Respiratory viruses are key in the development of exacerbations in COPD and asthma. We sought to characterise respiratory viruses to develop a model in which airway exposure to virus in addition to chronic allergen resulted in an exacerbated inflammatory and altered functional phenotype.2) of H3N2 treated animals was tested using pulse oximetry.BALB/c mice or human ICAM-1 transgenic mice (BALB/c background) were used in all assays. Animals were intranasally inoculated with either human rhinovirus 1B (HRV1B), human rhinovirus 16 (HRV16) or influenza A/Victoria/3/75 (H3N2). The kinetics of viral replication in naive animals and the lung inflammatory profiles in bronchoalveolar lavage (BAL) were assessed for up to seven days after single inoculations. The effects of HRV1B and H3N2 inoculation were further assessed in a model of chronic lung inflammation: animals were intranasally challenged with house dust mite extract (HDM) for up to seven weeks. Virus challenges were given after a robust HDM allergic phenotype developed. Some groups of animals were also treated with fluticasone propionate during and after the viral inoculation. After cessation of the challenge period, airway hyperresponsiveness (AHR) was assessed using whole body plethysmography, followed by BAL, serum and lung tissue analysis. In addition, the arterial oxygen saturation (POIn vitro cytopathic effect assays did not demonstrate that either HRV1B or HRV16 were able to replicate in lungs of relevant mice. After viral inoculation, a variable inflammatory response was detectable only in HRV1B treated animals up to 24 hours post inoculation. Animals treated with H3N2 developed a mixed inflammatory lung inflammation and a PO2 profile indicative of a viral effect on lung functionality. H3N2 also replicated well in the mouse. Single inoculations of either HRV1B or H3N2 were given to animals already challenged with HDM. There was no indication that addition of HRV1B to existing phenotype altered the HDM induced AHR or inflammatory profile. Combination H3N2/ HDM treatment resulted in an increase in BAL eosinophils and neutrophils, but no change to steroid responsiveness nor changes in PO2 readouts.Strains of human rhinovirus are variably able to elicit lung inflammation but not AHR changes following single exposure to mouse hosts. H3N2 infection resulted in viral replication and a strong inflammatory response, cleared in 7 days following infection. We were unable to demonstrate that rhinovirus infection resulted in an exacerbation type response when given to allergic animals, but steroid sensitive inflammatory exacerbation was noted in HDM/ influenza treated animals.


GJB2 and SLC26A4 genes for the presence of mutations, screened for the mitochondrial DNA (mtDNA) A1555G mutation, and screened for congenital CMV infection in DNA isolated from dried newborn blood spots. Results were obtained from 364 children. We established etiology for 60% of children. One or two known GJB2 mutations were present in 82 children. Twenty-four children had one or two known SLC26A4 mutations. GJB2 or SLC26A4 changes with unknown consequences on hearing were found in 32 children. The A1555G mutation was found in one child, and CMV infection was detected in 28 children. Auditory neuropathy spectrum disorder was confirmed in 26 children whose DNA evaluations were negative. A secondary objective was to investigate the relationship between etiology and audiological outcomes over the first 3 years of life. Regression analysis was used to investigate the relationship between hearing levels and etiology. Data analysis does not support the existence of differential effects of etiology on degree of hearing loss or on progressiveness of hearing loss.Hearing loss is an etiologically heterogeneous trait with differences in the age of onset, severity and site of lesion. It is caused by a combination of genetic and/or environmental factors. A longitudinal study to examine the efficacy of early intervention for improving child outcomes is ongoing in Australia. To determine the cause of hearing loss in these children we undertook molecular testing of perinatal “Guthrie” blood spots of children whose hearing loss was either detected via newborn hearing screening or detected later in infancy. We analyzed the  Permanent childhood hearing loss, which occurs in 1–2 per 1000 live births GJB2  gene, the SLC26A4 (pendred) gene and in the mitochondrial DNA at position 1555 (A1555G mutation) and congenital cytomegalovirus (CMV) infection have been considered as the major causes of congenital hearing loss in developed countries Childhood hearing loss is an etiologically heterogeneous condition, caused by environmental factors and/or genetic factors. Despite this heterogeneity, mutations in the The study was approved by institutional ethics review boards .The LOCHI study commenced in 2005, with recruitment completed in 2007. All families with children born in Australia between 2002 and 2007 and who presented for hearing services below 3 years of age at Australian Hearing (AH) were invited to participate. Parents of children enrolled in the LOCHI study provided written, informed consent to participate. All participants were invited to give additional informed consent for molecular testing. Of the 451 eligible children, written consent was obtained for 387 children for molecular testing. There was no significant difference in hearing loss  between the consent group and the non-consent group (p = 0.605). A total of 364 Guthrie card samples were obtained from the custodian authorities of health records. The sample comprised 280 whose hearing loss was detected via UNHS, 64 who did not have access to UNHS and 20 whose screening status was unknown.In Australia, the hearing of newborns is screened using automated auditory brainstem response (AABR). Infants who do not pass hearing screening are referred for diagnostic audiological assessment. These include tympanometry, middle ear muscle reflexes, otoacoustic emissions (OAEs), click-evoked auditory brainstem response (ABR) testing, and ABR or auditory steady-state responses (ASSR) testing using frequency-specific stimuli. Following diagnosis, children are referred to AH for assessment of hearing and provision of amplification, at no cost to families. Electrophysiological tests results are converted to estimated behavioral thresholds for fitting of hearing aids The hearing thresholds of participants from inception to 44 months of age were retrieved from clinical records held at the AH national database, with written permission from parents. Hearing thresholds at 0.25, 0.5, 1, 2, and 4 kHz for each ear were obtained. These audiometric thresholds were used to characterize evolution of children’s hearing loss over the first 3 years of life as fluctuating, progressive, or stable. In accordance with the audiological protocol of AH, fluctuating hearing loss is defined as a change in hearing threshold of 15 dB or greater at any octave frequency between 0.5 and 4 kHz, but subsequently recover over the period of investigation. Progressive hearing loss is defined as a decrease in 10 dB or greater at two or more adjacent frequencies between 0.5 and 4 kHz or a decrease in 15 dB at one octave frequency in the same frequency range over the period of investigation. Longitudinal audiograms that do not demonstrate the changes specified above are labeled as stable.ANSD is characterized by a dysfunction in neural/brainstem transmission of auditory stimuli in the presence of normal cochlear outer hair cell function. The clinical presentation is an absent or abnormal ABR responses and the presence of otoacoustic emissions or cochlear microphonics.Information about enlarged vestibular aqueduct (EVA) was obtained from clinical records held at the AH national database. The diagnosis of EVA was based on CT scan. Radiographic imaging was not part of routine clinical service offered to children identified with hearing loss.et al. 2; 1 mg/ml Proteinase K; 1% Tween-20) at 56°C for 1 hour. Following incubation at 100°C for 7 