[Towards personalized treatment with clozapine]. / Op naar een gepersonaliseerde clozapinebehandeling.

BACKGROUND: Clozapine is the most effective treatment for people with treatment-resistant schizophrenia. However, it is prescribed less often than guidelines indicate. AIM: To personalize clozapine treatment, we investigated the efficacy of clozapine as first- or second-line treatment and investigated whether there are factors that were associated with efficacy and side effects. METHOD: We collected a unique cohort of over 800 clozapine users diagnosed with a schizophrenia spectrum disorder. We meta-analyzed factors that were associated with response during clozapine treatment. Additionally, we conducted genetic association analyses to investigate the relations between side effects and symptom severity during clozapinetreatment. RESULTS: From our meta-analyses, we found that clozapine was more effective when used as a first- or second-line treatment. Furthermore, we found that younger age, less negative symptoms and the paranoid subtype of schizophreniawere associated with a better clozapine response. Several specific locations on genes (loci) were associated with clozapine-induced agranulocytosis and neutropenia, while polygenic risk scores were associated with symptom severity. CONCLUSION: We found that clozapine could be effective earlier in treatment and identified factors that could aid the prediction of< response to clozapine treatment in the future. These finding could contribute to the start of a personalized clozapine treatment.

Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4',6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.

New quantitative automated model to simulate bacterial dissemination in human tissue during irrigation of contaminated wounds.

This study presents a simple and cost-effective model using microparticles to simulate the bacterial distribution pattern in soft tissue after low- and high-pressure irrigation. Silica coated iron microparticles [comparable diameter (1 µm) and weight (0.8333 pg) to Staphylococcus aureus] were applied to the surface of twenty fresh human muscle tissue samples in two amputated lower legs. Particle dissemination into deep tissue layers as an undesired side effect was investigated in four measuring fields as positive control (PC) as well as after performing pulsatile high-pressure (HP, 8 measuring fields) and low-pressure flushing (LP, 8 measuring fields). Five biopsies were taken out of each measuring field to get a total number of 100 biopsies. After histological and digital image processing, the specimens were analysed, and all incomplete sections were excluded. A special detection algorithm was parameterised using the open source bioimage analysis software QuPath. The application of this detection algorithm enabled automated counting and detection of the particles with a sensitivity of 95 % compared to manual counts. Statistical analysis revealed significant differences (p < 0.05) in our three different sample groups: HP (M = 1608, S = 302), LP (M = 2176, SD = 609) and PC (M = 4011, SD = 686). While both HP and LP flushing techniques are able to reduce the number of bacteria, a higher effectiveness is shown for HP irrigation. Nevertheless, a challenge for the validity of the study is the use of dead tissue and therefore a possible negative influence of high-pressure irrigation on tissue healing and further dispersion of particles cannot be evaluated.

Translating Fear Circuitry: Amygdala Projections to Subgenual and Perigenual Anterior Cingulate in the Macaque.

Rodent fear-learning models posit that amygdala-infralimbic connections facilitate extinction while amygdala-prelimbic prefrontal connections mediate fear expression. Analogous amygdala-prefrontal circuitry between rodents and primates is not established. Using paired small volumes of neural tracers injected into the perigenual anterior cingulate cortex (pgACC; areas 24b and 32; a potential homologue to rodent prelimbic cortex) and subgenual anterior cingulate cortex (sgACC, areas 25 and 14c; a potential homologue to rodent infralimbic cortex) in a single hemisphere, we mapped amygdala projections to the pgACC and sgACC within single subjects. All injections resulted in dense retrograde labeling specifically within the intermediate division of the basal nucleus (Bi) and the magnocellular division of the accessory basal nucleus (ABmc). Areal analysis revealed a bias for connectivity with the sgACC, with the ABmc showing a greater bias than the Bi. Double fluorescence analysis revealed that sgACC and pgACC projections were intermingled within the Bi and ABmc, where a proportion were double labeled. We conclude that amygdala inputs to the ACC largely originate from the Bi and ABmc, preferentially connect to the sgACC, and that a subset collaterally project to both sgACC and pgACC. These findings advance our understanding of fear extinction and fear expression circuitry across species.

Effects of systematically varied thiourethane-functionalized filler concentration on polymerization behavior and relevant clinical properties of dental composites.

