Integrated exome and transcriptome analysis prioritizes MAP4K4 de novo frameshift variants in autism spectrum disorder as a novel disease-gene association.

The application of next-generation sequencing (NGS) to clinical practice is still hampered by the ability to interpret the clinical relevance of novel variants and the difficulty of evaluating their effect in specific tissues. Here, we applied integrated genomic approaches for interrogating blood samples of two unrelated individuals with neurodevelopmental disorders and identified a novel neuro-pathogenic role for the Mitogen-Activated Protein Kinase 4 gene (MAP4K4). In particular, we identified two novel frameshift variants in coding exons expressed in the blood and neuronal isoforms. Both variants were predicted to generate non-sense-mediated decay. By transcriptome analysis, we simultaneously demonstrated the deleterious effect of the identified variants on the splicing activity and stability of MAP4K4 mRNA. Therefore, we propose MAP4K4 as a novel causative gene for non-syndromic and syndromic neurodevelopmental disorders. Altogether, we prove the efficacy of an integrated approach of exome and transcriptome sequencing in the resolution of undiagnosed cases by leveraging the analysis of variants in genes expressed in peripheral blood.

Evaluation of tumor-infiltrating lymphocytes and association with prognosis in BRCA-mutated breast cancer.

BACKGROUND: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (<10%), intermediate (10-59%) and high (≥60%). Prognostic associations of sTils were evaluated as a continuous variable in univariate and multivariate models. Only follow-up time beyond date of BRCA mutation test was included. RESULTS: A large proportion had high sTils (27% in the full cohort, 36% in BRCA1-mutated, and 44% in ER negative breast cancers). Higher sTils were associated with BRCA1, ER negative breast cancer, high histological grade and medullary histology. In combined analysis for BRCA1 and BRCA2-mutated breast cancers, increasing sTils in 10% intervals were significantly associated with OS (HR 0.92, 95% CI 0.84-1.00, p = .05). For each 10% increment of sTils in BRCA1 breast cancers, a 10% reduction of mortality (adjusted HR 0.90 95% CI 0.81-0.99, p = .03) and a 13% reduction in risk of DFS-event (HR 0.87 95% CI 0.76-1.00, p = .05) was observed even after adjustment for ER status. No significant association with survival was of observed in the BRCA2 subgroup. Test for interaction of sTils and BRCA status was not statistically significant (p = .3). CONCLUSIONS: Breast cancer patients with a germline BRCA mutation had higher sTils than previously reported in sporadic breast cancers, and sTils were associated with favorable survival among BRCA carriers.

Clinical and molecular characterization of BRCA-associated breast cancer: results from the DBCG.

BACKGROUND: In breast cancer (BC) patients a cancer predisposing BRCA1/2 mutation is associated with adverse tumor characteristics, risk assessment and treatment allocation. We aimed to estimate overall- (OS) and disease-free survival (DFS) according to tumor characteristics and treatment among women who within two years of definitive surgery for primary BC were shown to carry a mutation in BRCA1/2 . MATERIAL AND METHODS: From the clinical database of the Danish Breast Cancer Group we included 141 BRCA1 and 96 BRCA2 BC patients. Estrogen receptor and HER2 status were centrally reviewed on paraffin-embedded tumor tissue. Information on risk reducing surgery was obtained from the Danish Pathology and Patient Registries and included as time-dependent variables in Cox proportional hazard models. RESULTS: Ten-year OS and DFS for BRCA1 BC patients were 78% (95% CI 69-85) and 74% (95% CI 64-81). Ten-year OS and DFS for BRCA2 BC were 88% (95% CI 78-94) and 84% (95% CI 74-91). BRCA1 BC patients as compared to BRCA2 BC patients had a higher risk of BC relapse or non-breast cancer within ten years of follow-up, independent of ER status (adjusted HR 2.78 95% CI 1.28-6.05, p = .01), but BRCA mutation was not associated with OS (adjusted HR 1.98, 95% CI 0.87-4.52, p = .10). In multivariate analysis, including both BRCA1 and BRCA2 carriers, no chemotherapy was associated with a higher risk of death (adjusted OS HR 3.58, 95% CI 1.29-9.97, p = .01) and risk reducing contralateral mastectomy (RRCM) was associated with a significantly reduced risk of death (adjusted OS HR 0.42, 95% CI =0.21-0.84, p = .01). CONCLUSION: Difference in OS between BRCA1 and BRCA2 BC patients could be ascribed to tumor-biology. BRCA1 BC patients may have a shorter ten-year DFS than BRCA2 BC patients. Chemotherapy and risk reducing contralateral mastectomy reduce mortality for both BRCA1 and BRCA2 BC patients.

