N = 20, 50), filtering incorporating of incomplete penetrance and phenocopy efficiently reduced the number of candidate SNVs. This suggests that filtering is useful when an assumption of no “genetic heterogeneity” is appropriate and could provide general guidelines for sample size determination.There has been recent success in identifying disease-causing variants in Mendelian disorders by exome sequencing followed by simple filtering techniques. Studies generally assume complete or high penetrance. However, there are likely many failed and unpublished studies due in part to incomplete penetrance or phenocopy. In this study, the expected number of candidate single-nucleotide variants (SNVs) in exome data for autosomal dominant or recessive Mendelian disorders was investigated under the assumption of “no genetic heterogeneity.” All variants were assumed to be under the “null model,” and sample allele frequencies were modeled using a standard population genetics theory. To investigate the properties of pedigree data, full-sibs were considered in addition to unrelated individuals. In both cases, particularly regarding full-sibs, the number of SNVs remained very high without controls. The high efficacy of controls was also confirmed. When controls were used with a relatively large total sample size (e.g.,  Understanding associations between human genetic variations and phenotypes, including risk of disease, is important for successful realization of personalized medicine. Such variants can be used as biomarkers. Recent advances in high-throughput sequencing technology (“next-generation DNA sequencing” (NGS)) enable exploration of human genetic variations on genome-wide and individual levels. The international “1,000-Genome Project,” which uses NGS technology, was launched in 2008. The project aims to create a detailed catalog of human genetic variations by sequencing at least 1,000 individuals .However, in practice it is often not known if the DNA type at a segregating site is mutant or ancestral. In exome analysis, DNA types are generally expressed as “reference . We defined MnA′ as the number of SNVs with nA alternative and n − nA reference alleles in the n haploid sequences. In a segregating site in n + 1 sequences, reference DNA is either mutant or ancestral. The expected number of SNVs in which reference DNA is mutant and nR  reference alleles in the n haploid sequences is derived by the product of the expected number of SNVs with nR + 1 mutant alleles in n + 1 sequences, θ/(nR + 1) based on /(n + 1). This is represented asIn addition to nR  reference alleles in n haploid sequences was obtained. The expectation is represented as the product of the expected number of SNVs with (n + 1) − (nR + 1) = (n − nR) mutant alleles in (n + 1) sequences, θ/(n − nR) based on /(n + 1). The resulting equation isSimilarly, the expected number of SNVs in which reference DNA is ancestral and with n +  − ,,MnA′, EnA can be equal to n at most in .The formula does not include sample size. Interestingly, this result is obtained by , assumin most in  , which is equivalent to 2N + 1 haploid exome sequences that are “randomly sampled” (2N + 1 times), followed by one sequence chosen as a reference from the 2N + 1 sequences. The remaining 2N sequences are randomly joined to form N diploids. The latter is used for illustrative purposes.Next, consider exome sequences of unrelated on model . The infnR and nA denote the number of reference and alternative alleles in a site, respectively. One hasnA or nR (and constant N), there is only one independent variable among nRR,  nRA and nAA. For example, if nA, and nAA are fixed, the other two variables, nRR and nRA, are automatically determined.Conditions of the variables were collected. As in K  be  the number of SNVs in which the number of reference and alternative alleles is nR and nA, respectively, and the number of individuals with genotypes RR, RA, and AA is nRR, nRA, and nAA, respectively, in total N individuals. The expected number of SNVs, E[K], is defined only when all conditions of (N haploid exome sequences and a reference to be “randomly sampled” (2N + 1 times). The number of SNVs with nA alternative and n − nA reference alleles in the 2N + 1 haploid samples can be readily obtained by  was determined given that a DNA site has nA alternative alleles was denoted as Prob. The number of distinct permutations of 2N is given by (2N)!/(nR!nA!). How many permutations result in the genotype configuration ? The number of ways to determine the genotype of each individual in distinct N individuals and generate a genotype configuration of  is equal to N!/(nRR!nRA!nAA!). The genotype RA can be generated from the two runs, RA and AR. Therefore, the number of permutations used to generate the genotype configuration  is derived from (2nRAN!)/(nRR!nRA!nAA!) and Prob = (2nRAN!)/(nRR!nRA!nAA!)×(nR!nA!)/(2N)!. The expression of Prob was shown elsewhere and used to perform the exact test of Hardy-Weinberg equilibrium [Let tions of  and 5b)K] = E[MnA′] ×Prob. E[K] = Ediff[Esamp[K | MnA′]], where Ediff is the expectation with respect to the diffusion model and Esamp is the expectation with respect to the binomial sampling. Binomial sampling is MnA′-times Bernoulli trial, addressing whether a site indicates genotype counts of . Probability of the Bernoulli trial is Prob. Therefore, Ediff[Esamp[K | MnA′]] = Ediff[MnA′Prob] = Ediff[MnA′]Prob. Ediff[MnA′] is represented by ] is not defined if (N = 2 (4 haploid sequences), the expected number of SNVs for  = , , , ,  satisfying ] is 13,333, 4,444.33, 2,222.17, 4,444.33, and 3,333.25, respectively. If both individuals are affected by a certain recessive disease with the genotype AA at a causal DNA site, we can use a filter to retain variants in which both individuals have the genotype AA. The expected number of SNVs after filtering is E[K] = 3,333.25. Similarly, when both individuals are affected by a dominant disease with genotypes AA or RA at a causal DNA site, the expected number of SNVs after filtering is E[K] + E[K] + E[K] = 4,444.33 + 4,444.33 + 3,333.25 = 12,221.91. In this way, by summing E[K] for all sets of  that satisfy E. Specifically, this can be expressed asAs noted, given tions of . In caseAA using , denotedE[K] is not defined if (X(≥X) of N affected individuals have AA is calculated byfined if  is not snAA+RA as the number of individuals with genotypes AA or RA (nAA+RA = nAA + nRA) can be expressed as E[K] as a function of N, nA, nAA+RA using ]. This results inIn cases of dominant disease, denoting RA using , denotedE[K] is not defined if (X(≥X) of N affected individuals have AA or RA is calculated byfined if  is not sN individuals consisting of Na affected individuals and Nc controls. In cases of recessive disease, we considered a filter to retain only SNVs in which at least QUOTE X(≥X) of Na affected and at most QUOTE Y(≤Y) of Nc control individuals have AA  of Na affected and at most QUOTE Y(≤Y) of Nc control individuals have AA or RA , in Na affected individuals follows a hypergeometric distribution. As a result, the expected number of SNVs, E[K2)], in which the number of alternative alleles is nA and the number of individuals with genotype AA is nAA(a) in Na affected individuals is represented asFirst we did not distinguish affected individuals from controls in total given by . Next weX(≥X) of Na affected individuals and at most Y(≤Y) of Nc control individuals have the AA genotype is obtained by summing E[K2]:nAA(a) is over the value satisfying the filtering condition, {nAA(a) : X ≤ nAA(a)∧nAA(c) = (nAA − nAA(a)) ≤ Y}. nAA(c) denotes the number of individuals with AA genotypes in the Nc controls.After filtering, the expected number of SNVs in which at least nA and the number of individuals with genotypes AA or RA (nAA+RA(a)) in Na affected individuals is represented bySimilarly, considering dominant diseases the expected number of SNVs in which the number of alternative alleles is X(≥X) of Na affected individuals and at most Y(≤Y) of Nc control individuals have the AA or RA genotypes is obtained by summing E[K2]:nAA+RA(a) is over the value satisfying the filtering condition, {nAA+RA(a) : X ≤ nAA+RA(a)∧nAA+RA(c) = (nAA+RA − nAA+RA(a)) ≤ Y}. Here, nAA+RA(c) denotes the number of individuals with AA or RA genotypes in Nc controls.After filtering, the expected number of SNVs in which at least N full-sibs with and without controls in a nuclear family ], of SNVs in which the number of sibs with genotypes RR, RA, and AA is nRR, nRA, and nAA, respectively, is represented asnRR + nRA + nAA = N, 00 = 1 and 01 = 02 = ⋯ = 0. Being simplified, this is shown asThe expected number, nRR,  nRA, nAA∈ nonnegative integers and N = nRR + nRA + nAA. Using (X(≥X) of N affected individuals have AA after filtering is calculated asnRR, nRA, nAA), satisfying the filter condition { : X ≤ nAA}. Similarly, in cases of dominant disease, the expected number of SNVs in which at least QUOTE X(≥X) of N affected individuals have an AA genotype after filtering is calculated using , satisfying the filter condition { : X ≤ nAA+RA}.Here, A. Using , the explated as∑E, of SNVs with the genotype configuration of sibs (nRR(a), nRA(a), nAA(a), nRR(c), nRA(c), nAA(c)) is represented asnRR = nRR(a) + nRR(c); nRA = nRA(a) + nRA(c); nAA = nAA(a) + nAA(c); nRR(a), nRA(a), nAA(a), nRR(c), nRA(c), nAA(c)∈ nonnegative integers; Na)( = nRR(a) + nRA(a) + nAA(a) and Nc)( = nRR(c) + nRA(c) + nAA(c). The expected number of SNVs after filtering is calculated as shown just below. In cases of recessive disease, the expected number of SNVs in which at least X(≥X) of Na affected and at most QUOTE Y(≤Y) of Nc control individuals have the genotype AA after filtering is obtained bynRR(a), nRA(a), nAA(a), nRR(c), nRA(c), nAA(c)), satisfying the filter condition {nAA(a) : X ≤ nAA(a)∧nAA(c) ≤ Y}. Similarly, in cases of dominant disease, the expected number of SNVs in which at least QUOTE X(≥X) of Na affected and at most QUOTE Y(≤Y) of Nc control individuals have AA or RA after filtering is calculated using (nA+RA(a) = nAA(a) + nRA(a) and nAA+RA(c) = nAA(c) + nRA(c), the summation is over (nRR(a), nRA(a), nAA(a), nRR(c), nRA(c), nAA(c)), satisfying the filter condition { : X ≤ nAA+RA(a)∧nAA+RA(c) ≤ Y}.We considered ented asE[Ksib2 was used, the number of SNVs appeared to decay exponentially with sample size N. However, the decrease in the number of SNVs was slower as N increased. As shown in N = 1, 2, 3, 4, 50, and 51 were 19999.50, 12221.92 (61.11%), 9333.10 (76.36%), 7761.71 (83.16%), 1808.56, and 1789.36 (98.94%), respectively, with ratios of the expected SNVs for N to those for N − 1 shown in parentheses. The first few individuals were highly effective in removing SNVs, but additional individuals were not. This was obvious when nonstringent filters  were used. In those cases, certain asymptotic values likely exist. For example, ≥90% of the filtered expected number of SNVs was 5540.39 for N = 50, but only 5306.36 for N = 100. From the perspective of identifying disease-causing variants, it is clear that nonstringent filters that take phenocopy into account do not work well even if the sample size is very large. However, using stringent filters, the expected number of SNVs remains high even if the sample size is large (1249.75 SNVs for N = 100). This shows that it is generally difficult to identify a disease-causing variant by filtering without a control.The expected number of SNVs after filtering in cases of dominant disease and unrelated individuals without controls is plotted in N = 14 and 0.001 at N = 20. Even with a single control, the situation changed drastically compared to cases without controls. For example, for N = 10, the expected number of SNVs was 4450.2 without a control, which dropped to 284.27 with one control. Using nonstringent filters that take phenocopy into account , an asymptotic value of approximately 700 may occur with one control, but filtering efficiency is improved if the number of controls totals 3 (21.74 SNVs for N = 53). In addition to phenocopy, filters that take reduced penetrance into account also work reasonably well if half of the exome samples (N/2) are controls. For example, the expected number of SNVs in which 80% of affected individuals and 20% of controls have the genotype AA/RA was 28.51 and 0.02 for N = 20 and 50, respectively.As shown in N = 100 without a control resulted in an expected number of SNVs of 1249.75 for dominant disease, but only 66.67 for recessive disease. Using stringent filtering, the expected number of SNVs for recessive