mins. the samples were rapidly cooled on ice. The samples were centrifuged  and the supernatants frozen for >4 hours at −80°C.Lysates for molecular screening were prepared from Guthrie blood spots, using a modification of the method described by Barbi GJB2 (exon 2) and the splice site mutation in intron 1 (IVS1+1 G>A). Exon 2 PCR products were sequenced in both directions. The IVS1+1 G>A mutation was screened for by Hph1 digestion of a PCR product which spans the splice site. If a single mutation in GJB2 was found, connexin-30 deletions del(GJB6-D13S1830) and del(GJB6d13s1854) were screened for as described by Castillo et al.Lysates were screened for mutations in the coding region of SLC26A4 gene exons was PCR amplified. Following nested PCR, the products were analyzed by High Resolution Melt (HRM) on the Corbett Rotorgene 6000 . Primary PCR products were sequenced when HRM variants were identified.Each of the in silico approaches to assess if unreported changes in the GJB2 and SLC26A4 genes are likely to affect protein function: PolyPhen-2 We used A1555G mutation introduces a HaeIII restriction enzyme site. PCR products were digested with HaeIII and analyzed by agarose gel electrophoresis.The mitochondrial 12S rRNA et al. et al. The presence of CMV DNA was determined in a multiplex, quantitative, real-time PCR assay, using the CMV and CPOL probes published in Sanchez GJB2, two mutations for GJB2, one mutation for SLC26A4 gene, two mutations for SLC26A4 gene, CMV infection, enlarged vestibular aqueduct (EVA), conductive loss) as two-category predictors, and the interactions between etiologies and age. We used two-tailed tests for all analyses and set statistical significance at p<0.05. The statistical analysis was performed using Statistica  and R The results are summarized as means and percentages. To investigate the relationship between etiology and hearing thresholds at 3 years of age (Hypothesis 1), multiple regression analysis was carried out using the four-frequency average hearing loss  as dependent variable and etiology as a predictor variable. To determine the evolution of hearing loss, children were grouped according to the AH national protocols into those with fluctuating, progressive, or stable hearing loss. To consider the relation between progression of hearing loss and etiology (Hypothesis 2), a mixed-effects model was fitted using the longitudinal 4FA HL  hearing level) data as dependent variable, the fixed effects were age (continuous variable), the seven etiologies (one mutation for Cx26 and SLC26A4 mutation databases We use the wording “reported mutations” for DNA and protein changes classified as causative mutations in GJB2 or SLC26A4 gene were detected in 31 (9%) children. We did not detect mutations or ambiguous changes or evidence of congenital CMV infection in 198 (54%) children. These findings were supplemented by information retrieved from clinical records and parental reports. The cause of hearing loss remained unknown for 146 children (40%).GJB2 were present in 82 children. Of these, 48 (58.5%) had two known mutations and 35 (41.5%) children had one mutation. The inheritance is in all cases compatible with a recessive inheritance pattern. Details of mutations and the evolution of hearing loss for individual children are shown in GJB2. Of the children with two mutations of Cx26, one child passed hearing screening at birth, but was diagnosed with moderate hearing loss at 19.4 months of age. Another child was diagnosed with auditory neuropathy spectrum disorder (ANSD), and two children had EVA. Ambiguous changes in the GJB2 gene were detected in 24 children, 22 of whom have one mutation and the remaining 2 have two mutations.Reported mutations of SLC26A4SLC26A4 mutation, two of whom also carried one uncertain mutation and four others also carried GJB2 mutations. For 8 other children, one ambiguous change of the SLC26A4 gene was detected. Details of mutations and evolution of hearing thresholds are shown in Reported mutations of SLC26A4 gene are associated with Pendred syndrome  and non-syndromic enlarged vestibular aqueduct . Three children who carried one mutation of SLC26A4 also had EVA, and one child who carried two mutations of SLC26A4 also had EVA. EVA was also found for 4 children who had Cx26 mutations, 1 who had CMV infection, and 8 who had normal results for the four causes tested. The number of children who had EVA could not be ascertained because information was available only for those with imaging results recorded in their clinical files.Mutations in the A1555G mutation was detected in one child. The mutation appeared homoplasmic on agarose gel electrophoresis. The child has bilateral ANSD with profound hearing loss. It is not known whether the child had been exposed to aminoglycosides.The mtDNA SLC26A4 gene. Details of the evolution of hearing loss for children who are CMV positive are presented in Cytomegalovirus infection was detected in 28 children. Of these, one child passed UNHS but was diagnosed with meningitis and hearing loss at 5.1 months of age. A second child with congenital CMV infection also had one mutation of Cx26, and was referred for unilateral hearing loss after screening. One CMV positive child also carried two mutations of the In the regression analysis examining the relation between etiology and hearing threshold levels at 3 years of age, the overall p-level for the regression model was 0.27. GJB2, SLC26A4, A1555G and CMV infection, and established a likely etiologic diagnosis for 166 (45.6%) of the 364 cases tested. Of the causes identified, a genetic cause was present in 138 children (83.1%) and an environmental cause in 28 children (16.9%).The incidence of permanent childhood hearing loss is 1 to 1.2 per 1000 at birth increasing to about 1.5 by 3 years of age et al for infants at a hospital in the US showing that 6% (16 of 256 infants with hearing loss) were CMV positive through urine cultures It has previously been suggested that hearing loss attributable to congenital CMV may be progressive in >50% of cases GJB2 gene accounted for 76.1% of the genetic cases. This frequency of occurrence is higher than the 50% previously reported for congenital, autosomal recessive, nonsyndromic, sensorineural hearing loss in the white population GJB2 mutations was not significantly different from those without the mutations is consistent with previous reports Looking specifically at the genetic causes of hearing loss in our population (n = 138), mutations in the GJB2 gene was detected in 35 children. The pathogenic effect of these single mutations are uncertain, but we have attributed the hearing losses to thise GJB2 gene changes, because the proportion of children with hearing loss and one detectable recessive GJB2 mutation is significantly higher than that of the general population (approximately 1%) GJB2 gene of 24 children are also ambiguous. We have classified the V27I and E114G changes, when together, as a mutation in accordance with the Connexins and Deafness HomepageGJB2 protein is unable to form functional gap junction channels in silico analysis suggests that is unlikely to cause hearing loss. Although SIFT Sequence analysis predicts that it is “likely to affect protein function”, PolyPhen-2 and SIFT BLink predict it is benign Click here for additional data file.Table S2Genotype-phenotype correlation in children with SLC26A4 mutations and evolution of hearing loss. Children with SLC26A4 mutations with additional diagnostic information regarding enlarged vestibular aqueduct (EVA), auditory neuropathy spectrum disorder (ANSD) and congenital cytomegalovirus infection (CMV) are also indicated.(DOC)Click here for additional data file.Table S3Hearing thresholds at time of diagnosis and evolution in children with hearing loss attributable to congenital CMV infection.(DOC)Click here for additional data file.Table S4PolyPhen-2, SIFT BLink and SIFT Sequence predictions of novel or controversial amino acid changes on GJB2 and SLC26A4 gene product functions.(DOCX)Click here for additional data file.