Acute hypoxia induces upregulation of microRNA-210 expression in glioblastoma spheroids.

AIM: Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. MATERIALS & METHODS: Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. RESULTS: MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. CONCLUSION: MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.

Erosion of the teeth: prevalence and distribution in a group of Danish school children.

AIM: This was to describe the prevalence of eroded tooth surfaces among 15-17 years old school children in a Danish city and to assess the reliability of diagnosis of erosions performed by 9 clinical examiners. METHODS: During a calendar year (2000) a total of 558 children in 4 public schools in Aarhus were examined by 7 examiners and 2 coordinators using the following criteria: intact; erosion confined to enamel; erosion exposing less than half of the dentine, and erosion involving more dentine than half the surface. RESULTS: It was found that 14.0% (95% CL: 11.3%; 17.1%) of the children had more than 3 surfaces eroded. In the maxilla lingual surfaces were more affected than the facial surfaces. No lesions exposing more than half of the dentine were observed. Despite a careful calibration, including a thorough discussion of the criteria and the experience gained during the study, a substantial inter examiner difference in diagnosis was found. CONCLUSION: The most frequently eroded sites were the lingual surfaces of maxillary incisors; the identification of eroded areas is often difficult and subject to a low inter examiner reliability.

Saturation of human saliva with respect to calcium salts.

It may be assumed that free ionic concentrations of calcium and phosphate in resting saliva tend to equilibrate with those in plaque fluid, and that salivary data can therefore be used to illustrate chemical conditions in both saliva and plaque. In the present study, salivary data collected from the literature or obtained in our laboratory were used to calculate degrees of super- and undersaturation with respect to apatites, brushite, beta-tricalcium phosphate, octacalcium phosphate, calcium carbonate and calcium fluoride in the pH range from 3 to 9. Concentrations of calcium, phosphate, fluoride, carbonate, and background ion strength of resting parotid saliva, resting submandibular saliva, and resting and stimulated whole saliva were entered into a computer program, and curves illustrating saturation in the pH range 3-9 constructed. It was found that oral fluids are supersaturated with respect to apatites above pH 5.3 and with respect to octacalcium phosphate and beta-tricalcium phosphate above pH 6. Parotid saliva was undersaturated with respect to brushite whilst submandibular saliva was supersaturated with respect to that salt in the pH range 6-8. Stimulated whole saliva with 25 mmol/l carbonate became supersaturated with respect to calcium carbonate only above pH 7.3, which may explain the absence of this salt in the human oral cavity. To maintain the saturation of oral fluids with respect to calcium fluoride, i.e. to ensure its survival in the mouth required 6 ppm fluoride in the aqueous phase. Therefore, this salt, the outcome of topical fluoride therapy, will inevitably dissolve in the oral fluids.

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle.

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations

Defluoridation of drinking water by boiling with brushite and calcite.

