In-vitro assessment of the forces generated by lingual fixed retainers

The aim of this article was to assess the effect of wire adaptation on the lingual surfaces of mandibular anterior teeth with 3 types of lingual retainers on the development of vertical and labiolingual forces.

A comparative assessment of torque generated by lingual and conventional brackets

The aim of this study was to assess the effect of bracket type on the labiopalatal moments generated by lingual and conventional brackets. Incognito™ lingual brackets (3M Unitek), STb™ lingual brackets (Light Lingual System; ORMCO), In-Ovation L lingual brackets (DENTSPLY GAC), and conventional 0.018 inch slot brackets (Gemini; 3M Unitek) were bonded on identical maxillary acrylic resin models with levelled and aligned teeth. Each model was mounted on the orthodontic measurement and simulation system and 10 0.0175 × 0.0175 TMA wires were used for each bracket type. The wire was ligated with elastomerics into the Incognito, STb, and conventional brackets and each measurement was repeated once after religation. A 15 degrees buccal root torque (+15 degrees) and then a 15 degrees palatal root torque (-15 degrees) were gradually applied to the right central incisor bracket. After each activation, the bracket returned to its initial position and the moments in the sagittal plane were recorded during these rotations of the bracket. One-way analysis of variance with post hoc multiple comparisons (Tukey test at 0.05 error rate) was conducted to assess the effect on bracket type on the generated moments. The magnitude of maximum moment at +15 degrees ranged 8.8, 8.2, 7.1, and 5.8 Nmm for the Incognito, STb, conventional Gemini, and the In-Ovation L brackets, respectively; similar values were recorded at -15 degrees: 8.6, 8.1, 7.0, and 5.7 Nmm, respectively. The recorded differences of maximum moments were statistically significant, except between the Incognito and STb brackets. Additionally, the torque angles were evaluated at which the crown torque fell well below the minimum levels of 5.0 Nmm, as well as the moment/torque ratio at the last part of the activation/deactivation curve, between 10 and 15 degrees. The lowest torque expression was observed at the self-ligating lingual brackets, followed by the conventional brackets. The Incognito and STb lingual brackets generated the highest moments.

A comparative assessment of forces and moments generated by lingual and conventional brackets

The aim of this study was to assess the effect of bracket type on the labiopalatal forces and moments generated in the sagittal plane. Incognito™ lingual brackets (3M Unitek), STb™ lingual brackets (Light Lingual System; ORMCO), and conventional 0.018 inch slot brackets (Gemini; 3M Unitek) were bonded on three identical maxillary acrylic resin models, with a palatally displaced right lateral incisor. The transfer trays for the indirect bonding of the lingual brackets were constructed in certified laboratories. Each model was mounted on the orthodontic measurement and simulation system and ten 0.013 inch CuNiTi wires were used for each bracket type. The wire was ligated with elastomerics and each measurement was repeated once after re-ligation. The labiopalatal forces and the moments in the sagittal plane were recorded on the right lateral incisor. One-way analysis of variance and post hoc Scheffe pairwise comparisons were used to assess the effect on bracket type on the generated forces and moments. The magnitude of forces ranged from 1.62, 1.27, and 1.81 N for the STb, conventional, and Incognito brackets, respectively; the corresponding moments were 2.01, 1.45, and 2.19 N mm, respectively. Bracket type was a significant predictor of the generated forces (P < 0.001) and moments (P < 0.001). The produced forces were different among all three bracket types, whereas the generated moments differed between conventional and lingual brackets but not between lingual brackets.

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

Synthesis and Structural Characterizations of New Coordination Polymers Generated by the Interaction Between the Trinuclear Triangular SBU [Cu 3 (μ 3 -OH)(μ-pz) 3 ] 2+ and 4,4′-Bipyridine. 3°

The reactions of 4,4′-bipyridine with selected trinuclear triangular copper(II) complexes, [Cu3(μ3-OH)(μ-pz)3(RCOO)2Lx], [pz = pyrazolate anion, R = CH3(CH2)n (2 ≤ n ≤ 5); L = H2O, MeOH, EtOH] yielded a series of 1D coordination polymers (CPs) based on the repetition of [Cu3(μ3-OH)(μ-pz)3] secondary building units joined by bipyridine. The CPs were characterized by conventional analytical methods (elemental analyses, ESI-MS, IR spectra) and single crystal XRD determinations. An unprecedented 1D CP, generated through the bipyridine bridging hexanuclear copper clusters moieties, two 1D CPs presenting structural analogies, and two monodimensional tapes having almost exactly superimposable structures, were obtained. In one case, the crystal packing makes evident the presence of small, not-connected pores, accounting for ca. 6% of free cell volume.

Prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media.