disease wasnA = 2N. In contrast, the expected number of SNVs for dominant disease is represented asThe number of SNVs after filtering in recessive disease shows a similar tendency to SNVs in dominant disease, as shown in Figures N, the expected number of SNVs for 100%, 90%, and 80% filtering was relatively similar compared to unrelated individuals. There was also a higher asymptotic value for 100%, 90% and 80% filtering. The asymptotic value was 3θ/4 = 9999.75, as explained below based on 100% filtering. When the sample size N is large, DNA sites in which the parents have genotypes RR × RA or RA × RA are removed by filtering because a certain proportion of sibs have the genotype AA , 28.51 (N = 20) and 0.02 (N = 50) when unrelated exomes were used and 512.68 (N = 10), 40.85 (N = 20), and 0.06 (N = 50) when full-sibs exomes were used.However, the situation drastically improved when we used controls . For a gθ/4 = 3333.25, which was obtained the same way as for dominant disease. The number of SNVs for recessive disease reached asymptotic values for 100%, 90%, and 80% filtering faster than for dominant disease. The effect of controls in recessive and dominant disease was high. For a given sample size N, the expected number of SNVs in sibs was comparable to that in unrelated individuals. For example, if half of the exome samples were controls, the expected SNVs in which at least 80% of affected individuals and at most 20% of controls have the genotype AA were 203.70 (N = 10), 11.86 (N = 20), and 0.01 (N = 50) when unrelated exomes were used and 200.09 (N = 10), 14.26 (N = 20), and 0.02 (N = 50) when full-sibs exomes were used.The number of SNVs after filtering in sibs for recessive disease shows a similar tendency to dominant disease, as shown in Figures n + 1 haploid sequences were randomly sampled from a population under the Wright-Fisher diffusion model with a constant population size, with n = 2N in N unrelated individuals and n = 2 in full-sibs (Figures n + 1 sequences is represented by formula (We assumed that  Figures . The infUsing a standard population genetics model, we modeled exome analysis for Mendelian disease and developed a method for calculating the expected number of candidate SNVs after filtering under a “no genetic heterogeneity” assumption. Exome sequences of unrelated individuals and full-sibs were considered with and without controls for dominant and recessive diseases. Without controls, particularly for full-sibs, the filtering approach had poor efficiency in reducing the number of candidate SNVs even when using a stringent filter Figures . With coFor rare dominant diseases, it is plausible that affected individuals in a pedigree share one disease-causing variant, even if the disease shows genetic heterogeneity. This indicates that the assumption of “no genetic heterogeneity” is appropriate because the frequencies of variants of the rare disease are also rare in a population, and only one founder in the pedigree should have one of the disease-causing variants (e.g., see Sobreira et al.  or Wang As described in


Schizophrenia patients exhibit deficits on visual processing tasks, including visual backward masking, and these impairments are related to deficits in higher-level processes. In the current study we used electroencephalography techniques to examine successive stages and pathways of visual processing in a specialized masking paradigm, four-dot masking, which involves masking by object substitution. Seventy-six schizophrenia patients and 66 healthy controls had event-related potentials (ERPs) recorded during four-dot masking. Target visibility was manipulated by changing stimulus onset asynchrony (SOA) between the target and mask, such that performance decreased with increasing SOA. Three SOAs were used: 0, 50, and 100 ms. The P100 and N100 perceptual ERPs were examined. Additionally, the visual awareness negativity (VAN) to correct vs. incorrect responses, an index of reentrant processing, was examined for SOAs 50 and 100 ms. Results showed that patients performed worse than controls on the behavioral task across all SOAs. The ERP results revealed that patients had significantly smaller P100 and N100 amplitudes, though there was no effect of SOA on either component in either group. In healthy controls, but not patients, N100 amplitude correlated significantly with behavioral performance at SOAs where masking occurred, such that higher accuracy correlated with a larger N100. Healthy controls, but not patients, exhibited a larger VAN to correct vs. incorrect responses. The results indicate that the N100 appears to be related to attentional effort in the task in controls, but not patients. Considering that the VAN is thought to reflect reentrant processing, one interpretation of the findings is that patients’ lack of VAN response and poorer performance may be related to dysfunctional reentrant processing. Patients with schizophrenia exhibit several visual processing impairments . The P100 is a positive wave peaking approximately 100 ms post stimulus onset. Several neural generators of the P100 have been identified, with activity largest over dorsal stream sites, with extrastriate and striate contributions  and through presentations at community residences. Patients met criteria based on the Structured Clinical Interview for DSM-IV Axis I Disorders  on a 17″ cathode ray tube monitor running at a 160 Hz refresh rate. Participants sat 1 m away from the monitor.Lmax−Lmin)/(Lmax + Lmin). We collected data on four target-mask stimulus onset asynchronies . However, we only analyzed data from the first three SOAs, as the mask terminated on or before the ERP components of interest for these SOAs, and there was little difference in performance between SOAs of 100 and 150. Fifty-four trials per SOA were presented in quasi-randomized fashion .The four-dot masking procedure was modified from similar tasks described elsewhere  at each SOA was analyzed.Electroencephalography was continuously recorded using a 64-channel Neuroscan SynAmps2 amplifier and a Neuroscan 64-channel QuickCap . Data were sampled at 500 Hz with a bandpass of 1–100 Hz. Horizontal electrooculogram  and vertical EOG (placed above and below the left eye) was also recorded. The reference during recording was a point halfway between electrodes Cz and CPz, and all sites were re-referenced offline to the average of the left and right mastoids. An electrode affixed to the forehead served to ground the array.Data were processed offline using Neuroscan Scan 4.3 and BrainVision Analyzer 2 software . Vertical eye blinks were removed using a regression-based algorithm , separately for each SOA. A time window was defined for each ERP component based on the peak activity observed by inspection of the mean global field power averaged across all subjects and SOAs. The width of the time window was selected to ensure coverage of each component and the mean activity within each window was the main dependent measure. The time windows were 68–108 and 134–174 ms for the P100 and N100, respectively. The P100 and N100 were examined in six parieto-occipital electrode sites where visual ERPs were largest based on visual inspection of the waveforms . These sites were also chosen as they overlap with sites used in previous examinations of the N100 and P100 response in visual processing studies in schizophrenia  24.1 (5.6) and 28.0 (5.5) trials for correct and incorrect, respectively, averaged over the two SOAs; for controls there were 28.6 (6.2) and 24.2 (6.1) trials for correct and incorrect, respectively. Activity at electrodes PO7, PO8, O1, and O2 were examined for the VAN. The VAN was measured as the mean activity within the time window of 250–310 ms. Additionally, the P100 and N100 (using the same time windows and electrodes described above) to correct and incorrect responses were examined.t-tests and chi-square tests. 2 (group) × 2 (hemisphere) × 3 (SOA) repeated measures ANOVAs were run separately for P100 and N100 for the entire sample, disregarding accuracy. To examine the VAN and accuracy effects on the P100 and N100 in the subsample, separate 2 (group) × 2 (accuracy) × 2 (SOA) repeated measures ANOVAs were run. In cases of repeated measures with more than one degree of freedom, we used Greenhouse–Geisser correction factors (ε). We report the uncorrected degrees of freedom, the corrected p-value, partial eta-square effect sizes  where appropriate.Demographic information is presented in Table 1, 139 = 19.76, p < 0.001, 2, 278 = 8.48, p <  0.001, ε = 0.92, p > 0.17). Overall, patients showed poorer performance compared to controls and both groups showed the expected decline in accuracy as SOA increased , 0.27 (1.25) vs. 0.59 (1.15) μV, correct vs. incorrect respectively (effect size = 0.27). In contrast, for the patients the t-tests showed no significant difference for correct vs. incorrect responses in patients , −0.15 (0.94) vs. −0.08 (1.03) μV, correct vs. incorrect respectively (effect size = 0.07). The group × accuracy interaction failed to reach significance  and an “old” patient group . The “young” patient group more closely matched the age of the healthy control group, with no statistical difference in age between these two groups. Two analyses were conducted. First, differences in ERPs and behavior between the “young” and “old” schizophrenia patient groups were examined; no significant group effects or interactions with group were seen . Second, differences in ERPs and behavior between only the “young” patients and the healthy controls were examined. Similarly, we performed this same split for the VAN analyses, with a “young” patient group  and an “old” patient group  being created. Again, there were no difference between the “young” and “old” patient groups and the pattern of results remained the same comparing the “young” patients to the healthy control sample. These analyses revealed the exact same pattern of results as when using the entire patient sample, indicating the presence of behavioral and ERP differences between schizophrenia patients and healthy controls regardless of age.Because analyses of covariance can sometimes misrepresent relationships among variables, we considered the potential effect of age on the behavioral and ERP data, without including a covariance analysis. We performed a median split, creating a “young” patient group  in reentrant processing along this pathway. Regardless of how patients correctly identified stimuli in the current study, the VAN results suggest a dysfunction in reentrant processing. However, the lack of a significant group × accuracy interaction in the VAN data means that we interpret these findings with caution.There also remains the possibility that other processes, such as consciousness of stimuli and attentional effort, may be accounting for our behavioral and EEG findings. For example, Del Cul et al.  found thAs with our behavioral study (Green et al., The current study has other limitations. First, nearly all patients were medicated at the time of testing, potentially affecting the results. However, there is evidence that antipsychotic medication does not affect masking performance (Cadenhead et al., Another limitation is that our design was not optimized for eliciting P300 or N2pc that have been examined in the four-dot backward masking studies conducted in healthy controls (Woodman and Luck, The results from this study help clarify findings from our previous behavioral study on object substitution masking (Green et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterogeneity among cells that constitute a solid tumor is important in determining disease progression. Our previous work established that, within a population of metastatic colonic tumor cells, there are minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (ΔΨm), and that these differences in ΔΨm are linked to tumorigenic phenotype. Here we expanded this work to investigate primary mammary, as well as colonic, tumor cell lines. We show that within a primary mammary tumor cell population, and in both primary and metastatic colonic tumor cell populations, there are subpopulations of cells with significant stable variations in intrinsic ΔΨm. In each of these 3 tumor cell populations, cells with relatively higher intrinsic ΔΨm exhibit phenotypic properties consistent with promotion of tumor cell survival and expansion. However, additional properties associated with invasive potential appear in cells with higher intrinsic ΔΨm only from the metastatic colonic tumor cell line. Thus, it is