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The purpose of this review was to summarize the evidence with regard to behavioral and psychosocial assessment of the periodontitis patient, the candidate for implant therapy, and the peri-implantitis patient. Periodontitis has an adverse effect on quality of life and its treatment can lead to significant improvements experienced by the patient. The latter is true for rehabilitation with dental implants, although patients harbor diverse expectations and perceptions of implant therapy, which can often interfere with satisfaction and/or influence long-term success. A thorough behavioral assessment of the candidate for implant therapy is essential, which should include, perceptions, expectations, as well as risk for behavioral disorders. Remedial action is essential to correct misperceptions and any identified risks. Finally, patients have limited awareness of limited ability to identify signs of peri-implantitis. The diagnosis of peri-implantitis can be a cause of significant distress, resentment, and loss of trust to the treatment and the caregivers. Despite documented value in clinical research, currently available instruments assessing patient-reported outcomes have little application in day-to-day clinical practice. Face-to-face patient to doctor open-ended communication remains the most effective way to comprehensively establish the long-term "therapeutic alliance" essential for the long journey for the periodontitis patient., (© 2024 The Author(s). Periodontology 2000 published by John Wiley & Sons Ltd.)

Aims/hypothesis: People with type 2 diabetes are heterogeneous in their disease trajectory, with some progressing more quickly to insulin initiation than others. Although classical biomarkers such as age, HbA 1c and diabetes duration are associated with glycaemic progression, it is unclear how well such variables predict insulin initiation or requirement and whether newly identified markers have added predictive value., Methods: In two prospective cohort studies as part of IMI-RHAPSODY, we investigated whether clinical variables and three types of molecular markers (metabolites, lipids, proteins) can predict time to insulin requirement using different machine learning approaches (lasso, ridge, GRridge, random forest). Clinical variables included age, sex, HbA 1c , HDL-cholesterol and C-peptide. Models were run with unpenalised clinical variables (i.e. always included in the model without weights) or penalised clinical variables, or without clinical variables. Model development was performed in one cohort and the model was applied in a second cohort. Model performance was evaluated using Harrel's C statistic., Results: Of the 585 individuals from the Hoorn Diabetes Care System (DCS) cohort, 69 required insulin during follow-up (1.0-11.4 years); of the 571 individuals in the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) cohort, 175 required insulin during follow-up (0.3-11.8 years). Overall, the clinical variables and proteins were selected in the different models most often, followed by the metabolites. The most frequently selected clinical variables were HbA 1c (18 of the 36 models, 50%), age (15 models, 41.2%) and C-peptide (15 models, 41.2%). Base models (age, sex, BMI, HbA 1c ) including only clinical variables performed moderately in both the DCS discovery cohort (C statistic 0.71 [95% CI 0.64, 0.79]) and the GoDARTS replication cohort (C 0.71 [95% CI 0.69, 0.75]). A more extensive model including HDL-cholesterol and C-peptide performed better in both cohorts (DCS, C 0.74 [95% CI 0.67, 0.81]; GoDARTS, C 0.73 [95% CI 0.69, 0.77]). Two proteins, lactadherin and proto-oncogene tyrosine-protein kinase receptor, were most consistently selected and slightly improved model performance., Conclusions/interpretation: Using machine learning approaches, we show that insulin requirement risk can be modestly well predicted by predominantly clinical variables. Inclusion of molecular markers improves the prognostic performance beyond that of clinical variables by up to 5%. Such prognostic models could be useful for identifying people with diabetes at high risk of progressing quickly to treatment intensification., Data Availability: Summary statistics of lipidomic, proteomic and metabolomic data are available from a Shiny dashboard at https://rhapdata-app.vital-it.ch ., (© 2024. The Author(s).)

Introduction: Type 2 diabetes (T2D) onset, progression and outcomes differ substantially between individuals. Multi-omics analyses may allow a deeper understanding of these differences and ultimately facilitate personalised treatments. Here, in an unsupervised "bottom-up" approach, we attempt to group T2D patients based solely on -omics data generated from plasma., Methods: Circulating plasma lipidomic and proteomic data from two independent clinical cohorts, Hoorn Diabetes Care System (DCS) and Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS), were analysed using Similarity Network Fusion. The resulting patient network was analysed with Logistic and Cox regression modelling to explore relationships between plasma -omic profiles and clinical characteristics., Results: From a total of 1,134 subjects in the two cohorts, levels of 180 circulating plasma lipids and 1195 proteins were used to separate patients into two subgroups. These differed in terms of glycaemic deterioration (Hazard Ratio=0.56;0.73), insulin sensitivity and secretion (C-peptide, p =3.7e-11;2.5e-06, DCS and GoDARTS, respectively; Homeostatic model assessment 2 (HOMA2)-B; -IR; -S, p=0.0008;4.2e-11;1.1e-09, only in DCS). The main molecular signatures separating the two groups included triacylglycerols, sphingomyelin, testican-1 and interleukin 18 receptor., Conclusions: Using an unsupervised network-based fusion method on plasma lipidomics and proteomics data from two independent cohorts, we were able to identify two subgroups of T2D patients differing in terms of disease severity. The molecular signatures identified within these subgroups provide insights into disease mechanisms and possibly new prognostic markers for T2D., Competing Interests: GR has received grant funding from, and is a consultant for, Sun Pharmaceuticals Inc. KS is CEO of Lipotype. KS and CK are shareholders of Lipotype. MG is an employee of Lipotype. MH is an employee of Janssen Research & Development. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Li, Dragan, Tran, Fung, Kuznetsov, Hansen, Beulens, Hart, Slieker, Donnelly, Gerl, Klose, Mehl, Simons, Elders, Pearson, Rutter and Ibberson.)

Obesity is considered by many as a lifestyle choice rather than a chronic progressive disease. The Innovative Medicines Initiative (IMI) SOPHIA (Stratification of Obesity Phenotypes to Optimize Future Obesity Therapy) project is part of a momentum shift aiming to provide better tools for the stratification of people with obesity according to disease risk and treatment response. One of the challenges to achieving these goals is that many clinical cohorts are siloed, limiting the potential of combined data for biomarker discovery. In SOPHIA, we have addressed this challenge by setting up a federated database building on open-source DataSHIELD technology. The database currently federates 16 cohorts that are accessible via a central gateway. The database is multi-modal, including research studies, clinical trials, and routine health data, and is accessed using the R statistical programming environment where statistical and machine learning analyses can be performed at a distance without any disclosure of patient-level data. We demonstrate the use of the database by providing a proof-of-concept analysis, performing a federated linear model of BMI and systolic blood pressure, pooling all data from 16 studies virtually without any analyst seeing individual patient-level data. This analysis provided similar point estimates compared to a meta-analysis of the 16 individual studies. Our approach provides a benchmark for reproducible, safe federated analyses across multiple study types provided by multiple stakeholders.