Introduction of thiourethane (TU) oligomer to resin-based dental restorative materials reduces stress and improves fracture toughness without compromising conversion. Localization of TU at the resin-filler interface via silanization procedures may lead to more substantial stress reduction and clinical property enhancements. The objective of this study was to evaluate composite properties as a function of TU-functionalized filler concentration. TU oligomers were synthesized using click-chemistry techniques and subsequently silanized to barium glass filler. Resin-based composites were formulated using varying ratios of TU-functionalized filler and conventional methacrylate-silanized barium filler. Material property testing included thermogravimetric analysis, real-time polymerization kinetics and depth of cure, polymerization stress, stress relaxation and fracture toughness. Clinical property testing included water sorption/solubility, composite paste viscosity, and gloss and surface roughness measured before and after subjecting the samples to 6 h of continuous tooth brushing in a custom-built apparatus using a toothpaste/water mixture. Increasing TU-filler in the composite resulted in as much as a 78% reduction in stress, coupled with an increase in fracture toughness. Conversion was similar for all groups. After simulated tooth brushing, gloss reduction was lower for TU-containing composites and surface roughness was less than or equal to the control.

[Validation of the Dutch Glasgow Anti- psychotic Side-Effect Scale for Clozapine]. / Glasgow Antipsychotica Bijwerkingen Schaal voor Clozapine; validatie van de Nederlandstalige versie.

BACKGROUND: To monitor the unique side effect pattern of clozapine, the Glasgow Antipsychotic Side-effect Scale for Clozapine (GASS-C) was developed in English and validated. This questionnaire was previously translated to Dutch, and revised, but not yet validated. AIM: The current study concerns the validation of the second revision of the GASS-C-NL-R2 for the Dutch language. METHOD: Two Spearman correlation tests were conducted to compare GASS-C-NL-R2 with the Dutch version of the Liverpool University Neuroleptic Side-Effect Rating Scale (LUNSERS) at two time p´oints. There was one week between these two time points. The test-retest reliability was determined using a Spearman correlation test and Cronbach's alpha on the GASS-C-NL-R2 between the two time points. In addition, a factor analysis was performed. RESULTS: Spearman's correlation coefficient between the GASS-C-NL-R2 and the LUNSERS was 0.830 (p < 0.001, n = 72) at the first time point and 0.684 (p < 0.001, n = 50) at the second time point. GASS-C-NL-R2 also had a strong test-retest reliability: Spearman's correlation coefficient was 0.680 (p < 0.001; n = 46), and Cronbach's alpha was 0.847, n = 78. Factor analysis showed that all questions were relevant. CONCLUSION: The current study shows that GASS-C-NL-R2 is a valid and reliable questionnaire to monitor side effects related to clozapine with a relatively high prevalence. Future studies should focus on the practical utility of GASS-C-NL-R2 with a larger sample size.

In vitro performance of 2-step, total etch adhesives modified by thiourethane additives.

OBJECTIVES: Thio-urethane oligomeric additives have been shown to improve the mechanical properties of dental composites and resin cements. To try to harness those same properties in dental adhesives, in this study, these oligomers (TU) were added to the matrix and/or as filler functionalization of experimental adhesives, and the effects on conversion and mechanical properties were analyzed. METHODS: BisGMA and HEMA (60/40 wt%) were used as the monomer matrix, made polymerizable by the addition of 0.2 wt% 2,2-dimethoxy-2- phenylacetophenone. 2,6-di-tert-butyl-4-methylphenol was added at 0.5 wt% as the inhibitor. This material was used as the unfilled control (BH). TU oligomers were added at 20 wt % to the matrix (BH+20%TU, unfilled) and/or used as filler functionalization (TF, 10 wt%). Fillers functionalized with methacrylate (MF, 10 wt%) served as the control. The experimental adhesives groups containing fillers were: BH+10%MF; BH+10%TF; BH+20%TU+10%MF; BH+20%TU+10%TF. Flexural properties were tested in three-point bending (wet and dry). Polymerization kinetics was followed in real-time in near-IR. Water Sorption/Solubility (WS/SL, ISO 4049) and Viscosity (rotational rheometry) were also evaluated. For Microtensile bond strength 40 vol% ethanol was added to adhesives, which was applied onto sound dentin from third human molars. The data were analyzed with one-way ANOVA and Tukey post-hoc test, and test t for the comparison between storage time of the microtensile bond strength test (alpha = 0.05). RESULTS: There was no significant difference between groups when yield strength (YS) and flexural modulus (FM) were evaluated in dry conditions. After water storage, all the groups containing TU in the matrix showed statistically lower YS/FM values. This was true in spite of the statistically higher conversion for those same groups. The maximum rate of polymerization (Rpmax) was higher for BH+10%TF and no significant difference was found for the groups BH and BH+10% MF. The lowest Rpmax values were found for BH+20%TU+10%TF and BH+20%TU. BH+20%TU+10%TF showed the highest viscosity values followed by BH+20%TU+10%MF and BH+20%TU, with statistically significant difference between them. For the microtensile bond strength test at 24h (p = 0.13) and 6 months (p = 0.11) and WS/SL (p > 0.05), no significant difference was found among groups. The storage time (24 h and 6 months) did not affect the microtensile bond strength results. CONCLUSION: In spite of improving the conversion, the addition of TU in the matrix reduced the mechanical properties of the adhesives tested after water storage. This did not affect the bond strength at 24 h or 6 months.