We and others have shown that subtype C HIV-1 isolates from patients failing on a regimen containing stavudine (d4T) or zidovudine (AZT) exhibit thymidine-associated mutations (TAMs) and K65R which can impair the efficacy of Tenofovir (TDF) at second line. Depending on the various studies, the prevalence of K65R substitution as determined by the Sanger method ranges from 4 to 30%. Our aim was to determine whether ultra-deep pyrosequencing (UDPS) could provide more information than the Sanger method about selection of K65R in this population of patients.27 subtype C HIV-1 isolates from treated patients failing on a regimen with d4T or AZT plus lamivudine (3TC) plus nevirapine (NVP) or efavirenz (EFV) and who had been sequenced by Sanger were investigated by UDPS at codon 65 of the reverse transcriptase (RT). 18 isolates from naïve patients and dilutions of a control K65R plasmid were analysed by Sanger plus UDPS.Analysis of Sanger sequences of subtype C HIV-1 isolates from naïve patients exhibited expected polymorphic substitutions compared to subtype B but no drug resistance mutations (DRMs). Quantitation of K65R variants by UDPS ranged from <0.4% to 3.08%. Sanger sequences of viral isolates from patients at failure of d4T or AZT plus 3TC plus NVP or EFV showed numerous DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) including M184V, thymidine-associated mutations (TAMs) plus DRMs to non- nucleoside reverse transcriptase inhibitors (NNRTIs). Two K65R were observed by Sanger in this series of 27 samples with UDPS percentages of 27 and 87%. Other samples without K65R by Sanger exhibited quantities of K65R variants ranging from <0.4% to 0.80%, which were below the values observed in isolates from naïve patients.While Sanger sequencing of subtype C isolates from treated patients at failure of d4T or AZT plus 3TC plus NVP or EFV exhibited numerous mutations including TAMs and 8% K65R, UDPS quantitation of K65R variants in the same series did not provide any more information than Sanger. We and others From a molecular point of view, it has been demonstrated that the RT KKK nucleotide motif at codons 64, 65, 66 in reverse transcriptase of subtype C HIV-1 appears to lead to template pausing that facilitates the selection of K65R, even in isolates from untreated patients The aim of the present study was to clarify the prevalence of K65R in these subtype C isolates from patients failing on a first line including d4T or AZT. Since we had the prevalence of K65R by the Sanger sequencing method, we investigated K65R variants by ultradeep pyrosequencing (UDPS) in the same samples as those already used for bulk sequencing and sought whether UDPS provided additional knowledge to the Sanger results.Subtype C HIV-1 isolates from Indian patients failing  on a first-line treatment including d4T or AZT plus 3TC plus NVP or EFV were sequenced on RT by the Sanger method, and the sequences were recorded in the Los Alamos database (GenBank JF895621–JF895673). Among these samples, 27 were randomly selected for investigation by UDPS using the Roche GS Junior equipment. RNA extracted previously from the samples was used to amplify a short region of RT with primers including specific sequences for the GS Junior system . The revSince the viral isolates obtained at initiation of first-line therapy were not available, the data obtained by Sanger and UDPS in isolates from patients at failure were compared to those of subtype C isolates from 18 naïve patients. Some of their bulk sequences have been previously published by our group Regarding the 27 isolates from treated patients at failure, the drug resistance mutations (DRMs) according to the French ANRS algorithm and following bulk DNA sequencing (Sanger) are listed in Although UDPS has limitations particularly with regard to polymerization and pyrosequencing errors The Sanger results of isolates from treated patients were as expected with a predominance of M184V, numerous TAMs of pathway1  and DRMs to NNRTIs . As mentioned above, 2 isolates of the series exhibited a K65R substitution.With regard to UDPS results at codon 70, the quantitative data were different from those recorded in naïve patients: 8 isolates exhibiting K70R with Sanger had UDPS K70R values above 34.60%, while 2 samples without K70R with Sanger (470 and 489) had K70R variants at quantities above the <0.4% background observed in naïve patients. We hypothesize that these isolates are undergoing a process of selecting K70R mutations. Regarding the K65R values apart the two samples with K65R by Sanger, the UDPS quantities of K65R variants were low and below those of isolates from naïve patients. Our results are not in accordance with those obtained by another group K65R substitutions are generated in subtype C isolates from naïve patients due to the 64–65–66 motif. There are some constraints in experienced patients failing on a suboptimal regimen with d4T or AZT plus 3TC plus NVP or EFV. We first hypothesize that 184V, which was the most prevalent mutation observed in our series of treated patients at failure, has dampened the emergence of 65R as noted by others


By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the jebLamc2 mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in jebLamc2 mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the jebLamc2 mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic  Lamc2) gene to address the possibility of genetic modifiers of JEB. We document the potent impact of differing genetic backgrounds on multiple facets of the JEB syndrome expressed in these mice and show that three neighboring amino acid changes within the non-collagenous domain 4 of the collagen XVII protein strongly modify their disease. The study provides a molecular explanation of how a primary mutation that weakens one component of the cutaneous basement membrane is influenced by normally innocuous allelic variants of another component to affect strength of dermal-epidermal adhesion and consequently, the severity of JEB. This approach may guide the genetic prognosis and diagnosis of human EB disorders.Epidermolysis bullosa (EB) is a group of rare genetic Mendelian disorders that result in mechanical fragility of the skin and mucosal membranes. Junctional EB is a subset caused by mutations that result in cleavage of the dermal-epidermal junction. All forms of EB demonstrate substantial variability in their clinical phenotype that is not readily explained. The possibility of genetic modifiers as the cause of this variability has been difficult to address in humans. We apply a mouse model carrying a hypomorphic allele of the laminin gamma 2 ( Epidermolysis bullosa (EB) is a group of rare heritable disorders that result in mechanical fragility of the skin and mucosal membranes. These disorders are