscles showed no change. In conclusion exercise might modify the measured apparent fat fraction. Trials using quantitative MRI should consider the timing of scanning sessions and physical examinations to avoid bias caused by the influence of exercise on measurements., (Copyright © 2012 Elsevier B.V. All rights reserved.)

• Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of tissues provides the means to analyse the spatial distributions of small molecules and proteins within tissues. This imaging technique is commonplace in medicinal and pharmaceutical research, but its application in plant science is very recent. Broader introduction requires specific adaptations for plant tissues. Sample preparation is of paramount importance in order to obtain high-quality spectra providing sufficient spatial resolution for compounds. Optimization is required for sectioning, choice of matrix and means of matrix deposition. • Here, we present our current protocols for the detection of small molecules in cryodissected immature barley (Hordeum vulgare) grains and tobacco (Nicotiana tabacum) roots. • Examples of MALDI-MSI measurements are provided, and the level of reproducibility across biological replicates is addressed. Furthermore, our approaches for the validation of distribution patterns and for the identification of molecules are described. • Finally, we discuss how MALDI-MSI can contribute to applied plant research., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

We demonstrate a 2 μm semiconductor disk laser emitting in a single longitudinal mode with a linewidth in the <10 kHz range. A heterodyne detection scheme was used for precise linewidth measurements. In these experiments, the output beams of two identical laser cavities were superposed in order to generate a beat note signal on a photodiode. In the absence of active frequency stabilization, a linewidth of 45 kHz was measured at an output power of 100 mW. When using a frequency stabilization consisting of a feedback loop with a Fabry-Perot interferometer as wavelength reference, the linewidth could be further reduced to 9 kHz.

Recent innovations in liquid chromatography-mass spectrometry (LC-MS)-based methods have facilitated quantitative and functional proteomic analyses of large numbers of proteins derived from complex samples without any need for protein or peptide labelling. Regardless of its great potential, the application of these proteomics techniques to plant science started only recently. Here we present an overview of label-free quantitative proteomics features and their employment for analysing plants. Recent methods used for quantitative protein analyses by MS techniques are summarized and major challenges associated with label-free LC-MS-based approaches, including sample preparation, peptide separation, quantification and kinetic studies, are discussed. Database search algorithms and specific aspects regarding protein identification of non-sequenced organisms are also addressed. So far, label-free LC-MS in plant science has been used to establish cellular or subcellular proteome maps, characterize plant-pathogen interactions or stress defence reactions, and for profiling protein patterns during developmental processes. Improvements in both, analytical platforms (separation technology and bioinformatics/statistical analysis) and high throughput nucleotide sequencing technologies will enhance the power of this method., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Voxel-based morphometry (VBM) is a widely applied method in computational neurosciences but it is currently recommended to compare only data collected at a single MRI scanner. Multi-site VBM would be a desirable approach to increase group size and, thus, statistical power. We aimed to assess if multi-site VBM is feasible on similar hardware and compare the magnitude of inter- and intra-scanner differences. 18 healthy subjects were scanned in two identical 3T MRI scanners using different head coil designs, twice in scanner A and once in scanner B. 3D T1-weighted images were processed with SPM8 and FSL4.1 and compared as paired t-test (scan versus re-scan) on a voxel basis by means of a general linear model (GLM). Additionally, coefficient-of-difference (coeffD) maps were calculated for respective pairs of gray matter segmentations. We found considerable inter-scanner differences clearly exceeding a commonly used GLM significance threshold of p<0.05 (FWE corrected). The spatial pattern of detected differences was dependent on whether SPM8 or FSL4.1 was used. The inclusion of global correcting factors either aggravated (SPM8) or reduced the GLM detected differences (FSL4.1). The coeffD analysis revealed markedly higher variability within the FSL4.1 stream both for the inter- and the intra-scanner comparison. A lowered bias cutoff (30 mm FWHM) in SPM8 improved the comparability for cortical areas. Intra-scanner scan/re-scan differences were generally weaker and did not exceed a p<0.05 (FWE corrected) threshold in the GLM analysis. At 3T profound inter-scanner differences are to be expected that could severely confound an unbalanced VBM analysis. These are like related to the receive bias of the radio-frequency hardware., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single-cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI-imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI-MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI-MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross-sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post-acquisition analysis) will enhance the relevance of MALDI-MSI technology in plant research., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We demonstrate an optically pumped semiconductor disk laser based on the (AlGaIn)(AsSb) material system, which operates at an emission wavelength of 2.8 μm. Up to 120 mW of output power were obtained in cw operation and more than 500 mW in pulsed mode. The performance of the present laser is discussed in comparison to shorter-wavelength semiconductor disk lasers based on the same materials system.

