The effects of supplementation of decapod zoea to Artemia basal diet on the digestive gland histology and the fatty acid composition in common octopus (Octopus vulgaris) paralarvae.

[EN]The present study aimed to evaluate the effect of the supplementation of different crab zoeas to enriched Artemia basal diet for O. vulgaris paralarvae during the first month of life. Paralarvae were fed using: enriched Artemia nauplii alone and Artemia co-fed with either first zoea stages of Grapsus adscensionis or Plagusia depressa. The experiment was carried out over a period of 28 days, in 0.12 m3 tanks with a flow-through rearing system. Growth in dry weightas well as mantle length and width were assessed weekly. Additionally, prey and paralarvae fatty acid composition and digestive gland (DG) histology were evaluated.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Residual visual processing following real or virtual lesions to primary visual pathways

Lesions to the primary geniculo-striate visual pathway cause blindness in the contralesional visual field. Nevertheless, previous studies have suggested that patients with visual field defects may still be able to implicitly process the affective valence of unseen emotional stimuli (affective blindsight) through alternative visual pathways bypassing the striate cortex. These alternative pathways may also allow exploitation of multisensory (audio-visual) integration mechanisms, such that auditory stimulation can enhance visual detection of stimuli which would otherwise be undetected when presented alone (crossmodal blindsight). The present dissertation investigated implicit emotional processing and multisensory integration when conscious visual processing is prevented by real or virtual lesions to the geniculo-striate pathway, in order to further clarify both the nature of these residual processes and the functional aspects of the underlying neural pathways. The present experimental evidence demonstrates that alternative subcortical visual pathways allow implicit processing of the emotional content of facial expressions in the absence of cortical processing. However, this residual ability is limited to fearful expressions. This finding suggests the existence of a subcortical system specialised in detecting danger signals based on coarse visual cues, therefore allowing the early recruitment of flight-or-fight behavioural responses even before conscious and detailed recognition of potential threats can take place. Moreover, the present dissertation extends the knowledge about crossmodal blindsight phenomena by showing that, unlike with visual detection, sound cannot crossmodally enhance visual orientation discrimination in the absence of functional striate cortex. This finding demonstrates, on the one hand, that the striate cortex plays a causative role in crossmodally enhancing visual orientation sensitivity and, on the other hand, that subcortical visual pathways bypassing the striate cortex, despite affording audio-visual integration processes leading to the improvement of simple visual abilities such as detection, cannot mediate multisensory enhancement of more complex visual functions, such as orientation discrimination.

High sensitivity analysis of BRAF mutations in neoplastic and non-neoplastic thyroid lesions

The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

ChronOS Inject in Children with Benign Bone Lesions – Does It Increase the Healing Rate?

Benign bone lesions in children although rare, can result in a pathological fracture. Although their etiology and pathogenesis are not yet entirely clear, the phenomenon of spontaneous healing is well known. Nevertheless, some benign bone lesions are unlikely to heal spontaneously due to the patient's age or high risk of fracture and deformity due to the lesion's location or size. The following study presents our results after treatment of these bone cysts with chronOS Inject.

Clinical activity of pemphigus vulgaris relates to IgE autoantibodies against desmoglein 3

Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disease and is primarily associated with IgG against desmoglein 3 (dsg3), a desmosomal adhesion protein. In light of the recent association of autoreactive T helper (Th) 2 cells with active PV, the present study sought to relate the occurrence of Th2-regulated dsg3-specific autoantibody subtypes, i.e. IgE and IgG4, in 93 well-characterized PV patients. Patients with acute onset PV (n=37) showed the highest concentrations of serum IgE and IgG4 autoantibodies, which were significantly lower in PV patients in remission (n=14). Furthermore, there was a strong correlation between dsg3-reactive IgE and IgG4 in acute onset, but not in chronic active (n=42) or remittent patients. Additionally, intercellular IgE deposits were detected in the epidermis of acute onset PV. Thus, dsg3-specific IgE and IgG4 autoantibodies are related to acute onset disease which provides additional support to the concept that PV is a Th2-driven autoimmune disorder.