likely that differences in the intrinsic ΔΨm among cells that constitute primary mammary tumor populations, as well as primary and metastatic colonic tumor populations, are markers of an acquired tumor phenotype which, within the context of the tumor, influence the probability that particular cells will contribute to disease progression. Cancer is a highly heterogeneous disease and diversity among cells that make up solid tumors likely influences the probability of tumor expansion and progression . AbnormaTo address the question of whether stable differences in intrinsic ΔΨm exist among cells in other tumor cell populations and, if so, whether these differences are similarly linked to tumorigenic phenotypes, we generated single cell subclones from SW480 cells, a cell line established from the primary colon tumor of the same patient from which SW620 cells were established  and fromHere we report that, consistent with our previous work, there are significant and stable differences in the intrinsic ΔΨm among a minor fraction of cells within a population of both SW480 and MCF7 cells and that these differences in ΔΨm are again linked to tumorigenic phenotype, with elevations in the intrinsic ΔΨm associated with decreased sensitivity to the chemoprotective agent butyrate and increased constitutive, hypoxia-independent VEGF secretion. Moreover, we demonstrate that unlike the differences in ΔΨm of clones from SW620 cells, the intrinsic ΔΨm of subclones derived from the SW480 primary colonic tumor cell population, or from the MCF7 primary mammary tumor cell population, do not impact invasive potential.Thus, our data establish significant stable heterogeneity in the intrinsic ΔΨm among subpopulations of cells of both a primary and metastatic colon tumor and from a primary mammary tumor and suggest that differences in intrinsic ΔΨm are not random variations or passive indices of heterogeneity, but rather markers of acquired behavior of subpopulations cells which, within the context of the tumor, are likely selected for, and contribute to, clinical progression of the disease .The ΔΨm was determined as previously described by staining cells with the ΔΨm-dependent fluorescent dye JC-1  and analyzing fluorescence emission by flow cytometry in detection channel 2 (FL-2) –17.2. Cell viability was then determined by the MTT assay [Cells grown to 80% confluence were incubated for 24 hours in medium alone or in medium containing 100 mM CoClTT assay .Cells at approximately 80% confluence were treated with medium alone or with medium containing 5 mM sodium butyrate (NaB) . Seventy-two hours later, cell viability was determined by the MTT assay .165 protein levels were quantified by ELISA  according to the manufacturer's protocol. Constitutive MMP7 and VEGF165 secretion levels were normalized by determining cells per well by the MTT assay and expressed relative to the population of cells.Cells were seeded into 96-well plates and grown to approximately 80% confluence. Conditioned tissue culture medium was then harvested, and MMP7 and VEGFμg of cell lysates were size-fractionated on 4–20% acrylamide SDS-PAGE gels  and blotted onto PVDF membranes . Replicate blots were probed with antibodies directed against fascin  or actin , followed by incubation with appropriate secondary antibodies. Reactions were detected by enhanced chemiluminescence and quantified by densitometry using Kodak IS4000R and Kodak Molecular Imaging Software.Cells grown to approximately 80% confluence were washed twice in ice cold PBS and lysed in buffer containing 0.5% NP-40, 1% Triton X-100, 0.2 mM sodium orthovanadate, 0.2 mM PMSF, 1 mM EDTA (pH 8), 1 mM EGTA (pH 8), 150 mM NaCl, and 10 mM Tris pH 7.4. Lysates were cleared by centrifugation and protein concentration determined . Thirty Cells were seeded into chambers consisting of a reconstituted basement membrane supported by an underlying polycarbonate membrane . Invasion through the basement membrane was determined by the staining and subsequent quantification by optical density at 560 nM, of cells adhering to the polycarbonate membrane.Data from at least 3 independent determinations of the intrinsic ΔΨm were analyzed by Bonferroni's Multiple Comparison Test. Mean data from other determinations were evaluated as a function of the relative intrinsic ΔΨm using linear regression analyses.JC-1 is a lipophilic, cationic fluorescent dye that exhibits ΔΨm-dependent uptake, accumulation, and aggregate formation in the mitochondria . The emiHere we investigated whether the distribution of JC-1 stained SW480 cells , a cell Similar to our previous work , suspensThe SW480 parental population and each of 149 derived subcloned cell lines were then stained with JC-1 and analyzed by flow cytometry. The intrinsic ΔΨm of each of subcloned lines was expressed relative to that of the unselected population of cells and plotted as a frequency distribution . The relL are between 0.17- and 0.50-fold lower than that of the population of SW480 cells; the ΔΨm of the subcloned lines in the ΔΨmE subgroup are comparable to that of the population of cells; and the ΔΨm of the subclones in the ΔΨmH subgroup are 1.60- to 2.30-fold higher than that of the population of cells. For subsequent investigations, we focused on 13 of the SW480 subcloned lines falling into 3 groups. As shown in Figures L subclones are statistically different from all of the ΔΨmH subclones (P ≤ 0.05). Similar to our previous work investigating intrinsic ΔΨm of SW620-derived clones , 20, theSimilarly, 83 subclones derived from the MCF7 population were expanded, the intrinsic ΔΨm of each was determined by JC-1, expressed relative to the population and plotted as a frequency distribution . The relP ≤ 0.05). We focused on 11 of the MCF7 subcloned lines; 2 with intrinsic ΔΨm at least 15% lower, 2 with ΔΨm comparable, and 7 with intrinsic ΔΨm at least 15% higher than that of the population of MCF7 cells . BonferrThus, these data extend the finding of minor subpopulations of cells with significant stable differences in intrinsic ΔΨm from SW620 metastatic colon tumor cells  to the pβ-oxidation [in vitro and in vivo [The unbranched short chain fatty acid butyrate is a natural constituent of the colonic contents, present at high concentrations due to its generation during fiber fermentation in the large intestine , 22. Rapxidation . In addixidation , NaB med in vivo –27 there in vivo –31.  in vivo metabolism, it is difficult to achieve and maintain effective serum levels even when butyrate salts are administered by continuous i.v. [Butyrate also has potent effects on a variety of other cell types, including normal and malignant mammary epithelial cells , 33. Howous i.v. , 35. We ous i.v. , 37, inious i.v.  and founP < 0.0001) or from MCF7 cells (P = 0.0005) with elevated ΔΨm linked to decreased sensitivity, consistent with our previous work [We have shown that the ΔΨm plays a critical role in NaB initiated cell cycle arrest and apoptotic pathways in SW260 cells , 17 and ous work , 14, 20.P = 0.0002 and P = 0.015, resp.) with elevated ΔΨm generally associated with increased levels of secretion . Whereas most MMPs are produced by stromal cells, MMP7 (matrilysin) is synthesized by tumor cells and its elevated expression, particularly at a tumor's invasive edge, is characteristic of metastatic cancer cells –45. We hP < 0.0001) [P = 0.303 and not shown, resp.).To investigate whether there is a similar relationship between constitutive MMP7 secretion and the intrinsic ΔΨm of SW480- or MCF7-derived subclones, MMP7 protein levels were quantified in harvested conditioned medium and plotted as a function of the ΔΨm of each cell line. Consistent with previous reports, we found that mean constitutive MMP7 secretion in the parental population of SW480 cells is approximately 50% lower than that of the metastatic SW620 cell population ( 0.0001) , 47, and 0.0001) , 20, theP = 0.007), with elevated ΔΨm linked to higher levels of fascin protein, similar to the association between elevated ΔΨm of SW620 subclones and MMP7 secretion [not correlated with the intrinsic ΔΨm of subclones derived from primary colon tumor cell line SW480 (P = 0.539) nor in the subclones derived from MCF7 mammary carcinoma cells .Because acquisition of an invasive phenotype in colonic carcinoma is linked to upregulation of the actin binding protein fascin , 50, we ecretion , 20. In in vitro [P < 0.0001). Furthermore, as suggested by the absence of an association between the intrinsic ΔΨm and MMP7 secretion or fascin expression levels . Finally, because we have shown that the intrinsic ΔΨm of subcloned cell lines derived from metastatic SW620 cells correlates with their ability to invade an artificial basement membrane in vitro , 20, we in vitro , 47, SW4 Figures , resp., Heterogeneity is a fundamental property of cellular systems, including solid tumors . Diversiβ-oxidation, including branched isobutyric acid and a fluorine-substituted analogue, heptafluorobutyric acid [in vitro [in vitro and that the differences in NaB sensitivity associated with intrinsic ΔΨm of subcloned cell lines derived from metastatic and primary colon tumors, and from a primary mammary tumor, may be related to alterations in mitochondrial metabolic activity. We show that in subclones derived from each of these cell lines, differences in intrinsic ΔΨm are linked to sensitivity to the chemoprotective agent NaB, with increased ΔΨm linked to decreased NaB-mediated cytotoxicity. Because malignant transformation is associated with a general elevation in the ΔΨm –9, theseric acid , are inein vitro , 52, 53.α or by accelerated mTOR-mediated translation of HIF1α [We also show that differences in intrinsic ΔΨm in subclones derived from primary mammary carcinoma cells, as well as primary and metastatic colon carcinoma cells, are linked to phenotypes consistent with expansion of solid tumors with elevated intrinsic ΔΨm associated with increased levels of constitutive, hypoxia-independent VEGF secretion. These results suggest the presence of pseudohypoxia, the activation of hypoxia-like pathway(s) under normoxic conditions, in cells with elevated intrinsic ΔΨm. Pseudohypoxia is achieved by either defective TCA cycle-mediated impaired degradation of HIF1of HIF1α –57. Actiof HIF1α , 59 and not linked to invasive potential in subclones derived from the paired primary colonic carcinoma cell line or from the primary mammary tumor cell line investigated here. Therefore, while subpopulations of cells with higher intrinsic ΔΨm from both primary and metastatic colonic tumors, and from a primary mammary tumor, acquire properties likely reflecting endogenous cellular resilience, demonstrated by decreased sensitivity to NaB-induced cytotoxicity, or are primed to rapidly adjust to alterations in microenvironment, demonstrated by constitutive hypoxia-independent VEGF secretion, other properties, such as enhanced invasive potential, appear only in subpopulations of cells with elevated ΔΨm that are components of a metastatic tumor. Thus, the relationship between the intrinsic ΔΨm and invasive potential may be secondary to its relationship with phenotypes associated with local solid tumor expansion. Whereas stable differences in the intrinsic ΔΨm of subclones derived from the metastatic colonic carcinoma cell line are linked to differences in invasive capacity , differe not identified in cells with decreased ΔΨm . There may also be additional heteroplasmic mtDNA populations in SW620 metastatic compared to SW480 primary colonic carcinoma cell lines [Although the mechanisms involved in generating and maintaining differences in ΔΨm are unclear, they may reflect alterations in the composition of mitochondrial membranes –65 (Marill lines . TherefoInterestingly, in patients with stage III colon cancers, resistance to fluorouracil-based chemotherapy has been linked to specific somatic mtDNA mutations , and we The steep electrochemical proton gradient across the mitochondrial inner membrane is mainly accounted for by its matrix-side-negative electrical component, the ΔΨm. Based on Nernst's law, cations capable of permeating biological membranes can accumulate in the mitochondrial matrix at a 10-fold higher level with each 60 mV of transmembrane voltage . TherefoIn summary, our data suggest that stable differences in intrinsic ΔΨm among cells that comprise colon and mammary tumors reflect cellular heterogeneity and identify cells that are important in tumor progression and potential predictors of response to, and targets of, chemoprotection and chemotherapy.