Both the nucellar projection (NP) and endosperm transfer cells (ETC) of the developing barley grain (harvested 8 days after flowering) were isolated by laser capture micro-dissection combined with pressure catapulting. Protein extracts were analyzed by nanoUPLC separation combined with ESI-Q-TOF mass spectrometry. The majority of the ~160 proteins identified were involved in translation, protein synthesis, or protein destination. The NP proteome was enriched for stress defense molecules, while proteins involved in assimilate transport and the mobilization of nutrients were common to both the NP and the ETC. The combined qualitative and quantitative protein profiling allowed for the identification of several proteins showing tissue specificity in their expression, which underlines the distinct biological functions of these two tissues within the developing barley grain.

Autophagy is a highly regulated trafficking pathway that leads to selective degradation of cellular constituents such as protein aggregates and excessive and damaged organelles. Atg1 is an essential part of the core autophagic machinery, which triggers induction of autophagy and the Cvt pathway. Although changes in Atg1 phosphorylation and complex formation are thought to regulate its function, the mechanism of Atg1 kinase activation remains unclear. Using a quantitative mass spectrometry approach, we identified 29 phosphorylation sites, of which five are either upregulated or downregulated by rapamycin treatment. Two phosphorylation sites, threonine 226 and serine 230, are evolutionarily conserved and located in the activation loop of the amino terminal kinase domain of Atg1. These phosphorylation events are not required for Atg1 localization to the phagosome assembly site (PAS), or the proper assembly of the multisubunit Atg1 kinase complex and binding to its activator Atg13. However, mutation of either one of these sites results in a loss of Atg1 kinase activity and its function in autophagy and the Cvt pathway. Taken together, our data suggest that phosphorylation of Atg1 on multiple sites provides critical mechanisms to regulate Atg1 function in autophagy and the Cvt pathway.

We describe here the effect of UV-B irradiation on the proteome and flavonoid content of the barley seedling leaf epidermis and mesophyll. Flavonoid analysis was performed using UPLC-PDA/-MS. The major flavonoid molecule responding to UV-B radiation was saponarin, and this accumulated in the epidermis, but not in the mesophyll. Changes in protein expression were determined using two-dimensional gel electrophoresis (2-DE) and identified 11 responsive proteins (seven up-regulated and two down-regulated in the epidermis; and one up- and one down-regulated in the mesophyll). A label-free LC-MS/MS(E) approach was applied for a subset of samples consisting of epidermis tissue and was able to detect a further 15 (11 up-regulated and four down-regulated) proteins. Most of the proteins with changed expression pattern after UV-B treatment were involved in initial responses characteristic for oxidative stress. Others were primary metabolism proteins involved in the supply of precursors for secondary metabolites. A separate analysis of epidermis and mesophyll tissue is important to give a spatially resolved picture of the response to UV-B treatment. The label-free LC-MS/MS(E) approach is complementary to the more conventional two-dimensional gel electrophoresis, as there was no overlap between the spectra of proteins identified by the two techniques.

Patients with trophic ulcers of lower extremities are relatively frequently diagnosed with diabetes mellitus with venous aetiology being the cause of these defects in up to 70% of patients. Chronic venous insufficiency most frequently results from primary venous insufficiency (mostly lower limb varices) or occurs secondarily as a result of deep vein thrombosis. Lower limb varices are common in human population; they affect 20-25% of women and 10-15% of men, and the incidence increases with age. With respect to radicality of treatment approaches, surgical management is unequivocally considered as the most effective. Apart from the traditional open surgery, clinical practice of the recent years has seen the advance of mini-invasive endovascular methods to treat varices of the main (stem) veins--radiofrequency and laser thermocoagulation. The main principle behind radiofrequency ablation is thermocoagulation of insufficient saphenous vein by bipolar radiofrequency current; endovenous laser uses, for the same purpose, energy of a beam of light. Both methods inactivate the insufficient veins while leaving them in the patient body. Thermal damage of the pathological venous wall leads to contraction and obliteration of the vein and, gradually, to its full resorption. Apart from minimizing recurrence, the technique is also valuable with respect to its cosmetic effect and gentleness of the procedure, allowing fast return to full activity. Studies comparing endovascular interventions with the traditional surgeries confirmed that radiofrequency or laser techniques are safe and well tolerated with the outcomes fully comparable to open surgery in its modern form.

