Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

FDPS finish line design and tooth preparation technique.

The purpose of this research is to perform and verify the “modified 50° preparation” suggested by the authors. This procedure was performed with a scrupulous standardization of the tooth’s preparation and then of the laboratory techniques to produce a metal-ceramic crown, and the same for the crown cementation technique of on the preparation of the original sample. After the esthetic evaluations, the obtained sample was included in EPON resin and sectioned in the lingual-vestibular sense. The sections were then observed with an optical microscope in different magnifications for the final evaluations. The geometric design proposed presents the advantages of two great preparation techniques: 50° preparation and circumferential shoulder. The 50° bevel designed in first phase of preparation and the second phase of the circumferential 27° shoulder, together allowed to satisfy the following requirements: aesthetic, marginal accuracy, periodontal compliance, conservation and stability. However, this type of prosthetic preparation is not the only one, but it is one of the marginal designs in prosthetic dentistry.

Anatomic relationship between temporomandibular joint and middle ear.

The present study was designed to identify a relationship between temporomandibular joint and tympanic cavity and potentially justify the possible clinical correlations between these two anatomical entities. For this reason the authors conducted an anatomic-pathological study about the temporomandibular joint (TMJ) and the neighboring anatomical areas of the middle ear by autopsy finds of human adults.

Temporomandibular joint and related structures: anatomical and Histological aspects.

In literature, there is no unanimous agreement about the anatomical and functional characteristics of the temporomandibular joint (TMJ) and its components. The aim of this work is to increase the knowledge about components of the temporomandibular joint, starting from the revision of classical anatomy, through macroscopic and microscopic study, 20 samples of human dissected temporomandibular joints.

TMJ’s capsule histological and macroscopical study: relationship between ligamentous laxity and TMI dysfunctions.

In this study, the authors examined the capsular structures of 20 human temporomandibular joints (TMJ) macroscopically and microscopically, in order to improve knowledge of these structures, as part of their possible participation in the genesis of TMJ dysfunctions.

A comparative study of antibacterial and anti-inflammatory effects of mouthrinse containing tea tree oil.

The study evaluated the antibacterial and anti-inflammatory efficacy, domiciliary oral hygiene, of a mouthrinse containing Tea Tree Oil (TTO) comparing it with two mouthrinses containing chlorhexidine 0,12% respectively and essential oils, and a placebo. MATERIALS AND METHODS: A pilot study, randomized 4 × 4, controlled, cross-over, double-blind. 16 subjects with gingivitis (7 males and 9 females) aged 21-37 years, were randomly divided into four groups based on mouthwash that had to be used for domiciliary oral hygiene: mouthwash with essential oils, mouthwash with chlorhexidine 0,12 %, mouthwash containig tea tree oil and mouthwash placebo. Clinical evaluation was performed by: Full Mouth Plaque Score (FMPS), Full Mouth Bleeding Score (FMBS), Gingival Index (GI), discolorations, language examination and alteration of taste. The data were recorded before and 2 weeks after treatment. RESULTS: Statistical analysis shows that treatments with tea tree oil, essential oils and chlorhexidine are effective. Comparing treatments should be noted that the tea tree oil gives a greater improvement in the GI and FMBS, while it is the least effective in the control of bacterial plaque. CONCLUSION: Although further studies are needed, the anti-inflammatory properties of the mouthwash made from TTO would seem to be a valuable non-toxic adjunct in the management of gingivitis.

Cad-cam procedure and implant-prosthetic rehaBilitation. Case report.

OBJECT: The aim of this paper is to present a clinical case in which the CAD-CAM procedure was applied for a prosthetic rehabilitation on implants. MATERIALS: Digital CAD-CAM production by laboratory of the final restoration. RESULTS: CAD-CAM production offers the opportunity to easily collaborate with laboratory. This prosthetic production gives better and demonstrated clinical results for the patient. CONCLUSION: CAD-CAM production is a very important instrument for prosthetic team. This work-flow compared with traditional methods is faster, precise and predictable.

A novel workflow for computer guided implant surgery matching digital dental casts and CBCT scan.