Pemphigus herpetiformis: analysis of the autoantibody profile during the disease course with changes in the clinical phenotype

Pemphigus herpetiformis (PH) is a rare dapsone-responsive variant of pemphigus, characterized by annular and vesiculopustular cutaneous lesions. Most PH serum samples contain autoantibodies against desmoglein (Dsg)1, but not Dsg3, and the presence of the latter is almost invariably associated with mucosal involvement, as predicted based on the 'Dsg compensation theory'. We describe a patient with features characteristic of PH with histologically eosinophilic spongiosis who repeatedly tested positive for anti-Dsg3 but not anti-Dsg1 autoantibodies by ELISA. To investigate whether the peculiar clinical phenotype was due to a distinct immunological profile, the patient's serum was tested by ELISA and immunoblotting using recombinant forms of Dsg3. Serum samples were found to have low and high reactivity against the EC1 and the EC4 domains of Dsg3, respectively, whereas the autoantibodies belonged predominantly to the IgG1 and IgG4 subclasses. The overall immunological profile was typical of pemphigus vulgaris. The patient finally developed isolated oral erosions 22 months after initial presentation, without significant changes in the autoantibody profile and of the targeted antigenic sites. Our patient presented features characteristic of PH. Although circulating anti-Dsg3 antibodies were present, the patient had only cutaneous involvement for a long period. Our findings indicate that the proposed Dsg compensation theory cannot always explain the clinical phenotype, changes in which may occur without apparent modification of the autoantibody profile and antibody specificity. Hence, additional factors, such as Fcgamma-dependent neutrophil activation, may critically affect the clinical presentation of pemphigus.

PPAR-gamma expression in peritoneal endometriotic lesions correlates with pain experienced by patients

Endometriosis is a significant gynecologic condition that can cause both pain and infertility and affects up to 15% of women during their reproductive years. In peritoneal endometriotic lesions, the expression of peroxisome proliferation-activated receptor gamma, a nuclear receptor with antiinflammatory and neuroprotective roles, is positively correlated with the pain reported by patients.

PET/CT-guided biopsies of metabolically active bone lesions: applications and clinical impact

In a minority of cases a definite diagnosis and stage grouping in cancer patients is not possible based on the imaging information of PET/CT. We report our experience with percutaneous PET/CT-guided bone biopsies to histologically verify the aetiology of hypermetabolic bone lesions.

Remineralization of initial carious lesions in deciduous enamel after application of dentifrices of different fluoride concentrations

The aim of the present study was to evaluate the remineralization potential of five dentifrices with different fluoride concentrations. Initial caries lesions were created in 72 cylindrical enamel blocks from deciduous teeth. The specimens were randomly distributed among six experimental groups corresponding to six experimental periods. Each of the six volunteers carried two deciduous enamel specimens fixed in an intraoral appliance for a period of 4 weeks. They brushed their teeth and the enamel blocks at least two times a day with dentifrices containing 0 ppm (period 1), 250 ppm (period 2), and 500 ppm fluoride (period 3), respectively. A second group of volunteers (n = 6) used dentifrices with a fluoride content of 0 ppm (period 4), 1,000 ppm (period 5), or 1,500 ppm (period 6). At the end of the respective period, the mineral content was determined by transversal microradiography (TMR). The use of dentifrices containing 500 ppm fluoride (38% MR), 1,000 ppm fluoride (42% MR), and 1,500 ppm fluoride (42% MR) resulted in a statistically significant higher mineral recovery compared to the control group (0 ppm fluoride). Mineral recovery was similar after use of dentifrices containing 0 and 250 ppm fluoride (24%; 25%). It is concluded that it is possible to remineralize initial carious lesions in deciduous enamel in a similar way as it has been described for enamel of permanent teeth.