Background: An alveolar ridge preservation (ARP) procedure is commonly performed to preserve ridge width and height for optimal dental implant placement. Mixing different sources of bone grafts may help maximize their properties to obtain predictable ARP results., Objective: The aim of this case series is to highlight the clinical and radiographic outcomes of a combined allograft and xenograft approach for a predictable ARP procedure., Materials and Methods: Eleven clinical cases that required tooth extraction and future implant placement were selected for this case series. All surgeries were performed by a single clinician in a private practice setting. All cases underwent an ARP procedure, which included atraumatic extraction, degranulation, assessment of the socket, and the use of a combination of allograft and xenograft fibers/granules in a 1:1 mixing approach. Based on the socket defect, either a type I or type III collagen membrane was used during the procedure. In all cases CBCT imaging was done prior to surgical implant placement, and digital implant planning was performed integrating interdisciplinary best practices. Implant placement was carried out using manufacturer's recommendations., Results: Five male and five female patients were included in the study, with one of the male patients having two teeth extracted for a total of 11 cases. The reasons for teeth extraction included fracture, endodontic failure, periodontal infection, and subgingival decay. Healing was uneventful after the ARP procedure for all patients. A minimum of 120 days elapsed between ARP and the surgical implant procedure. Complete radiographic bone fill was observed in the CBCT image for all extraction sites, and no additional bone augmentation was needed at the time of implant placement., Conclusion: This case series demonstrates that based on clinical and radiographic outcomes, a combination of allogenic and xenogenic bone substitute can be utilized to obtain predictable results following ARP. Future randomized controlled trials that can compare different ratios between the two biomaterials are indicated to guide clinicians in the mixing protocols and their outcomes.

We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression., (© 2023. The Author(s).)

Epigenetic regulation of gene expression using histone deacetylase (HDAC) inhibitors is a promising strategy for developing new anticancer agents. The most common HDAC inhibitors are hydroxamates, which, though highly potent, have limitations due to their poor pharmacokinetic properties and lack of isoform selectivity. Trifluoromethylketones (TFMK) developed as alternatives to hydroxamates are rapidly metabolized to inactive trifluoromethyl alcohols in vivo, which prevented their further development as potential drug candidates. In order to overcome this limitation, we designed trifluoropyruvamides (TFPAs) as TFMK surrogates. The presence of an additional electron withdrawing group next to the ketone carbonyl group made the hydrate form of the ketone more stable, thus preventing its metabolic reduction to alcohol in vivo. In addition, this structural modification reduces the potential of the TFMK group to act as a covalent warhead to eliminate off-target effects. Additional structural changes in the cap group of the inhibitors gave analogues with IC 50 values ranging from upper nanomolar to low micromolar in the cytotoxicity assay, and they were more selective for cancer cells over normal cells. Some of the most active analogues inhibited HDAC enzymes with low nanomolar IC 50 values and were found to be more selective for HDAC8 over other isoforms. These molecules provide a new class of HDAC inhibitors with a metabolically stable metal-binding group that could be used to develop selective HDAC inhibitors by further structural modification., 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 © 2022 Elsevier Masson SAS. All rights reserved.)

With coronavirus disease 2019 (COVID-19) leading to ubiquitous changes across the education system, Tufts University School of Dental Medicine took advantage of their new, fast-changing environment to foster engagement among faculty members regarding curricular modifications and their impact on assessment outcomes. A virtual curricular retreat was planned, where adaptations could be discussed through the lens of Miller's Pyramid. The retreat provided an opportunity for faculty to participate in a guided dialogue via a "think-pair-share" activity that resulted in documenting the outcomes of recent curriculum changes while allowing for reflection for future improvement., (© 2021 American Dental Education Association.)

Type 2 diabetes is a multifactorial disease with multiple underlying aetiologies. To address this heterogeneity, investigators of a previous study clustered people with diabetes according to five diabetes subtypes. The aim of the current study is to investigate the etiology of these clusters by comparing their molecular signatures. In three independent cohorts, in total 15,940 individuals were clustered based on five clinical characteristics. In a subset, genetic ( N = 12,828), metabolomic ( N = 2,945), lipidomic ( N = 2,593), and proteomic ( N = 1,170) data were obtained in plasma. For each data type, each cluster was compared with the other four clusters as the reference. The insulin-resistant cluster showed the most distinct molecular signature, with higher branched-chain amino acid, diacylglycerol, and triacylglycerol levels and aberrant protein levels in plasma were enriched for proteins in the intracellular PI3K/Akt pathway. The obese cluster showed higher levels of cytokines. The mild diabetes cluster with high HDL showed the most beneficial molecular profile with effects opposite of those seen in the insulin-resistant cluster. This study shows that clustering people with type 2 diabetes can identify underlying molecular mechanisms related to pancreatic islets, liver, and adipose tissue metabolism. This provides novel biological insights into the diverse aetiological processes that would not be evident when type 2 diabetes is viewed as a homogeneous disease., (© 2021 by the American Diabetes Association.)

Aims/hypothesis: Five clusters based on clinical characteristics have been suggested as diabetes subtypes: one autoimmune and four subtypes of type 2 diabetes. In the current study we replicate and cross-validate these type 2 diabetes clusters in three large cohorts using variables readily measured in the clinic., Methods: In three independent cohorts, in total 15,940 individuals were clustered based on age, BMI, HbA 1c , random or fasting C-peptide, and HDL-cholesterol. Clusters were cross-validated against the original clusters based on HOMA measures. In addition, between cohorts, clusters were cross-validated by re-assigning people based on each cohort's cluster centres. Finally, we compared the time to insulin requirement for each cluster., Results: Five distinct type 2 diabetes clusters were identified and mapped back to the original four All New Diabetics in Scania (ANDIS) clusters. Using C-peptide and HDL-cholesterol instead of HOMA2-B and HOMA2-IR, three of the clusters mapped with high sensitivity (80.6-90.7%) to the previously identified severe insulin-deficient diabetes (SIDD), severe insulin-resistant diabetes (SIRD) and mild obesity-related diabetes (MOD) clusters. The previously described ANDIS mild age-related diabetes (MARD) cluster could be mapped to the two milder groups in our study: one characterised by high HDL-cholesterol (mild diabetes with high HDL-cholesterol [MDH] cluster), and the other not having any extreme characteristic (mild diabetes [MD]). When these two milder groups were combined, they mapped well to the previously labelled MARD cluster (sensitivity 79.1%). In the cross-validation between cohorts, particularly the SIDD and MDH clusters cross-validated well, with sensitivities ranging from 73.3% to 97.1%. SIRD and MD showed a lower sensitivity, ranging from 36.1% to 92.3%, where individuals shifted from SIRD to MD and vice versa. People belonging to the SIDD cluster showed the fastest progression towards insulin requirement, while the MDH cluster showed the slowest progression., Conclusions/interpretation: Clusters based on C-peptide instead of HOMA2 measures resemble those based on HOMA2 measures, especially for SIDD, SIRD and MOD. By adding HDL-cholesterol, the MARD cluster based upon HOMA2 measures resulted in the current clustering into two clusters, with one cluster having high HDL levels. Cross-validation between cohorts showed generally a good resemblance between cohorts. Together, our results show that the clustering based on clinical variables readily measured in the clinic (age, HbA 1c , HDL-cholesterol, BMI and C-peptide) results in informative clusters that are representative of the original ANDIS clusters and stable across cohorts. Adding HDL-cholesterol to the clustering resulted in the identification of a cluster with very slow glycaemic deterioration., (© 2021. The Author(s).)

