The greater residuals in the deeper waters could result from diss

The greater residuals in the deeper waters could result from dissolution of carbonate minerals and contributions from water masses with different TA–SAL relationships. As a result, the TA–SAL relationship in (2) should only selleck chemicals llc be used for mixed layer waters of the Pacific study area where

nitrate concentrations are less than 15 μmol kg− 1. Data used to derive the TA–SAL relationship in (2) were collected over a number of years covering El Niño and non-El Niño events (Table 1). We investigated how the time and location of sampling for TA might influence the calculated TA values by classifying measured TA surface values as collected in El Niño or non-El Niño conditions using the Oceanic Niño Index (ONI). The ONI is a three-month running mean of NOAA ERSST.v3 SST anomalies in the Niño 3.4 (5°N:5°S, 170°W:120°W) region based on the 1971–2000 period (http://www.cpc.ncep.noaa.gov/data/indices/). Data collected within the Niño 3.4 and Niño 4 (5°S:5°N, 160°E:150°W) regions were identified and assigned to El Niño events when the SST anomalies where above ALK inhibitor 0.5 °C for 3 consecutive months, or La Niña events when the SST anomalies where below 0.5 °C for 3 consecutive months. If the ONI was less than 0.5 °C over the 3 consecutive

months, these values were assigned a “neutral” condition. All data collected outside of the Niño 4 and Niño 3.4 regions were considered less likely to be influence by La Niña and El Niño events and in Table 1 have been assigned as “outside”. For all samples, 13% were collected during an El Niño condition, 11% during a non-El Niño condition (5% of La Niña and 6% of neutral events), and 76% were outside the Niño 3.4 and Niño 4 regions (Table 1). The TA–SAL relationship of (2) was found to be independent of the El Niño or non-El Niño conditions in the study area (Fig. 3). Thus, the Eq. (2) relationship appears to be applicable for all phases of ENSO. The earlier relationships used to estimate TA of Chen and Pytkowicz (1979) and Lee et al. (2006) covered a greater region of

the ocean and include temperature and salinity terms. Y-27632 in vivo The greater range of the residuals of the Chen and Pytkowicz equations (− 45 to 20 μmol kg− 1, Fig. 3a) compared to (2) is likely due to their relationship using a limited amount of data collected between August 1973 and June 1974 during the Pacific Geochemical Ocean Section Study. The Lee et al. (2006) relationships were based on more data than Chen and Pytkowicz and the calculated TA residuals compared to measured values are smaller. However, the variance of the fit over the study region as indicated by the slopes of the lines in Fig. 3b was greater than the Eq. (2) fit (Fig. 3c). Eq. (2) is an updated version of the relationship of Christian et al. (2008), which only used data reported in the GLODAP database (http://cdiac.ornl.gov/oceans/glodap/) and (2) also includes more recent data from the CARINA database (http://cdiac.ornl.gov/oceans/).

Subjects then performed the following tasks, each for 30 s; i) qu

Subjects then performed the following tasks, each for 30 s; i) quiet standing with eyes open (QS EO); ii) quiet standing with eyes closed (QS EC); iii) one-leg standing with eyes open (OLS EO) and; iv) one-leg standing eyes closed (OLS EC). One-leg standing was performed on the dominant leg. For each task the subject was asked to remain with their feet positioned on specific points marked on the floor and to remain as still as possible for 30 s; the timer was started once the subject had established their balance. If the subject lost their balance

during the task (and moved their feet from the specific points), the trial was terminated and restarted until they were able to remain balanced for the full 30 s trial. For each MVC, the root mean JQ1 square (RMS) value was calculated over 0.2 s intervals of the raw EMG data, using an automated script Galunisertib datasheet in Spike2 software. The greatest 0.2 s interval RMS value from the 3 MVCs was taken. For each muscle, the RMS of the EMG voltage over 0.2 s intervals was calculated throughout each 30 s task. To allow comparison of muscle activity between subjects this was normalised to the peak RMS value during an MVC for that muscle. The normalised RMS

values were averaged, disregarding the first and last 3 s of data. This gave one normalised value per muscle for each task. Co-contraction of antagonistic muscles (RF-ST and TA-GL) was calculated using Equation (1) (Rudolph et al., 2001). equation(1) Co-contraction Index = (lower EMG/higher EMG)∗(lower EMG + higher EMG)where; lower EMG and higher EMG represent the average normalised RMS value of the agonist and antagonist muscles. Statistical analysis was performed using SigmaPlot statistical 17-DMAG (Alvespimycin) HCl package. Two-way analysis of variance (ANOVA) was used to compare tasks and between the hypermobile and control groups for each muscle. Where data was not normally distributed, a logarithm transformation was used. Post-hoc analysis involved an all pairwise multiple comparison procedure using either the Holm-Sidak method or Tukey Test. A p-value of <0.05 was taken as significant. All subjects were able to complete