Existing methods for defluoridating drinking water involve expensive high technology or are slow, inefficient and/or unhygienic. A new method is now suggested, encompassing brushite and calcite suspension followed by boiling. Our aim was to examine the efficiency of the method and the chemical reactions involved. Brushite, 0.3-0.5 g, and an equal weight of calcite were suspended in 1 litre water containing 5-20 ppm fluoride. The suspensions were boiled in an electric kettle, left to cool and the calcium salts to sediment. Solution ion concentrations were determined and sediments were examined by X-ray diffraction. In distilled water initially containing 5, 10 and 20 ppm fluoride the concentration was reduced to 0.06, 0.4 and 5.9 ppm, respectively. Using Aarhus tap water which contained 2.6 mmol/l calcium the final concentrations were 1.2, 2.5 and 7.7 ppm, respectively, and runs without calcite gave results similar to those with calcite. Without boiling the fluoride concentration remained unaltered, as did the brushite and calcite salts, despite occasional agitation by hand. All solutions were supersaturated with respect to fluorapatite and hydroxyapatite and close to saturation with respect to brushite. Boiling produced well-crystallised apatite and traces of calcite, while boiling of brushite alone left a poorly crystallised apatite. We conclude that boiling a brushite/calcite suspension rapidly converts the two salts to apatite which incorporates fluoride if present in solution, and that this process may be exploited to defluoridate drinking water.

Fluoride is unable to reduce dental erosion from soft drinks.

The main aim of the present study was to compare the erosive capabilities of some fruit-flavoured drinks, fresh or saturated with CaF2, with their content of acids and with previous results from some carbonated soft drinks. The other aim was to measure and compare the rates of dissolution of CaF2 in some carbonated and non-carbonated drinks and water. Seven commercially available fruit-flavoured drinks were diluted for drinking. Two human molars, each with two approximately 4 x 4 mm windows, were exposed continuously to 500 ml of each drink with or without prior equilibration with CaF2 under gentle agitation for 48 h. The depths of the erosions were then measured on microradiographs made from sections. Dissolution rate of CaF2 was measured by suspending 0.5 g of the salt in 0.5 litre of the drinks for 2, 10 and 60 min followed by solution analysis. The pH of the drinks was 2.83-3.51. The amount of NaOH required to bring pH to 5.5 ranged from 12-42 mmol/l, which is more than the amount necessary for most carbonated soft drinks. Equilibration with CaF2 gave total fluoride concentrations of 3-8 ppm. The depths of the lesions induced by the drinks without added fluoride were 450-625 microm whilst those developed by the drinks equilibrated with CaF2 were 350-625 microm. The dissolution of CaF2 was faster in the carbonated drinks and in distilled water than in the non-carbonated drinks. In conclusion, non-carbonated fruit-flavoured drinks contain considerable amounts of acids which, in vitro, induce erosions in teeth similar to those induced by carbonated soft drinks. Saturation with CaF2 reduced the in vitro development of erosions by 28% induced by drinks with pH above 3; in drinks with pH below 3, erosions were not affected by pH, despite total fluoride concentrations of up to 20 ppm.

Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells.

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.

Prevention by means of fluoride of enamel erosion as caused by soft drinks and orange juice.

Fluoride has been suggested to prevent erosion of the teeth, either after a topical treatment of the teeth or by addition of fluoride to the acidic drink. The main aim of the present study was to describe the dissolution of calcium fluoride in some soft drinks and orange juice and compare it with the amounts of calcium fluoride left on the enamel surfaces after a topical treatment. A further aim was to describe the dissolution of enamel in soft drinks and juice saturated for 3 days with solid calcium fluoride. Solid calcium fluoride was suspended in each of 10 soft drinks and orange juices and gently agitated for 72 h, after which the drinks were analyzed for calcium, phosphate and fluoride and pH was determined. To examine the erosion-preventive effect of the calcium fluoride-rich drink, intact teeth were exposed to the drinks with or without calcium fluoride. It was found that from 6 to 45 mg of calcium fluoride was dissolved per liter of drink. The more acidic the drink, the more calcium fluoride was dissolved, presumably due to HF formation. The teeth exposed to the soft drinks all showed erosion-like lesions. Very little effect of the 4-6 ppm ionic fluoride dissolved in the soft drinks was observed. In orange juice, however, the dissolved calcium fluoride established a saturation with respect to fluorapatite and consequently, the erosion-like lesion was replaced by a caries-like lesion. In conclusion, the acidic soft drinks are capable of dissolving considerable amounts of calcium fluoride and the erosion-preventive effect of even high fluoride concentrations is limited.

The influence of saliva on the formation of calcium fluoride-like material on human dental enamel.