Clozapine as a first- or second-line treatment in schizophrenia: a systematic review and meta-analysis.

OBJECTIVE: No consensus exists on whether clozapine should be prescribed in early stages of psychosis. This systematic review and meta-analysis therefore focus on the use of clozapine as first-line or second-line treatment in non-treatment-resistant patients. METHODS: Articles were eligible if they investigated clozapine compared to another antipsychotic as a first- or second-line treatment in non-treatment-resistant schizophrenia spectrum disorders (SCZ) patients and provided data on treatment response. We performed random-effects meta-analyses. RESULTS: Fifteen articles were eligible for the systematic review (N = 314 subjects on clozapine and N = 800 on other antipsychotics). Our meta-analysis comparing clozapine to a miscellaneous group of antipsychotics revealed a significant benefit of clozapine (Hedges' g = 0.220, P = 0.026, 95% CI = 0.026-0.414), with no evidence of heterogeneity. In addition, a sensitivity analysis revealed a significant benefit of clozapine over risperidone (Hedges' g = 0.274, P = 0.030, 95% CI = 0.027-0.521). CONCLUSION: The few eligible trials on this topic suggest that clozapine may be more effective than other antipsychotics when used as first- or second-line treatment. Only large clinical trials may comprehensively probe disease stage-dependent superiority of clozapine and investigate overall tolerability.

Achilles tendon elastic properties remain decreased in long term after rupture.

PURPOSE: Rupture of the Achilles tendon results in inferior scar tissue formation. Elastography allows a feasible in vivo investigation of biomechanical properties of the Achilles tendon. The purpose of this study is to investigate the biomechanical properties of healed Achilles tendons in the long term. MATERIALS AND METHODS: Patients who suffered from Achilles tendon rupture were recruited for an elastographic evaluation. Unilateral Achilles tendon ruptures were included and scanned in the mid-substance and calcaneal insertion at least 2 years after rupture using shear wave elastography. Results were compared to patients' contralateral non-injured Achilles tendons and additionally to a healthy population. Descriptive statistics, reliability analysis, and correlation analysis with clinical scores were performed. RESULTS: Forty-one patients were included in the study with a mean follow-up-time of 74 ± 30; [26-138] months after rupture. Significant differences were identified in shear wave elastography in the mid-substance of healed tendons (shear wave velocity 1.2 ±1.5 m/s) compared to both control groups [2.5 ±1.5 m/s (p < 0.01) and 2.8 ±1.6 m/s (p < 0.0001) contralateral and healthy population, respectively]. There was no correlation between the measurements and the clinical outcome. CONCLUSIONS: This study shows that the healed Achilles tendon after rupture has inferior elastic properties even after a long-term healing phase. Differences in elastic properties after rupture mainly originate from the mid-substance of the Achilles tendon, in which most of the ruptures occur. Elastographic results do not correspond with subjective perception. Clinically, sonoelastographical measurements of biomechanical properties can be useful to provide objective insights in tendon recovery.

Timing of anterior cruciate ligament reconstruction within the first year after trauma and its influence on treatment of cartilage and meniscus pathology.