each task for 30 s on their first attempt. Fig. 1 shows normalised EMG RMS amplitudes of the 6 muscles measured during the 4 tasks for both groups. ANOVA revealed a significant effect of task on muscle activity (P < 0.001). Post-hoc analysis revealed that TA activity was significantly greater during task 4 compared with tasks 1 and 2 for both groups (P < 0.001; Fig. 1). GM activity was significantly greater during task 4 compared with tasks 1 and 2 (P < 0.05; Fig. 1) within the control group only; although it was observed to increase in the hypermobile group, this did not reach statistical significance. A co-contraction index was calculated for antagonistic muscles (RF-ST and TA-GL). ANOVA revealed a significant effect of task on TA-GL co-contraction (P < 0.001).

The refinement or coarsening of the mesh is still guided by the c

The refinement or coarsening of the mesh is still guided by the curvature

Alectinib price of the field. However, a scaling by the local magnitude of the field is now included in the metric. The final metric is obtained by consideration of the interpolation error in the LpLp norm, p∈[1,∞)p∈[1,∞). The general metric, denoted MpMp, has the form equation(9) Mp(x)=1∊(x)(det(|H(x)|))-12p+n|H(x)|=(det(|H(x)|))-12p+nM∞,(Chen et al., 2007 and Loseille and Alauzet, 2011b), where n   is the spatial dimension of the problem. Since det|H|=∏i|λi|det|H|=∏i|λi|, a scaling by a measure of the magnitude of the curvature of the field is included in the metric. The extent to which det|H|det|H| influences the metric is determined by the choice of p  . As p   is reduced, smaller scales are given more weight in the metric and as a result are better represented ( Loseille and Alauzet, 2011b). In the limit p→∞p→∞, M∞M∞ is recovered. The work of Loseille and Alauzet (2011b) shows that the influence of smaller scales in the metric rapidly decreases

as p   increases and their good results for p=2p=2 motivates the use of this value here. Hence, the third and final metric is given by equation(10) M2(x)=1∊(x)(det(|H(x)|))-16|H(x)|=(det(|H(x)|))-16M∞. In Fluidity-ICOM, the user chooses which solution fields a metric will be formed for and, therefore, which fields the mesh will adapt to. If the user chooses selleck inhibitor to adapt to multiple solution fields, a metric, MfMf, is formed for each chosen solution field, f  . The final metric, M  , is then obtained from a superposition of the metrics for individual fields M=⋂fMfM=⋂fMf ( Castro-Díaz et al., 1997). The user must also specify minimum and maximum edge lengths and this information is Ponatinib cost included through a restriction

on the eigenvalues of |H||H| (e.g. Pain et al., 2001). In addition, the user can provide an upper and/or lower bound on the number of mesh vertices. If the adaptive algorithm is configured appropriately, this bound should not be reached. Given a metric, the aim of the mesh optimisation step is to satisfy the criteria, Eq. (5) and thereby optimise the mesh for the current system state. The mesh is modified through a series of local topological and geometrical operations which, in two dimensions in Fluidity-ICOM, are performed using the algorithms of Vasilevskii and Lipnikov (1999). The operations include edge-collapsing, edge-splitting, edge-swapping and vertex-movement. More details and diagrams can be found in Pain et al., 2001, Piggott et al., 2009 and Vasilevskii and Lipnikov, 1999. Once the mesh optimisation stage has been performed, solution fields have to be interpolated between the pre- and post-adapt meshes. The interpolation methods available in Fluidity-ICOM fall into two categories. The first is referred to as consistent interpolation ( Applied Modelling and Computation Group, 2011).

Corticospinal axons originating from the contralesional cortex ha

Corticospinal axons originating from the contralesional cortex have been found to suffer lesion-induced sprouting and cross the spinal cord midline, innervating the deafferented side contralateral to the lesion (Benowitz and Carmichael, 2010 and Liu et al., 2011). Moreover, inhibition of neuronal activity of contralesional sensorimotor cortex leads to impairment of recovered reach-to-grasp movement (Biernaskie et al., 2005). Recruitment of other motor regions, such as red nucleus, might also be involved (Jarratt and Hyland, 1999 and Morris et al., 2011).