The New York Times, “the invisible yet toxic brew of gases and particles clinging to smokers’ hair and clothing, not to mention cushions and carpeting, that lingers long after secondhand smoke [SHS] has cleared from a room.”You may never have heard of thirdhand smoke, or THS, but chances are you’ve smelled it. THS is, in the words of So how dangerous might THS really be? The answer, still to be pronounced, will depend on many factors.THS was a topic of interest long before it received its present name. The seed of the idea that cigarette smoke toxicants might linger on room and car surfaces long after the smoke itself was gone was planted in 1953, when it was reported that smoke condensate painted onto mice caused cancer.10In 1991 the house dust of smokers’ homes was first found to be contaminated with nicotine.12In 2008 similar findings were reported for cars.A 2010 study showed THS also remains after smokers move out of their homes, even after being vacant for two months and being prepared for new residents, sometimes with new carpeting and paint.16thirdhand smoke may have first appeared in print in 2006,Pediatrics.The term N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), 4-(N-nitrosomethylamino)-1-(3-pyridinyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN).22A new development emerged when Mohamad Sleiman, a chemist with the Indoor Environment Department of the Lawrence Berkeley National Laboratory (LBNL) Environmental Energy Technologies Division, and colleagues reported that nicotine adsorbed onto surfaces reacted with nitrous acid—an air pollutant found in vehicle exhaust and produced by improperly vented gas stoves and burning tobacco—to form tobacco-specific nitrosamines (TSNAs) including 1- NNA.For NNK, divide this figure by 5 (only 1 ng/cm−2 NNK was found on the dashboard): 8/5 = 1.6 μgAssuming the passenger weighs 80 kg, the potential doses received are:NNA: 8 μg over 80 kg body weight = 0.1 μg/kg for 3 days, or 0.033 μg/kg/dayNNK: 1.6 μg over 80 kg body weight = 0.02 μg/kg for 3 days, or 0.0066 μg/kg/dayBut although the predicted figures clearly could be lower, Sleiman says at least some of the input figures seem reasonable. He points out, “The quantities of TSNAs on the paper were only those collected after three days of one person smoking ten cigarettes per day. How much more might be accumulated after months of smoking, perhaps by more than one smoker smoking more than ten per day?”“Different materials absorb different amounts of nicotine [therefore producing different amounts of TSNAs],” adds coauthor Hugo Destaillats, also of the LBNL. “We only examined paper and stainless steel; other materials in cars and homes absorb other quantities.” For instance, wool, cotton, silk, linen, acetate, and polyester all absorb SHS compounds,29Further, although nitrous acid levels typically reach 5–15 ppb by volume indoors and 30 ppb by volume in vehicles, concentrations as high as 100 ppb by volume have been measured indoors.a]pyrene, 1,3-butadiene, benzene, formaldehyde, cadmium, arsenic, and lead—that the researchers did not consider in their studies. “Alongside NNK and other TSNAs, they could increase the dangers of thirdhand smoke residue,” Gundel says. Moreover, she says, the dermal CPF for at least one compound in SHS—benzo[a]pyrene—is actually about 15-fold higher than its oral counterpart.31Gundel adds that SHS contains many more toxic and carcinogenic compounds—such as benzo cars and rooms is very expensive, and real-estate agents know that smoking affects property values.”Regardless of whether THS is conclusively shown to cause illnesses, it is already changing attitudes, behaviors, norms, expectations, purchasing behavior, and the economic value of personal property and real estate, Matt says. In combination, these are powerful factors that have the potential to reduce tobacco use and lower the health risks associated with smoking itself as well as SHS and THS exposure.“The most important impact of the efforts to prevent exposure to thirdhand smoke,” Matt says, “may be . . . the reduction of health risks from active smoking and secondhand smoke exposure.” For these forms of tobacco smoke exposure, at least, the discussion about whether they may be dangerous is well and truly concluded.


Because the sulfate/PM2.5 ratio dropped among six localities but the PM2.5 mortality coefficient did not “substantially” increase, the authors concluded that sulfate must be “about as toxic” as average PM2.5. In a two-pollutant world, perhaps.2.5 species, and a specific species is emitted from several sources, chemical-specific associations might not reflect inherent toxicity but rather status as a marker of harmful coemissions  may occur unrelated to any inherent toxicity , which would include both secondary sulfate and its reaction products.Human panel studies can examine effects of PMAny conclusions regarding sulfate toxicity are premature until consistent results from advanced models , which a


Alterations in the composition of the gut microbiome and/or immune system function may have a role in the development of autism spectrum disorders (ASD). The current study examined the effects of prenatal and early life administration of lipopolysaccharide (LPS), a bacterial mimetic, and the short chain fatty acid, propionic acid (PPA), a metabolic fermentation product of enteric bacteria, on developmental milestones, locomotor activity, and anxiety-like behavior in adolescent male and female offspring. Pregnant Long-Evans rats were subcutaneously injected once a day with PPA (500 mg/kg) on gestation days G12–16, LPS (50 µg/kg) on G15–16, or vehicle control on G12–16 or G15–16. Male and female offspring were injected with PPA (500 mg/kg) or vehicle twice a day, every second day from postnatal days (P) 10–18. Physical milestones and reflexes were monitored in early life with prenatal PPA and LPS inducing delays in eye opening. Locomotor activity and anxiety were assessed in adolescence (P40–42) in the elevated plus maze (EPM) and open-field. Prenatal and postnatal treatments altered behavior in a sex-specific manner. Prenatal PPA decreased time spent in the centre of the open-field in males and females while prenatal and postnatal PPA increased anxiety behavior on the EPM in female rats. Prenatal LPS did not significantly influence those behaviors. Evidence for the double hit hypothesis was seen as females receiving a double hit of PPA  displayed increased repetitive behavior in the open-field. These results provide evidence for the hypothesis that by-products of enteric bacteria metabolism such as PPA may contribute to ASD, altering development and behavior in adolescent rats similar to that observed in ASD and other neurodevelopmental disorders. Autism spectrum disorders (ASD) are neurodevelopmental disorders with roughly 4 males diagnosed for every 1 female. ASD comprise a number of behavioral symptoms, including impairments in communication, social behavior, sensory abnormalities, and restricted and repetitive behavior It is becoming well established that both genetics and environmental factors contribute to the development and expression of ASD. A number of genes involved in immune function, mitochondrial function, and neural circuit formation have been implicated Clostridia, Bacteroidetes, and Desulfovibrio in the GI tract of autistic children. Many of these bacteria are antibiotic-resistant. As such, repeated early infections in postnatal life treated with antibiotics may provide an enteric environment that promotes overgrowth of these bacteria resulting in intestinal inflammation The gastrointestinal tract (GI) is home to over a trillion commensal bacteria, known as the microbiome, that have a bidirectional relationship with the central nervous system and contribute to normal immune system development and homeostasis in both humans and rodents. GI dysbiosis has been implicated in inflammatory diseases and neuropsychiatric health Byproducts of these enteric bacteria (from carbohydrate and some protein metabolism) include the short chain fatty acid, propionic acid (PPA) Immune dysfunction may increase the risk for ASD with alterations in the adaptive and innate cellular immune responses having been observed in children  has shown to result in changes in anxiety-like and exploratory behavior in adult and adolescent male and female rats. Increased anxiety in the elevated plus maze have been shown in male and female adolescent and adult offspring Results of a number of studies have also shown that the effects of postnatal LPS on subsequent behavior do not manifest themselves unless a second environmental insult  is experienced in adulthood The present study investigated the effects of prenatal treatment with the immune stimulant, LPS, and the microbiome associated gastrointestinal factor, PPA, on postnatal developmental milestones, open-field activity and anxiety-like behavior in adolescent male and female offspring. In addition, the effects of a second ‘hit’ of PPA in the second postnatal week were examined. It was hypothesized that prenatal LPS would increase anxiety-like behavior in offspring and that prenatal PPA would increase locomotor activity and anxiety-like behavior in offspring. Combinations of prenatal and postnatal treatments, prenatal LPS with postnatal PPA and prenatal PPA with postnatal PPA, were included to assess whether behavioral effects were magnified compared to that seen after either treatment alone. Developmental delay was observed in male and female rats with prenatal PPA and LPS, while increased anxiety-like behavior was sex- and treatment-specific.Procedures were approved by the University of Western Ontario Animal Care Sub-Committee and were in accordance with the Canadian Council of Animal Care (CCAC) guidelines (Protocol Number: 2008-063).ad libitum access to both food (ProLab RMH 3000) and water. A 12∶12 h light:dark cycle (lights on at 0700 h) was maintained in a temperature controlled colony room (21±2°C). Litters were born on G22  0), toe-clipped for identification, and were weaned at P21 . On P21, pups were weaned and randomly culled to a maximum of 10 animals per litter . Weaned rats were housed in same-sex, same-postnatal treatment groups of 2 or 3 in standard polypropylene cages under the same conditions as dams. All behavioral testing took place during the light phase and animals were monitored  during testing.Twelve primiparous female Long-Evans rats weighing between 270–310 g were mated with adult male Long-Evans rats  for a total of 12 litters. Females were paired overnight with a male the night before behavioral estrus. Sperm present on a vaginal smear (hematoxylin & eosin stain) the morning after pairing indicated successful mating and this was designated gestational day 0 (G0). Dams were housed individually in standard polypropylene cages (45×22×20 cm) with E. coli serotype 0111:B4, L2630, Sigma Chemical, St. Louis, MO, USA) was dissolved in 0.1 M phosphate buffered saline and administered SC at a low dose of 50 µg/kg on G15 and G16 for a total of 2 injections. Prenatal LPS administered at this time has been previously shown to increase anxiety-like behavior Sodium propionate  was dissolved in 0.1 M phosphate buffered saline and administered at a dose of 500 mg/kg (0.26 M) subcutaneously  once a day on G12–16 for a total of 5 injections. This dose was selected based on previous studies and is meant to resemble states of metabolic dysfunction, thus a larger dose of PPA than that found in normal human colon was used 20 