Consistency for the tree bootstrap in respondent-driven sampling.

Respondent-driven sampling is an approach for estimating features of populations that are difficult to access using standard survey tools, e.g., the fraction of injection drug users who are HIV positive. Baraff et al. (2016) introduced an approach to estimating uncertainty in population proportion estimates from respondent-driven sampling using the tree bootstrap method. In this paper we establish the consistency of this tree bootstrap approach in the case of [Formula: see text]-trees.

MED13L-related intellectual disability: involvement of missense variants and delineation of the phenotype.

Molecular anomalies in MED13L, leading to haploinsufficiency, have been reported in patients with moderate to severe intellectual disability (ID) and distinct facial features, with or without congenital heart defects. Phenotype of the patients was referred to "MED13L haploinsufficiency syndrome." Missense variants in MED13L were already previously described to cause the MED13L-related syndrome, but only in a limited number of patients. Here we report 36 patients with MED13L molecular anomaly, recruited through an international collaboration between centers of expertise for developmental anomalies. All patients presented with intellectual disability and severe language impairment. Hypotonia, ataxia, and recognizable facial gestalt were frequent findings, but not congenital heart defects. We identified seven de novo missense variations, in addition to protein-truncating variants and intragenic deletions. Missense variants clustered in two mutation hot-spots, i.e., exons 15-17 and 25-31. We found that patients carrying missense mutations had more frequently epilepsy and showed a more severe phenotype. This study ascertains missense variations in MED13L as a cause for MED13L-related intellectual disability and improves the clinical delineation of the condition.

A randomised clinical study to determine the effect of a toothpaste containing enzymes and proteins on plaque oral microbiome ecology.

The numerous species that make up the oral microbiome are now understood to play a key role in establishment and maintenance of oral health. The ability to taxonomically identify community members at the species level is important to elucidating its diversity and association to health and disease. We report the overall ecological effects of using a toothpaste containing enzymes and proteins compared to a control toothpaste on the plaque microbiome. The results reported here demonstrate that a toothpaste containing enzymes and proteins can augment natural salivary defences to promote an overall community shift resulting in an increase in bacteria associated with gum health and a concomitant decrease in those associated with periodontal disease. Statistical analysis shows significant increases in 12 taxa associated with gum health including Neisseria spp. and a significant decrease in 10 taxa associated with periodontal disease including Treponema spp. The results demonstrate that a toothpaste containing enzymes and proteins can significantly shift the ecology of the oral microbiome (at species level) resulting in a community with a stronger association to health.

The role of Triclosan in dentifrice formulations, with particular reference to a new 0.3% Triclosan calcium carbonate-based system.

AIM: To discuss the role of Triclosan in dentifrice systems and demonstrate the enhanced efficacy of Triclosan in calcium carbonate-based systems when the level of the antimicrobial agent is raised from 0.2% to 0.3%. Triclosan is the most commonly used antimicrobial agent in oral care products, being compatible with a wide range of ingredients found in toothpaste formulations, whilst having no negative sensory features (e.g. taste, staining) that are associated with some other antimicrobial/anti-plaque agents. Triclosan is a broad spectrum antimicrobial agent, with additional anti-metabolic and anti-inflammatory properties. When delivered to the mouth in oral care products, Triclosan can selectively inhibit Gram negative anaerobic bacteria implicated in gingivitis and periodontal diseases, while leaving species associated with oral health relatively unaffected. Worldwide, attempts have been made to boost delivery/activity of Triclosan, either by use of copolymers or by combination with other agents such as zinc citrate. However, Triclosan has also been shown to maintain clinical efficacy against plaque and gingivitis when present as the sole antimicrobial in toothpaste formulations.

Regulation of [Ca2+]c oscillations by plasma membrane Ca2+ fluxes: a role for natriuretic peptides.

We have investigated the effects of natriuretic peptides on oscillations in [Ca(2+)](c) (cytosolic free Ca(2+) concentration) in two different cell types: freshly isolated rat hepatocytes and the ECV304 cell line. Our data, from both cell types, suggest that natriuretic peptides modulate the frequency of [Ca(2+)](c) oscillations through alterations in plasma membrane Ca(2+) fluxes. Here, we review evidence for a role for plasma membrane Ca(2+) fluxes in the control of the frequency of [Ca(2+)](c) oscillations. We describe a hypothetical mechanism through which this might be achieved. We propose a physiological role for regulated control of Ca(2+) efflux in modulating [Ca(2+)](c) oscillations.

Plaque antibacterial levels following controlled food intake and use of a toothpaste containing 2% zinc citrate and 0.3% Triclosan.