Liu et al. (2011) have found significant recovery of success rate induced by treatment with MSCs after middle cerebral artery occlusion (MCAo) in mice. This observation is not in agreement to the present results, but the several differences in experimental approaches should explain Akt inhibitor this discrepancy, i.e., regions affected by Linsitinib solubility dmso ischemia, protocol of pre- and post-ischemic accompaniment, cell type and animal species used. Some hypotheses might explain the absence of significant recovery promoted by BMMCs. First, opposite to MSCs, BMMCs might not be able to promote enough neuroprotection in cortical tissue to permit cortical recruitment for compensatory recovery of reach-to-grasp movement. We observed no effect of BMMCs treatment in the extension of ischemic lesion. This quantitative analysis confirms gross analysis made in a previous study (Giraldi-Guimarães

et al., 2009). However, a significant decrease of neurodegeneration has been observed after the same protocol of treatment (Giraldi-Guimarães et al., FER 2009). Since we have observed recovery of unsophisticated sensorimotor functions (Giraldi-Guimarães et al., 2009, present study), the results suggest that the rescue of a small number of neurons can be sufficient to promote some functional recovery, although it should be unable to result in macroscopic reduction of damage and increase recovery of skilled movements. Nonetheless, every effort should be made to save neurons, even though

a small number. Second, complete recovery of sophisticated movements would not be able to occur after large sensorimotor cortical lesions, except for therapies that might promote reconstruction of lost cortical tissue, e.g., the use of embryonic or induced pluripotent stem cells (Polentes et al., 2012). As discussed above, complete recovery of success rate in reach-to-grasp task has been only found after small focal lesion of motor cortex (Alaverdashvili and Whishaw, 2008). Thus, the present study opens the question about BMMCs capability to recovery skilled movements. To evaluate the deepness of sensorimotor recovery promoted by BMMCs and other cell therapies, time window of cell administration after lesion induction, cell dose, location and extension of brain lesion are some of the experimental approaches that need to be tested in further studies.

The overall potency estimate of the candidate standard 86/500 bas

The overall potency estimate of the candidate standard 86/500 based on the laboratories performing bioassays is 211.3 IU, with 95% confidence interval from 189.4 to 235.7 IU. Samples A and B (86/500) and sample C (86/564) were all included in the original collaborative study that was conducted to establish the 1st IS 86/504 (Kirkwood,

1979). Based on the data presented in that study, the estimated potency of 86/500 relative to 86/504 was 204 IU, in excellent agreement with the results from the current study, and providing further evidence of the long-term stability of 86/500. The potency of 86/564 relative to 86/504 in the original study was 225 IU, in reasonable agreement with http://www.selleckchem.com/hydroxysteroid-dehydrogenase-hsd.html the results from the current study. The potency of sample C (86/564) was also calculated relative Crizotinib to sample A (86/500), the candidate

replacement IS, assuming a hypothetical value of 200 IU for 86/500. These calculations were performed for each assay, and the laboratory means, within-laboratory between-assay %GCVs, and overall means, were calculated in the same way as for potencies relative to 86/504 above. The individual laboratory mean estimates are shown in Table 8, along with the within laboratory %GCVs. The overall mean estimate, and between-laboratory %GCV, are also shown in Table 8. The overall mean is 235 IU, consistent with the overall mean of 236 IU calculated relative to 86/504 (Table 4). The between laboratory and within laboratory variation, as measured by the %GCVs, are comparable to the values obtained for sample C relative to 86/504. For this, samples of 86/500 stored at − 70 °C, − 20 °C, + 4 °C and + 20 °C were assayed, subsequently analysed and potencies either expressed relative to the samples stored at − 70 °C. The mean potency estimates

of the candidate A (coded 86/500) stored at different temperatures (expressed as a percentage of the − 70 °C sample) are shown in Table 9. There is no detectable degradation, even after 26 years at + 20 °C. It is not possible to apply the usual Arrhenius model to obtain predictions of % loss per year, as there is no degradation. Clearly 86/500 is very stable, and suitable to serve as a standard. Although samples had also been stored at + 37 °C, it was not possible to properly reconstitute these samples after such a long period at high temperature. Therefore, to confirm the stability at + 37 °C, an additional assay was performed on a sample that had been stored for 1 month at + 37 °C, and this was indistinguishable from the − 20 °C sample (data not shown). Further studies showed that the potency of 86/500 is not diminished after 1 week of storage at either + 4 °C or + 20 °C following reconstitution. However, it is recommended that 86/500 is used soon after reconstitution.