mM, . InjectiAs synaptogenesis occurs during the first 3 weeks of postnatal life in rats A timeline showing drug administration and behavioral testing is provided in The body weights of pups were monitored daily for the first 20 days of life. The following developmental milestones were assessed for day of appearance: Righting reflex (P2–6): pups were placed on their back and given 30 s to turn onto stomach with all limbs outstretched from body; Pinna detachment (P2–4): bilateral pinna unfolding completely from head; Incisor eruption (P7–13): both upper and lower; Eye opening (P12–16): scored as 0 =  both eyes closed, 1 =  one eye open, 2 =  both eyes open; Negative geotaxis (P7–10): time (s) to rotate 180° on a 30° incline when placed head down (assesses vestibular function and motor development) The EPM was made of wood and painted grey with non-toxic paint. The apparatus consisted of two opposite open arms (54×12 cm) with no sides or ends and orthogonal to two enclosed arms with sides and ends (54×12×48 cm). The four arms extended from a centre platform (12×12 cm) and the apparatus was raised 50 cm from the floor. An overhead camera connected to a television and DVD-R recorded behavior for later scoring.Testing took place on the afternoon of P40. Animals were recorded for 5 minutes and placed on the centre platform facing an open arm to begin the test. After each animal, the maze was cleaned with a 20% alcohol solution. Measures assessed included the number of entries onto open and closed arms and time spent (s) on open and closed arms. Percent time in open arms was taken as a measure of anxiety (time spent in open arms/time spent in open arms+time spent in closed arms×100).Locomotor activity was monitored using eight Versamax Animal Activity Monitors , each consisting of a Plexiglas open field chamber (40 cm×40 cm×30.5 cm), and a Plexiglas lid with air holes. Infrared beams surrounding each chamber recorded horizontal and vertical locomotor activity as beam breaks, from which locomotor measures were compiled Animals were placed in the novel open-field for 60 min on P42 to assess any changes in locomotor activity. Horizontal activity measures analyzed were: total distance (TD) − total horizontal distance (cm); horizontal movement time (MT) − amount of time (s) an animal was engaged in horizontal movement; number of horizontal movements (NM) − number of horizontal movements separated by 1 s stop time. Vertical activity measures analyzed were: vertical movement time (VT) − amount of time (s) an animal spent in a vertical position; number of vertical movements (VM) − number of vertical movements separated by 1 s stop time. Repetitive activity was measured using number of revolutions (clockwise and counterclockwise) - number of times an animal runs in a clockwise or counterclockwise circle of at least 2 inches in diameter. Duration spent (s) in the periphery and centre was measured, and locomotor activity was corrected for time spent in each zone .All analyses were performed with IBM Statistics 20 . As pups within a litter are not independent samples, the effects associated with belonging to a litter and being raised in a litter must be accounted for. To do this, linear mixed models were used for each of the dependent variables, with Litter used as a subject variable. Fixed factors in all models were: Sex, Prenatal drug, Postnatal drug, with litter size used as a covariate. For body weight, negative geotaxis, and eye opening, Day was also included as a factor. LSD post-hocs were performed. Significance was set to α = 0.05.2(11) = 4.49, p>.05. There were also no significant differences in the number of male to female pups in each of the prenatal treatment groups, F = 1.88, p = 0.211.A Chi-square test was performed to verify that litter sizes did not significantly differ across groups, χF = 3077, p<0.001, and male pups weighed significantly more than female pups, F = 88.02, p<0.001  = 6.20, p<0.001. Birth weight and weight over the first 12 days of life did not significantly differ among prenatal treatments (p<0.001) on P13–19 in the prenatal PPA and 2VEH groups, ps <0.05 (p<0.001) with individual days failing to reach significance in the prenatal LPS group (Weight was monitored daily for the first 20 days of life (P0–P19). All pups gained weight across days,  p<0.001 . There weatments . Postnatps <0.05 . PostnatPS group .F = 298.04, p<0.001, with males weighing significantly more than females, F = 1841.06, p<0.001. A significant Sex×Prenatal drug×Postnatal drug interaction, F = 3.36, p = 0.019, indicated that in prenatal 2VEH animals, postnatal PPA adolescent males weighed significantly more than postnatal VEH males, p<0.001 , with no significant effect of postnatal drug in adolescent females . All animals gained weight across days,  females .F = 13.0, p<0.001, indicated that prenatal PPA and LPS treated male and female pups were significantly different from both 5VEH (ps <0.01) and 2VEH treated pups (ps <0.05) on P14, while on P15, prenatal PPA treated male and female pups were significantly different from 5VEH and LPS (ps <0.05).There was no developmental delay and no sex differences in pups as a result of either prenatal PPA or LPS treatment for eruption of top and bottom incisors, and pinna detachment –C. ThereF = 6.70, p = 0.011, with males performing the reflex significantly earlier than females, and prenatal drug treatment having no significant effect on this reflex  = 3.66, p = 0.012  = 4.95, p = 0.003. Males receiving prenatal LPS and postnatal PPA had significantly higher scores on the free-fall righting reflex test than males receiving prenatal LPS and postnatal VEH, p = 0.002. Females receiving prenatal LPS and postnatal PPA had significantly lower scores on the reflex test than females receiving prenatal LPS and postnatal VEH (p = 0.031) and lower scores than females receiving prenatal 2VEH and postnatal PPA (p = 0.029) or prenatal PPA and postnatal PPA  = 8.55, p = 0.004, horizontal movement time, F = 6.31, p = 0.014, and number of revolutions, F = 11.86, p = 0.001). Significant main effects of Prenatal drug were found for total distance traveled, F = 3.23, p = 0.026, and number of horizontal movements, F = 4.37, p = 0.006, but not for horizontal movement time. Animals prenatally exposed to PPA or 5VEH moved a significantly greater total distance than animals in both the LPS and 2VEH control group, ps <0.05 , with the same effect in both male (5VEH significantly greater than 2VEH p = 0.014) and female offspring  = 2.68, p = 0.051. Further analysis showed that a double hit of prenatal and postnatal PPA increased the number of revolutions made in the female offspring, but not in the male offspring (p = 0.001), prenatal PPA-postnatal VEH treated females (p = 0.046), and female offspring exposed to prenatal LPS or 2VEH and postnatal PPA (ps <0.05).There were no effects of prenatal LPS or PPA, or postnatal PPA treatment on vertical activity measures . For number of revolutions, the Sex×Prenatal drug×Postnatal drug interaction was nearly significant, ffspring . Female Overall, prenatal PPA, prenatal LPS, and postnatal PPA alone did not produce hyper- or hypo-activity in male and female adolescent offspring. Prenatal PPA and postnatal PPA combined significantly increased the number of revolutions in female, not male offspring.F = 4.83, p = 0.004, for percent time spent in the centre . There were no significant effects of treatment on the number of entries into the centre or perimeter of the open-field and postnatal PPA as compared to postnatal VEH had no significant effect on percent time in the centre or perimeter of the open-field  = 11.53, p = 0.001, and spent more time moving horizontally than males, Sex F = 8.41, p = 0.005.Locomotor activity measures were corrected for the amount of time spent in the perimeter or centre of the open-field. Prenatal LPS, prenatal PPA, and postnatal PPA did not significantly affect locomotor activity in the perimeter of the open-field on any horizontal or vertical activity measures  = 5.82, p = 0.018, and Prenatal drug, F = 5.14, p = 0.002, for total distance traveled in the centre  = 2.82, p = 0.043, for horizontal movement time in the centre . This effect was present in both females  and in males . For horizontal movement time, prenatal PPA treated animals spent significantly more time moving in the centre than 5VEH treated animals, p = 0.005.Postnatal PPA and prenatal LPS also did not affect central locomotor activity. However, prenatal PPA resulted in increased activity in the centre of the open-field. There were significant main effects of Sex, e centre  and a sie centre . AnimalsF = 6.56, p = 0.012, and spent more time engaged in rearing than males, Sex F = 4.48, p = 0.037. A significant Sex×Prenatal drug interaction for number of vertical movements in the centre of the open-field, F = 2.89, p = 0.040, indicated that females prenatally exposed to PPA performed a significantly greater number of vertical movements than prenatal PPA treated males, p = 0.001  = 2.99, p = 0.035. Postnatal PPA treated animals in the prenatal 5VEH group entered the open arm significantly fewer times than postnatal VEH animals (p = 0.01). A significant Sex×Prenatal drug interaction for both open arm entries, F = 9.45, p<0.001, and percent time in the open arm, F = 7.14, p<0.001, showed there were no differences in open arm entries among male offspring. Female offspring prenatally exposed to PPA entered the open arm significantly less often (p = 0.001) and spent significantly less time in the open arm than prenatal 5VEH treated female offspring . The significant Sex×Prenatal drug interaction, F = 4.10, p = 0.009, for time spent in the closed arm showed that female offspring in the prenatal 5VEH group spent significantly less time in the closed arm than female offspring in the other 3 prenatal groups  = 6.74, p = 0.011, but this was only in female offspring (p = 0.037).For number of entries into the open arm, there was a Prenatal drug×Postnatal drug interaction, p<0.001, . PrenataIn summary, females in the prenatal PPA group made significantly less open arm entries, and spent less percent time in the open arm with more time in the closed arm than controls. Postnatal PPA significantly increased closed arm time in female offspring.Until relatively recently, limited attention has been given to possible developmental effects of microbiome associated, GI metabolic products in rodents. Likewise, investigations of the effects of prenatal and postnatal immune activation with LPS and poly I:C have been mostly limited to adult male rodents. The present study investigated the effects of prenatal PPA and prenatal LPS in the emergence of postnatal developmental milestones, locomotor activity and on anxiety-like behavior in both male and female adolescent offspring. Additionally, a PPA regimen in the second week of life was used to evaluate if a subsequent postnatal insult would exacerbate any behavioral effects of prenatal treatment.Prenatal LPS and PPA treatment resulted in developmental delays in male and female offspring, suggesting altered neurodevelopmental effects. Prenatal PPA alone did not influence open-field activity, but as expected, prenatal PPA produced increased anxiety-like behavior as evidenced by decreased time spent in the centre of the open-field in male and female adolescent rats, and less time on the open arm of the elevated plus maze (EPM) in females. Prenatal LPS did not alter behavior in the open-field and EPM. Postnatal PPA alone did not alter