The secretome of a tobacco cell suspension culture was investigated by a combined proteomic and metabolomic approach. Protein analysis from 2-DE gels led to identification of 32 out of 60 spots from culture medium. Identified proteins were mainly involved in stress defence and cell regeneration processes. Among them three putative new isoforms, e.g. for chitinase, peroxidase and beta-1,4-xylosidase were identified, not yet present in available protein databases for the genus Nicotiana. GC-MS analysis of time course experiments revealed significant changes for metabolites involved in energy transport, signalling and cell development. Among them, the most significant increase was found for putrescine in the medium of cultures entering the exponential phase. Results showed strong abundance of stress associated proteins and metabolites in the absence of elicitors or additional stress treatments.

Introduction: Periproctal abscess is a routine diagnosis of everyday surgical practice. Fournièr's gangrene with progress to the sepsis is one of the most serious complication of this disease., Case Report: The authors present a case review of fulminant surgical infection run as a Fournièr's gangrene and a necrotizig fasciitis of a right lower limb and body with the progress of severe septic status, which started on the strength of a contusion with formation of a periproctal absceding haematoma. A patienthood led to the long-term intensive care with a necessity of multiple surgeries and with subsequent rehabilitative care. This case is rare by the origin of thus extensive infection of soft tissue and its illustration of the heftiness of the patient's treatment with such a disease., Conclusion: Fournièr's gangrene and necrotizing fasciitis are loaded by a high-grade of mortality. It is one of the most serious surgical infection, which ends by death frequently. The therapy is primarily surgical, but it cannot be sufficient without aggressive intensive care of a patient.

Aims: Endovascular techniques of saphenous ablation are the miniinvasive alternatives of the radical surgical treatment. This study summarizes our own clinical experience with endovenous laser., Material and Methods: During 6 year period we performed in total 723 endovenous laser procedures of trunk varicose veins of lower extremities in 630 patients. Every patient was preoperatively examined clinically and with color duplex machine. Primary varicose veins were operated on in 664 cases, in 59 cases the procedure was performed in recurrent varicose veins with reflux in the residual saphenous trunk. Post-operative follow-up (clinical and duplex ultrasound) was performed after 5 days and 1 month, 6 months and yearly thereafter. The results were evaluated by comparison of CEAP clinical class and quality of life (QoL) pre- and post-operatively, by the percentage of recanalizations and also using Kaplan-Meier life-table method., Results: No thrombosis, nor pulmonary embolism were diagnosed in the post-operative period; from the whole cohort of 723 laser procedures, the postoperative data were available during different time periods in 718 cases (99.3%). Saphenous occlusion was verified in 97.3% after 1 month, non-occlusion or early reopening was seen in 2.7%. In total, 44 non-occluded trunk veins were found during the whole follow-up period (1-72 months, mean 15 months) which represents the final occlusion result of 93.9%. With stronger Kaplan-Meier analysis, we reached 88.9% occlusion rate during the follow-up period of 6 years. Cox regression analysis of factors influencing non-occlusion and early or late recanalisation of saphenous vein found 2 factors with statistical importance: energy per centimeter of vein length (p = 0.04) and laser power (p = 0.04). Cumulative rate of occlusions in 72 months horizon is significantly higher (94%) in patients treated with more than 50 J/cm compared to less than 50 J/cm (87%), log-rank test 0.039. When comparing the influence of laser power on the quality of saphenous occlusion, the treshold of 13W was set arbitrary based on median values in occluded and non-occluded cohorts and using the Kaplan-Meier survival method, the results of the treatment with power less than 13W and more than 13W were analysed. Using the power values less than 13W, the results were significantly better (p = 0.031) compared to power values of 13 W or more. Mean clinical CEAP classification improved from 2.22 (before operation) to 0.24 (1 month after) and 0.48 (last visit) and also QoL was significantly better in laser group compared to traditional surgery group (p < 0.001). The sick leave was also significantly shorter in the laser group ( median 0 days) compared to traditional group (median 40 days), p < 0.01., Conclusions: The results of endovenous laser ablation of varicose veins depend mainly on meticulous pre-operative ultrasound examination and sufficient technical parameters of the therapy. The present study supports the concept of "slow heating" during the endovenous laser treatment of varicose veins to achieve sufficient energy per centimetre of the vein and the optimal clinical outcome.

