Giuseppe Perinetti

Intraflagellar transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse

Most eukaryotic cells have a primary cilium which functions as a sensory organelle. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly, was found to colocalize with both the microtubule organizing centre (MTOC) and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse. IFT20 knockdown resulted in impaired T-cell receptor/CD3 (TCR/CD3) clustering and signalling at the immune synapse, due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57 and IFT88) and the TCR. The results identify IFT20 as a new regulator of immune synapse assembly in T cells and provide the first evidence to implicate IFT in membrane trafficking in cells lacking primary cilia, thereby introducing a new perspective on IFT function beyond its role in ciliogenesis.

The closure of Pak1‐dependent macropinosomes requires the phosphorylation of CtBP1/BARS

Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C‐terminal‐binding protein‐1/brefeldinA‐ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin‐independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21‐activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS‐induced fission to human viral infection.

Dental occlusion, body posture and temporomandibular disorders: where we are now and where we are heading for

The aim of this investigation was to perform a review of the literature dealing with the issue of relationships between dental occlusion, body posture and temporomandibular disorders (TMD). A search of the available literature was performed to determine what the current evidence is regarding: (i) The physiology of the dental occlusion-body posture relationship, (ii) The relationship of these two topics with TMD and (iii) The validity of the available clinical and instrumental devices (surface electromyography, kinesiography and postural platforms) to measure the dental occlusion-body posture-TMD relationship. The available posturographic techniques and devices have not consistently found any association between body posture and dental occlusion. This outcome is most likely due to the many compensation mechanisms occurring within the neuromuscular system regulating body balance. Furthermore, the literature shows that TMD are not often related to specific occlusal conditions, and they also do not have any detectable relationships with head and body posture. The use of clinical and instrumental approaches for assessing body posture is not supported by the wide majority of the literature, mainly because of wide variations in the measurable variables of posture. In conclusion, there is no evidence for the existence of a predictable relationship between occlusal and postural features, and it is clear that the presence of TMD pain is not related with the existence of measurable occluso-postural abnormalities. Therefore, the use instruments and techniques aiming to measure purported occlusal, electromyographic, kinesiographic or posturographic abnormalities cannot be justified in the evidence-based TMD practice.

Clinical, microbiologic, and biochemical effects of subgingival administration of a xanthan-based chlorhexidine gel in the treatment of periodontitis: a randomized multicenter trial.

BACKGROUND The use of locally delivered antibacterials containing chlorhexidine (CHX) was proposed to improve the effectiveness of non-surgical periodontal treatment. The present multicenter randomized study investigated the effects of a xanthan-based chlorhexidine (Xan-CHX) gel used as an adjunct to scaling and root planing (SRP) in the treatment of chronic periodontitis. METHODS Ninety-eight systemically healthy subjects with moderate to advanced periodontitis were recruited in four centers (59 females and 39 males; aged 24 to 58 years). For each subject, two experimental sites located in two symmetric quadrants were chosen with probing depths (PD) >or=5 mm and positive for bleeding on probing (BOP). These two sites were randomized at the split-mouth level with one receiving a single SRP treatment and the other receiving a single SRP + Xan-CHX gel treatment. Supragingival plaque, modified gingival index, PD, clinical attachment level (CAL), and BOP were evaluated at baseline (prior to any treatment) and after 3 and 6 months. At the same times, subgingival microbiologic samples and gingival crevicular fluid (GCF) were collected for the analysis of total bacterial counts (TBCs), including the identification of eight putative periodontopathogens, and alkaline phosphatase (ALP) activity, respectively. RESULTS The Xan-CHX treatment group showed greater improvements compared to the SRP group for PD and CAL at 3 and 6 months (P <0.001). The differences in PD reduction between the treatments were 0.87 and 0.83 mm at 3 and 6 months, respectively (P <0.001); for CAL, these were 0.94 and 0.90 mm, respectively (P <0.001). Similar behavior was seen when the subgroup of pockets >or=7 mm was considered. The percentage of sites positive for BOP was similar between the treatments at each time point. For the comparisons between the treatment groups, no differences were seen in the TBCs and GCF ALP activity at baseline and 6 months; in contrast, slightly, but significantly, lower scores were recorded for the Xan-CHX treatment group at 3 months (P = 0.018 and P = 0.045, respectively). Moreover, greater reductions in the percentages of sites positive for the eight putative periodontopathic bacteria were generally seen for the Xan-CHX treatment group compared to SRP alone. CONCLUSIONS The adjunctive use of Xan-CHX gel promoted greater PD reductions and CAL gains compared to SRP alone. These results were concomitant with better microbiologic and biochemical outcomes when Xan-CHX gel use was added to SRP, particularly up to 3 months after treatment.