open-field activity, yet increased anxiety-like behavior in the EPM. Evidence for the double hit hypothesis was present in female adolescent rats, with the combination of PPA treatments increasing repetitive behavior, while no effect of a prenatal LPS and postnatal PPA double insult was observed. A double hit of prenatal LPS and postnatal LPS was beyond the scope of this study, but given evidence supporting two hits of LPS  and adolescent male rats in an open-field General activity in a novel open-field was greater in prenatal PPA and 5VEH treated offspring compared to LPS and 2VEH treated offspring. Receiving five injections may have induced a mild stress response and been enough of a stressor to affect development. Prenatally stressed rats have been shown to be hyperactive in a novel open-field, as well as displaying anxiety-like behavior General activity in an open-field does not seem to be altered by MIA alone in adolescent or adult offspring, as observed in this and prior studies The low dose of LPS (50 µg/kg) administered during mid-late gestation (G15–16) may help explain why there were no effects of prenatal LPS on anxiety measures in the open-field and EPM. Increased anxiety-like behavior in the EPM was observed in adolescent male rats prenatally exposed to LPS on G16 or G17 at higher doses (100 and 150 µg/kg), and in adult mice offspring exposed to a similar dose of LPS as this study, but earlier prenatally (G10), or postnatally Campylobacter jejuni) increased anxiety behavior and was associated with increased early gene expression in brain regions implicated in anxiety C. jejuni, is not a pathogen and may not alter developmental processes in the same way. It is possible that similar immune processes are activated  as central PPA induces an innate neuroinflammatory response in the brain When the open field was divided into centre and perimeter zones, prenatal PPA treatment increased anxiety-like behavior in both male and female offspring. Reduced time in the centre is indicative of increased anxiety, as the open space acts as an aversive space. Adolescent males and females prenatally exposed to PPA were also hyper-active when in the centre of the open-field, which may suggest a level of aversiveness. Adult rats fed a carbohydrate-rich diet exhibited anxiety and elevated levels of SCFAs in the gut There were sexually dimorphic effects of prenatal and postnatal PPA treatment, with female rats displaying increased anxiety-like behavior in the EPM. Compared to 5VEH treated control females, time in the open arm and open arm entries were decreased in prenatal PPA treated females and closed arm time was increased in prenatal PPA and postnatal PPA treated females. An increase in anxiety is consistent with previous reports of prenatal VPA and enteric infection producing anxiety-like behavior in male and female adult rat offspring A sex difference was present in the 5VEH control group, with males exhibiting similar levels of behavior as prenatal PPA treated males and females, suggesting anxiety in these control males. Brief daily periods of maternal stress resulted in increased anxiety in male adolescent rats, but not females Similar to prenatal 5VEH treated males, both males and females treated with LPS and 2VEH displayed low levels of activity on the EPM. There may have been a ceiling effect in the current study that prevented an effect of LPS from being evident. It appears that this effect may be specific to these animals as behavioral testing occurred in a paired fashion , and prenatal 5VEH treated females performed as expected. It is unclear why 2VEH and LPS treated animals produced such low levels of maze exploration. It has been shown that exposure to additional stressors has been required before increased anxiety-like behavior on the EPM was observed in rats treated with postnatal LPS An imbalance between excitation and inhibition in the brain has been implicated in autism and anxiety, with changes in GABA and possibly serotonin suggested to be critical. Modifications in GABAergic systems have been reported in various brain regions of patients with ASD While no behavioral effects of LPS were found in the current study, acute and chronic LPS during gestation induces a proinflammatory response and alters the placental barrier, which may allow external agents and/or cytokines to gain access to the fetus In summary, these results are the first to demonstrate that prenatal PPA, and one of a few to demonstrate that postnatal PPA and a low dose of LPS, alters developmental processes and subsequent behavior in male and female adolescent rats, resembling alterations observed in ASD and previous animal models. Prenatal LPS and prenatal PPA produced delay in eye opening and prenatal and postnatal PPA increased anxiety-like behavior in both male and female offspring, with a greater effect observed in female offspring. Developmental delay and altered temperament are observed in children with ASD, as reduced communication and motor skills or inappropriate emotional responses  are observed Figure S1Body weight (g) for male and female offspring from postnatal days 0–9. A–B: Males. C–D: Females. Rats were prenatally exposed to either lipopolysaccharide (LPS) on G15–16, propionic acid (PPA) on G12–16, or their respective phosphate buffered saline controls (2VEH and 5VEH). There were no significant differences between prenatal treatment groups in body weight at birth or over the first 9 days of life. Error bars represent S.E.M. Refer to (TIF)Click here for additional data file.Figure S2Body weight (g) for adolescent male and female offspring (P39–51). A–B Males. C–D Females. Rats were prenatally exposed to either lipopolysaccharide (LPS) on G15–16, propionic acid (PPA) on G12–16, or their respective phosphate buffered saline controls (2VEH and 5VEH). Postnatal drug treatment, either PPA or VEH, was administered 2x/day every other day from P10–18. Males weighed significantly more than females , while males receiving postnatal PPA weighed significantly more than postnatal VEH treated males in the prenatal 2VEH group (2VEH-VEH vs. 2VEH-PPA in Panel A), * ps <0.05. Error bars represent S.E.M. Refer to (TIF)Click here for additional data file.Figure S3Developmental milestones and reflexes for male and female offspring. A: Pinna detachment. B: Top incisor eruption. C: Bottom incisor eruption. D: Righting reflex. Males performed the righting reflex slightly earlier than females . E. Negative geotaxis. There were no significant differences between prenatal treatment groups in emergence of milestones and reflexes. Error bars represent S.E.M. Refer to (TIF)Click here for additional data file.Figure S4Additional thigmotaxis measures (P42) in male and female offspring. Activity measures were time corrected. A: Total distance traveled in the perimeter (cm/s). B: Horizontal movement time in the perimeter (s). Females were significantly greater than males for both total distance and movement time, ps <0.01. C: Number of vertical movements in the perimeter. D: Vertical time in the perimeter. There were no significant differences in vertical measures. E. Horizontal movement time in the centre (s). Prenatal PPA treated females spent significantly more time moving than prenatal 5VEH treated females. F: Vertical time (s) in the centre. Error bars represent S.E.M. Refer to p<0.05.(TIF)Click here for additional data file.


Hemodynamic imaging results have associated both gender and body weight to variation in brain responses to food-related information. However, the spatio-temporal brain dynamics of gender-related and weight-wise modulations in food discrimination still remain to be elucidated. We analyzed visual evoked potentials (VEPs) while normal-weighted men (n = 12) and women (n = 12) categorized photographs of energy-dense foods and non-food kitchen utensils. VEP analyses showed that food categorization is influenced by gender as early as 170 ms after image onset. Moreover, the female VEP pattern to food categorization co-varied with participants' body weight. Estimations of the neural generator activity over the time interval of VEP modulations (i.e. by means of a distributed linear inverse solution [LAURA]) revealed alterations in prefrontal and temporo-parietal source activity as a function of image category and participants' gender. However, only neural source activity for female responses during food viewing was negatively correlated with body-mass index (BMI) over the respective time interval. Women showed decreased neural source activity particularly in ventral prefrontal brain regions when viewing food, but not non-food objects, while no such associations were apparent in male responses to food and non-food viewing. Our study thus indicates that gender influences are already apparent during initial stages of food-related object categorization, with small variations in body weight modulating electrophysiological responses especially in women and in brain areas implicated in food reward valuation and intake control. These findings extend recent reports on prefrontal reward and control circuit responsiveness to food cues and the potential role of this reactivity pattern in the susceptibility to weight gain. The prevalence of eating disorders and, in particular, overweight is increasing throughout the world population causing long-term health problems such as diabetes or cardiac disorders. Obesity is linked to a loss in the ability to adjust food intake for maintaining the energetic balance of the body. Although both genders are affected, women have been found to be more susceptible to developing eating disorders Differences in food perception between genders  have so far mainly been investigated with hemodynamic neuroimaging. DelParigi and colleagues Apart from gender, body weight (as quantified by BMI) was found to be an additional factor influencing cerebral responsiveness to food viewing even in normal-weight women. Killgore and Yurgelun-Todd Female socialization often results in stronger preoccupation with nutritional aspects and body image 2, mean = 20.67 [s.e.m. ±0.54]; men = 20–25 kg/m2, mean = 23.0 [s.e.m. ±0.63]), the BMI range being similar between genders. None of the participants reported current or prior neurological or psychiatric illnesses or a history of dieting or eating disorders. All participants had normal or corrected-to-normal vision. All of the EEG recording sessions started between 13:00h–14:00h to control for circadian modulations of hunger. Further, participants were instructed to have eaten lunch to satiety before the recording sessions. They likewise explicitly reported what they had eaten for both breakfast and lunch that day in a questionnaire. All participants provided written, informed consent to the procedures, which were approved by the Ethics Committee of the Faculty of Biology and Medicine of the University of Lausanne and the Vaudois University Hospital Center (CHUV).Twenty-four  remunerated volunteers, aged 19–36 years (mean±s.e.m. = 25.8±1.1 yrs), participated in the study www.pstnet.com/eprime).Participants were presented with four blocks of trials each comprising 50 non-complex colorful photographs of energy-dense high-fat foods and 50 non-food kitchen utensils see  in pseudhttp://www.biosemi.com/pics/zero_ref1_big.gif). All analyses were conducted using the CarTool software (http://sites.google.com/site/fbmlab/cartool) and the Sten toolbox (www.unil.ch/fenl/Sten). To calculate VEPs, epochs of EEG from 98 ms pre- to 488 ms post- stimulus onset (i.e. 50 data