Para cada paciente foram registadas 10 deglutições As variáveis

Para cada paciente foram registadas 10 deglutições. As variáveis estudadas foram as ondas (peristálticas, simultâneas, retrógradas e não transmitidas, em percentagem), a amplitude das ondas (em mmHg) www.selleckchem.com/products/ABT-888.html e o pico médio e máximo das ondas manométricas (em mmHg/seg) Foi considerado normal o valor de amplitude maior ou igual a 30 mmHg. O programa informático que faz a análise computacional

dos dados fornece os valores isolados e a média para cada variável estudada em cada indivíduo. Fornece também o valor percentual das ondas registadas, de acordo com as suas características. Os indivíduos foram divididos em 2 grupos, de acordo com a glicemia em jejum. O primeiro com a glicemia menor ou igual a 7 mmol/l tinha 11 indivíduos. O segundo tinha

14 indivíduos com glicemia > 7 mmol/l. A duração da doença, a média de idades e a distribuição por género, em ambos os grupos, foram Angiogenesis inhibitor semelhantes. O número relativamente pequeno de indivíduos incluídos neste estudo é uma das suas mais importantes limitações. Foi utilizado o Teste t de Student SPSS 17 para a análise estatística dos dados. Os resultados são apresentados pela média com a significância estatística para um valor de p < 0,05. No grupo de pacientes estudado, vimos que a percentagem de ondas peristálticas no corpo esofágico foi maior nos pacientes com glicemia em jejum inferior a 7 mmol/l do que nos pacientes com glicemia > 7 mmol/l, 84,9 vs 80,1%, mas a diferença não foi estatisticamente significativa (p > 0,05). A percentagem de ondas retrógradas, 3,5 vs 2,0% e simultâneas, 6,2 vs 1,0% eram ligeiramente mais elevadas em pacientes com glicemia em jejum < 7 mmol/l mas, em todos os casos, a diferença não foi estatisticamente significativa (p > 0,05). No entanto, a percentagem de ondas não transmitidas foi

significativamente maior nos diabéticos com glicemia em jejum > 7,0 mmol/l 16,3%, do Lepirudin que nos diabéticos com glicemia basal  0,05). Quando analisado o pico médio das ondas manométricas esofágicas, os resultados de cada grupo (glicemia 7 vs glicemia > 7 mmol/l) foram, nos 3 canais de registo, os seguintes: P1 – 22,8 vs 25,5 mmHg/seg; P2 – 29,6 vs 31,4 mmHg/seg; P3 – 28,8 vs 31,2 mmHg/seg; média do pico médio 27,1 vs 28,9 mmHg/seg; p > 0,05. Em relação ao pico máximo das ondas manométricas, também não se encontraram diferenças estatisticamente significativas.

gondii infection profoundly alters the manner in which rodents pe

gondii infection profoundly alters the manner in which rodents perceive and respond to stressful stimuli ( Webster,

2007), only two previous studies have investigated whether T. gondii is related to human anxiety ( Groer et al., 2011 and Miman et al., 2010). Groer et al. assessed whether T. gondii seropositivity FK228 order and serointensity were associated with anxiety among a cohort of pregnant women enrolled in a study of postpartum thyroiditis, as assessed by the Profile of Mood Disorder States (POMS), a non-clinical diagnostic screening instrument ( Groer et al., 2011). Similar to our study, the authors found a positive correlation between T. gondii serointensity and the POMS tension-anxiety subscale score (r = 0.31, p < 0.04). However, use of the POMS limited Groer et al. to scoring participants on a 5-point anxiety scale, whereas our study utilized selleckchem a validated survey instrument that enabled us to assign subjects clinical diagnoses of GAD. In addition, generalizability of their findings were limited to pregnant women enrolled in a study of postpartum thyroiditis ( Groer et al., 2011), whereas we included a subset of individuals drawn from a population-based sample in our study. To our knowledge, only one prior study has examined associations between T. gondii and any anxiety disorder as diagnosed by DSM-IV criteria ( Miman et al., 2010). In a case-control study of 142