Aims and Objectives: There is an increased interest to incorporate interprofessional educational models in the academic dental institutions to serve both student education and patient care. The aim of this report is to integrate evidence-based dentistry (EBD) with an interprofessional approach to treat methadone maintenance therapy (MMT) patients. Current example illustrates the case of a 26-year-old man receiving MMT who presented at Tufts University School of Dental Medicine with the chief complaint of "I want to fix my teeth." He presented with a collapsed vertical dimension of occlusion, extensive partial edentulism on both arches with bone loss, and a past history of drug abuse since the age of 16 years., Materials and Methods: The 5-step (ask, acquire, appraise, apply, assess) framework for the EBD process was used. First, the PICO (population, intervention, comparison, and outcome) question was asked, and then the literature was acquired and appraised. An expert librarian assisted in finding articles on the effects of methadone on the oral cavity and consequences that will affect dental treatment. The search was conducted on PubMed, using the following keywords: oral health, dentistry, dental health, and methadone. The search was performed from 1/1/2005 to 1/1/2018. After appraisal, the studies were applied in the clinical setting and treatment outcomes were assessed both subjectively and objectively., Results: The initial search identified there is sparse evidence on the topic. Only 34 articles were acquired. Based on the scientific evidence published, the interprofessional expertise of the clinical care team, and patient's perspective, 4 treatment plan options were proposed. The selected treatment plan was considered the best option considering an EBD person-centered approach. Progress of treatment, outcomes, and lessons learned were assessed., Conclusions: This study demonstrates that incorporating EBD concepts and an interprofessional approach, MMT patients can be successfully treated. Future studies on this topic are recommended, specially considering the growth of the opioid epidemic in the past years and the need to treat the MMT patients and educate students., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.

Aim: Holoprosencephaly (HPE) is the most common brain malformation. A wide spectrum of anatomical variants are characterized by a lack of midline separation of the cerebral hemispheres. The aim of this study was to assess the ultrasound diagnostic criteria for HPE., Material and Method: A database of 175 fetuses with central nervous system anomalies identified by ultrasound was collected retrospectively from 2006 to 2016 in this multicenter, retrospective, observational study. Among them 18 cases (10.2%) with HPE were identified., Results: The prevalence of HPE was 2.5:10.000 with the sex distributionmale:female of 1:1.6. Six cases were alobar subtype, 3 were semilobar, 7 were lobar and 2 were middle interhemispheric variant. In the second trimester, we consider that the abnormal fusion of the lateral ventricles and the absence of the cavum septum pellucidum are the most important landmarks for HPE. Facial abnormalities varied considerably., Conclusion: This study illustrates the heterogeneity of HPE with different cerebral and facial appearances.

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans. Comparative proteomic analysis of consecutively collected susceptible and resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1 H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar. Together these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.

Advancements in research and technology are transforming our world. The dental profession is changing too, in the light of scientific discoveries that are advancing biological technology-from new biomaterials to unravelling the genetic make-up of the human being. As health professionals, we embrace a model of continuous quality improvement and lifelong learning. Our pedagogical approach to incorporating the plethora of scientific-technological advancements calls for us to shift our paradigm from emphasis on skill acquisition to knowledge application. The 2017 ADEE/ADEA workshop provided a forum to explore and discuss strategies to ensure faculty, students and, ultimately, patients are best positioned to exploit the opportunities that arise from integrating new technological advances and research outcomes. Participants discussed methods of incorporating the impact of new technologies and research findings into the education of our dental students. This report serves as a signpost of the way forward and how to promote incorporation of research and technology advances and lifelong learning into the dental education curriculum., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Various studies reported beneficial effects of dehydroepiandrosterone (DHEA) and its sulphate (DHEAS), the neurosteroids involved in various brain functions, on synaptic plasticity, neuronal survival, memory, learning and behavior. This study aimed to investigate the behavioral profile of acute DHEA treatment by using active avoidance (AA) task, primarily predictive of the effects on the retrieval-based learning, and by applying forced swim test (FST), for assessment of antidepressant-like potential. Adult male Wistar rats received intraperitoneal injections of either DHEA (2, 10, 20mg/kg) or solvent, 30min prior to testing. DHEA, in a manner resembling an inverted U shape, influenced the retrieval imposed to rats in AA paradigm. The significant improvement of the performance in the retention session was observed following 10mg/kg DHEA treatment and it was not due to the changes in the motor activity, as indicated by unaltered locomotor parameters (inter-trial crossing). Moreover, 10mg/kg of DHEA significantly decreased the duration of immobility in FST, demonstrating antidepressant-like effects. The capability of bicuculline (2mg/kg) to antagonize the effects of DHEA has been evaluated simultaneously. The retrieval-facilitating as well as antidepressant-like effects of 10mg/kg DHEA were counteracted by bicuculline, a competitive antagonist of GABA A receptors, suggesting involvement of GABAergic system. These results support administration of DHEA as potential therapeutic strategy for treating depression and related cognitive impairments, but emphasized the importance of adequate dosing, as DHEA levels that are too high or too low may not be beneficial., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Wheat gluten confers superior baking quality to wheat based products but elicits a pro-inflammatory immune response in patients with celiac disease. Transamidation of gluten by microbial transglutaminase (mTG) and tissue transglutaminase (tTG) reduces the immunogenicity of gluten; however, little information is available on the minimal modification sufficient to eliminate gliadin immunogenicity nor has the effectiveness of transamidation been studied with T-cell clones from patients. Here we demonstrate that mTG can efficiently couple three different acyl-acceptor molecules, l-lysine, glycine ethyl ester, and hydroxylamine, to gliadin peptides and protein. While all three acyl-acceptor molecules were cross-linked to the same Q-residues, not all modifications were equally effective in silencing T-cell reactivity. Finally, we observed that tTG can partially reverse the mTG-catalyzed transamidation by its isopeptidase activity. These results set the stage to determine the impact of these modifications on the baking quality of gluten proteins and in vivo immunogenicity of such food products.

Objective: To estimate the patient-specific risk of pre-eclampsia (PE) at 30-34 weeks' gestation by a combination of maternal characteristics and medical history with multiples of the median (MoM) values of mean arterial pressure (MAP), uterine artery pulsatility index (UtA-PI), serum placental growth factor (PlGF) and serum soluble fms-like tyrosine kinase-1 (sFlt-1), and stratify women into high-, intermediate- and low-risk management groups., Methods: This was a prospective observational study in women attending a third-trimester ultrasound scan at 30-34 weeks as part of routine pregnancy care. Patient-specific risks of delivery with PE at < 4 weeks from assessment and at < 40 weeks' gestation were calculated using the competing-risks model to combine the prior risk from maternal characteristics and medical history with MoM values of MAP, UtA-PI, PlGF and sFlt-1. On the basis of these risks, the population was stratified into high-, intermediate- and low-risk groups. Different risk cut-offs were used to vary the proportion of the population stratified into each risk category and the performance of screening for delivery with PE at < 4 weeks from assessment and delivery with PE from 4 weeks after assessment and up to 40 weeks' gestation was estimated., Results: The study population of 8128 singleton pregnancies included 234 (2.9%) that subsequently developed PE. Using a risk cut-off of 1 in 50 for PE delivering at < 4 weeks and a risk cut-off of 1 in 150 for PE delivering at < 40 weeks' gestation, the proportion of the population stratified into high, intermediate and low risk was about 3%, 26% and 71%, respectively. The high-risk group contained 90% of pregnancies with PE at < 4 weeks and 40% of those with PE at 4 weeks from assessment to 40 weeks' gestation. The intermediate-risk group contained a further 49% of women with PE at 4 weeks from assessment to 40 gestational weeks. In the low-risk group, none of the women developed PE at < 4 weeks and only 0.3% developed PE at 4 weeks to 40 gestational weeks., Conclusion: The study presents risk stratification of PE by the combined test at 30-34 weeks, aiming to identify a high-risk group in need of intensive monitoring from the time of the initial assessment and up to 40 weeks' gestation and an intermediate-risk group in need of monitoring from 4 weeks after the initial assessment and up to 40 weeks' gestation. All pregnancies would need to be reassessed at 40 weeks' gestation. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd., (Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.)