caused by defects in any one of at least 18 genes whose encoded proteins control the integrity of the dermal and epidermal layers LAMA3), laminin β3 (LAMB3), and laminin γ2 (LAMC2); in either integrin α6 (ITGA6) or β4 (ITGB4), which form the α6β4 integrin heterodimer; or in COL17A1, which forms the collagen XVII homotrimer COL17A1 and can be subcategorized based on generalized or localized manifestations as well as their clinical presentations ITGA6 or ITGB4 and is classified separately based on the additional phenotype of pyloric atresia JEB typically results from mutations in any one of the three genes encoding subunits of laminin 332, a heterotrimeric macromolecule comprised of laminin α3  as a major genetic modifier of JEB in jebLamc2 mice. Ultrafine recombination mapping limited the causal variations to three neighboring amino acid changes within the non-collagenous domain 4 (NC4) of this protein. This identification of a genetic modifier provides a molecular explanation of how epistasis between a primary mutation that weakens one component of the cutaneous basement membrane, laminin 332, and normally innocuous allelic variants of another component, collagen XVII, affects the strength of dermal-epidermal adhesion and consequently modifies the severity of JEB.While a unifying feature of all subforms of EB is adherence to simple Mendelian patterns of inheritance, considerable phenotypic variation in disease severity can persist even within subtypes of EB and within affected families. Genetic modifiers may account for this incomplete penetrance and the “vastly different” clinical phenotypes that are commonly observed jebLamc2 mutation develop a progressive form of JEB characterized most obviously by blistering and scarring of the ears and tails as the result of separations within the dermal-epidermal BM jebLamc2 allele arose on strain 129X1/SvJ and was backcrossed 10 generations onto four other strain backgrounds: C57BL/6J (B6), DBA/1J (DBA1), FVB/NJ (FVB), and MRL/MpJ (MRL). Cohorts of jeb/jebLamc2 males from each strain were aged, visually inspected weekly, and quantitatively scored for the severity of ear and tail lesions (jeb/jebLamc2 progeny from an F1 cross of the most susceptible (MRL) and most resistant (FVB) strain backgrounds. These results were consistent with co-dominant inheritance - jeb/jebLamc2 F2 and (B6 × FVB)-jeb/jebLamc2 F2, with >200 mice per group. Both sets of mice were monitored for the age at which ear lesions were first visually detectable and genome wide scans of their DNAs were performed using single nucleotide polymorphism (SNP) markers distributed through the autosomal and X chromosomes. For analysis, the F2 datasets were combined to increase the statistical power 2 3.9×10−11). Three QTLs were significant in multiple regression tests: chr19@42cM; chr11@32cM; and sex by QTL interaction (Sex*chr7@4cM). The sex*chr7@4cM QTL was observed only among the B6/FVB heterozygous mice. The chr11@32cM QTL dropped below the significance threshold if the sex*chr7@4cM QTL was excluded from the model, indicating it being a marginal QTL candidate. The most robust QTL (Prob>chi2 7.96×10−7), replicated in both mouse crosses independent of sex and explaining 5.6% of the genetic variance, was located on chr19 with a 95% confidence interval of 34–50 cM peaking at 42 cM.Crosses were made between phenotypically disparate strains to map the modifiers. Two groups of F2jebLamc2 homozygotes. Grouping the mice by the D19Dcr40 SSLP marker mapping to ∼47 Mb on chr19 revealed a strong but incomplete concordance between D19Dcr40 genotype and disease onset , we performed genetic association studies on cohorts of (MRL × FVB) FjebLamc2 allele was transferred onto existing chr19 consomic stocks B6.PWDchr19 and B6.A/Jchr19 and a B6.129S1chr19 congenic strain. Ear and tail scores and tail tension test comparisons indicated that the A/Jchr19 and 129S1chr19 alleles moderately attenuated disease and the PWDchr19 allele strongly attenuated disease as compared to the B6chr19 allele  F2 mapping cross in which a chr19 QTL exists but the pattern is inverse to the overall strain phenotype: 129X1 is later onset than DBA1, but the 129X1chr19 allele confers earlier onset  to evaluate haplotype relationships in the region surrounding Col17a1 (Col17a1 genes. cDNAs from 7 of the 8 strains (except 129S1) were also sequenced from exon 1 through most of exon 53. A comprehensive list of polymorphisms among the 8 strains is provided as To broadly evaluate the candidacy of sequence polymorphisms in  Col17a1 . These rCol17a1 predicted to result in nonsense changes or altered splicing patterns. With the exception of PWD, there were no missense changes outside of the candidate 1085 bp interval of Col17a1. Limiting the analysis to the 1085 bp candidate interval defined by the PWD/B6 recombinants identified 3 non-synonymous coding SNPs , 5 synonymous coding SNPs (4 in exon 49 and 1 in exon 50) and 1 intronic SNP that distinguished B6 from PWD (PWDchr19 recombinant strains R03F and R03L (None of the strains showed variation in from PWD . Aside ffrom PWD . They weand R03L . The onl1275 N1277 T1292) shared by B6 and 129X1; Haplo 2 (S1275 N1277 I1292) unique to MRL; Haplo 3 (S1275 S1277 I1292) shared by A/J, 129S1, DBA1 and FVB; and Haplo 4 (G1275 S1277 I1292) unique to PWD (Translation of the three candidate missense SNPs identify amino acid (AA) positions 1275, 1277, and 1292 within the non-collagenous 4 (NC4) subdomain of collagen XVII. Four related haplotypes are evident: Haplo 1  and the 1000 Genomes Project  human databases to assess the potential for coding variants in COL17A1 - in populations that were unbiased in that that did not including cases of documented EB - that may be candidates for imparting EB modifier effects. From the combined EVS+1000GP databases, representing ∼7600 individuals not known to be affected by EB, missense polymorphisms were identified for 13% (195 of 1497) of the codons of COL17A1. The minority allele frequencies of 135 missense polymorphisms among ∼6500 individuals (those reported in EVS) are shown in Having identified certain coding variants of mouse COL17A1 mutations reported in EVS, 15% were ‘possibly damaging’ and 47% ‘probably damaging’. However, such predictions can be misleading. The most common minority allele, leading to the change T210M, found in 42% of the population and homozygous in 20% of the individuals surveyed, is predicted by Polyphen to be ‘probably damaging’, but 20% of the population does not have EB. Substitutions for Glycine, Proline and Arginine were most frequent overall . No JEB-associated nonsense mutations were found in the EVS or 1000GP databases, perhaps due to their deleterious effects. However, three known JEB-causing missense transpositions  segregating in the general population at low frequency in a heterozygous state. EVS and 1000GP also record missense variants in 6 of 15 AA positions that overlap with JEB-causing nonsense mutations ; the consequences of this overlap are not known.jeb/jebLamc2 mice. A number of JEB causing mutations have been reported to locate to exons 51 and 52 jebLamc2 mutation and varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on a form of JEB-nH. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we unambiguously show that the gene Col17a1 is a strong genetic modifier of the non-Herlitz form of JEB that develops in jebLamc2 mice. This modifier is explained concisely by variations in 1 to 3 neighboring AA in the NC4 domain of the collagen XVII protein that are normally innocuous but act epistatically to alter the strength of dermal-epidermal adhesion in the context of the jebLamc2 mutation and broadly impact the clinical severity of JEB.We have addressed the prospect of genetic modifiers of a heritable, Mendelian mechanobullous disorder. Using mice homozygous for the jebLamc2 mutation was originally described in the context of the129X1/SvJ genetic background on which the mutation was first detected jebLamc2 homozygotes in the context of multiple genetic backgrounds, a remarkable range of variation in their age of onset and overall severity was apparent. In the most extreme cases, the MRL background presented with maximal, life threatening disease scores by 10 weeks of age, while FVB did not achieve maximum scores until >47 weeks of age. Other strains demonstrated intermediate phenotypes, all supporting a typical, complex genetic model in which several allelically variant loci contribute to overall disease.The pathophysiology of the spontaneously arising UitLamc2 allele. This dichotomy likely reflects a common feature of ‘monogenic’ disorders: mutations that result in profoundly life threatening consequences will overshadow potentially modulating factors, whereas mutations in disease causing genes that result in more attenuated forms, such as the JEB-nH syndrome of jebLamc2 mice, are more susceptible to the effects of background modifiers.However, a unifying feature was their modes of action. Genetic variables envisioned to impact JEB include: 1) those that integrally contribute to the structural elements of the basement membrane; and 2) those that act secondarily through inflammation or repairing skin lesions . The fact that measurements of the strength of dermal-epidermal adhesion was predictive of disease onset and severity well before the appearance of dermal lesions and that the excellent wound healing strain background, MRL Col17a1: 1) mapping in two F2 crosses (DBA1 × 129X1- jeb/jebLamc2 F2 and B6 × FVB-jeb/jebLamc2 F2) identified a strong QTL overlapping the Col17a1 locus; 2) phenotype to Col17a1 genotype correlations of F2 mice of the most polarized strain backgrounds (MRL and FVB) were highly significant; 3) phenotypic analysis of chr19 congenics and sub-congenics consistently framed Col17a1 as the modifier locus. Limitation of the disease modifying effects to neighboring coding changes within exon 50 of Col17a1 was strongly indicated by fortuitous recombinants from crosses of B6 and B6.PWDchr19 mice that limited the chr19 candidate interval to 1085 bp and included three non-synonymous coding SNPs.The results described herein identify relatively conservative AA changes limited to the NC4 domain of collagen XVII that are able to modulate a JEB syndrome caused by a hypomorphic mutation in the LAMC2 component of the laminin 332 complex. Multiple lines of evidence triangulated to Col17a1 using the Persikov – Singh algorithm This deep genetic resolution is unusual. PRDM9 is a long-array zinc-finger and chromatin-modifier protein that binds DNA sequences and promotes their meiotic recombination jeb/jebLamc2 mice. Moreover, it is likely that the modulatory effects of these variations are not limited to the cutaneous layer because the PWD chr19 conferred more resistant to non-cutaneous manifestations of JEB, including pulmonary functions and bone mineralization. Thus, seemingly subtle AA changes in the collagen XVII NC4 domain modify multiple pathophysiological manifestations of a JEB syndrome caused by a primary defect in a component of laminin 332.The candidacy of these missense SNPs as the cause of the JEB-modifying chr19 QTL among a number of mouse strains analyzed is strongly supported by sequence comparisons. The missense SNPs predict variation in neighboring AA positions 1275, 1277, and 1292 in the NC4 domain . CompariOur results support a model of functional epistasis in which a mutationally crippled form of laminin 332 permits normally innocuous AA changes to become functionally significant modifiers of JEB. The components of laminin 332 form an extracellular matrix heterotrimer that tethers the epidermal and dermal layers. The heterotrimer is secreted by keratinocytes assembled as N-terminal branching from a central coiled-coil structure with globular domains at its C-terminus. The standard model is that this heterotrimer bridges the lamina lucida and lamina densa by binding of its C-terminal globular domains to membrane bound α6ß4 integrin of basal keratinocytes and its N-terminus β3 and γ2 arms to collagen VII anchoring fibrils in the dermis COL17A1 gene can by themselves result in clinical manifestations of JEB-nH with varying severity. The carboxyl-terminal half of the collagen XVII extracellular domain has been shown to interact with laminin 332 COL17A1 and LAMB3 has also been reported where the proband was a compound heterozygote for L855X and R1226X COL17A1 nonsense mutations and heterozygous for the R635X recessive LAMB3 nonsense mutation Col17a1 modifiers may not be limited in their effect to diseases caused by mutant forms of laminin 332. In this context, it is of interest that QTL genetic analysis of mice that are experimentally induced to develop a form of EB Acquisita after immunization with a collagen VII polypeptide identified a strong QTL whose position overlaps with Col17a1Col17a1 modifiers of JEB may also be able to impact other forms of blistering diseases.A product of basal keratinocytes, collagen XVII forms a homotrimeric type II transmembrane molecule whose extracellular portion is comprised of 15 COL domains that presumably provide rigidity separated by 16 NC domains COL17A1 gene in the EVS and the 1000GP databases, unbiased by cases of documented EB, identifies numerous missense SNPs, including those that encode AA that map within and around the functionally-documented NC4 domain of collagen XVII. Such candidate modifier SNPs would be likely bypassed by conventional mutation detection-biased methods, but may act epistatically with the disease causing mutation as genetic modifiers. Thus, informed by mouse studies