PURPOSE: Anterior cruciate ligament (ACL) ruptures are often associated with primary meniscal and cartilage lesions. Late reconstruction of ACL-deficient knees may increase the risk of developing secondary meniscal and cartilage lesions; hence, the timing of ACL repair is of the utmost importance. Because meniscus outcome is also a potential predictor for osteoarthritis (OA), this study compared ACL repair within the first 6 months after injury to that of surgery conducted 7-12 months after injury with regard to the incidence of meniscal and cartilage lesions. METHODS: This prospective cross-sectional study included all complete isolated primary ACL ruptures treated in our institution within 1 year after trauma over a 12-month period. Exclusion criteria were revision ACL, complex ligament injuries, previous knee surgery, and missing injury data. Cartilage lesions were classified according to the score established by the International Cartilage Repair Society (ICRS score) and meniscal tears according to their treatment options. RESULTS: Two hundred and thirty-three of 730 patients (162 men, 71 women) with ACL repair met the inclusion criteria. 86.3 % of surgical interventions were conducted within 6 months and 13.7 % after 6 months of trauma. Severe cartilage lesions grade III-IV did not significantly differ between the different time points of ACL repair (<6 months 39.9 %; >6 months 31.3 %; p = n.s.). Medial meniscus lesions received significantly higher meniscal repair in early compared to delayed ACL repair. Significantly higher rate of meniscal repair of the medial meniscus was seen in cases of early ACL repair compared to delayed (<6 months 77.2 %, >6 months 46.7; p = 0.022). The rate of medial meniscal repair in early ACL repair was significantly higher for women (89.5-0 %; p = 0.002), however, not for men (73.3-53.8 %; p = n.s.). No differences were found for lateral meniscal lesions, with regard to neither the different time points (p = n.s.) nor the sex (p = n.s.). CONCLUSIONS: Because of the significantly higher rate of prognostically advantageous meniscal repair, the recommendation for an ACL reconstruction within 6 months after trauma was made to preserve the meniscus and reduce the risk of developing OA. LEVEL OF EVIDENCE: Prospective cross-sectional cohort study, Level II.

Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM.

We have applied serial block-face scanning electron microscopy (SBF-SEM) to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs that secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9±5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections.

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells.

A combination of two-dimensional (2D) and three-dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and ß cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a ß cell.

Posterolateral fusion in acute traumatic thoracolumbar fractures: a comparison of demineralized bone matrix and autologous bone graft.

UNLABELLED: INTRODUCTION Alternative fusion expanders are in clinical use for instrumented posterolateral fusion (PLF) to avoid donor site morbidity in autologous bone graft (ABG) harvesting. Purpose of this study was to evaluate demineralized bone matrix (DBM) in PLF as alternative to the gold standard of ABG in acute traumatic vertebral body fractures of the thoracolumbar spine. MATERIAL AND METHODS We retrospectively identified 101 patients with acute traumatic vertebral body fractures of the thoracic and lumbar spine who were treated with instrumented PLF in our level one trauma center between 2005 and 2011. Patients with a primary paraplegia, osteodepriving disease or loss to follow-up had been excluded. Until August 2008, autologous bone graft harvested from the posterior iliac crest was used in PLF (control group n = 46). Starting September 2008, DBM was used as fusion expander in PLF (study group n = 16). Clinical and radiological evaluation was performed with a minimum followup of 18 months to assess the clinical and radiological outcome. RESULTS We found a fusion rate of 94% in patients undergoing PLF with the use of DBM and 100% with the use of ABG. There was one major complication of deep infection in the DBM group and two cases of superficial wound infection in the ABG group. We discovered a trend of reduced operating time with the use of DBM. CONCLUSIONS DBM leads to a similar fusion rate as the use of ABG in patients undergoing PLF for acute traumatic vertebral body fractures of the thoracic and lumbar spine. DBM is associated with reduced operating time. LEVEL OF EVIDENCE III: case-control study Key words: demineralized bone matrix instrumented posterolateral fusion, acute traumatic vertebral body fracture, thoracolumbar spine, autologous bone graft.

Mechanistic study of the stabilization of dentin-bonded restorative interfaces via collagen reinforcement by multi-acrylamides.