Objective: To compare the performance of screening by soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio > 38 for the prediction of delivery with pre-eclampsia (PE) at < 1 week and < 4 weeks from assessment when the test is carried out at 31-34 vs 35-37 weeks' gestation., Methods: This was a prospective observational study in women attending a third-trimester ultrasound scan as part of routine pregnancy care; the visit was at 30-34 weeks' gestation in the first phase of the study and at 35-37 weeks in the second phase. Serum sFlt-1 and PlGF were measured and their ratio calculated. We estimated the detection rate (DR) and false-positive rate (FPR) of sFlt-1/PlGF ratio > 38 for predicting delivery with PE at < 1 week and < 4 weeks after assessment and compared the performance of screening when the test was carried out at 31 + 0 to 33 + 6 vs 35 + 0 to 36 + 6 weeks' gestation., Results: The study population included 8063 singleton pregnancies that were examined at 31-34 weeks and 3703 at 35-37 weeks. Delivery with PE occurred at < 1, < 4 and ≥ 4 weeks from assessment in five (0.1%), 29 (0.4%) and 202 (2.5%) women assessed at 31-34 weeks, respectively, and in seven (0.2%), 39 (1.1%) and 21 (0.6%) of those assessed at 35-37 weeks. In women without PE, the median sFlt-1/PlGF ratio increased with gestational age at screening and a ratio of 38 was just below the 99 th percentile at 32 weeks' gestation and just below the 90 th percentile at 36 weeks. In the two gestational windows, the DR of PE delivering < 4 weeks from assessment was similar (75.9% (95% CI, 56.5-89.7%) vs 79.5% (95% CI, 63.5-90.7%)), but the FPR was substantially lower at 31-34 weeks than at 35-37 weeks (1.7% (95% CI, 1.4-2.0%) vs 9.6% (95% CI, 8.7-10.6%)). The number of cases with PE delivering < 1 week from assessment was small, but similarly, in the two gestational windows, the DR was comparable (80.0% (95% CI, 28.4-99.5%) vs 85.7% (95% CI, 42.1-99.6%)), and the FPR was substantially lower at 31-34 weeks than at 35-37 weeks (1.9% (95% CI, 1.6-2.2%) vs 10.2% (95% CI, 9.3-11.3%))., Conclusion: The performance of sFlt-1/PlGF ratio > 38 in the prediction of delivery with PE at < 1 and < 4 weeks from assessment is substantially different when the assessment is at 31-34 weeks' gestation compared to at 35-37 weeks. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd., (Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.)

Objective: To evaluate a soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) ratio cut-off of 38 for the prediction of pre-eclampsia (PE) in routine assessment in singleton pregnancies at 30-37 weeks' gestation., Methods: This was a prospective observational study in women attending a third-trimester ultrasound scan at 30-37 weeks as part of routine pregnancy care. Serum sFlt-1 and PlGF were measured and their ratio was calculated. We estimated the detection rate (DR), false-positive rate (FPR), positive predictive value (PPV) and negative predictive value (NPV) of sFlt-1/PlGF ratio >38 for the prediction of delivery with PE at < 1, < 4 and ≥ 4 weeks after assessment., Results: The study population of 12 305 singleton pregnancies was examined at a median of 32.4 (range, 30.0-36.9) weeks and included 14 (0.11%), 77 (0.63%) and 227 (1.84%) cases that subsequently delivered with PE at < 1, < 4 or ≥ 4 weeks' after assessment, respectively. The DR, FPR, PPV and NPV of sFlt-1/PlGF ratio > 38 in the prediction of delivery with PE at < 1 week were 78.6%, 4.5%, 1.9% and 99.97%, respectively; the values for delivery with PE at < 4 weeks were 76.6%, 4.1%, 10.4% and 99.85% and for delivery with PE ≥ 4 weeks were 20.7%, 4.3%, 8.3% and 98.47%., Conclusion: In routine screening of singleton pregnancies, the performance of a sFlt-1/PlGF ratio > 38 is modest for the prediction of delivery with PE at < 1 and at < 4 weeks after assessment and poor for the prediction of delivery with PE at ≥ 4 weeks after assessment. A sFlt-1/PlGF ratio > 38 predicted 79% of cases delivering with PE at < 1 week after assessment, at a FPR of 4.5%; consequently, a policy of hospitalizing patients with a ratio > 38 would potentially lead to unnecessary hospitalization in 4.5% of pregnancies and a ratio of ≤ 38 would falsely reassure one fifth of women who will deliver with PE within 1 week of assessment. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd., (Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.)

Background: Although most postmenopausal women diagnosed with endometrial cancer usually present with vaginal bleeding, when complete cervical stenosis is present, this sign may be missing. In these cases, the patient usually complaints for pelvic or abdominal pain while the transvaginal ultrasonography might reveal the presence of an intrauterine fluid collection in association with a thickened endometrial lining., Case Report: We present the case of a 65-year-old patient who presented with association of pelvic pain, enlarged uterine cavity with an underlying hematometra and an irregular, thickened endometrium who was submitted to surgery for total histerectomy, bilateral adnexectomy, pelvic and para-aortic lymph node dissection., Conclusion: Histopathological studies revealed the presence of a well-differentiated endometrial adenocarcinoma. At three years of follow-up, the patient is free of any recurrent disease., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro-Pro endopeptidase (PPEP-1). PPEP-1 cleaves C. difficile cell surface proteins (e.g. CD2831). Here, we confirmed that PPEP-1 has a unique preference for prolines surrounding the scissile bond. Moreover, we show that it exhibits a high preference for an asparagine at the P2 position and hydrophobic residues at the P3 position. Using a PPEP-1 knockout C. difficile strain, we demonstrate that the removal of the collagen binding protein CD2831 is fully attributable to PPEP-1 activity. The PPEP-1 knockout strain demonstrated higher affinity for collagen type I with attenuated virulence in hamsters., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In 2010, the Romanian Angel Appeal Foundation launched a 3-year national training and development programme to develop and deliver a model of diagnostic and therapeutic services aimed at promoting social inclusion for children and young people with autism spectrum disorders. The project adopted a number of strategies aimed at developing knowledge and skills among professionals and increasing awareness in political and public spheres: (a) a three-stage training programme designed to increase knowledge of autism spectrum disorders and promote best practice among professionals working in services providing for children with autism spectrum disorders and their families, on a nationwide basis; (b) two online courses for general practitioners and psychiatrists, with content relating to the identification, diagnosis and treatment of autism spectrum disorders; (c) a total of 40 counselling and assistance centres for people with autism spectrum disorders were launched in partnership with local authorities; (d) a national strategy for social and professional integration of people with autism spectrum disorders developed through consultation with political, statutory and voluntary sector partners; and (e) a nationwide media campaign to raise awareness of the needs of children and young people with autism spectrum disorders that reached over eight million people. The project provides a transferable model to achieve important improvements in the quantity and quality of services on a national level within a brief time frame., (© The Author(s) 2014.)

Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90β, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.