and focusing genetic modifier searches on allelic variants within functionally-important interaction domains of dermal-epidermal adhesion proteins may aid in the genetic prognosis and diagnosis of mechanobullous blistering disorders. Furthermore and as exemplified by this study, the elucidation of modifier loci may provide new clues into the functionally relevant interactions among these proteins that could direct new treatments for EB.The existence of remarkably potent genetic modifiers of JEB in mice raises the possibility that genetic modifiers may also contribute to the spectrum of clinical variation noted for many subforms of EB in humans. At the highest level, the considerable phenotypic and clinical variability of EB is explained by the fact that mutations in at least 18 genes can lead to these mechanoblistering disorders. The second level of variation is caused by the nature of each mutation and its penetrance. The third level, which is the focus of our study, is the extent to which missing heritability is caused by genetic modifiers. Potent modifiers of JEB are evident among strains of laboratory mice despite their simple genetic structure in which public allelic variation prevails because of related heritages and inbreeding. Humans, in contrast, have a much deeper haplotype structure with a spectrum of variation ranging from public to rare alleles and confounding genotype/phenotype associations. This is further complicated by the rarity of human cases for any given EB disease-causing mutation and lack of reliable and comparable phenotype information concerning those affected. Our survey of the All animal protocols were reviewed and approved by The Jackson Laboratory Institutional Animal Care and Use Committee .jeb/jebLamc2 and B6-jeb/jebLamc2 homozygous for the hypomorphic jebLamc2 allele were described previously jeb/jebLamc2, FVB-jeb/jebLamc2 and MRL-jeb/jebLamc2 were produced after backcrossing the 129X1-jebLamc2 allele for 10 generations onto DBA/1J, FVB/NJ and MRL/MpJ strain backgrounds and selecting to be homozygous for a congenic interval including the 129X1-defined jebLamc2 allele framed by markers D1Dcr2 and D1Dcr15 located at 150 and 160 Mb on chr1. jeb/jebLamc2 mice consomic for chromosome 19 were produced by crossing the jebLamc2 allele from B6-jeb/jebLamc2 mice to C57BL/6J-A/Jchr19/NaJ and C57BL/6J-PWD/Phchr19/ForeJ to produce strains B6.A/J Lamc2jeb/jebchr19 and B6.PWD Lamc2jeb/jebchr19. The chr19 congenic strain B6.129S1 Lamc2jeb/jebchr19 was similarly produced by a cross of B6-jeb/jebLamc2 to B6.129(Cg)-tm1DyhPnlip/J mice. The tm1DyhPnlip targeted mutation was originally made using (129X1/SvJ × 129S1/Sv)F1-+Kitl-derived R1 embryonic stem cells 129S1 Lamc2jeb/jebchr19 mice carry a large 129S1-derived chr19 congenic segment  on chr1 at ∼150 and 160 Mb, respectively. Primers were also developed to identify both Lamc2 wt and jeb mutant bands to confirm homozygosity. Lamc2Dcr1a F- CCTGTCTCATTTCTGGTAGGCTTT and R-CACACGTCACCACACCTGCT oligonucleotide primers mapping to intron 18, and Lamc2Dcr1b- F-GCGCCAGTCCTCCGATAGA mapping to the retroviral insert were used in a three primer reaction, giving bands of 121 bp for wild-type and 218 bp for jebLamc2. Primers used to identify tm1UitLamc2 wild-type and mutant bands were as previously described All crosses which required identification of P<0.05.Mice were typically scored weekly. Ears were scored from 0 (unaffected) to 6 (severe) based on limited or overall inflammation and ear erosion. Tails were scored from 0 (unaffected) to 6 (severe) based on the generalized loss of epithelial structure, scarring, and scabbing. Time to event survival analysis using Cox proportional hazards model was used to identify strain/genotype differences on ear and tail score across different cohorts of mice. Time to event was defined as age at which subjects first reached an ear/tail score of 4. When multiple levels of strains/genotype existed within a given cohort, risk ratios were calculated across all pairs of strains to determine significant differences among strains/genotypes at P<0.05.Determination of the mechanical force in Newtons necessary to cause the dermal-epidermal cleavage of tail skin was performed on euthanized mice as previously described 2jeb/jebLamc2 and 213 (B6 × FVB/NJ) F2jeb/jebLamc2 mice were analyzed in this study. Age of onset in weeks was identified for each animal with censored values for those ‘unaffected’ by 12 months of age. Genetic cohorts were genotyped for 147 or 136 SNP markers across the autosomal and X chromosomes spaced approximately ∼13 Mb through the Jackson Laboratory Fine Mapping Service by KBiosciences . Data from the two crosses were combined in Pseudomarker2.02 P values for terms in the multiple regression model were calculated, and terms were dropped sequentially until all of the terms in the model were significant at the 1% level for main QTL effects and 0.1% for interaction effects.For QTL analysis, a combined cross of 243 (DBA/1J × 129X1) FRadial tail sections were cut with a razor blade, frozen in OCT on dry ice, and then stored at −40°C. Samples were sectioned between 10 and 12 µm thick onto super frost plus slides . The sectioned tissues were fixed in ice-cold acetone for 10 minutes, washed three times in phosphate-buffered saline, and then allowed to dry. Primary antibodies were added to slides pre-blocked with 3% horse serum for 2 hours at room temperature in a humid chamber. The slides were then washed 3× in phosphate-buffered saline, incubated in the dark with fluorescence labeled secondary antibodies for 1 hour, washed 3× and cover-slipped using anti-fade gel mounting media . Rabbit anti-mouse collagen VII  diluted 1∶50 and rat anti-mouse integrin α6  diluted 1∶100 were used for immunostaining. Secondary antibodies used at a dilution of 1∶250 were goat anti-rabbit FITC and goat anti-rat Alexa Fluor 546 . Imaging was performed using a SP5 Leica confocal microscope at 63× .−1. Once the mouse was anesthetized, a small incision was made between the third and fifth tracheal rings and a tracheal cannula was inserted and secured in place with suture. Each mouse was ventilated at 200 breaths per minute with a tidal volume of 10 ml kg−1 body weight using a flexiVent ventilator . Once the mouse was breathing passively, two consecutive sigh breaths were performed to open the airways and lungs. Sigh breaths were performed throughout the experiment to insure that the airways remained open. Pressure-volume loops, elastance and compliance measurements were recorded and analyzed using SCIREQ flexiVent 5.1 software. Comparisons between cohorts were performed using the Student's two-tailed heteroscedastic t-test.Mice were anesthetized with ketamine/dormitor at doses based on body weight. To prevent the mice from breathing against the respirator, they were treated with pancuronium bromide in NaCl as a muscle relaxant at 0.2 mg kgPeripheral dual-energy X-ray absorptiometry performed using a Lunar PIXImus densitometer  was used to assess bone and non-bone tissue parameters. Bone parameters of mineral density (BMD) and mineral composition (BMC), and non-bone parameters of percent fat and total tissue mass (lean+fats) were determined after head exclusion and analyzed using Lunar PIXImus 2.1 software, as described Col17a1 specific oligonucleotide primers pairs for RT-qPCR , stored at room temperature for 24 hours, and then frozen at −40°C until ready to process. Total RNA was isolated using the standard Trizol reagent method (Invitrogen). Total RNA (500 µg) was converted to cDNA using the MessageSensor RT kit (Ambion/Applied Biosystems).  