Background: Vein shrinkage is a surrogate marker for successful laser treatment of varicose veins. However, many controversies still remain concerning the best laser parameters to use. The aim of this study was standardisation of intraoperative energy dosages and pull-back rates to achieve optimal clinical results., Design: Ex-vivo study in surgically removed saphenous trunks., Material and Methods: Great saphenous veins were removed by Babcock stripping and irradiated with laser energy delivered by a laser diode emitting at 980 nm. In total, 279 vein segments (5 cm long) were treated using powers from 5-15 W. Vein segments were opened longitudinally and the circumference measured in the treated and untreated regions to assess thermal shrinkage., Results: The greatest shrinkage and minimum number of perforations was achieved using lower or medium power (8 to 12 W) with longer exposure to administer laser energy. The median percentage vein shrinkage was 50% (power 5 W), 45% (8 W), 40% (10 W), 45% (12 W) and 59% (15 W). When a higher power was used (15 W), the perforations were more frequent and carbonisation was marked., Conclusions: Our data suggests that similar efficacy with fewer vein perforations may be obtained with low or medium power settings and increased exposure when undertaking laser obliteration of saphenous trunks. This may result in fewer adverse events such as ecchymosis following treatment in patients.

Aims: Crossectomy (extended saphenofemoral or saphenopopliteal junction ligation) and stripping of refluxing saphenous trunk represent the basis of the traditional surgical therapy of varicose veins. Endovascular techniques of saphenous ablation are the mini-invasive alternatives of the radical surgical treatment. The objective of this study is the comparison of the endovenous laser ablation with and without crossectomy through open groin access., Material and Methods: Retroprospective study compares the results of the group of patients treated with simple endovenous laser ablation (HVL - 329 limbs) and HVL completed with crossectomy (35 limbs). Both groups were comparable in terms of basic demographic and preoperative clinical data (p > 0.05). In both cohorts, subgroups with identical laser parameters were selected (p > 0.05). All procedures were performed according the same standard protocol, EVL patients received LMWH in the postoperative period. The results were evaluated by the comparison of CEAP clinical class pre and postoperatively, the percentage of recanalizations and also using the Kaplan-Meier life-table method., Results: No thrombosis, nor pulmonary embolism were diagnosed in the post-operative period. During the follow-up (98% treated limbs), venous occlusion was observed in 88% of them (91.05 % in the endovenous group and 65.71% in the combined group). These differences are not statistically significant (p = 0.24). Same results were found using the Kaplan-Meier method (p = 0.086). Treatment significantly reduced CEAP clinical class in both groups but the results are better (C = 0.41 vs. 0.8) in the endovenous group (p = 0.004)., Conclusions: Endovenous ablation of the refluxing saphenous vein represents good alternative of crossectomy and stripping. Combination of both procedures is not effective and, on the contrary, can cause short and long-term complications.

Aims: The endovenous laser treatment of varicose veins has been using for several years throughout the world with clinical results comparable to traditional surgery. Nevertheless, many controversies still exist in the world literature in terms of parameters of laser generator and procedure itself. The aim of this laboratory study was the standardisation of the procedure and set-up of the optimal technical parameters to achieve maximal vein shrinkage as basic marker of successful long-term result., Material and Methods: The insufficient trunks of the long saphenous veins which were stripped during the traditional Babcock's stripping procedure, were irradiated with the laser energy delivered by the diode generator emitting 980 nm laser beam in the laboratory settings. In total, 279 vein segments were treated. We used the power of 5W, 8W, 10W, 12W and 15W during the maximal time possible to achieve the maximal shrinkage of the saphenous vein with minimal number of perforations. The study cohort consisted of two groups -in the first group the veins were filled with the blood (n = 139), in the other one the veins were empty (n = 140) to simulate the patient's position on the operating table. After the procedure, every vein segment was cut longitudinally, unfolded and its inner circumference was measured and compared to inner circumference of untreated part of the same venous segment., Results: Maximal shrinkage and minimal number of perforations were achieved using lower or medium power (8 to 12 W). Circumference of shrunken vein compared to normal venous circumference (100%) was as follows: 50% (power 5W), 45% (power 8W), 40% (power O1W), 45% (power 12W) and 58.6% (power 15W). These differences are statistically significant (p < 0.001). When higher power was used (15W), the perforations and carbonisations were more frequent and total energy was lower but the difference in amount of energy delivered was not significant (p = 0.379)., Conclusions: Shrinkage of the vein depends on laser power. Based on our experiments, we recommend photocoagulation with lower or medium power (8 to 12 W) and slower pull-back (0.2 to 2 mm/s) to achieve the sufficient energy per centimeter of the vein and the optimal long-term outcome.