Immunoglobulin E (IgE) is a heterodimeric glycoprotein involved in antiparasitic and allergic immune reactions. IgE glycosylation is known to exhibit significant interindividual variation, and several reports have indicated its relevance in determining IgE activity. Here, we present site-specific glycosylation analysis of IgE from three different sources: IgE from the serum of a hyperimmune donor, from the pooled serum of multiple nondiseased donors, and from the pooled serum of 2 patients with IgE myeloma. The heavy chains were isolated and digested with either trypsin, proteinase K, or chymotrypsin, which permitted coverage of all seven potential N-glycosylation sites. The resulting (glyco-)peptides were analyzed by nano-reversed-phase-LC-MS/MS and MALDI-TOF/TOF-MS/MS. Site Asn264 was shown to be unoccupied. In all three samples, site Asn275 contained exclusively oligomannosidic structures with between 2 and 9 mannoses, whereas sites Asn21, Asn49, Asn99, Asn146, and Asn252 contained exclusively complex-type glycans. For the nonmyeloma IgE, the majority of these glycans were biantennary and core-fucosylated and contained one or two terminal N-acetylneuraminic acids. In contrast, myeloma IgE showed a higher abundance of triantennary and tetraantennary glycan structures and a low abundance of species with a bisecting N-acetylglucosamine. Our approach allows comparison of the glycosylation of IgE samples in a site-specific manner.

Aim: To determine correlation between central corneal thickness (CCT) and applanation intraocular pressure (IOP) in normal patients with primary open angle glaucoma and patients with ocular hypertension., Methods: Two-year retrospective study designed to determine correlation between CCT and IOP. Records of 121 patients were included in the study. Inclusion criteria were: IOP higher than 22 mm Hg for a group with ocular hypertension but without functional, morphological damage and progression, diagnosis of glaucoma for a group with open-angle glaucoma and normal ophthalmological results for a control group. Patients who were mono-oculus, patients with secondary glaucoma and who had surgical treatment were excluded. The patients were selectively grouped according to types of glaucoma, by gender and age. The IOP values were measured by Goldman's applanation tonometer, CCT values were determined using ultrasonic pachymeter, in the period from January 2011 to December 2012., Results: Of total 121 subjects, 51 had primary open angle glaucoma (POAG), 40 had ocular hypertension (OHT) and 30 had normal ophthalmological findings (control group). The CCT values in OHT group 529.37+/-25.18 μm were greater than of both POAG, 501.02+/-14.04 μm and control group, 497.37+/-14.90 μm. The IOP values in OHT group were 20.25+/- 3.62 mm Hg, and in POAG group were 18.25+/-2.70 mm Hg, while in the control group they were 13.53+/- 3.62 mm Hg., Conclusion: Determination of CCT in patients with glaucoma is crucial, considering its impact on IOP values, which represents a parameter in glaucoma diagnosis, as well as monitoring further progress of ocular hypertension and patients diagnosed with glaucoma.

Introduction: The trans-isomer of resveratrol is the active ingredient of Poligonum cuspidatum, known for its medicinal properties and traditionally used in the treatment of neuropsychiatric disorders. It is also found abundantly in the skin of red grapes and red wine. Previous studies have suggested that trans-resveratrol demonstrates a variety of pharmacological activities including antioxidant, anti-inflammatory, as well as neuroprotective properties and procognitive effects., Objective: The goal of the present study was to examine the influence of trans-resveratrol on behavior in rats and its antidepressant properties., Methods: Male Wistar rats were treated intraperitoneally (i.p.) with the increasing doses of trans-resveratrol (5, 10 and 20 mg/kg) or vehicle (dimethyl sulfoxide--DMSO), 30 minutes before testing of the spontaneous locomotor activity or forced swimming. For the experiments, the behavior of the animals was recorded by a digital camera, and the data were analyzed by one-way ANOVA, followed by Tukey post-hoc test., Results: Testing of spontaneous locomotor activity, after the application of vehicle or increasing doses of trans-resveratrol, showed no statistically significant difference between groups (p > 0.05). In the forced swim test, one-way ANOVA indicated statistically significant effects of trans-resveratrol (p < 0.001).Tukey post-hoc test showed that resveratrol significantly decreased immobility time at the doses of 10 mg/kg and 20 mg/kg, manifesting the acute antidepressant-like effects. There were no statistically significant differences between the resveratrol treatment of 5 mg/kg and vehicle (p > 0.05)., Conclusion: The results from our study suggest that transresveratrol produces significant effects in the central nervous system. After single application, it has acute antidepressant effects, but without influence on locomotor activity.

Apolipoprotein-CIII (apoCIII) is an abundant blood glycoprotein associated with lipoprotein particles. Three different glycoforms have been described, all containing a mucin-type core-1 O-glycosylation with either zero, one or two sialic acids. Changes in the relative abundance of these glycoforms have been observed in a variety of different pathologies. In this study, ultrahigh resolution 15T MALDI Fourier transform ion cyclotron resonance (FTICR) MS was used to analyze apoCIII isoforms in serum protein profiles. For this purpose, serum proteins were purified using both a fully automated RPC18-based magnetic bead method and an RPC4 cartridge-based solid phase extraction method. Six new apoCIII isoforms were identified with low-ppm mass measurement errors and ultrahigh precision. These were characterized by more complex glycan moieties that are fucosylated instead of sialylated. To confirm the glycan moiety and localize the glycosylation site, top-down ESI-FTICR-MS/MS and bottom-up LC-ion trap MS/MS were used. A large variation in the presence and abundance of the fucosylated isoforms was found in a set of 96 serum samples. These findings of fucosylated apolipoprotein-CIII isoforms warrant further research to elucidate the implications these glycoforms may have for the plethora of studies where alterations in apoCIII have been linked to the development of many different pathologies.

Conflict of Interest: none declared., Introduction: Authors are from reference documentation to archive at Organization of Federation of blind and visually impaired in central Serbia (Kragujevac), by retrograde analysis, of 2007-2012, comprehend two groups by etiology-clinical characteristics of congenital blindness and visually impaired, caused infection or non infection example., Aim: to analyze relationship between infectious and non infectious of congenital blindness and visually impaired in our referent region and compare with world references., Material/methods: With 6-years analysis included the most frequency cases of congenital blindness and visually impaired in two groups, according to presence or absence infectious causes. From infectious causes of congenital blindness and visually impaired are included: CMV - infection, congenital rubella syndrome, congenital toxoplasmosis, congenital syphilis and rare mixed syndrome. From non infectious causes are included: retinitis pigmentosa, retinopathy prematurity, primary congenital glaucoma, Leber's congenital amaurosis and rare syndrome., Results: From total number of registered blind and visually impaired - 1308 (100%), over the last 6 years, the registration was 349 (26.68%) with congenital blindness and visually impaired. From recorder with the number of the most common congenital blindness and visually impaired-194 (55.59%) with infections cause, and 155 (44.41%) non infection cause., Conclusion: Congenital blindness has shown permanent increase in past 6 years, in group with infectious and with non infectious causes. Congenital blindness and visually impaired of the most common etiology among registered members of our association in Kragujevac is subject of our correlation and global trends mentioned observation of these diseases.

Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, β, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein's O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC-ESI-ion trap (IT)-MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS3 spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process.