After a topical fluoride application two resulting features can be observed and measured: the formation of calcium fluoride and a decrease in caries incidence. For the formation of solid calcium fluoride the source of calcium may be either the enamel itself or remaining saliva or--in plaque--plaque fluid and calculus. The aim of the present study was to examine whether saliva through its calcium content has any influence on the amount of calcium fluoride formed by a topical fluoride treatment of enamel. The roots and the fissure systems of 64 freshly extracted intact third molars were covered with nail varnish and allocated to eight groups of 8. Half of the groups were put in fresh paraffin-stimulated saliva for 5 min after which surplus of saliva was shaken off whilst the other groups remained in distilled water. The teeth were given a 2-min topical treatment with either distilled water (control), neutral 0.2% NaF, neutral 2% NaF or 2% NaF acidified with 0.1 mol/l H3PO4. After the topical treatment the teeth were rinsed in running tap water for 5 min, dried and revarnished before determination of calcium fluoride. It was found that the amount of calcium fluoride formed depended on the concentration of fluoride (the higher the fluoride concentration, the higher was the amount of calcium fluoride), the acidity of the solution (presumably due to the increase in available calcium through enamel dissolution) and the presence of saliva (presumably due to its calcium content and its mucinous nature). As there was always more calcium fluoride formed in the presence of saliva, thorough drying of the teeth prior to topical treatments may be superfluous.

A laboratory study evaluating the release of hydroxyl ions from various calcium hydroxide products in narrow root canal-like tubes.

AIM: The aim of the present study was to describe pH changes in a variety of buffering solutions within a narrow test tube containing either a gutta-percha point with incorporate calcium hydroxide, a commercial calcium hydroxide paste (Calcicur) or a freshly mixed paste of calcium hydroxide in distilled water. METHODOLOGY: The test material was placed centrally in a test tube of 2 mm inner diameter. Saline (1%) was placed at one end, whilst the buffering solutions were introduced at the other. The pH of the buffering solutions was monitored using electrodes placed at each end of the test tube. RESULTS: It was found that the pH 4.01 buffer strongly resisted pH changes at levels below 6.0, whilst saliva and bovine serum was buffered less and more evenly in the whole range up to pH 11.5. The calcium hydroxide containing gutta-percha points caused the pH to increase quickly in the sodium chloride solution to levels above 11.5. However, in bovine serum, in saliva and in the pH 4.01 buffer the pH remained below 8.5, 8.0 and 6.0, respectively, 1 mm from the point. In contrast, the release of hydroxyl ions from the two calcium hydroxide pastes brought pH above pH 11.5 irrespective of the buffering of the solutions 5 mm from the paste. CONCLUSION: It is concluded that Calcicur and the calcium hydroxide-water mixture contained substantially more available calcium hydroxide than did the calcium hydroxide containing gutta-percha points, with the result that the release of hydroxyl ions from the points was limited in comparison to that from the pastes.

Plaque pH and associated parameters in relation to caries.

Intensified plaque acidogenicity in caries-prone subjects was reported many years ago, but emerging evidence has suggested that the relationship may not be as strong as once thought. We have now determined a range of acidogenicity variables in subjects having both caries prevalence and incidence data, and have included plaque mineral data in the analysis. pH measurements were made in 20 randomly selected subjects from a high-caries group (mean DMFS = 8. 95) and 20 from a caries-free group of Beijing children aged 12 years participating in a caries prediction study. Subgroups with a 12-month DMFS increment >/=2 or = 0 were also formed from the two groups, respectively. Measurements were made with an iridium oxide electrode inserted between teeth 13/14, 23/24, 34/35 and 44/45, before and every 5 min for 30 min after rinsing with 10% sucrose, and the 4 resulting 'Stephan curves' averaged using a plaque pH analysis program. Supragingival plaque was collected from buccal and lingual smooth surfaces of posterior and upper anterior teeth and its acid extract analysed for Ca, P and F. Caries-free subjects (based on past experience) had a significantly higher maximum plaque pH and pH value after 30 min (reflecting a faster return to resting pH), a lower minimum enamel dissolution capacity of plaque and recorded less time below pH 7.0 than did high-caries subjects. No other differences were significant, including those of the principal acidogenic parameters 'minimum pH attained after a sugar rinse', 'curve area below the critical pH of 5.5' and 'time below the critical pH'. Selection of the caries groups on the basis of both experience and incidence did not reveal significant differences in more parameters. Upper arch plaque was significantly more acidogenic than lower arch plaque, and there was a consistently strong association between upper and lower arch values in individuals. Ca, P and F in the subjects' plaque had little or no influence on the principal acidogenic parameters. Our failure to find a relationship between caries prevalence or activity and these principal acidogenicity parameters may be related to differences between fissure and smooth surface plaque, temporal variations in acidogenicity and/or to use of F toothpaste during the 1-year observation period. These results support the view that factors such as the frequency of acidogenic episodes may be more important in caries progression than the degree of acidogenicity during any one episode.