Aims: Following surgical therapy, new varicose veins may develop, in particular at the site of the previous saphenofemoral junction. The objective of this study is the appreciation of the causes of the bad results of surgical procedures in the treatment of varicose veins and the proposal of the possibilities of their prevention., Material and Methods: Retrospective study of the group of 404 patients (573 procedures) with the diagnosis of the recurrence after previous radical surgery was performed. After exclusion of perforator refluxes, the most frequently, the great saphenous vein territory was affected (86%), followed by the small saphenous vein territory (14%). The group of 30 consecutive patients (35 limbs) with saphenofemoral or saphenopopliteal recurrence was selected for this study. Based on preoperative ultrasound and per-operative morphological findings, in 12 patients (14 limbs) - 8 women and 4 men, meticulous histopathological examination of the venous tissue block from the saphenofemoral or saphenopopliteal region, VEGF (Vascular endothelial growth factor) and protein S-100 investigation were performed., Results: In 14 limbs with sonographic and clinical suspition for neovascularisation, this entity was confirmed in 11 of them (79%) which represents 31.42% of the whole group., Conclusions: The varicose veins recurrences can be observed not only after technically or tactically imperfect primary procedure (which is surely the most common cause of the varicose veins recurrence), but also after the operations performed by the experienced venous surgeon in the perfect way. Neovascularisation explains a certain number of postoperative recurrences despite correctly conducted disconnection of the saphenous termination. The endovascular procedures (endovenous laser or radiofrequency saphenous obliteration) without open groin access can minimise the possibility of this complication.

Failed open reduction internal fixation (ORIF) of the proximal femur can render patients severely disabled. This study analyzed the short-term functional results and complications of total hip arthroplasty (THA) for complications of ORIF after hip fracture. A retrospective arthroplasty database review identified 36 patients treated with a THA for complications of ORIF of the hip. These patients were matched to a cohort of 36 patients who underwent primary unilateral THA for osteoarthritis. Study results indicate that THA for failed ORIF of the hip is a successful procedure despite concerns of increased intraoperative difficulty and risk of fracture.

Background: Intra-articular steroid hip injection (IASHI) has been prescribed for painful hip arthritis since the 1950s, but with advances in medical and surgical management its role is less certain today. There are very few published data on the utility or prescribing patterns of IASHI., Methods: We developed a questionnaire to seek expert opinion on IASHI that we distributed to practising Ontario-based members of the Canadian Orthopaedic Association. We systematically describe the current practices and expert opinion of 99 hip surgeons (73% response rate), focusing on indications, current use and complications experienced with IASHI., Results: Only 56% of surgeons felt that IASHI was therapeutically useful, with 72% of surgeons estimating that 60% or less of their patients achieved even transient benefit from IASHI. One-quarter of the surgeons believe that IASHI accelerates arthritis progression, most of whom had stated that it would be no great loss if IASHI was no longer available. Nineteen percent of the surgeons believed that the infection rate related to total hip arthroplasty (THA) may be increased after IASHI, and this was associated with fewer IASHIs ordered per year, compared with the number prescribed by those who did not feel that infection rates would increase., Conclusions: This systematic collection of expert opinions demonstrates that substantial numbers of surgeons felt that, in their patients, IASHI was not therapeutically helpful, may accelerate arthritis progression or may cause increased infectious complications after subsequent THA.