There are several modulatory sites at GABA(A) receptors, which mediate the actions of many drugs, among them benzodiazepine. Three kinds of allosteric modulators act through the benzodiazepine binding site: positive (agonist), neutral (antagonist), and negative (inverse agonist). The goal of the present study was to examine the influence of the inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acting on α GABA(A) receptor and compare its dose-response effects on memory and depression-like behavior. We independently studied the effects of DMCM (0.05-1.0 mg/kg) on retention versus acquisition of active avoidance and depression-like behavior in the forced swim test. Throughout the study, drugs were given intraperitoneally, 30 min before testing. ANOVA has showed that treatment with DMCM significantly affected retrieval of avoidance response (p<0.05), exerted promnesic effects in inverted U-shape manner. Dunnett's test indicated that the DMCM avoidance-facilitatory dose was 0.1mg/kg. At the dose facilitating retrieval of avoidance memory, DMCM significantly (p<0.05, comparison of regression coefficients by Student's t-test) and progressively increased acquisition rate during 5 days training, compared to the saline group. In forced swim test, ANOVA indicated statistically significant effects of DMCM (p<0.05). Dunnett's analysis showed that DMCM significantly decreased immobility time at the dose of 0.1mg/kg, exerted acute antidepressant-like effects. Our results experimentally support the findings that under certain circumstances, nonselective benzodiazepine site inverse agonists, produce memory-enhancing and antidepressant-like effects. The molecular and neuronal substrates linking the actions of specific GABA-benzodiazepine receptor complex subunits remains to be further elucidated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.

Background: Analysis of injury and illness prevalence in elite sport provides the basis for the development of prevention programmes., Objectives: To analyse the frequency and characteristics of injuries and illnesses occurring during the 13th Federation Internationale de Natation (FINA) World Championships 2009., Design: Prospective recording of newly incurred injuries and illnesses., Methods: The 13th FINA World Championships hosted 2592 athletes from 172 countries in the disciplines of swimming, diving, synchronised swimming water polo and open water swimming. All team physicians or physiotherapists were asked to complete daily a standardised reporting form for all newly incurred injuries and illnesses for their teams. To cover teams without medical staff, the physicians of the Local Organizing Committee also submitted daily report forms., Results: 171 injuries were reported resulting in an incidence of 66.0 per 1000 registered athletes. The most affected body parts were the shoulder (n=25; 14.6%), and head (n=21; 12.3%). Half of the injuries occurred during training. The most common cause of injury was overuse (n=61; 37.5%). 184 illnesses were reported resulting in an incidence of 71.0 per 1000 registered athletes. The respiratory tract was most commonly affected (n=91; 50.3%) and the most frequently classified cause was infection (n=81; 49.2%). The incidence of injuries and illnesses varied substantially among the five disciplines, with the highest incidence of injury in diving and the lowest in swimming., Conclusions: As the risk of injury varied with the discipline, preventive measures should be discipline specific and focused on minimising the potential for overuse. As most of the illnesses were caused by infection of the respiratory and gastrointestinal tract, preventive interventions should focus on eliminating common modes of transmission.

Dental education is regarded as a complex, demanding and often stressful pedagogical procedure. Undergraduates, while enrolled in programmes of 4-6 years duration, are required to attain a unique and diverse collection of competences. Despite the major differences in educational systems, philosophies, methods and resources available worldwide, dental students' views regarding their education appear to be relatively convergent. This paper summarizes dental students' standpoint of their studies, showcases their experiences in different educational settings and discusses the characteristics of a positive academic environment. It is a consensus opinion that the 'students' perspective' should be taken into consideration in all discussions and decisions regarding dental education. Moreover, it is suggested that the set of recommendations proposed can improve students' quality of life and well-being, enhance their total educational experience and positively influence their future careers as oral health physicians. The 'ideal' academic environment may be defined as one that best prepares students for their future professional life and contributes towards their personal development, psychosomatic and social well-being. A number of diverse factors significantly influence the way students perceive and experience their education. These range from 'class size', 'leisure time' and 'assessment procedures' to 'relations with peers and faculty', 'ethical climate' and 'extra-curricular opportunities'. Research has revealed that stress symptoms, including psychological and psychosomatic manifestations, are prevalent among dental students. Apparently some stressors are inherent in dental studies. Nevertheless, suggested strategies and preventive interventions can reduce or eliminate many sources of stress and appropriate support services should be readily available. A key point for the Working Group has been the discrimination between 'teaching' and 'learning'. It is suggested that the educational content should be made available to students through a variety of methods, because individual learning styles and preferences vary considerably. Regardless of the educational philosophy adopted, students should be placed at the centre of the process. Moreover, it is critical that they are encouraged to take responsibility for their own learning. Other improvements suggested include increased formative assessment and self-assessment opportunities, reflective portfolios, collaborative learning, familiarization with and increased implementation of information and communication technology applications, early clinical exposure, greater emphasis on qualitative criteria in clinical education, community placements, and other extracurricular experiences such as international exchanges and awareness of minority and global health issues. The establishment of a global network in dental education is firmly supported but to be effective it will need active student representation and involvement.

Classical benzodiazepines (BZs) exert anxiolytic, sedative, hypnotic, muscle relaxant, anticonvulsive, and amnesic effects through potentiation of neurotransmission at GABA(A) receptors containing alpha(1), alpha(2), alpha(3) or alpha(5) subunits. Genetic studies suggest that modulation at the alpha(1) subunit contributes to much of the adverse effects of BZs, most notably sedation, ataxia, and amnesia. Hence, BZ site ligands functionally inactive at GABA(A) receptors containing the alpha(1) subunit are considered to be promising leads for novel, anxioselective anxiolytics devoid of sedative properties. In pursuing this approach, we used two-electrode voltage clamp experiments in Xenopus oocytes expressing recombinant GABA(A) receptor subtypes to investigate functional selectivity of three newly synthesized BZ site ligands and also compared their in vivo behavioral profiles. The compounds were functionally selective for alpha(2)-, alpha(3)-, and alpha(5)-containing subtypes of GABA(A) receptors (SH-053-S-CH3 and SH-053-S-CH3-2'F) or essentially selective for alpha(5) subtypes (SH-053-R-CH3). Possible influences on behavioral measures were tested in the elevated plus maze, spontaneous locomotor activity, and rotarod test, which are considered primarily predictive of the anxiolytic, sedative, and ataxic influence of BZs, respectively. The results confirmed the substantially diminished ataxic potential of BZ site agonists devoid of alpha(1) subunit-mediated effects, with preserved anti-anxiety effects at 30 mg/kg of SH-053-S-CH3 and SH-053-S-CH3-2'F. However, all three ligands, dosed at 30 mg/kg, decreased spontaneous locomotor activity, suggesting that sedation may be partly dependent on activity mediated by alpha(5)-containing GABA(A) receptors. Hence, it could be of importance to avoid substantial agonist activity at alpha(5) receptors by candidate anxioselective anxiolytics, if clinical sedation is to be avoided.

Benzodiazepine site inverse agonists may increase or decrease locomotor activity in rodents, depending on the experimental settings. We have compared the behavioral responses to environmental novelty of rats treated with the non-selective inverse agonist DMCM (2 mg/kg) and the alpha1-subunit affinity-selective inverse agonist 3-EBC (15 mg/kg). The behavior in spontaneous locomotor assay (during 45 min) and elevated plus maze (EPM) was automatically recorded. In the EPM, general activity-related parameters were similarly decreased, whereas only DMCM inhibited open-arm activity. In the locomotor assay, both compounds depressed locomotion in the first 15 min and activity in the central zone of the chamber. However, the influence of 3-EBC was less pronounced. The alpha1-subunit selective antagonist beta-CCt (15 mg/kg) attenuated locomotor depression, but not the central-zone avoidance elicited by DMCM. When habituated to the chamber, DMCM-treated animals emitted a plateau phase of activity, which disappeared by adding beta-CCt. Hence, inhibition of activity in exposed areas may be mediated by non-alpha1-subunits, whereas both alpha1 and non-alpha1-subunits may participate in suppression of activity in more protective areas of an apparatus. Hyperlocomotion in habituated animals may depend primarily on the alpha1-subunit. Moreover, the bimodal influence of inverse agonists on locomotion can be biphasic, observable in the same experiment.