OBJECTIVES: To measure the levels of zinc and Triclosan present in plaque 12 hours post-brushing and following two weeks home use of a toothpaste formulation containing 2% zinc citrate and 0.3% Triclosan. To measure the levels of zinc and Triclosan in plaque following two weeks home use of the test toothpaste formulation together with a further morning's brushing and a day of controlled food intake. METHODS: A total of 104 subjects completed the study. Plaque samples were taken before use of the test toothpaste and again after a specified regime of product use and food intake. The samples were analysed for zinc or Triclosan. RESULTS: Levels of zinc and Triclosan in plaque 12 hours after last brushing and following a 2-week home usage of product, were 149.1 microg/g and 8.6 microg/g respectively. Following a morning brushing and a day of controlled food intake zinc and Triclosan levels were 94.7 microg/g and 4.1 microg/g respectively. These levels of agents were found to reduce pH drop in vitro. CONCLUSIONS: Regular use of a toothpaste containing 2% zinc citrate and 0.3% Triclosan can lead to a build-up of antibacterial agents in plaque that continue to work even after controlled food intake.

The effect of a 2% zinc citrate, 0.3% Triclosan dentifrice on plaque acid production following consumption of a snackfood.

OBJECTIVES: A) To assess plaque lactate production following consumption of three foods (cake, chocolate/caramel bar, sweetened coffee), and B) To measure the effect of a fluoride dentifrice containing 2% zinc citrate and 0.3% Triclosan on plaque lactate and pH drop following consumption of cake. METHODS: A) 10 subjects completed the first study. Plaque samples taken before and at 8,15 and 30 minutes after eating. Samples were analysed for lactate via Capillary Electrophoresis. B) 30 subjects completed the second study. Plaque samples were taken before and after cake and use of test dentifrice or no treatment control. Plaque pH and lactate content were assessed. RESULTS: A) Plaque lactate levels increased after all three foods; peak lactate levels occurred 8 minutes after eating. B) Plaque lactate concentrations after eating cake were 39.2mM for the control treatment and a significantly lower value, 23.6mM, for the test 2% zinc citrate, 0.3% Triclosan dentifrice. After food challenge, pH values were 5.53 for the no treatment group and a significantly higher value of 5.79 for the test dentifrice group. CONCLUSIONS: A toothpaste containing 2% zinc citrate, 0.3% Triclosan can significantly reduce plaque lactate generation and pH drop induced by cake, compared to no treatment control.

Ornithine carbamoyltransferase deficiency: improved sensitivity of testing for protein tolerance in the diagnosis of heterozygotes.

The most direct test of functional capacity of the liver in nitrogen disposal is to stress the urea cycle with a high protein load. This has been used in the diagnosis of heterozygosity for ornithine carbamoyltransferase deficiency for many years by measuring the subsequent excretion of orotic acid in urine. Reports have shown some ambiguity in both this and the more recent allopurinol test. We investigated the effects of different foods as the protein load and of different analytical methods. A standardized protocol was developed, giving 35 g protein per m2 surface area as steamed fat-free chicken breast to be eaten within 30 min. Urine was collected at zero time and over 0-2, 2-4 and 4-6 h. Compliance was checked by assessing excretion of amino acids. Diagnostic sensitivity was improved by reference to the change in excretion, i.e. the ratio of excretions 2-4 h/0-2 h. Extension of the test to 6 h gave no diagnostic advantage over a 4 h test. Comparison of the analysis of total orotic acids by the photometric method of Harris and Oberholtzer, the reference method for this study, with that by the method of Goldstein and colleagues showed that the latter gave erratic results with some false positives. However, comparison of the method of Harris and Oberholtzer with specific orotic acid analysis by a modification of the stable-isotope internal standard method of Rimoldi and colleagues yielded the same diagnoses. The improved protein load test gave a clearly positive result in all 16 obligate heterozygotes and 2 possible heterozygotes tested from 14 kindred, and a clearly negative result in all 18 control subjects and all 6 of the possible heterozygotes who were later shown by DNA studies not to carry the family mutation. The test appears at least as sensitive and specific as the allopurinol test, and is more convenient because of the short period of sample collection.

Switching from simple to complex oscillations in calcium signaling.

We present a new model for calcium oscillations based on experiments in hepatocytes. The model considers feedback inhibition on the initial agonist receptor complex by calcium and activated phospholipase C, as well as receptor type-dependent self-enhanced behavior of the activated G(alpha) subunit. It is able to show simple periodic oscillations and periodic bursting, and it is the first model to display chaotic bursting in response to agonist stimulations. Moreover, our model offers a possible explanation for the differences in dynamic behavior observed in response to different agonists in hepatocytes.

Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors.