points before and 250 data points after stimulus onset) were separately averaged for each image category and each participant. In addition to an automatic ±80 µV artifact rejection criterion, EEG epochs containing eye blinks or other noise transients were removed by trial-to-trial inspection of the data. Prior to EEG epoch averaging as a function of gender and image category, data from artifact electrodes of each participant were interpolated using 3-D splines Continuous EEG was acquired at 512 Hz though a 160-channel Biosemi ActiveTwo system  referenced to a ground circuitry . This circuitry functions as a feedback loop driving the average potential across the montage as close as possible to the amplifier zero. Details of this circuitry, including a diagram can be found on the Biosemi website . We provide only the essential details on the methods here but see  we refraIn order to objectively determine stable VEP intervals that reveal response modulations as a function of image category and gender, the collective post-stimulus group-average VEP periods were subjected to a topographic cluster (i.e. VEP map) analysis based on a hierarchical clustering algorithm We further computed VEP means for each participant, condition and electrode sensor over the time interval of stable VEP clustering (here: 170–213 ms). An electrode-wise ANOVA with the within-subject factor of image category (food and non-food) and the between-subject factor of gender then served to visualize local VEP interactions between factors. The result of this analysis is presented as an intensity plot representing time, electrode location, and significant p-values. We would like to stress, however, that our main conclusions are based on global measures of the electric field, i.e. incorporating data points from all electrode sensors in a conjoined and reference-independent manner.Modulations in the strength of the electric field at the scalp were assessed by a time point-wise ANOVA on the Global Field Power GFP;  with the3) at each node. Prior fundamental and clinical research have documented and discussed in detail the spatial accuracy of this inverse solution, which are on the order of the grid size of the solution points  as in former reports from our group Intracranial sources underlying the surface electric fields were estimated for each image category and gender using the local auto-regressive average (LAURA) distributed linear inverse solution 3) of each source node and to each image category (food and non-food) with the respective BMI of individuals were calculated for each gender separately. Only nodes showing reliable correlations (r(10)±0.58; p≤0.05) and exceeding the spatial criterion of ≥15 contiguous nodes were considered significant. In addition, the interaction term obtained by the abovementioned ANOVA served to restrict the obtained correlations to brain regions in which source node activity significantly differed as a function of the viewed image category and gender. The results of the correlation analyses were rendered on the MNI brain with the Talairach and Tournoux coordinates Further, two-tailed Pearson correlations between the current density value  = 4.95; p≤0.05), but no main effect of gender or interaction between category and gender.The 24 participants readily performed the discrimination between images of foods and kitchen utensils. Women responded correctly to 96.79% (s.e.m. ±0.86) of the food images and to 95.41% (s.e.m. ±1.14) of the non-food images. Men answered correctly to 96.54% (s.e.m. ±0.96) of the foods and to 95.27% (s.e.m. ±1.50) of the non-foods. An ANOVA with the within-subject factor image category and the between-subject factor gender revealed an effect of category  = 5.62; p≤0.05). Post-hoc unpaired t-tests between genders showed that men were reliably faster than women in categorizing the food images (t(22) = 2.62; p≤0.05) as well as the non-food images (t(22) = 3.81; p≤0.01). We would remind the reader, however, that reaction times were cued by a question mark that appeared subsequent to image presentation so as to avoid contamination of the VEP by motor-related activity. Thus, the above reaction times likely do not reflect the true latency of decision-related processes. We nonetheless include them here for completion.Women responded with a mean (±s.e.m.) reaction time of 406±27 ms to food images and 422±25 ms to non-food images. Men responded with a mean (±s.e.m.) reaction time of 309±26 ms to food images and 291±24 ms to non-food images. The 2×2 ANOVA revealed an interaction between category and gender  = 9.61; p≤0.01; η2 = 0.32) as well as interactions of category×gender  = 10.04; p≤0.01; η2 = 0.33), category×BMI  = 9.22; p≤0.01; η2 = 0.32), category×map  = 3.75; p≤0.05; η2 = 0.16), category×gender×BMI  = 10.32; p≤0.01; η2 = 0.34) and category×map×BMI  = 3.58; p≤0.05; η2 = 0.15). A marginally significant interaction was furthermore found between category×map×gender  = 3.47; p = 0.057; η2 = 0.15). These results indicate that the VEP pattern over the 170–213 ms interval is modulated by gender and BMI and also by whether participants viewed food or non-food objects.We conducted an ANCOVA on these GEV values with the within-subject factors image category and map, the between-subject factor gender and the covariate BMI. All reported values were adapted by Huynh-Feldt corrections. The ANCOVA revealed an effect of category  = 21.60; p≤0.01; η2 = 0.68) as well as interactions of category×BMI  = 19.67; p≤0.01; η2 = 0.66), category×map  = 7.24; p≤0.01; η2 = 0.42) and category×map×BMI  = 6.13; p≤0.05; η2 = 0.38) in women only. In men, no effects or interactions were observed . In summary, these results show that the modulation in VEP topography over the 170–213 ms interval is associated with BMI when women, but not men, discriminate food from non-food.Separate ANCOVAs for each gender revealed an effect of category . This ANOVA revealed no time intervals of gender effects or interactions between image category and gender. This latter finding argues against a simple explanation based on heightened arousal/attention in women vs. men, which would have resulted in stronger VEPs (particularly during early processing stages).In addition to the global VEP analyses, To this point, our analyses indicate that VEPs in men and women resulted in stable but differing response topographies between 170–213 ms post-stimulus onset when categorizing food and non-food images. Furthermore, the female response pattern during food viewing co-varied with BMI despite the fact that participants' weight was within normal ranges.As topographic modulations are indicative of changes in the neural generator activity underlying the surface VEP pattern, we estimated the active neural sources in women and men over the time interval showing topographic variations (i.e. 170–213 ms post-image onset) when viewing either image category see . Signifi(10) = −0.80; p<0.01). Furthermore, even when excluding the responses of female participants with lowest BMIs (i.e. below 20 kg/m2), the negative correlation remained reliable (p<0.05 for both parametric and non-parametric testing).In succession, we calculated correlations between all source nodes showing the interaction effect and individuals' BMI. Significant (negative) correlations between neural source strength and BMI were only observed in the female responses to food viewing , restricIn the current study, we investigated whether gender and variations in BMI within the normal range influence the spatio-temporal brain dynamics in the visual categorization of food-related objects. We found that gender and BMI both modulate the topography of VEPs and neural source activity over the time interval from 170–213 ms after image onset. However, only in women the VEP topography during food – non-food discrimination and neural source strength in ventromedial prefrontal cortex when viewing food objects was associated with BMI over the derived time period.The obtained time course of gender influences in our study coincides with previous findings on gender differences between ∼100–200 ms when biologically relevant objects such as faces Further, we found evidence that the global VEP topography during food-related object categorization co-varies with BMI in normal-weight women, but not in men, over an interval from 170–213 ms after image onset. On the other hand, we did not observe interactions between image category and gender in global VEP strength (i.e. GFP) in our group of normal-weight subjects. That is, interactions between image category and gender were more apparent in qualitative than in quantitative analyses of the VEP topography. These findings thus complement previous VEP studies that focused more on local differences in response amplitudes between normal- and overweight subjects during food-related object perception without special regard to gender variations In accordance with the topographic pattern observed over the 170–213 ms time interval, we also observed interactions between image category and gender in neural source activation strength in frontal and temporo-parietal regions. These areas have previously been implicated in food discrimination at a similar timing Associations between weight and brain activation patterns have hitherto been shown by several hemodynamic imaging studies comparing brain responses between normal-weight and obese subjects during food viewing The ventral PFC is an area in which several sensory, cognitive and affective inputs converge, and signals are computed that guide behavioral goal-directed values and choices Yet, our study also points to a decreased responsiveness of prefrontal regions implicated in reward evaluation and food intake control with increasing weight, but only in women. The time course of the obtained association in women indicates that neural response attenuation is already effective within 200 ms after food cue onset, i.e. over a time interval showing initial food categorization effects modulated by viewer's gender. That is, varying food cue responsiveness between genders as a function of weight may present an indication towards an elevated vulnerability of women to potential weight gain. As most studies have hitherto addressed this issue in women only, prospective studies on the relation between responses of food reward/control circuits and weight gain in both genders are needed to further elucidate gender influences on weight gain susceptibility. Furthermore, previous reports evinced an elevated responsiveness of prefrontal and striatal regions in overweight participants, potentially indicating a switch in food reward and control mechanisms along a weight continuum that might mediate a change in eating style from a more ‘cognitive’ reflective mode to a more ‘automatic’ reflexive mode Figure S1Results of the neural source estimations over the 170–213 ms interval during food viewing (upper panels) and non-food viewing (lower panels) in women (left panels) and men (right panels) rendered on the MNI template brain. In both genders, viewing energy-dense foods addressed temporal, parietal and occipital regions in both hemispheres. In women, the active neural sources were more widely distributed than in men, also comprising inferior prefrontal areas. Neural sources during the viewing non-food images in both genders were less disseminated than during food viewing. Results on the statistical differences in activation patterns as a function of image category and gender are shown in (TIF)Click here for additional data file.