Individual variations of pH, buffer capacity, and concentrations of calcium and phosphate in unstimulated whole saliva.

In order to evaluate the risk of development of dental caries and/or of formation of dental calculus, salivary variables have often been used, but not with particular success. A reason for the apparent lack of association could be that the individual temporal variation of a characteristic was so substantial relative to the overall variation that it is not possible to characterize an individual by a single salivary measurement. The aim here was to examine the individual variation of pH, buffer capacity, and concentrations of calcium and phosphate and to compare it with the overall variation of the characteristics in order to shed light on the above problem. Eight weekly samples of up to 4 ml of unstimulated whole saliva were collected from 11 dental students before tooth brushing on their arrival at 8 a.m. in the dental school. Calcium was determined by atomic absorption spectroscopy, phosphate colorimetrically, and pH electrometrically. The buffer capacity was assessed by titration of the saliva sample from the pH initially observed to pH 3. It was found that within each individual the concentration of calcium and of phosphate, pH, the hydroxyapatite ion product and the buffer capacity varied considerably over the 7 weeks. The individual range frequently covered more than a third of the total range. Further, within each of the variables, single individuals could be found whose samples covered 60% or more of the overall range, whilst others covered less than 10% of the range. It was therefore concluded that, although collected at the same time of the day, pH, buffer capacity and concentrations of calcium and phosphate in unstimulated whole saliva in the single individual vary so much that characterization of individuals and of their saliva based on a single salivary analysis is unreliable and hazardous.

Enamel erosion by some soft drinks and orange juices relative to their pH, buffering effect and contents of calcium phosphate.

The capability of a soft drink or a juice to erode dental enamel depends not only on the pH of the drink, but also on its buffering effect. As the latter is the ability of the drink to resist a change of pH it may add to the effects of the actual pH. The aim of the present study was to compare the pH and the buffering effect of various soft drinks with their erosive effects and the solubility of apatite. In 18 soft drinks, mineral waters and juices available on the Danish market, pH and the concentrations of calcium, phosphate and fluoride were determined. The buffering effect was determined by titration with NaOH. Human teeth (n = 54) covered with nail varnish except for 3x4-mm windows were exposed to 1.5 liters of the drink for either 7 days or 24 h under constant agitation. The depth of the erosions was assessed in longitudinal sections. The depth was found to vary greatly from 3 mm eroded by the most acidic drinks and fresh orange juice to only slightly affected surfaces by most of the mineral waters. The dissolution of enamel increased logarithmically inversely with the pH of the drink and parallel with the solubility of enamel apatite. Orange juice, pH 4.0, supplemented with 40 mmol/l calcium and 30 mmol/l phosphate did not erode the enamel as the calcium and phosphate saturated the drink with respect to apatite. Generally, the lower the pH the more NaOH was necessary to bring the pH to neutrality. In particular the buffering effect of the juice was high. For all drinks, no effect of their low fluoride concentrations was observed.

Pharmacology of FMRFamide-related peptides in helminths.

Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide-related peptides (FaRPs), with more yet to be identified. To date, few non-FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free-living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand-receptor recognition features among the Nematoda. Structure-activity studies on nematode FaRPs have revealed that structure-activity relationship (SAR) "rules" differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross-phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross-phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non-neuropeptidergic systems, also remains to be elucidated.