RT-qPCR  were desCol17a1 specific oligonucleotide primers using a Qiagen LongAmp PCR kit according to the manufacturer's protocols. Genomic DNA obtained from blood processed similarly was used to obtain intron-spanning sequences. PCR products were magnetic bead purified and sequenced on an Applied Biosystems 3730xl by the JAX DNA Sequencing Service. Sequence data were analyzed using Applied Biosystems Sequencing Analysis software version 5.2 and Sequencher software version 4.10. The Sanger Mouse Genomes Project (sanger.ac.uk) was used as the source of complete genomic sequence comparisons of strains including C57BL/6J, A/J, 129S1/SvImJ, DBA/2J and PWK/PhJ. The Mouse Phenome Database (phenome.jax.org) CGD-MDA1 dataset was utilized to compare strain polymorphism patterns in Col17a1 and its flanking regions. These data were combined with our sequence and SSLP typing data related to 129X1, DBA/1J and PWD to identify all Col17a1 polymorphisms among 8 strains  in mice 10 weeks of age for the same strains . *, p≤0.05; ***; ≤0.001; ****, ≤0.0001. Statistical significance of tail scoring data is not provided because most groups of mice were euthanized before achieving a score of 4 due to the severity of their ear lesions.Mouse strain background causes substantial variation in the onset and severity of JEB-nH in (TIF)Click here for additional data file.Figure S2PWDchr19 recombinants. * indicates the recombination breakpoints that frame the candidate interval.Gel validation of B6/B6.(TIF)Click here for additional data file.Table S1Col17a1 expression among mouse strains surveyed.RT-qPCR fails to identify transcriptional alterations in tail skin (DOCX)Click here for additional data file.Table S2Col17a1 among relevant mouse strains.Strain relationships of the genomic region including (DOCX)Click here for additional data file.Table S3Col17a1 among the mouse strains used in this study.Sequence-based polymorphisms of (XLSX)Click here for additional data file.Table S4Amino acid polymorphisms of human Collagen XVII based on the EVS and 1000 genome databases.(DOCX)Click here for additional data file.Table S5Col17a1.Localization of a PRDM9 binding site in the 1085 bp recombinant interval of (DOCX)Click here for additional data file.Table S6Chr1 and Chr19 markers used in this study.(XLSX)Click here for additional data file.


Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments. Over the last 40 years, 5 year survival rates for cancer patients have risen dramatically, but total death rates have remained stubbornly high in vitro tissue culture techniques. 2D tissue culture plastic and 3D extracellular matrices (ECM), as scaffolds for cell growth, provide the cell with very different biochemical and mechanical environments. Cells growing in 3D matrices have mechano-transducers and ECM adhesion proteins globally expressed on their surfaces; however, cells grown in 2D only interact with a solid substrate on their basal surface in vitro systems are poor indicators of in vivo processesAn important aspect of chemoresistance is the role of the tumor microenvironment. Tumor cells exist within a dynamic 3D environment which is not completely replicated by standard in vitro.Many investigations have shown that ECM interacting proteins can be used as predictors of tumor behavior and patient outcomes50 values on the order of 10–100 nM for various isoforms of mTOR and PI3K The phosphatide inositol 3 kinase (PI3K)/AKT pathway is an important intracellular signaling cascade which affects cell growth, migration, protein expression and survival To help understand and quantify the effects of the tumor ECM on cell behavior, we have compared proliferation, migration and protein expression of U2OS osteosarcoma cells between 2D and 3D collagen gels, and investigated the effect of changes in the ECM on inhibition of the PI3K pathway.2. PI103 Experiments were performed on the pediatric osteosarcoma cell line U2OS or breast adenocarcinoma cell line MCF7. Cells were cultured in complete RPMI media supplemented with 10% fetal calf serum and 1% penicillin-streptomycin solution . Cell cultures were maintained in 2D monolayers in a humidified incubator at 37°C, 5% C02) for 2 hours to allow for complete polymerization. The polymerization time was kept consistent as previous experiments indicated that polymerization time can significantly affect pore size (unpublished data). After polymerization, 100 µl of media was added on top of the gels.Collagen Type 1 gels were prepared as described previously For cell culture experiments, cells were stained with 5 µM CMRA (Molecular Probes). Cells in 2D experimental groups were plated on tissue culture plastic while cells in 3D conditions were added to the unpolymerized collagen solution to a final concentration of 100,000 cells/ml for the proliferation assay and 30,000 cells/ml for migration trials. The gels were then polymerized as described above, resulting in cell-embedded collagen gels. Media was replaced with fresh media daily. When applicable, experiments were exposed to 250 nM of PI103. For these experiments, PI103 was added to the collagen solutions and the quenching media. Additional drug was added with each subsequent media change to maintain the initial drug concentration.http://www.micro-manager.org). Confocal images were analyzed using IMARIS 7.3.2 . Cells were analyzed using the spot tracking and surface detection routines. Spot tracking gives the centroid position of each cell at each time point and was used to track cell number and cell movement over time. Surface detection outlined the 3D surface of each cell, and was used for cell size and shape measurements. Cell size was used as an excluding measurement to reject overly small cells from being included in cell number and cell movement measurements.Images were acquired using a DMI600B microscope  and ImagEM EM-CCD Camera  using a spinning disc confocal setup . Imaging was done using Micro-Manager 1.4 Software  at 37°C for one hour. Cells