Several mechanisms underlying the molecular basis of ARVD/C type 1 have been proposed. Evaluating previously described mechanisms might elucidate how TGFβ3 contributes to disease progression in ARVD/C type 1. Here we review how TGFβ3 can induce fibrogenesis through Smad and/or β-catenin signaling. Moreover, the role of apoptosis is addressed. Finally the extent to which the immune system has been demonstrated to be a modulating and amplifying agent in the onset and progression of ARVD/C in general is discussed.Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited cardiomyopathy associated with cardiac arrhythmias originating in the right ventricle, heart failure, and sudden cardiac death. Development of ARVD/C type 1 has been attributed to differential expression of transforming growth factor beta 3 (TGF Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), also known as ARVD, ARVC, and ARVC/D, is an inherited cardiomyopathy. It is associated with cardiac arrhythmias originating in the right ventricle, heart failure, and sudden cardiac death . DemandiV1–V3), are major criteria. In addition, depolarization or conduction anomalies can be observed, for example, epsilon waves and late potentials, as well as arrhythmias, such as ventricular tachycardia [To help establish a diagnosis, Task Force Criteria have been proposed by McKenna et al.  and revihycardia . Fibrofahycardia .DSP gene), plakophilin 2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC-2), and junctional plakoglobin (JUP). The nondesmosomal proteins related to ARVD/C are desmin (DES), transmembrane protein 43 (TMEM43), transforming growth factor β-3 (β3TGF), lamin-A/C (LMNA), titin (TTN), phospholamban (PLN), and α-T-catenin (CTNNA3). Even though these genes are associated with the different types of ARVD/C, often the underlying mechanisms resulting in the disease phenotype remain uncertain.As part of the Task Force Criteria, genetics may be a helpful tool in establishing or confirming a diagnosis. Several mutations associated with ARVD/C have been described to date. Five proteins, out of a total of twelve that are related to ARVD/C, are part of the desmosomal complex , and thuOver the years, it has become clear that ARVD/C is not a structural defect present at birth, explaining the original term dysplasia, and that the disease can also be biventricular or even left dominant . The afoβ3. The genetic origin of the disease was first mapped in 1994 [β3 gene [β3 [β3 expression in vivo has not been verified. Nevertheless, it seems reasonable to presume that TGFβ3 overexpression causes ARVD/C type 1.The present review will focus on potential mechanisms involved in the development of ARVD/C type 1 (OMIM: 107970). ARVD/C type 1 is a rare form of ARVD/C , found o in 1994 , but the in 1994 . For two[β3 gene . An earlgene [β3 . As the gene [β3  demonstrβ3.In the more prominent forms of ARVD/C, resulting from mutations in desmosomal proteins, occasionally multiple mutations were observed in a single patient , 29. Preβ3 (GeneID: 7043) is a secreted protein involved in tissue development and differentiation as well as fibrogenesis [β3 overexpression in fibrogenesis, cell-cell adhesion abnormalities, apoptosis, and the role of inflammatory injury in general in ARVD/C will be discussed.TGFogenesis . Howeverβ3 is directly involved in promoting fibrofatty infiltration in the myocardium, or that the replacement of myocardial cells is an indirect result of other processes, such as apoptosis, remains to be established.The extent of fibrosis in the heart is correlated with the severity of conduction anomalies, such as delayed activation . Howeverβ3 is known to be involved in tissue regeneration, accelerating healing and improving tissue strength, and it has been used to reduce scar tissue formation in wound healing [β3 in extracellular matrix organization. Active TGFβ is thought to affect fibroblasts through TGFβ receptor 1 and 2 (TGFBR1 and 2), activating the Smad signaling pathway, as reviewed by Leask [β receptor complex, and Smad 2 and 3 subsequently relay the signal to the nucleus, altering gene expression, as illustrated in β1) overexpression induces cardiac fibrosis, resulting in an increased collagen content in the heart [β3 exerts its function through the same signaling pathway, it seems possible that TGFβ3 overexpression will also induce fibrosis in the myocardium. This is further corroborated by the presence of both TGFβ 1 and 3 during cardiac fibrogenesis [β overexpression might excessively activate cardiac fibroblasts, resulting in fibrosis.TGF healing . This wa healing , implyinby Leask , and Shiby Leask . In thishe heart . As TGFβogenesis . Moreoveogenesis , 40. Thuper se might be insufficient to lead to ARVD/C. Persistent fibrosis resulting from TGFβ overexpression requires cofactors [However, Smad activation ofactors , which mAn alternative explanation for excessive fibrofatty tissue formation in the myocardium is episodes of apoptosis/necrosis. This would provide cofactors required for persistent fibrosis, as corroborated by several works, in which expression of these factors was observed after heart failure, dilated cardiomyopathy, and myocardial infarction , 43. Morβ3-activated Smad signaling, on one side and the biochemical environment created in the tissue to sustain fibrosis on the other side. Disturbing this equilibrium by excessive TGFβ3 expression may result in the augmented levels of fibrosis seen in ARVD/C.Thus, it is likely that the myocardial fibrosis is the result of a complex interplay of intracellular signaling pathways, among which TGFβ-catenin is an integral part of the adherens junctions, loss of cell-cell adhesion inhibits Wnt/β-catenin signaling. This perturbed cell signaling pathway can result in induced cardiac myocyte apoptosis and development of fibrofatty tissue [Clear signs of gap junction remodeling have been observed in ARVD/C patients –47, whicy tissue , 45. Yety tissue . Furthery tissue , 24.β3 and desmosomal proteins affect β-catenin signaling [β-catenin is stabilized through Wnt signaling [β-catenin translocates to the nucleus, regulating gene transcription  and subsequently Nemo-like kinase (NLK) in the heart. In turn, NLK inhibits Wnt/β-catenin signaling by counteracting β-catenin-induced gene transcription [β 1 and 3 have been shown to decrease E-cadherin expression via the Smad signaling pathway [β-catenin signaling, as was found in ARVD/C caused by mutations in DSP and JUP. Thus, TGFβ inhibited β-catenin signaling could stimulate adipogenesis in cardiac tissue.TGFcription . Moreove pathway , 60. Thiβ3 overexpression might be expected to have an activating role in the Wnt/β-catenin pathway. In human renal proximal tubular cells, TGFBR2 was shown to dissociate from adherens junctions upon TGFβ1 activation [β3 is known to bind to the same receptor, possibly eliciting a similar process in the heart. Incubation with TGFβ1 yielded increased levels of β-catenin, and dissociation of β-catenin from adherens junction complexes was observed [β-catenin was stabilized by TGFβ activity, thereby promoting Wnt/β-catenin signaling [β-induced fibrosis [Alternatively, TGFtivation . TGFβ3 iobserved . In addiignaling , 59. Thiignaling  or mightfibrosis .β3 overexpression. Several pathways have been associated with TGFβ signaling, sometimes with counteracting effects, as illustrated in β-catenin signaling, as the effects of Wnt signaling are strongly dependent on the cell type and signaling context [β overexpression on Wnt signaling remain obscure. Further research is necessary to elucidate the complex interplay of TGFβ signaling and Wnt signaling.At this point, the molecular mechanisms that might be involved in organ fibrogenesis are not properly defined . There i context . For thiDsp and Jup deficient mice, increased levels of apoptosis were observed [β-catenin signaling, which has been shown to be involved in regulation of apoptosis [β-catenin signaling might be, at least in part, responsible for the increased levels of apoptosis observed in ARVD/C [Finally, in both observed , 53. Thipoptosis . Hence, n ARVD/C –66.β3 might be exerted through its influence on cell-cell adhesion integrity, thereby altering β-catenin signaling in a variety of ways. Ultimately this can result in fibrofatty tissue development and apoptosis.Therefore, one of the effects of TGFMyocardial damage is one of the hallmarks of ARVD/C. Signs of increased levels of apoptosis in myocardial tissue are frequently observed in patients suffering from ARVD/C –67. Evenβ3 remains to be established. TGFβ signaling has been implicated in both cell growth and apoptosis, dependent upon the concentration of the cytokine and the signaling context. Myocardial cells of patients suffering from ARVD/C were shown to express and overexpress proapoptotic caspase 3 (CASP3) and Bax, respectively [β-induced apoptosis in other tissues, as reviewed by Schuster and Krieglstein [β to a heterodimer of TGFBR1 and 2, Smad proteins are activated. Ultimately, CASP3 and subsequently the caspase cascade are activated through this pathway. Thus, TGFβ3 overexpression might induce CASP3 expression, thereby lowering the threshold to apoptosis. Alternatively, altered Wnt/β-catenin signaling might also be involved, as discussed earlier.However, the origin of the increased level of apoptosis remains uncertain. Whether apoptosis in ARVD/C type 1 is a direct or secondary effect of the increased levels of TGFectively , 66. Expeglstein . Throughβ3 overexpression. Unraveling the source of myocardial degeneration is complicated by the subtle interplay of signaling pathways, and the multiple functions of TGFβ3 [β3 remains an enigma, as most other tissues seem to be unaffected [Nevertheless, at this point it remains uncertain whether the aforementioned pathways are the main processes involved in myocardial apoptosis resulting from TGFof TGFβ3 . Moreoveaffected . More reAnother contributor to the onset and progression of ARVD/C in general is inflammatory injury. Infiltration of inflammatory cells in necrotic or degenerative cardiac tissue has been reported in ARVD/C patients . For exaA frequent finding upon pathological examination of ARVD/C biopsies is myocarditis , 71. It α (TNF-α) and interleukin 1-β (IL1-β) converting enzyme result in production of nitric oxide synthase (iNOS) [Nevertheless, the role of viruses cannot entirely be excluded. Multiple studies report the presence of the enteroviral genome in the myocardium of ARVD/C patients with myocarditis, thereby supporting the possibility of its role in ARVD/C , 72. Seve (iNOS) . Ultimate (iNOS) .Genetic predispositions may amplify damage to myocytes caused by myocarditis, which in turn might quicken the progression of ARVD/C. However, it remains unclear whether inflammation and/or infection could be a primary cause of myocyte damage or a secondary response to degenerative loss of myocytes. Moreover, inflammation of cardiac tissue is not limited to the infiltration of inflammatory cells. Cardiac myocytes themselves produce inflammatory mediators and might thus contribute to immune-independent degeneration of cardiac tissue . As discβ3 overexpression, as in ARVD/C type 1, can be amplified. Such interference might contribute to severe myocardial injury, as observed in ARVD/C patients [Another possible mechanism by which inflammation plays a role in progression of ARVD/C might be the effect of inflammatory mediators on desmosomal proteins. As set out in the introduction, ARVD/C is predominantly caused by mutations in genes associated with cardiac desmosomes , 14. Intpatients .β3 overexpression has not been established. Finally, inflammatory mediators in ARVD/C patients may be useful in risk stratification and possibly the development of anti-inflammatory therapies to ameliorate quality of life.Taken all together, inflammation and/or infection may play a significant role in ARVD/C, possibly as a modulating and amplifying agent in disease progression. However, further research is required to elucidate the order in which certain pathogenic events occur; that is, whether inflammatory effects are a primary causal agent or a secondary response to already affected cardiac tissue. Moreover, a direct link to TGFβ3 overexpression, which is associated with ARVD/C type 1, might excessively activate cardiac fibroblasts through Smad signaling, resulting in fibrosis. Furthermore, TGFβ3 overexpression may affect cell-cell adhesion integrity by dissociating β-catenin, thereby interfering with cell signaling pathways, resulting in fibrogenesis and apoptosis. Concerning the latter, overexpression of TGFβ3 may stimulate CASP3 expression, resulting in a lowered threshold for apoptosis. Finally, inflammatory injury seems to play a significant role in progression of ARVD/C in general. The putative mechanisms seem to be interrelated, but at this point it is unknown whether these processes occur in concert, all induced by TGFβ3 individually, or that some of the observations result indirectly from TGFβ3 overexpression.The myocardial damage observed in ARVD/C is to a large extent due to the fibrofatty replacement and apoptosis of ventricular myocardium. TGFβ3TGF gene, one may question whether TGFβ3 overexpression is the actual molecular basis of ARVD/C type 1. One concern is that in vivo data on the (over) expression of TGFβ3 in ARVD/C type 1 patients are lacking. Another concern involves the unsuccessful efforts to identify mutations in β3TGF in the two families of the original linkage analysis by Rampazzo et al. [PKP2 gene was identified. A further concern is the apparent cardioselective phenotype of ARVD/C type 1, despite the widespread tissue expression of β3TGF. The recently generated Tgfb3 conditional knockout mouse [Although Beffagna et al.  demonstro et al. . Of noteo et al.  recentlyut mouse  may provβ3 and signaling pathways, their role is largely speculative. Therefore, further research is required to fully elucidate and verify the specific role TGFβ3 induced signaling and the immune system in ARVD/C type 1 disease progression. A better understanding of ARVD/C can ultimately lead to better means for diagnosis and possibly it can offer possibilities for treatment of the disease.Revealing the molecular basis for ARVD/C remains to be a challenging task. In this review, an overview of putative mechanisms associated with onset and progression of ARVD/C, in particular type 1, has been provided, defining different directions to better understand the aforementioned molecular basis of the disease. However, research on ARVD/C type 1 is hindered by the small number of patients. This explains, at least in part, that the role of the aforementioned mechanisms has not been established. Even though the putative mechanisms are based on observations in ARVD/C patients as well as known interactions between TGF