Immunosuppression following intra-articular injections of steroid into the hip may interfere with asepsis in a subsequent total hip arthroplasty (THA). We have undertaken a retrospective, matched, cohort study of infective complications after THA, in 40 patients who had received such an injection and 40 who had not. In the injection group there were five revisions, four of which were for deep infection. There were none in the matched group. The overall rate of revision in our database of 979 primary THAs was 1.02%. Six additional patients who had received injections underwent investigation for infection because of persistent problems in the hip as compared with one in the control group.

Study Design: This repeated-measures biomechanical study evaluated the tensile force required to cause bipedicular (hangman's) fractures in isolated porcine C2 specimens, and the subsequent force to failure after direct fracture repair with bipedicular lag screws., Objectives: To assess the pullout strength of direct lag screw fixation of hangman's fracture, relative to the strength of the intact specimens., Summary of Background Data: Clinical studies have reported successful treatment of hangman's fractures by direct screw repair followed by a collar after surgery. However, to the authors' best knowledge, there has not yet been a biomechanical analysis of the force required for screw pullout after direct repair., Methods: Of 60 mounted porcine C2 specimens subjected to tensile anteroposterior force, hangman's fracture occurred in 15 cases, 12 of which were fixed with bipedicular 4.0-mm lag screws. These fixed specimens were retested to failure in the same manner., Results: Most specimens had laminar fractures after the application of tensile force, with 15 of 60 (25%) showing bipedicular fractures. The force to bipedicular failure was 3259.1 +/- 148.5 N (mean +/- standard error of the mean). After screw fixation, the force to failure of the same specimens was 882.0 +/- 108.5 N (mean +/- standard error of the mean), or 27.3% of the intact bone., Conclusions: The pullout strength was substantial (882 N), although the relative strength of fixation was only 27.3% of the fracture strength shown by the intact specimens. The findings are discussed in relation to previous studies. To the authors' best knowledge, this is the first study to examine screw pullout forces after direct repair of hangman's fracture.

The authors present their experiences with new miniinvasive treatment of varicose veins--endovenous diode laser system. The results of 60 procedures treating truncal varices are reported with special concern to percutaneous access technique under ultrasound guidance. This new concept based on haemodynamic ultrasound findings is compared to the traditional surgical technique of cross-sectomy and stripping.

The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compounds. We recently showed that the separated periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a K(d) of 5.5 microM at pH 7. To identify positively charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titration calorimetry (ITC). In 12 cases, the K(d) for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 microM). In one case (R98A), the K(d) was 6-fold decreased (0.8 microM), whereas in four cases (R66A, H69A, R107A, and K109A) the K(d) was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV circular dichroism (CD) spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the melting point (T(m)) of wild-type CitAP and mutein R98A by 6.2 and 9.5 degrees C, respectively, but had no effect on the T(m) of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.

Multiple specialized exposures have been described for revision total hip arthroplasty. In this article, we report on a series of 7 patients who underwent major isolated acetabular revision surgery through a modified iliofemoral approach. The use of this approach was deemed desirable because of the individual pathology present in each case, particularly major acetabular bone loss (Paprosky 2C/3B). However, successful reconstruction was achieved in all cases with the most frequent complication being meralgia paresthetica., (Copyright 2003, Elsevier Science (USA). All rights reserved.)

Unlabelled: The authors evaluate the results achieved in two groups of patients operated on for varicose veins in small saphenous vein (SSV) territory in 3 year interval. In the first group gathered from January to December 1998 (114 patients--36 men and 78 women, 123 procedures--118 primary and 5 re-do) the operative indication was based on the clinical examination and continual doppler evaluation. These patients were operated on in general, spinal or local and flash general anaesthesia. In the second group gathered in the same period of the year 2001 (72 patients--21 men and 51 women) 75 procedures were performed (3 patients with bilateral operation). In this group, 49 patients with 50 procedures were selected. The diagnosis was based not only on clinical and continuous doppler examination, but mainly on colour flow duplex mapping. The operation was performed on strictly ambulatory basis using pure local anaesthesia completed with small dose of sedation. Any patient needed complementary sclerotherapy one month after procedure. In mid-term follow-up complementary conservative treatment was necessary in 51 p.c. of the whole series., Conclusion: Use of colour coded duplex ultrasound in preoperative evaluation of varicose veins patients enabled us to precise preoperative diagnosis, to diminish the number of aggressive surgical procedures in favour of less traumatic operations and to perform this surgery on ambulatory basis. Nevertheless, small sahenous vein surgery still remains delicate and sometimes also hazardous.