In the previous study of the rat frontal cortex slices oxygen consumption (QO2), polarographically determined using the biological oxygen monitor, a moderate respiratory depressant action of midazolam ex vivo (1.0 mg/kg) has been observed. Antagonist of the benzodiazepine binding site, flumazenil, blocked the effect of the agonist. However, midazolam-gamma-aminobutyric acid (GABA) interactions pointed to the possibility that a part of midazolam action is independent of the classical GABA potentiation. To test this presumption, GABAA receptor antagonists bicuculline and picrotoxin were administered. Both blockers antagonized the QO2 reducing effect of the combination of per se effective doses of midazolam (1.0 mg/kg) and GABA (5 x 10(-4) mol/l), as well as of GABA (5 x 10(-4) mol/l) itself. However, neither effects of midazolam (1.0 mg/kg) on its own, nor those of midazolam in presence of the physiological, per se ineffective, concentration of GABA (10(-6) mol/l), were susceptible to antagonism. These results show that ex vivo influence of midazolam on cerebral metabolic activity should be partly ascribed to some of its cellular mechanisms probably associated to the GABA modulation, but distinct from the standard GABA-potentiating effects of benzodiazepines.

Rationale: The pharmacological approach, using subtype selective ligands, complements genetic studies on the specific contribution of individual receptor subtypes to the various effects of benzodiazepines., Objective: The aim of this study was to examine the relative significance of alpha1-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site on anxiety and memory processes., Methods: We tested the effects of the nonselective antagonist flumazenil, the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt), the nonselective agonist midazolam, the preferential alpha1-subunit selective agonist zolpidem, and the nonselective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in a two-way active avoidance task in rats. The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of the two agonists were also examined. In the schedule 2 x 30 trials, drugs were administered i.p. 20 min before the training session. Avoidance responses in the training session are an anxiety-mediated behavior, whereas performance in the retention session relates to the effects on memory., Results: Flumazenil and beta-CCt did not affect behavior. Midazolam (2.0 mg/kg) facilitated acquisition performance, while DMCM (1.0 and 2.0 mg/kg) induced the opposite effect. Flumazenil antagonized both effects. Beta-CCt potentiated the effect of midazolam, and partly antagonized the effect of DMCM. Midazolam (0.5 and 1.0 mg/kg) and zolpidem (1.0-3.0 mg/kg) impaired, while DMCM (0.1 mg/kg) facilitated the subjects' performance in the retention test. The amnesic effects were attenuated but not fully reversed, while the effect of DMCM was counteracted by both antagonists., Conclusion: The results indicate the alpha1-subunit interferes with the anxiolytic effect of a benzodiazepine site agonist and may contribute to the DMCM-induced anxiogenic effect. It is also substantially involved in the bidirectional memory processing in the active avoidance paradigm.

Recent research on genetically modified mice has attributed the amnesic effect of benzodiazepines mainly to the alpha1-containing GABA(A) receptor subtypes. The pharmacological approach, using subtype selective ligands, is needed to complement genetic studies. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) (0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha1-subunit selective agonist zolpidem (0-3.0 mg/kg), and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (0-2.0 mg/kg) in the one-trial step-through passive avoidance task in rats. The compounds were administered intraperitoneally, before the acquisition test. Flumazenil and beta-CCt did not affect retention performance. Midazolam and zolpidem induced amnesia in a dose-dependent manner. The complete reversal of amnesia was unattainable. The effects of zolpidem were significantly attenuated by the both, flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg); by contrast, only flumazenil was considerably effective when combined with midazolam. DMCM exerted promnesic effects at 0.2mg/kg, in an inverted U-shape manner. Both antagonists tended to abolish this action. The results indicate that some other alpha-subunit(s), in addition to the alpha1-subunit, contribute to the amnesic actions of non-selective benzodiazepine site agonists in the passive avoidance task. On the other hand, a significant part of the DMCM-induced promnesic effect could involve the alpha1-subunit and/or other putative beta-CCt-sensitive binding site(s).

Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the alpha1 and alpha5 subunits, whereas the effects on procedural memory can be mainly mediated by the alpha1 subunit. The pervading involvement of the alpha1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of alpha5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states.

Recent research using genetically modified mice has pointed to the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. The aim of this study was to examine the relative significance of alpha(1)-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site in the elevated plus-maze (EPM) under dim red light in rats. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha(1)-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt, 0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha(1)-subunit selective agonist zolpidem (0-2.0 mg/kg) and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0-2.0 mg/kg). The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of both kinds of agonists were also examined. The standard spatio-temporal parameters reflecting anxiety (percentage of open arm entries and time) and locomotion (closed and total arm entries) were analyzed. beta-CCt did not affect behavior, while flumazenil at the highest dose (20.0 mg/kg) decreased indices of open arm activity and total arm entries. Midazolam at the dose of 1.0 mg/kg significantly increased the percentage of open arm time, whereas at 2.0 mg/kg both anxiety-related parameters were increased. In contrast to the open arm entries, the open arm time was independent of the decreased closed arm entries, observed at 2.0 mg/kg. Flumazenil abolished these effects, whereas beta-CCt partially potentiated the anxiolytic actions of midazolam. Zolpidem significantly increased both open-arm indices at 1.0 mg/kg, but the effect was dependent on the decreased closed arm entries. The selectivity of the anxiolytic-like effects of zolpidem was further checked under brighter white illumination. In these settings, the influence on anxiety-related, but not activity-related parameters, was absent. All of the activity-related effects of midazolam and zolpidem were mainly counteracted by both antagonists. DMCM produced significant anxiogenic effects at 1.0 mg/kg (open arm time) and 2.0 mg/kg (both parameters). beta-CCt (30.0 mg/kg) and flumazenil at higher dose (20.0 mg/kg) antagonized the effects of DMCM. The results indicate the anxiolytic effects of a non-selective benzodiazepine site agonist involve a predominant role of subunits other than alpha(1), whereas the behavioral indices of the anxiolytic-like properties of an alpha(1)-selective ligand, if observed, depend on the experimental settings and the changes in locomotor activity, and hence were behaviorally non-specific. The present results generally correspond well to the behavioral findings with the genetically modified mice. On the other hand, the relative significance of the alpha(1)-subunit in the anxiogenic effects of DMCM could not be clearly deduced.

The purpose of the present study was to examine the influence of midazolam on the retrieval and acquisition rate of two-way active avoidance in rats. In the schedule 2 x 100 trials, the effects of midazolam (0.5-5.0 mg/kg), benzodiazepine binding site antagonist flumazenil (2.5-10.0 mg/kg), specific antagonist of GABA(A) receptor, bicuculline (0.5-4.0 mg/kg), and the blocker of GABA(A) receptor containing Cl(-) channels, picrotoxin (1.0-5.0 mg/kg), on the second day retrieval of avoidance performance were investigated, as well as the influence of the used blockers of GABA(A) receptor function on midazolam effects. Furthermore, the effect of midazolam (1.0 mg/kg) on acquisition rate in the 5 x 50 schedule, as well as the effects of third day treatment changing in that paradigm, was examined. Throughout the study, drugs were given intraperitoneally, 30 min before testing. Midazolam at the dose of 1.0 mg/kg facilitated avoidance retrieval, whereas flumazenil and bicuculline did not significantly change behavior. Picrotoxin (5.0 mg/kg) diminished performance. All three kinds of blockers used abolished facilitatory action of midazolam, confirming GABAergic mediation of the effect of benzodiazepine. Midazolam (1.0 mg/kg) increased acquisition rate during five consecutive days relative to saline, but without significant effect on the first day acquisition. In the case of third day changing of treatments, the intersection of regression rate lines was detected. Results from active avoidance paradigm experimentally support the findings from human studies that in certain circumstances, benzodiazepines, potentiating GABAergic neurotransmission, could produce retrieval-enhancing effects in memory tasks.