Hydrolytically and enzymatically-stable multi-acrylamides have been proposed to increase the long-term durability of dental adhesive interfaces as alternatives to methacrylates. The aim of this study was to investigate the mechanical and biochemical properties of experimental adhesives containing multi-functional acrylamides concerning collagen reinforcement and metalloproteinases (MMP) activity. Multi-functional acrylamides, TMAAEA (Tris[(2-methylaminoacryl) ethylamine) and DEBAAP (N,N-Diethyl-1,3-bis(acrylamido) propane), along with the commercially available DMAM (N,N-dimethylacrylamide) (monofunctional acrylamide) and HEMA (2-Hydroxyethyl methacrylate) (monofunctional methacrylate - control) were tested for stability against enzymatic hydrolysis by cholesterol esterase/pseudocholinesterase (PC/PCE) solutions for up to 30 days. Collagen-derived substrate and gelatin zymography were performed to examine the effect of the compounds on the biological activity of human recombinant and dentin-extracted gelatinases MMP-2 and MMP-9. In situ zymography was carried out by fluorescent collagen degradation combined with confocal microscopy analysis. Hydroxyproline content was measured in collagen derived from dentin extracts though reaction with Ehrlich's reagent p-dimethylaminobenzaldehyde (DMAB), generating a stable chromophore measured at 550 nm. Storage shear modulus of demineralized dentin discs treated with the tested compounds was measured by oscillatory rheometry, in order to investigate potential collagen reinforcement. FT-IR was performed to determine qualitative differences in collagen based on observed changes in amide bands. The results were analyzed by ANOVA/Tukey's test (α = 0.05). Multi-acrylamides survived 30 days of incubation in cholinesterase/pseudo-cholinesterase (PC/PCE) solutions, while HEMA showed approximately 70 % overall degradation. Incubation with multi-acrylamides reduced collagen degradation as evidenced by the reduced hydroxyproline levels and by the 30 % increase inshear storage modulus. Biochemical and zymography assays showed no noticeable inhibition of recombinant and extracted MMPs enzymatic activity. The infra-red spectroscopy results for multi-functional acrylamides treated samples demonstrated shifts of the amide II bonds and marked increase in intensity of the bands 1200 cm-1, which may indicate partial collagen denaturation and some degree of cross-linking of the compounds with collagen, respectively. The multi-acrylamides exhibited not only comparable mechanical properties but also demonstrated significantly enhanced biochemical stability when compared to the widely used methacrylate control. Clinical relevance: These findings highlight the potential of multi-acrylamides to increase the bonding stability to tissues and, ultimately, contribute to the longevity of dental restorations.

Nanosilver/DCOIT-containing surface coating effectively and constantly reduces microbial load in emergency room surfaces.

BACKGROUND: Colonization of near-patient surfaces in hospitals plays an important role as a source of healthcare-associated infections. Routine disinfection methods only result in short-term elimination of pathogens. AIM: To investigate the efficiency of a newly developed antimicrobial coating containing nanosilver in long-term reduction of bacterial burden in hospital surfaces to close the gap between routine disinfection cycles. METHODS: In this prospective, double-blinded trial, frequently touched surfaces of a routinely used treatment room in an emergency unit of a level-I hospital were treated with a surface coating (nanosilver/DCOIT-coated surface, NCS) containing nanosilver particles and another organic biocidal agent (4,5-dichloro-2-octyl-4-isothiazolin-3-one, DCOIT), whereas surfaces of another room were treated with a coating missing both the nanosilver- and DCOIT-containing ingredient and served as control. Bacterial contamination of the surfaces was examined using contact plates and liquid-based swabs daily for a total trial duration of 90 days. After incubation, total microbial counts and species were assessed. FINDINGS: In a total of 2880 antimicrobial samples, a significant reduction of the overall bacterial load was observed in the NCS room (median: 0.31 cfu/cm2; interquartile range: 0.00-1.13) compared with the control coated surfaces (0.69 cfu/cm2; 0.06-2.00; P < 0.001). The nanosilver- and DCOIT-containing surface coating reduced the relative risk of a critical bacterial load (defined as >5 cfu/cm2) by 60% (odds ratio 0.38, P < 0.001). No significant difference in species distribution was detected between NCS and control group. CONCLUSION: Nanosilver-/DCOIT-containing surface coating has shown efficiency for sustainable reduction of bacterial load of frequently touched surfaces in a clinical setting.

Triacrylamide-Based Adhesives Stabilize Bonds in Physiologic Conditions.