Nowadays computer-guided "flap-less" surgery for implant placement using stereolithographic tem-plates is gaining popularity among clinicians and patients. The advantages of this surgical protocol are its minimally invasive nature, accuracy of implant placement, predictability, less post-surgical discomfort and reduced time required for definitive rehabilitation. Aim of this work is to describe a new protocol (Smart Fusion by Nobel Biocare), thanks to which is now possible to do a mini-invasive static guided implant surgery, in partially edentulous patients with at least 6 remaining teeth, without the use of a radiographic guide. This is possible thanks to a procedure named surface mapping based on the matching between numerous points on the surface of patient's dental casts and the corresponding anatomical surface points in the CBCT data. The full protocol is examined focusing the attention on the clinical and laboratory procedures. CONCLUSIONS: Also with some critical points and needing an adequate learning curve, this protocol allows to select the ideal implant position in depth, inclination and mesio-distal distance between natural teeth and or other implants enabling a very safe and predictable rehabilitation compared with conventional surgery. It represents a good tool for the best compromise between anatomy, function and aesthetic, able to guarantee better results in all clinical situations.

Her2/neu extracellular domain shedding in uterine serous carcinoma: implications for immunotherapy with trastuzumab.

BACKGROUND: We evaluated shedding of epidermal growth factor type II receptor (Her2/neu) extracellular domain (ECD) in primary uterine serous carcinoma (USC) cell lines and in the serum of USC patients and its biological effects in experiments of trastuzumab-induced cytotoxicity in vitro. METHODS: Her2/neu expression was evaluated by immunohistochemistry (IHC), real-time PCR and flow cytometry, while c-erbB2 gene amplification was assessed using fluorescent in situ hybridisation (FISH). Her2/neu ECD levels in the supernatants of USC cell lines and in the serum of 38 USC patients and 19 controls were tested using ELISA. The biologic effect of Her2/neu ECD on trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated in 5-h chromium-release assays. RESULTS: Five out of ten USC cell lines overexpressed Her2/neu by IHC and showed amplification of the c-erbB2 gene. High levels of Her2/neu ECD were found in supernatants of all FISH-positive tumours. In contrast, FISH-negative USC was negative for Her2/neu ECD shedding. Serum Her2/neu ECD levels in patients harbouring 3+Her2/neu tumours were higher than those found in healthy women (P=0.02) or USC patients with 2+ or 1+/negative Her2/neu expression (P=0.02). In cytotoxicity experiments, trastuzumab-mediated ADCC was significantly decreased by the addition of Her2/neu ECD-containing supernatants (P=0.01). CONCLUSION: FISH-positive c-erbB2 USC cell lines shed high levels of Her2/neu ECD. High levels of Her2/neu ECD in USC patients may reduce trastuzumab-mediated ADCC in vitro and potentially neutralise its therapeutic effect in vivo.

The inhibitory effects of wine phenolics on lysozyme activity against lactic acid bacteria.

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating.

[Prevention of postoperative adhesions]. / Aderenze postchirurgiche: attuali strategie preventive.

Postoperative adhesions represent a common consequence in patients who underwent abdominal or pelvic surgery. Such adhesions can be asymptomatic, but they can cause complications such as chronic abdomino-pelvic pain, secondary infertility, an increase in bowel obstruction risk and more complexity for future surgery, including longer surgery times and an increase in morbidity. Normally, adhesions appear after offences against the peritoneum, causing flogosys, and develop both in new sites, previously not involved, and in sites already interested in adhesiolysis. Previous laparotomy is an important risk factor, as after laparatomy a minimum of 93% of patients present adhesions during a following surgery. Furthermore, the rate of recurrence after adhesiolysis is 85%. Among several strategies employed, valid prevention methods are: using minimally invasive surgery techniques, reducing the incision area, containing tissue dehydration during surgery and an accurate hemostasis. Also, for preventing and reducing adhesions, the usage of NSAIDs, fibrinolytics and anticoagulants, as well as the application of substances acting as a physical barrier, have been proposed. Recently, crystalloid solutions have been introduced, using the hydro-flotation principle for intraperitoneal organs. This research aims to analyze causes and epidemiology for postoperative adhesions, with particular regard to gynecological operations and to describe and compare the means available to prevent them.

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine.

AIMS: To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum) in wine fermentation. METHODS AND RESULTS: Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose(-) and Melibiose(+) strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and beta-glucosidase activities were assayed on whole cells during fermentation at 13 degrees and 20 degrees C. Saccharomyces uvarum displayed higher esterase activity at 13 degrees C, while S. cerevisiae displayed higher beta-glucosidase activity at both temperatures. CONCLUSIONS: The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.

[Use of bulking agents in urinary incontinece]. / L'utilizzo dei bulking agents nel trattamento dell'incontinenza urinaria.