Diagnostic performance of dental maturity for identification of skeletal maturation phase

The objective of this study is to analyse the diagnostic performance of the circumpubertal dental maturation phases for the identification of individual-specific skeletal maturation phases. A total of 354 healthy subjects, 208 females and 146 males (mean age, 11.1 ± 2.4 years; range, 6.8-17.1 years), were enrolled in the study. Dental maturity was assessed through the calcification stages from panoramic radiographs of the mandibular canine, the first and second premolars, and the second molar. Determination of skeletal maturity was according to the cervical vertebra maturation (CVM) method on lateral cephalograms. Diagnostic performances were evaluated according to the dental maturation stages for each tooth for the identification of the CVM stages and growth phases (as pre-pubertal, pubertal, and post-pubertal) using positive likelihood ratios (LHRs). A positive LHR threshold of 10 or more was considered for satisfactory reliability of any dental maturation stage for the identification of any of the CVM stages or growth phases. The positive LHRs were generally less than 2.0, with a few exceptions. These four teeth showed positive LHRs greater than 10 only for the identification of the pre-pubertal growth phase, with values from 10.8 for the second molar (stage E) to 39.3 for the first premolar (stage E). Dental maturation assessment is only useful for diagnosis of the pre-pubertal growth phase, and thus, precise information in relation to the timing of the onset of the growth spurt is not provided by these indices.

Dental malocclusion and body posture in young subjects: a multiple regression study

OBJECTIVES: Controversial results have been reported on potential correlations between the stomatognathic system and body posture. We investigated whether malocclusal traits correlate with body posture alterations in young subjects to determine possible clinical applications. METHODS: A total of 122 subjects, including 86 males and 36 females (age range of 10.8–16.3 years), were enrolled. All subjects tested negative for temporomandibular disorders or other conditions affecting the stomatognathic systems, except malocclusion. A dental occlusion assessment included phase of dentition, molar class, overjet, overbite, anterior and posterior crossbite, scissorbite, mandibular crowding and dental midline deviation. In addition, body posture was recorded through static posturography using a vertical force platform. Recordings were performed under two conditions, namely, i) mandibular rest position (RP) and ii) dental intercuspidal position (ICP). Posturographic parameters included the projected sway area and velocity and the antero-posterior and right-left load differences. Multiple regression models were run for both recording conditions to evaluate associations between each malocclusal trait and posturographic parameters. RESULTS: All of the posturographic parameters had large variability and were very similar between the two recording conditions. Moreover, a limited number of weakly significant correlations were observed, mainly for overbite and dentition phase, when using multivariate models. CONCLUSION: Our current findings, particularly with regard to the use of posturography as a diagnostic aid for subjects affected by dental malocclusion, do not support existence of clinically relevant correlations between malocclusal traits and body posture.

The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse.

ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. Highlighted Article: The ciliary regulator Rab8 is co-opted by T-cells during the assembly of the immune synapse to promote the VAMP-3-dependent step of polarized T-cell receptor recycling.

Correlation of 4Pi and electron microscopy to study transport through single Golgi stacks in living cells with super resolution.

Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited resolution and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern high‐resolution light microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electron microscopy in cell biology applications. In this study, we provide the first comparative correlative analysis of three‐dimensional structures obtained by 4Pi microscopy and segmented by a zero‐crossing procedure with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein procollagen 3 was mapped by both 4Pi microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for the structures, indicating the particular accuracy of the 4Pi microscopy. Furthermore, for the first time, transport of a cargo molecule (vesicular stomatitis virus G protein‐pEGFP) through individual Golgi stacks (labeled by galactosyl transferase‐venusYFP) was visualized by 4Pi microscopy. Following the procedures validated by the correlative analysis, our transport experiments show that (i) VSVG‐pEGFP rapidly enter/exit individual Golgi stacks, (ii) VSVG‐pEGFP never fills the GalT‐venusYFP compartments completely and (iii) the GalT‐venusYFP compartment volume increases upon VSVG‐pEGFP arrival. This morphological evidence supports some previous TEM‐based observations of intra‐Golgi transport of VSVG‐pEGFP and provides new insights toward a better understanding of protein progression across Golgi stacks. Our study thus demonstrates the general applicability of super‐resolution fluorescence microscopy, coupled with the zero‐crossing segmentation procedure, for structural studies of suborganelle protein distributions under living cell conditions.