subjects, Miman et al. found that individuals with psychiatrist-diagnosed

obsessive–compulsive disorder (OCD) were more likely to be seropositive for T. gondii than were healthy controls (chi-square 12.12, p < 0.01). However, the authors did not report continuous or categorical antibody levels. Overall, our study is the first to demonstrate that, in addition to a positive association between T. gondii seropositivity and GAD, there may be a graded relationship between T. gondii IgG antibody levels and odds of GAD. While the underlying Vildagliptin mechanisms by which T. gondii specifically affects GAD but not PTSD or depression remain uncertain, potential anxiogenic pathways include histopathological, immunological, and neuromodulatory alterations ( Webster, 2007). Rodent studies have failed to uncover a highly selective tropism of T. gondii for a specific brain region; tissue cysts have been detected throughout the brain, with observed distribution patterns varying both between ( Berenreiterova et al., 2011, Haroon et al., 2012 and Vyas et al., 2007) and within ( Berenreiterova et al., 2011) studies. However, cyst density does not appear homogenous across brain regions ( Berenreiterova et al., 2011), while a recent study suggests that cysts may preferentially persist and increase in number in limbic regions known to mediate anxiety, including the amygdala and hypothalamus ( Haroon et al., 2012). In vivo studies of chronically infected rodents indicate that T.

Countries that should improve their data collection and reporting

Countries that should improve their data collection and reporting systems are mainly found in Africa, Asia and among the island states in Oceania and the Caribbean (Table 1). The quality of the statistics included in the FAO capture databases http://www.selleckchem.com/products/PD-0332991.html is mostly dependent upon the accuracy and reliability of the data collected

and provided by countries. When analyzing aggregated or global trends, the number of countries, the size of FAO fishing areas and the extended species coverage included in the database often play a buffer effect. Despite significant annual variations by country, fishing area and species, recent global total catch trend has been quite stable in the last four years (2006–2009) for which statistics are available at the time of writing, ranging between 88.9 and 90.1 million tonnes. On the other hand, in some cases disaggregated data series may be biased or disrupted due to a range of reasons: • erroneous reporting: magnitudes of reported catches may be erroneous due to shortcomings in the data collection system, wrong procedures applied in raising sample data, 20 or for political reasons, e.g. countries with a centrally planned economy which report continuously growing catches to match targets

set in yearly or multi-year national plans; As already noted in Section 3.2.1, trends in the data series also reflect political

and natural events that greatly impacted the fishery sector in a country. For example, striking decreases of capture production in the 1960s for the Democratic Republic MAPK Inhibitor Library of the Congo and in 1996 for Burundi and Rwanda were due to political crises and civil wars, while the drop of Spanish catches in the Southeast Atlantic was a consequence of the Namibian independence. old Hurricane Katrina struck the US Gulf Coast at the end of August 2005 and, although the Western Central Atlantic fishing area covers the US coast from North Carolina to the Mexican border, total catches by the United States in that year decreased by almost 20% in comparison to the previous year. Serious catch reductions are also expected as a consequence of the April 2010 oil spill in the Gulf of Mexico and the March 2011 tsunami in Japan. Unexpectedly, other natural disasters, like the December 2004 tsunami that affected many important Asian fishing countries and the cyclone Nargis that in May 2008 caused the worst natural disaster in the recorded history of Myanmar, did not result in significant catch decreases as it would have been expected due to the magnitude of the devastations. FAO requested clarifications to the most involved countries. Indonesia replied that damages in Banda Aceh due to the tsunami were compensated by increased catches in other regions.

Severity of GBS was scored on admission using (arm and leg) motor

Severity of GBS was scored on admission using (arm and leg) motor disability grading functional scale [9]. Overall disability sum score = arm disability scale (range 0–5) + leg disability scale (range 0–7); overall range: 0 (no signs of disability) to 12 (maximum disability).Clinical improvement was noted by improvement in the motor functional

PD-332991 grading scale and successful weaning (decrease of mechanical ventilator parameters) and extubation from mechanical ventilation following therapy. Management was started immediately by intravenous immunoglobulins (IVIG) in a standard dose of 400 mg/kg/day for 5 successive days. If IVIG failed to do any clinical improvement, five sessions of plasmapheresis for 5 successive days was performed using 5% albumin as replacement fluid and exchange volume 40–60 ml/kg per session. Nerve conduction studies were performed at the end of the first 2 weeks from the onset of neurologic symptoms. Nerve conduction studies that showed unclassified