Urinary incontinence consist in voluntary urine leakage. Female affected in the world are about 200 thousand. Urinary incontinence affect severely women quality of life. There are different kinds of urinary incontinence that can be treated in different ways. We can use pelvic floor rehabilitation, drug therapy, invasive and non-invasive surgical treatment. Different treatments are used for different incontinence types. Periurethral injection is the most common procedure between non-invasive surgery. The most recent bulking agents occasionally determine severe adverse reaction or complication. Frequently we can have just pain during injection and a temporary urine retention. During the latest years we used a lot of bulking agents: bovine collagen, autologous fat, carbon particles, macroplastique, calcium hydroxylapatite, ethylene vinyl alcohol copolymer, dextranomer. Urethral injection have success in 40-90%. We can assert that macroplastique is the most efficacy and safe on the basis of literature data and of our experience data. This surgical procedure, in fact, has good percentage of success in accurately selected patients. In our experience Macroplastique can also be used in oncological patients, in elderly women, in patients with important comorbidity and with high surgical risk with good objective and subjective results.

Outbreak of African tick-bite fever in six Italian tourists returning from South Africa.

In May 1999, a cluster of cases of African tick-bite fever was detected in six Italian tourists who had returned from South Africa. All of the patients had moderate fever and cutaneous eschars. Regional lymphangitis was observed in three of the patients and skin rash in two. By comparing the number of eschars with the number of detectable bite sites it was suggested that at least two-thirds of the biting vectors were capable of transmitting Rickettsia africae. The clinical course of disease was mild in all cases, and all but one of the patients recovered spontaneously before antibiotic treatment was initiated. The diagnosis of African tick-bite fever was confirmed serologically using both microimmunofluorescence and Western blot tests.

Isolation and characterization of Borrelia burgdorferi sensu lato strains in an area of Italy where Lyme borreliosis is endemic.

Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to "San Martino" Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.

KDC1, a novel carrot root hair K+ channel. Cloning, characterization, and expression in mammalian cells.

Potassium is an essential nutrient which plays an important role in many aspects of plant growth and development. Plants have developed a number of highly specific mechanisms to take up potassium from the soil; these include the expression of K(+) transporters and potassium channels in root cells. Despite the fact that root epidermal and hair cells are in direct contact with the soil, the role of these tissues in K(+) uptake is not well understood. Here we report the molecular cloning and functional characterization of a novel potassium channel KDC1 which forms part of a new subfamily of plant K(in) channels. Kdc1 was isolated from carrot root RNA and in situ hybridization experiments show Kdc1 to be highly expressed in root hair cells. Expressing the KDC1 protein in Chinese hamster ovary cells identified it as a voltage and pH-dependent inwardly rectifying potassium channel. An electrophysiological analysis of carrot root hair protoplasts confirmed the biophysical properties of the Kdc1 gene product (KDC1) in the heterologous expression system. KDC1 thus represents a major K(+) uptake channel in carrot root hair cells.

A new FISH protocol with increased sensitivity for physical mapping with short probes in plants.

Fluorescence in situ hybridization (FISH) is a well-established technique used for the detection of specific DNA regions, that has been applied to interphase nuclei, pachytene and metaphase chromosomes as well as to extended DNA fibres. This technique allows the physical mapping of specific DNA sequences both on individual chromosomes and extended fibres. A new FISH protocol is described here that enhances the sensitivity of the method. Probes for small unique DNA sequences of less than 2 kb give high signal-to-noise ratio with this method, and can be visualized easily by means of conventional fluorescence microscopy.

Expression pattern of the carrot EP3 endochitinase genes in suspension cultures and in developing seeds

Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11. Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results support the hypothesis that the EP3 endochitinase has a "nursing" function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos.

The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.

The secretory nature of the lesion of carrot cell variant ts11, rescuable by endochitinase.

The carrot cell variant ts11 is unable to form somatic embryos at the non-permissive temperature of 32 degrees C, but the block can be overcome by the addition of a 32-kDa acidic endochitinase to the medium. In this work we conducted a cyto-histological analysis of the blocked embryo forms. The morphology of the endomembrane system is altered; in particular, the ER is dilated and may show electron-dense precipitates and continuity with the plasma membrane. These morphological alterations do not occur in the presence of externally-added endochitinase. We also noticed modifications of the culture medium that are probably related to the morphological observations: the total amount of secreted proteins is reduced and pulse-chase experiments revealed that, compared with wild-type cells, the secretion of major polypeptides is reduced while new minor polypeptides are secreted. Western blot analysis revealed the presence of the binding protein BiP, a resident of the ER and of glutamine synthase, a cytosolic protein, in the medium of ts11 but not wild-type cells. These results indicate that ts11 is altered in the secretory pathway but do not clarify the role of endochitinase.