In this study, an acrylamide-based adhesive was combined with a thiourethane-based composite to improve bond stability and reduce polymerization stress, respectively, of simulated composite restorations. The stability testing was conducted under physiologic conditions, combining mechanical and bacterial challenges. Urethane dimethacrylate was combined with a newly synthesized triacrylamide (TMAAEA) or HEMA (2-hydroxyethyl-methacrylate; control) to produce a 2-step total-etch adhesive system. Methacrylate-based composites (70 wt% silanized filler) were formulated, containing thiourethane oligomers at 0 (control) or 20 wt%. Standardized preparations in human third molars were restored; then, epoxy replicas were obtained from the occlusal surfaces before and after 7-d storage in water or with Streptococcus mutans biofilm, which was tested after storage in an incubator (static) or the bioreactor (mechanical challenge). Images were obtained from the replicas (scanning electron microscopy) and cross sections of the samples (confocal laser scanning microscopy) and then analyzed to obtain measurements of gap, bacterial infiltration, and demineralization. Microtensile bond strength of specimens stored in water or biofilm was assessed in 1-mm2 stick specimens. Data were analyzed with analysis of variance and Tukey's test (α = 0.05). HEMA-based materials had greater initial gap measurements, indicating more efficient bonding for the acrylamide materials. When tested in water, the triacrylamide-based adhesive had smaller gaps in the incubator or bioreactor. In the presence of biofilm, there was less difference among materials, but the acrylamide/thiourethane combination led to statistically lower gap formation in the bioreactor. HEMA and TMAAEA-based adhesives produced statistically similar microtensile bond strengths after being stored in water for 7 d, but after the same period with biofilm-challenged specimens, the TMAAEA-based adhesives were the only ones to retain the initial bond strength values. The use of a stable multiacrylamide-based adhesive led to the preservation of the resin-dentin bonded interface after a physiologically relevant challenge. Future studies will include a multispecies biofilm model.

Genome-wide association analyses of symptom severity among clozapine-treated patients with schizophrenia spectrum disorders.

Clozapine is the most effective antipsychotic for patients with treatment-resistant schizophrenia. However, response is highly variable and possible genetic underpinnings of this variability remain unknown. Here, we performed polygenic risk score (PRS) analyses to estimate the amount of variance in symptom severity among clozapine-treated patients explained by PRSs (R2) and examined the association between symptom severity and genotype-predicted CYP1A2, CYP2D6, and CYP2C19 enzyme activity. Genome-wide association (GWA) analyses were performed to explore loci associated with symptom severity. A multicenter cohort of 804 patients (after quality control N = 684) with schizophrenia spectrum disorder treated with clozapine were cross-sectionally assessed using the Positive and Negative Syndrome Scale and/or the Clinical Global Impression-Severity (CGI-S) scale. GWA and PRS regression analyses were conducted. Genotype-predicted CYP1A2, CYP2D6, and CYP2C19 enzyme activities were calculated. Schizophrenia-PRS was most significantly and positively associated with low symptom severity (p = 1.03 × 10-3; R2 = 1.85). Cross-disorder-PRS was also positively associated with lower CGI-S score (p = 0.01; R2 = 0.81). Compared to the lowest tertile, patients in the highest schizophrenia-PRS tertile had 1.94 times (p = 6.84×10-4) increased probability of low symptom severity. Higher genotype-predicted CYP2C19 enzyme activity was independently associated with lower symptom severity (p = 8.44×10-3). While no locus surpassed the genome-wide significance threshold, rs1923778 within NFIB showed a suggestive association (p = 3.78×10-7) with symptom severity. We show that high schizophrenia-PRS and genotype-predicted CYP2C19 enzyme activity are independently associated with lower symptom severity among individuals treated with clozapine. Our findings open avenues for future pharmacogenomic projects investigating the potential of PRS and genotype-predicted CYP-activity in schizophrenia.

Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation.

Gene therapy holds great potential for the treatment of various acquired and inherited pulmonary diseases. Among various viral vectors, adeno-associated viral (AAV) vectors have been most frequently used in different clinical trials of pulmonary gene therapy. In the present study, we examined the kinetics and duration of transgene expression, vector biodistribution and development of neutralizing antibodies (NAB) in mice after pulmonary application of AAV2/9 vector. The pulmonary route of application did not affect any of the measured parameters. Transgene expression and biodistribution analysis at day 450 post-application confirmed the systemic spread of the vector after pulmonary delivery. Using SPB(-/-) mice, the study shows that AAV2/9-mediated gene expression is influenced by animal gender but not mouse genotype and is insensitive to the presence of lung inflammation. Lower expression levels were observed in male compared with female mice, and transient immunosuppression with dexamethasone significantly reduced the development of NAB in both genders of mice. The study thus advances this serotype for further development and use as a therapeutic vector.