Assisted reproductive technologies (ART) induce vascular dysfunction in humans and mice. In mice, ART-induced vascular dysfunction is related to epigenetic alteration of the endothelial nitric oxide synthase (eNOS) gene, resulting in decreased vascular eNOS expression and nitrite/nitrate synthesis. Melatonin is involved in epigenetic regulation, and its administration to sterile women improves the success rate of ART. We hypothesized that addition of melatonin to culture media may prevent ART-induced epigenetic and cardiovascular alterations in mice. We, therefore, assessed mesenteric-artery responses to acetylcholine and arterial blood pressure, together with DNA methylation of the eNOS gene promoter in vascular tissue and nitric oxide plasma concentration in 12-wk-old ART mice generated with and without addition of melatonin to culture media and in control mice. As expected, acetylcholine-induced mesenteric-artery dilation was impaired (P = 0.008 vs. control) and mean arterial blood pressure increased (109.5 ± 3.8 vs. 104.0 ± 4.7 mmHg, P = 0.002, ART vs. control) in ART compared with control mice. These alterations were associated with altered DNA methylation of the eNOS gene promoter (P < 0.001 vs. control) and decreased plasma nitric oxide concentration (10.1 ± 11.1 vs. 29.5 ± 8.0 μM) (P < 0.001 ART vs. control). Addition of melatonin (10(-6) M) to culture media prevented eNOS dysmethylation (P = 0.005, vs. ART + vehicle), normalized nitric oxide plasma concentration (23.1 ± 14.6 μM, P = 0.002 vs. ART + vehicle) and mesentery-artery responsiveness to acetylcholine (P < 0.008 vs. ART + vehicle), and prevented arterial hypertension (104.6 ± 3.4 mmHg, P < 0.003 vs. ART + vehicle). These findings provide proof of principle that modification of culture media prevents ART-induced vascular dysfunction. We speculate that this approach will also allow preventing ART-induced premature atherosclerosis in humans.

Assessing stool quantities generated by three specific Kato-Katz thick smear templates employed in different settings.

BACKGROUND The Kato-Katz technique is recommended for the diagnosis of helminth infections in epidemiological surveys, drug efficacy studies and monitoring of control interventions. We assessed the comparability of the average amount of faeces generated by three Kato-Katz templates included in test kits from two different providers. METHODS Nine hundred Kato-Katz thick smear preparations were done; 300 per kit. Empty slides, slides plus Kato-Katz template filled with stool and slides plus stool after careful removal of the template were weighed to the nearest 0.1 mg. The average amount of stool that was generated on the slide was calculated for each template, stratified by standard categories of stool consistency (i.e. mushy, soft, sausage-shaped, hard and clumpy). RESULTS The average amount of stool generated on slides was 40.7 mg (95 % confidence interval (CI): 40.0-41.4 mg), 40.3 mg (95 % CI: 39.7-40.9 mg) and 42.8 mg (95 % CI: 42.2-43.3 mg) for the standard Vestergaard Frandsen template, and two different templates from the Chinese Center for Disease Control and Prevention (China CDC), respectively. Mushy stool resulted in considerably lower average weights when the Vestergaard Frandsen (37.0 mg; 95 % CI: 34.9-39.0 mg) or new China CDC templates (37.4 mg; 95 % CI: 35.9-38.9 mg) were used, compared to the old China CDC template (42.2 mg; 95 % CI: 40.7-43.7 mg) and compared to other stool consistency categories. CONCLUSION The average amount of stool generated by three specific Kato-Katz templates was similar (40.3-42.8 mg). Since the multiplication factor is somewhat arbitrary and small changes only have little effect on infection intensity categories, it is suggested that the standard multiplication factor of 24 should be kept for the calculation of eggs per gram of faeces for all investigated templates.

Specific processing of tenascin-C by the metalloprotease meprinbeta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprinbeta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprinbeta. In chicken tenascin-C, meprinbeta processed all three major splicing variants by removal of 10kDa N-terminal and 38kDa C-terminal peptides, leaving a large central part of subunits intact. A similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15kDa) and two C-terminal fragments (40 and 55kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprinbeta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprinbeta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprinbeta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprinbeta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprinbeta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Cathepsin D primes caspase-8 activation by multiple intra-chain proteolysis

During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.

Activation of T cells by carbamazepine and carbamazepine metabolites

BACKGROUND: T-cell-mediated hypersensitivity is a rare but serious manifestation of drug therapy. OBJECTIVES: To explore the mechanisms of drug presentation to T cells and the possibility that generation of metabolite-specific T cells may provoke cross-sensitization between drugs. METHODS: A lymphocyte transformation test was performed on 13 hypersensitive patients with carbamazepine, oxcarbazepine, and carbamazepine metabolites. Serial dilution experiments were performed to generate drug (metabolite)-specific T-cell clones to explore the structural basis of the T-cell response and mechanisms of antigen presentation. 3-Dimensional energy-minimized structures were generated by using computer modeling. The role of drug metabolism was analyzed with 1-aminobenzotriazole. RESULTS: Lymphocytes and T-cell clones proliferated with carbamazepine, oxcarbazepine, and some (carbamazepine 10,11 epoxide, 10-hydroxy carbamazepine) but not all stable carbamazepine metabolites. Structure activity studies using 29 carbamazepine (metabolite)-specific T-cell clones revealed 4 patterns of drug recognition, which could be explained by generation of preferred 3-dimensional structural conformations. T cells were stimulated by carbamazepine (metabolites) bound directly to MHC in the absence of processing. The activation threshold for T-cell proliferation varied between 5 minutes and 4 hours. 1-Aminobenzotriazole, which inhibits cytochrome P450 activity, did not prevent carbamazepine-related T-cell proliferation. Substitution of the terminal amine residue of carbamazepine with a methyl group diminished T-cell proliferation. CONCLUSION: These data show that carbamazepine and certain stable carbamazepine metabolites stimulate T cells rapidly via a direct interaction with MHC and specific T-cell receptors. CLINICAL IMPLICATIONS: Some patients with a history of carbamazepine hypersensitivity possess T cells that cross-react with oxcarbazepine, providing a rationale for cross-sensitivity between the 2 drugs.