Formation of caries-like lesions in vitro on the root surfaces of human teeth in solutions simulating plaque fluid

Lesion formation on root surfaces of human posterior teeth was studied in acetate/lactate buffers with a background electrolyte composition based on plaque fluid analyses. Lesion depth after 28 days at 37 degrees C was measured in relation to: the presence or absence of cementum; the concentration of undissociated buffer; the presence or absence of magnesium ions at plaque fluid concentration. Each factor was evaluated at several values of -log(ion activity product for hydroxyapatite): pI(HA). Solutions were formulated to minimize variation in pH, which varied by < or =0.03 for a given comparison (individual pI(HA)) and by 0.42-0.82 over the range of pI(HA) within experiments. Lesions on surfaces from which cementum had been ground were significantly deeper than on intact surfaces, but this is considered to be due to subsurface mechanical damage and not to a solubility difference. Neither the concentration of undissociated buffer nor the presence of magnesium ions significantly affected lesion depth. Lesion depth was strongly influenced by the correlated variations in pI(HA) and pH. At pI(HA) 54 and 55, only extremely shallow lesions formed. From pI(HA) 56, lesion depth increased with increasing pI(HA). The results confirm that the solubility of the mineral of root tissues is higher than that of hydroxyapatite, but indicate that it is probably lower than suggested by Hoppenbrouwers et al. [Arch Oral Biol 1987;32:319-322]. For calcium concentrations of 3-12 mM, the critical pH for root tissue mineral was calculated as 5.22-5.66 assuming solubility equivalent to pI(HA) 54 and 5.08-5.51 assuming pI(HA) 55.

Late Infiltration of Post-orthodontic White Spot Lesions

White spot lesion (WSL) infiltration has been recommended immediately after debonding of orthodontic brackets. It is however not clear if established inactive WSLs can also be masked through infiltrationOrthodontic treatment of a 19-year-old patient had to be terminated prematurely due to development of multiple WSLs of varying severity. Three months after debonding, the patient presented for lesion infiltration. After etching with 15% HCl gel and re-wetting of the dried surfaces it seemed that a good outcome could be expected. Lesion infiltration led to complete masking of less severe WSLs. The visual appearance of moderate and severe WSLs was improved but they were still visible after treatment.Inactive WSLs may not represent an increased caries risk, but patients are often bothered esthetically. Infiltration by repeated etching might be a viable approach even for inactive WSLs. Controlled clinical trials are needed to investigate the long-term performance of this technique.

CO2 and diode laser for excisional biopsies of oral mucosal lesions. A pilot study evaluating clinical and histopathological parameters

PURPOSE: The present pilot study evaluates the histopathological characteristics and suitability of CO2 and diode lasers for performing excisional biopsies in the buccal mucosa with special emphasis on the extent of the thermal damage zone created. PATIENTS AND METHODS: 15 patients agreed to undergo surgical removal of their fibrous hyperplasias with a laser. These patients were randomly assigned to one diode or two CO2 laser groups. The CO2 laser was used in a continuous wave mode (cw) with a power of 5 W (Watts), and in a pulsed char-free mode (cf). Power settings for the diode laser were 5.12 W in a pulsed mode. The thermal damage zone of the three lasers and intraoperative and postoperative complications were assessed and compared. RESULTS: The collateral thermal damage zone on the borders of the excisional biopsies was significantly smaller with the CO, laser for both settings tested compared to the diode laser regarding values in pm or histopathological index scores. The only intraoperative complication encountered was bleeding, which had to be controlled with electrocauterization. No postoperative complications occurred in any of the three groups. CONCLUSIONS: The CO2 laser seems to be appropriate for excisional biopsies of benign oral mucosal lesions. The CO2 laser offers clear advantages in terms of smaller thermal damage zones over the diode laser. More study participants are needed to demonstrate potential differences between the two different CO2 laser settings tested.