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.

Thimerosal enhances agonist-specific differences between [Ca2+]i oscillations induced by phenylephrine and ATP in single rat hepatocytes.

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the stimulating agonist; the differences lie in the rate of fall of [Ca2+]i from its peak. We considered that differential sensitivity of the InsP3 receptor may underlie agonist specificity. The thiol reagent, thimerosal, is known to increase the sensitivity of the Ca2+ stores to InsP3 by increasing the affinity of the InsP3 receptor for InsP3 in rat hepatocytes. We show here that a low dose of thimerosal (1 microM), insufficient alone to elevate [Ca2+]i, potentiates [Ca2+]i oscillations induced by phenylephrine or ATP in single, aequorin-injected, rat hepatocytes. Moreover, thimerosal enhances both the frequency and amplitude of phenylephrine-induced oscillations, whereas, in contrast, ATP-induced oscillations undergo an increase in the duration of the falling phase of individual [Ca2+]i transients. Thimerosal, therefore, enhances, rather than eliminates, agonist-specific differences in the hepatocyte [Ca2+]i oscillator.

Effects on the hepatocyte [Ca2+]i oscillator of inhibition of the plasma membrane Ca2+ pump by carboxyeosin or glucagon-(19-29).

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the receptor species activated; the variability results in differences in the rate of fall of [Ca2+]i from its peak. It is conceivable that the plasma membrane Ca(2+)-ATPase (PM Ca2+ pump) may have an important role in the mechanism underlying agonist specificity. It has recently been shown that an esterified form of carboxyeosin, an inhibitor of the red cell PM Ca2+ pump, is suitable for use in whole cell studies. Glucagon-(19-29) (mini-glucagon) inhibits the Ca2+ pump in liver plasma membranes, mediated by Gs. We show here that carboxyeosin and mini-glucagon inhibit Ca2+ efflux from populations of intact rat hepatocytes. We show that carboxyeosin and mini-glucagon enhance the frequency of oscillations induced by Ca(2+)-mobilizing agonists in single hepatocytes, but do not affect the duration of individual transients. Furthermore, we demonstrate that inhibition of the hepatocyte PM Ca2+ pump enables the continued generation of [Ca2+]i oscillations for a prolonged period following the removal of extracellular Ca2+.

Bovine growth hormone induces oscillations in cytosolic free Ca2+ in single rat hepatocytes.

Single rat hepatocytes microinjected with the photoprotein aequorin generate oscillations in the cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. We show here that, in single rat hepatocytes, bovine growth hormone (bGH) is able to induce [Ca2+]i oscillations which display similarities with oscillations induced by phenylephrine. Thus the rate of rise of intracellular Ca2+ in each oscillation closely resembles that induced by Ins(1,4,5)P3-mediated agonists. However, the duration of bGH-induced oscillations increases with agonist concentration, in contrast to phenylephrine-induced oscillations, which undergo an increase in frequency as the agonist concentration is raised, without any increase in the duration of individual oscillations.

Actions of ADP, but not ATP, on cytosolic free Ca2+ in single rat hepatocytes mimicked by 2-methylthioATP.

1. Aequorin-injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin-injected hepatocytes to ADP and ATP. 2. The effects of the phosphorothioate analogue of ATP, 2-methylthioATP (2-meSATP), have been examined on single rat hepatocytes. This analogue is belived to be the most potent agonist at the P2Y1 subclass of purinoceptor. 3. The [Ca2+]i transients induced by 2-meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. 4. At hig concentrations, 2-meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall. 5. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2-meSATP- and ADP-induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP-induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i. 6. ATP-induced transients were specifically potentiated by the co-addition of alpha, beta-methyleneATP, whereas 2-meSATP- and ADP-induced transients were unaffected by this treatment. 7. We conclude that 2-meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from teh receptor(s) mediating the effects of ATP.

Cytosolic free Ca2+ oscillations induced by diadenosine 5',5"'-P1,P3-triphosphate and diadenosine 5',5"'-P1,P4-tetraphosphate in single rat hepatocytes are indistinguishable from those induced by ADP and ATP respectively.

Diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) induce distinctive patterns of [Ca2+]i oscillations in single rat hepatocytes. We show here that [Ca2+]i oscillations induced by Ap3A and ADP are indistinguishable and that [Ca2+]i oscillations induced by Ap4A closely resemble those induced by ATP. These similarities embrace the following: (1) ADP and Ap3A invariably induce [Ca2+]i transients of short duration (approx. 9 s). Ap4A, like ATP, can induce, depending upon the individual cell, either transients of short duration (approx. 9 s), transients of much longer duration or a mixture of short and long transients within a single response. We show here that the pattern of oscillations induced by Ap4A is similar to that induced by ATP in the same hepatocyte. (2) Elevated intracellular cyclic AMP concentration modulates Ap3A-induced transients, like ADP-induced transients, through an increase in both the peak [Ca2+]i and the frequency of the transients. In contrast, Ap4A-induced transients, like ATP-induced transients, develop an increased duration or a sustained rise in [Ca2+]i, with no rise in peak [Ca2+]i. (3) Ap3A-induced transients, like ADP-induced transients, are abolished by low concentrations of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB; 5-10 nM), whereas long Ap4A-induced transients, like long ATP-induced transients, are refractory to high concentrations of PDB (100 nM). We propose that the [Ca2+]i oscillations induced in rat hepatocytes by Ap3A are mediated by the same purinoceptor that mediates the effects of ADP, whereas the oscillations induced by Ap4A are mediated by the same purinoceptor(s) that mediate the effects of ATP.

Oscillations in cytosolic free Ca2+ induced by ADP and ATP in single rat hepatocytes display differential sensitivity to application of phorbol ester.

We have previously described differences in the oscillatory responses of cytosolic free Ca2+ concentration ([Ca2+]i) in hepatocytes to ADP and ATP, which we have interpreted as evidence that these two nucleotides are acting at distinct receptors. We show here that ADP- and ATP-induced oscillations are differentially sensitive to application of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB). ADP-induced [Ca2+]i oscillations are abolished by low concentrations of PDB (5-10 nM), whereas ATP-induced oscillations of long duration are refractory to PDB, even at greatly elevated concentrations (100 nM). The data illustrate a further difference in the actions of ADP and ATP, strengthening the argument that these agonists are not acting at the same receptor on rat hepatocytes.

Elevated intracellular cyclic AMP exerts different modulatory effects on cytosolic free Ca2+ oscillations induced by ADP and ATP in single rat hepatocytes.

Single aequorin-injected hepatocytes respond to agonists acting via the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]free). The duration of [Ca2+]free transients is characteristic of the stimulating agonist. We have previously reported that ADP and ATP, which are believed to act through a single P(2y)-purinoceptor species, induce very different oscillatory [Ca2+]free responses in the majority of hepatocytes. We have interpreted these data as evidence for two separate Ca(2+)-mobilizing purinoceptors for these nucleotides. We show here that the elevation of intracellular cyclic AMP concentration, by the co-application of either dibutyryl cyclic AMP or 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)butyryl]- forskolin (L858051), exerts different modulatory effects on [Ca2+]free oscillations induced by ADP and ATP in single rat hepatocytes. Elevated intracellular cyclic AMP levels enhance the frequency and peak [Ca2+]free of transients induced by ADP. In contrast, the elevation of intracellular cyclic AMP levels in hepatocytes producing [Ca2+]free oscillations in response to ATP stimulates either an increase in the duration of transients or a sustained rise in [Ca2+]free. The data illustrate a further difference between the oscillatory [Ca2+]free responses of hepatocytes to ADP and ATP, thus further arguing against ADP and ATP acting via a single purinoceptor species.

Adenosine 5'-[alpha beta-methylene]triphosphate potentiates the oscillatory cytosolic Ca2+ responses of hepatocytes to ATP, but not to ADP.

Single rat hepatocytes microinjected with aequorin generate oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. The duration of these transients has been shown to be characteristic of the stimulating agonist, so that transients of very different duration can be induced in the same individual hepatocyte by different agonists. In a previous study we have shown that ADP and ATP, which are believed to act through a single P2y-purinoceptor species, elicit very different [Ca2+]i responses in most of the hepatocytes. We have interpreted this as evidence for two Ca(2+)-mobilizing purinoceptors. The methylated derivative of ATP, adenosine 5'-[alpha beta-methylene]-triphosphate (pp[CH2]pA), is only a weak P2y-purinoceptor agonist. When 100 microM pp[CH2]pA was supplied to aequorin-injected hepatocytes, there was no effect on [Ca2+]i. However, 25 microM pp[CH2]pA co-supplied with ATP causes a potentiation of the [Ca2+]i response in most of the hepatocytes. The effect was specific for ATP-induced transients; [Ca2+]i transients induced by other agonists, and importantly by ADP, were not affected by addition of pp[CH2]pA. This further illustrates differences in the actions of ADP and ATP, strengthening the argument for separate receptors for these nucleotides.

Adenine dinucleotide-mediated cytosolic free Ca2+ oscillations in single hepatocytes.

Single rat hepatocytes microinjected with aequorin respond to Ca(2+)-mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+ oscillations which resemble those induced by ADP and ATP.