Dissolution of powdered human enamel suspended in acid solutions at a high solid/solution ratio under a 5% CO2 atmosphere at 20 degrees C.

The aim was to examine the nature of enamel dissolution in aqueous suspensions with a high solid/solution ratio and in a CO2-rich atmosphere. Before experimentation, a water-saturated mixture of 95% N2-5% CO2 was passed through the acid solutions for 24 hr. Samples of 2 g of powdered enamel were suspended in 7 ml of either 5 or 10 mmol/l HClO4, with or without 2 parts/10(6) fluoride and kept gently agitated for 24 hr in the above atmosphere. The same enamel samples were repeatedly exposed to fresh acid for 26 runs. All experiments were duplicated. The aqueous phase was analysed after 20 min and 24 hr for calcium, phosphate, fluoride, chloride, sodium and magnesium. It was found that after 20 min the fluoride was invariably taken up in the enamel and the solution was supersaturated with respect to hydroxyapatite with pH ranging 6.7-5.6. During the following 24 hr pH increased further, the supersaturation remained unchanged and the concentrations of calcium and phosphate in solution decreased. In contrast, sodium, magnesium and chloride were released from enamel during the entire period. In the later runs, the supersaturation with respect to hydroxyapatite was only modest and the decrease of calcium and phosphate concentrations limited, as were the release of sodium, magnesium and chloride. It is concluded that despite a CO2-rich atmosphere, calcium, phosphate and carbonate were released from enamel and quickly established a supersaturation with respect to hydroxyapatite with a secondary reprecipitation of mineral. It indicates that within the dental caries lesion in vivo, lesion fluid cannot exist undersaturated with respect to enamel apatite.

A computer program for correlating dental plaque pH values, cH+, plaque titration, critical pH, resting pH and the solubility of enamel apatite.

A computer program was written in Visual Basic (Microsoft) to calculate (a) the area between a plaque pH curve (as seen after a sucrose rinse) and either a resting pH (around pH 7) or a critical pH value (around 5.5) above at least parts of the pH curve; (b) the solubility of apatite at the pH values in plaque; (c) the area between the plaque pH solubility curve and the apatite solubility at the resting pH/critical pH; (d) the area between plaque cH+ curve and the cH+ value at resting pH/ critical pH; and (e) the area between a plaque pH curve and a cut-off pH value below the curve, e.g. pH 3. It was found that because both the cH+ and the solubility of apatite increased logarithmically with a pH drop, the two latter area functions (d, e) were basically different from those based directly on pH curves. Thus, pH changes around the resting pH value had little effect on areas calculated from concentrations of H+ and solubility. In contrast, a small pH change around pH 4 had a strong impact on both demineralization potential and areas based on cH+. Also, because of the logarithmic nature, demineralization potentials were generally large in comparison to remineralization potentials, a point that has hitherto received little attention.

Human supragingival in vivo calculus formation in relation to saturation of saliva with respect to calcium phosphates.

The aim was to establish if there is a correlation between the rate of formation of supragingival calculus and the degree of supersaturation of saliva with respect to apatite, brushite and other calcium phosphates. The rate of calculus formation was estimated by measuring the calculus formed on lingual surfaces of the lower incisors in 15 individuals during 30 days. Submandibular saliva stimulated with citric acid and resting whole saliva, alter pH determination and ultrafiltration to remove protein was analysed for calcium, phosphate, sodium, potassium and carbonate. The degree of saturation with respect to calcium phosphates was calculated using a computer program. The rate of calculus formation among the 15 participants ranged widely, from a score of 0.5 to a high of 15. A close correlation (r = 0.91) between salivary pH and degree of supersaturation was found A weak correlation between calcium and calculus formation was also found. But neither for unstimulated nor stimulated saliva was a significant relation between supersaturation with respect to any of the calcium phosphates and the rate of calculus formation observed. Therefore, taking into consideration the background of the wide range of rates of calculus formation and of degrees of supersaturation, it was concluded that relation between them is unlikely, and the use of degree of saturation as a diagnostic tool seems unreliable.