results were excluded from subsequent study analysis. Motor conduction studies were performed on median, ulnar, tibial and peroneal nerves in both sides, using conventional techniques. Sensory nerve conduction studies were performed on median, ulnar and sural nerves using conventional studies. Patients were classified as having AMAN or AIDP on the basis of the electrodiagnostic criteria proposed by Ho et al. [10]. Pretreatment serum sample was taken on admission and frozen JNJ-26481585 at −80 °C until sending for antiganglioside antibodies assay. The serum IgG antibodies against gangliosides GM1, GM2, GD1a and GD1b subtypes were tested by ELISA on admission. This test is a qualitative enzyme-linked immunosorbent assay (ELIZA) for in vitro human antibodies assay in serum. When at least one for of the tested antibodies was positive (>1:500), patients were regarded as antiganglioside positive. Patients were initially grouped by positivity of antiganglioside antibodies and then by the electrodiagnoses of AIDP and AMAN. Summary

statistics were constructed using frequencies and proportions for categorical data, and means and SDs for continuous variables. We compared positive and negative groups as well as AMAN and AIDP groups using the χ2 test for categorical data, and t testes for continuous variables. P value < 0.05 was considered to be statistically significant. All statistical analysis was performed using the SPSS software program version 16 (SPSS, Chicago, USA). A total of 47 patients with GBS were admitted to the PICU within the first two weeks from the onset of neuropathy during the period of the study. There were 30 (64%) males and 17 (36%) females. The mean age was 7.272 ± 2.5 years. Thirty-six (77%) children had antecedent illness; acute respiratory infection in 13 patients (27.

DNA sequence analysis was performed using the BigDye Terminator v

DNA sequence analysis was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit and migrated on capillary 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence similarity was performed using BLASTN [22]. Putative mutations were identified after multiple sequence alignment using Clustal W [23] and electropherogram analysis. The existence of each putative mutation was confirmed by sequencing

DNA from both parents, as well as by a secondary validation method, i.e. restriction enzyme digestion of DNA or amplification using specific primers. PCR was used additionally to confirm check details identified gene mutations (described above). Primers were designed to amplify alleles with suspected gene deletion (1318_20delAAC), by using forward primer sequences designed with (native TNAP: 5′-GCCCACAGCTCACAACAAC-3′)

or without (1318_20delAAC: 5′-GCCCACAGCTCACAACTAC-3′) the three base pair AAC deleted. PCR reactions were performed using 5 ng of DNA template, 0.4 μM each forward and reverse (5′-GTCCACGAGCAGAACTACG-3′) primers and LightCycler® FastStart DNA MasterPLUS SYBER Green I kit 1X (Roche Diagnostics, Penzberg, Germany) MG-132 price in the LightCycler® 2.0 Instrument (Roche Diagnostics). Three dimensional (3D) models of the native TNAP protein and mutants (p.N440del, p.R152C and p.N440del/p.R152C) were constructed based on the previously determined 3D structure of human placental alkaline phosphatase (PLAP) (PDB ID: 1EW2) [17] using SWISS-MODEL software (http://swissmodel.expasy.org/) [24], [25] and [26]. These models were aligned, visualized, and analyzed

using the open source software PyMOL Graphics System Molecular (Version 1.2r3pre, Schrödinger, LLC) (http://www.pymol.org). Internal contacts for native and mutant residues in the TNAP structure were analyzed using STING Millennium software [27] from the Brazilian Enterprise for Agricultural Research (EMBRAPA) (http://www.nbi.cnptia.embrapa.br/SMS/index_s.html). Atazanavir Primary dental pulp cells from both probands A and B (genotype: p.[N440del];[R152C]) and four control individuals (native TNAP) were obtained as previously reported [20]. Briefly, extracted teeth were placed in biopsy media, and pulp was harvested by cracking open the teeth using a dental chisel and hammer and removing the soft tissue with sterile forceps. Pulp cells were obtained by enzymatic digestion with 3 mg/mL collagenase type I and 4 mg/mL dispase (Gibco®, Invitrogen™, Life Technologies) for 1 h at 37 °C. Cells at passage four were seeded on coverslips in 24-well cell culture plate (2 × 104 cells per well) and were cultured in DMEM with FBS 5% for 24 h. After two washes in phosphate-buffered saline (DPBS, Invitrogen™, Life Technologies), cells were fixed in 2% paraformaldehyde in DPBS for 20 min at room temperature (RT). Blocking for non-specific binding was performed by incubating with blocking buffer solution (10% normal donkey serum in DPBS) for 45 min at RT.