Acute effects on the behaviour of the organophosphate insecticide dichlorvos and its standard antidotes possessing behavioural activity, atropine and diazepam, were studied separately and in combinations in male Wistar rats. In the spontaneous locomotor activity test, dichlorvos and diazepam decreased, whereas atropine increased performance. The effect of dichlorvos was obtained at a dose (5 mg/kg) that induced overt intoxication, and could not be reversed during first half hour-period after administration of any combination of drugs. In the other two tests, active avoidance learning and rotarod performance, the effective dose of dichlorvos (2 mg/kg) was devoid of somatic signs of intoxication. In these more sensitive tests, the effective atropine dose (40 mg/kg) completely reversed dichlorvos-induced incapacitation. In the rotarod test, diazepam (0.5 mg/kg) contributed to the incapacitating effect of dichlorvos, and impeded desirable influence of atropine as well. In the active avoidance test, diazepam (2.5 mg/kg) contributed to failure to escape; it did not influence the dichlorvos-induced decrease of avoidance performance, nor did it impair the completely reversing effects of atropine. The results point to the possible summation of acute incapacitating effects of organophosphates and diazepam on motor performance, which seems to be, at least partly, antagonized by sufficiently high doses of atropine. However, taking into account the long-term neuroprotective role of the anticonvulsant diazepam, and hence its delayed beneficial influences on behaviour, the immediate testing of atropine/diazepam treatment of organophosphate intoxication in active avoidance paradigm could possess beside sensitivity the predictive value as well.

This study investigated the impact of benzodiazepine receptor agonist, midazolam and antagonist, flumazenil, on the rat frontal cortex slices oxygen consumption (QO(2)), in presence and absence of gamma-aminobutyric acid (GABA). QO(2) was polarographically determined, using the biological oxygen monitor. As it was previously shown, GABA on its own decreases QO(2) moderately. Midazolam decreased QO(2) at 1.0mg/kg, whereas flumazenil had no effect. In combination with per se ineffective GABA (10(-6)mol/l), flumazenil showed respiratory depressant action, presumably revealing partial agonistic activity at some of GABA(A) receptor subtypes. However, it completely antagonized effects of midazolam on QO(2), on its own and in presence of GABA. Our results show that in vivo well-established effects of midazolam on cerebral metabolic activity could be reproduced in in vitro settings. Moreover, flumazenil antagonized this action, indicating the role of GABA(A)-benzodiazepine receptor complex activation in QO(2) regulation.

The management of pediatric cataracts is, by far, more complex than the management of cataracts in adults and depends not only on deterioration of visual acuity, but also on the status of the other eye, the general health of the child, the motivation and the financial situation of the parents and last, but not least, on the surgeons' experience and skill, and the technical support. The therapeutically approach involves: the preoperative evaluation to establishes the indication and the timing of surgery the adaptation of the surgical technique for each case the choice of the aphakic correction the visual rehabilitation in the context of amblyopia management.

Purpose: To study the technical difficulties using the MONARCH II injector, intraoperative incidents, postoperative astigmatism., Methods: Retrospective study which included 135 eyes with cataract who underwent cataract surgery between March 2002-August 2003. The surgical intervention consisted of lens extraction by phacoemulsification technique and IOL implantation with the MONARCH II injector. We recorded the following parameters: problems during the loading of the IOL in the cartridge, difficulties during the implant, problems due to the damage of the IOL during the passage in the cartridge, IOL centration in the capsular bag, induced postoperative astigmatism., Results: We had no major difficulties using this type of injector. We did not recorded important differences between the hydrophobic and hydrophilic IOLs. We had a number of 17 intraoperative incidents (12.6%). We met the following intraoperative incidents: the last haptic capture in the tunnel of the cartridge in 5 cases (3.7%), small marks on the optic surface left by the tip of the injector in 3 cases (2.2%), the leading haptic stuck on the optic surface in 2 cases (1.4%), breaking of the last haptic (multi-piece IOL) in 1 case (0.7%), the turning up side down of IOL during unfolding (hydrophilic IOLs) in 6 cases (4.4%). Postoperative astigmatism was less than 1D cylinder in 88% of cases., Conclusions: The IOL implantation using MONARCH II injector is easy and doesn't need learning curve. The technique is superior compared with forceps implantation (especially for acrylic hydrophilic IOLs). No significant differences between implantation of acrylic IOLs. IOL centration was good. The design of the injector tip preserved optic surface and haptic from damage. The conical tip of the cartridge didn't change the architecture of the corneal wound. The postoperative astigmatism was small.

This study investigated the impact of modulating the gamma-aminobutyric acid(A) (GABA)(A)-benzodiazepine receptor complex activity on the rat frontal cortex slices oxygen consumption (QO(2)), polarographically determined using the biological oxygen monitor. Throughout the study, diazepam, flumazenil and picrotoxin were administered i.p. 30 min before sacrificing animals and obtaining slice preparations, while GABA was added directly into the medium in the reaction chamber. GABA decreased QO(2) in concentrations of 5 x 10(-4), 10(-2) and 5 x 10(-2)mol l(-1), while 10(-5) and 10(-6)mol l(-1) GABA had no effect, as well as diazepam, flumazenil and picrotoxin. All diazepam doses (1, 2.5 and 5 mg kg(-1)) increased action of 5 x 10(-4)mol l(-1) GABA, whereas 2.5 mg kg(-1) dose amplified the effect of 10(-6)mol l(-1) GABA. Flumazenil and picrotoxin (5 mg kg(-1) both) blocked diazepam's effects. Flumazenil augmented 10(-6)mol l(-1) GABA effects, while picrotoxin and flumazenil abolished the effects of 5 x 10(-4)mol l(-1) GABA. To our knowledge, this is the first study to examine the influence of modulation of GABA(A)-benzodiazepine receptor function on cerebral metabolism of oxygen in in vitro settings. The results are in accordance with those obtained in numerous in vivo studies, pointing to the moderate level of influence of GABA(A)-benzodiazepine receptor complex on QO(2) regulation.

The aim of the study was to investigate the morphology of mesothelial cells of the peritoneum of patients with terminal renal failure (TRF), taken by the biopsy immediately before the onset of peritonal dialysis (PD), and to compare it with the findings in patients with PD. The samples were prepared in the way standard for light microscopy and transmission electron microscopy. In patients with TRF intracytoplasmatic inclusions could be observed, unusual protrusions of mesothelial apical surfaces, deformation of mesothelial cells and their detachment from the basal membrane, as well as the dilatated cisternae of granulated endoplasmatic reticulum with filamentous structures in some of them. In patients on PD cytoplasmic protrusions of different shapes and contents were observed at the surface of mesothelial cells, multiplication of basal membrane, occurrence of young forms of mesothelial cells as well as the detachment of those cells from the basal lamina.