Probing stress relaxation behavior in glassy methacrylate networks containing thio-carbamate additives.

The incorporation of thiourethane prepolymer (TU) into either the organic phase or as a surface treatment for filler particles in composites reduces polymerization stress and improves fracture toughness. The aim of this study was to gain insight into the influence of the inclusion of thiourethanes on the resulting network of methacrylate-based materials polymerized via free-radical mechanisms. Dynamic mechanical analysis was used to elucidate network parameters and potential stress relaxation behavior of these networks. TU oligomers were synthesized using a combination of trimethylol-tris-3-mercaptopropionate and dicyclohexylmethane 4,4'-diisocyanate and added into composite formulations at 20 wt% replacing part of the organic matrix and/or as TU-silanes used to functionalize filler particles (TU-matrix, TU-Sil or TU-matrix/sil). Materials not containing any form of TU were used as the control (in those cases, 3-(trimethoxysilyl)propyl methacrylate was used as the silane agent). Filler was added at 50 wt%. Degree of conversion was evaluated by near-IR spectroscopy, mechanical properties by 3-point bending and rotational rheometry. Dynamic mechanical analysis was used to obtain network parameters (glass transition temperature (Tg), storage modulus, cross-link density, and breadth of tan delta a proxy for network homogeneity - temperature sweep experiments) and to evaluate the potential for network relaxation (stress relaxation). TU-containing formulations showed 10% higher DC than the control. The time to reach storage/loss modulus crossover in the rheometer experiments was significantly longer for TU-matrix and TU-matrix/sil in comparison with the control (21.6, 27.9, and 5.1 s, respectively). TU-matrix and TU-matrix/sil presented significant lower Tg than the control (151.5, 153.8, and 161.3 °C, respectively). There were no statistical differences among the groups in terms of shear modulus, cross-link density, breadth of tan delta, flexural strength/modulus, and toughness. For at least one group (TU-matrix/sil), the relaxation time was four times faster than for the control at 105 °C. The addition of TU additives into dental polymers resulted in a stark reduction in the stress relaxation time. This behavior, in tandem with the network characterization and mechanical properties seems to indicate the TU networks undergo a variety of reversible associative and dissociative chemical reactions which facilitate enhanced stress relief.

Thiourethane filler functionalization for dental resin composites: Concentration-dependent effects on toughening, stress reduction and depth of cure.

OBJECTIVES: The aim of this study was to modify the surface of fillers used in dental composites by the synthesis of two novel thiourethane oligomeric silanes, used to functionalize the silica-containing inorganic particles. Several thiourethane silane concentrations were tested during the silanization process to systematically assess the effect of silane coverage on experimental composite conversion, polymerization stress and fracture toughness. MATERIALS AND METHODS: Two different thiourethane silanes were synthesized based either on 1,6-hexanediol-diissocynate (HDDI), or 1,3-bis(1-isocyanato-1-methylethyl) benzene (BDI). Conventional 3-(Trimethoxysilyl)propyl methacrylate was used as the control. Glass fillers were silanized with 1, 2 or 4 wt% of each thiourethane silane, then evaluated by thermogravimetrical analysis. Photopolymerizable resin composites were prepared with Bis-GMA/UDMA/TEGDMA and 50 wt% silanized glass filler. Polymerization kinetics and degree of conversion were tested using Near-IR. Bioman was used to test polymerization stress. Data were analyzed with two-way ANOVA/Tukey's test (α = 5%). RESULTS: The mass of silane coupled to the filler increased with the concentrations of thiourethane in the silanizing solution, as expected. Thiourethane-containing groups exhibited significantly higher degree of conversion compared to control groups, except for BDI 4%. HDDI 4%, BDI 2% and BDI 4% showed significantly lower polymerization stress than control groups. HDDI 4% exhibited significantly higher fracture toughness. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Novel filler functionalization with thiourethane silanes may be a promising alternative for improving dental composites properties by significantly increasing the degree of conversion, fracture toughness and reducing the polymerization stress.