In vitro evaluation of fracture resistance and failure mode of internally restored endodontically treated maxillary incisors with differing heights of residual dentin

STATEMENT OF PROBLEM Some of the associated effects of different restorative systems placed in endodontically treated teeth with varying heights of residual dentin have yet to be examined in a comprehensive manner. There is a need for additional information regarding fracture resistance and mode of failure. PURPOSE The purpose of this in vitro study was to evaluate the effect of 3 different restorative techniques with varying amounts of remaining dentin heights on the fracture resistance and failure mode of endodontically treated teeth. MATERIAL AND METHODS Three groups of 40 human maxillary incisors were subdivided into 4 subgroups (n=10) with respect to the uniform height of the residual coronal dentin, defined as 0-, 2-, 4-, or 5-mm from the cemento-enamel junction, and then restored internally using a composite resin (Z100 MP) (control group), a cobalt-chromium ceramic alloy custom-made cast post and core (IPS d.SIGN 30; CCPC group), or a carbon fiber post system (Tech Xop 2000; CFP group). All specimens were then restored with nonprecious cast crowns. Static loading tests were performed on each specimen until failure (crack without a complete fracture). The data were analyzed with 2-way ANOVA and Bonferroni-corrected t test for independent samples (alpha=.05). Failure was classified as either favorable (allowing repair) or catastrophic (not allowing repair). RESULTS The fracture resistance values (N) for the 0-, 2-, 4-, and 5-mm residual dentin heights were: 88, 143, 154, and 202 for the control group, 230, 264, 364, and 383 for the CCPC group, and 153, 235, 346, and 357 for the CFP group, respectively. Generally, all the differences tested were statistically significant. The failure mode was catastrophic for no control specimens, for 36 CCPC specimens, and for 4 CFP specimens. CONCLUSIONS The highest and lowest fracture resistances were recorded for the CCPC and control groups, respectively, at each residual dentin height. An increased height of residual dentin generally provided greater fracture resistance. The fracture resistance of the CCPC group was, however, similar or only slightly higher than that of the CFP group when 2, 4, or 5 mm of residual dentin height was present. In contrast, the failure mode was favorable for almost all of the CFP and control groups, while it was catastrophic in most of the CCPC group.

Treatment Effects of Removable Functional Appliances in Pre-Pubertal and Pubertal Class II Patients: A Systematic Review and Meta-Analysis of Controlled Studies

Background Treatment effects of removable functional appliances in Class II malocclusion patients according to the pre-pubertal or pubertal growth phase has yet to be clarified. Objectives To assess and compare skeletal and dentoalveolar effects of removable functional appliances in Class II malocclusion treatment between pre-pubertal and pubertal patients. Search methods Literature survey using the Medline, SCOPUS, LILACS and SciELO databases, the Cochrane Library from inception to May 31, 2015. A manual search was also performed. Selection criteria Randomised (RCTs) or controlled clinical trials with a matched untreated control group. No restrictions were set regarding the type of removable appliance whenever used alone. Data collection and analysis For the meta-analysis, cephalometric parameters on the supplementary mandibular growth were the main outcomes, with other cephalometric parameters considered as secondary outcomes. Risk of bias in individual and across studies were evaluated along with sensitivity analysis for low quality studies. Mean differences and 95% confidence intervals for annualised changes were computed according to a random model. Differences between pre-pubertal and pubertal patients were assessed by subgroup analyses. GRADE assessment was performed for the main outcomes. Results Twelve articles (but only 3 RCTs) were included accounting for 8 pre-pubertal and 7 pubertal groups. Overall supplementary total mandibular length and mandibular ramus height were 0.95 mm (0.38, 1.51) and 0.00 mm (-0.52, 0.53) for pre-pubertal patients and 2.91 mm (2.04, 3.79) and 2.18 mm (1.51, 2.86) for pubertal patients, respectively. The subgroup difference was significant for both parameters (p<0.001). No maxillary growth restrain or increase in facial divergence was seen in either subgroup. The GRADE assessment was low for the pre-pubertal patients, and generally moderate for the pubertal patients. Conclusions Taking into account the limited quality and heterogeneity of the included studies, functional treatment by removable appliances may be effective in treating Class II malocclusion with clinically relevant skeletal effects if performed during the pubertal growth phase.