harvested from standard tissue culture plastic were also treated with collagenase to ensure this treatment did not affect results. Liberated cell suspensions were normalized by cell count and lysed in lysis buffer  for 15 minutes on ice and centrifuged for 10 min at 15,000×g. Whole cell lysates were then subjected to 10% SDS-PAGE followed by electrotransfer onto PVDF membranes. Membranes were blocked overnight at 4°C in 5% non-fat dry milk (BioRad) in Tris buffered saline with 0.1% Tween-20  before primary antibody incubation in 0.5% non-fat dry milk in TBST (Santa Cruz) for one hour at room temperature. After washing in TBST, membranes were incubated in secondary (horseradish-peroxidase conjugated) antibodies (AbCam) in TBST for one hour at room temperature. Membranes were visualized with Pierce ECL substrate (Thermo Scientific). All protein bands were normalized to GAPDH content and analyzed via densitometry using ImageJ.in vitro experiments. The cells are also very robust and react well to being embedded in collagen gels.In order to investigate the effects of the ECM on cell behavior the proliferation, migration, protein expression and response to PI3K inhibition of U2OS and MCF7 cells on tissue culture plastic and in Type 1 collagen gels of different collagen concentrations was investigated. U2OS cells were chosen due to their high native PI3K/Akt pathway activity 2, and were therefore seeded at 1000 cells/cm2.Because the 3D culture conditions introduce inherent differences in local cell density, the growth of cells seeded at different densities within collagen gels was analyzed. As can be seen in Next, the effect of the live cell dye on the proliferation of U2OS and MCF7 cells in 3D collagen gels was investigated. The CMRA fluorophore has been used previously without effect on cell behavior The proliferation of U2OS and MCF7 cells was examined in type I collagen gels of varying densities. U2OS cells proliferated significantly slower in 3D collagen gels than in 2D monolayer . Cells g−5)   This is This evaluation of the proliferation and migration of U2OS and MCF7 cells clearly shows that these cells behave very differently in 3D collagen gels than on flat 2D substrates. Because the PI3K pathway is critical to multiple cellular processes, the levels of key proteins in the PI3K/Akt pathway were compared between U2OS cells grown in 2D and 3D . U2OS ceCulturing U2OS and MCF7 cells in 3D collagen gels produces very different behavior than on standard tissue culture plastic. Further, in U2OS cells the move to a 3D culture platform yields very different levels of activity in the important PI3K/MTOR pathway. How does this difference in behavior and PI3K pathway activation affect the response of U2OS cells to pharmacological inhibition of the PI3K/mTOR pathway? As can be seen from We performed the same experiment on MCF7 cells to show that these results can be obtained from multiple cell lines . After 8Given the reduction in motility in U2OS cells grown in type I collagen gels in comparison with that in 2D monolayer the effect of PI3K pathway inhibition on cellular motility in these different conditions was also investigated. As can be seen in In an attempt to explain the observed difference in drug sensitivity between the two different collagen concentrations the effect of PI103 treatment on the levels of various phosphoproteins in the PI3K pathway was assessed. As can be seen from in vitro investigations of cell behavior. The gels are easy to create, inexpensive and are conducive to live cell manipulation and imaging. The gels are also customizable, with the ability to control pore size, ligand density and stiffness by either changing the concentration of collagen in vivo and can contribute to many kinds of pathologies.Our study provides quantitative understanding of the role of ECM in altering cellular behavior, in particular the response of PI3K pathway, in 2D and 3D environments. Our data indicates that the presence of a three-dimensional ECM significantly affects the proliferation and motility of U2OS and MCF7 cells and is consistent with emerging research into the role of ECM in cell behavior Our study demonstrates that the presence of a 3D ECM is sufficient to alter the activation of the PI3K/Akt pathway in U2OS cells. This reduced activation was consistent with an observed reduction in cellular proliferation and migration speed in cells growing in 3D matrix. Future investigations are needed to understand how a mechano-chemical signal from the ECM is transduced to intracellular signaling cascades.Previous work has shown that the presence of ECM proteins is sufficient to confer resistance to cancer cells against anti-tumor agents The fact that the ECM is sufficient to alter cell behavior, protein expression and drug response must be taken into account when designing and executing drug candidate validation studies and clinical trials. Currently, the vast majority of novel cancer therapeutics are screened for their efficacy against well-established cancer cell lines on high throughput, plate-based 2D systems


In approximately 50% of all malignancies this is achieved by inactivating mutations in the p53 gene. However, in several cancer entities, including melanoma, p53 mutations are rare. It has been recently proposed that tyrosinase related protein 2 (TRP2), a protein involved in melanin synthesis, may act as suppressor of the p53 pathway in melanoma. To scrutinize this notion we analyzed p53 and TRP2 expression by immunohistochemistry in 172 melanoma tissues and did not find any correlation. Furthermore, we applied three different TRP2 shRNAs to five melanoma cell lines and could not observe a target specific effect of the TRP2 knockdown on either p53 expression nor p53 reporter gene activity. Likewise, ectopic expression of TRP2 in a TRP2 negative melanoma cell line had no impact on p53 expression. In conclusion our data suggest that p53 repression critically controlled by TRP2 is not a general event in melanoma. ARF, which binds MDM2 and inhibits its E3 ubiquitin ligase activity Caenorhabditis elegans that secretion of the TRP2 homolog Tyr2 by sensory neurons inhibits p53 expression of germ cells. Moreover, TRP2 knockdown in WM266-4 melanoma cells lead to increased p53 expression sensitizing the cells to cisplatin-induced apoptosis.Advanced melanoma is a cancer that is largely resistant to cytotoxic drugs or irradiation; this had been at least in part attributed to an impaired p53 dependent apoptosis response In this report we further scrutinize a possible role of TRP2 in the regulation of p53 in five different melanoma cell lines and do not find such a relation.Tumor samples from primary and metastatic melanomas were obtained by surgical excision for either therapeutic or diagnostic purposes and had undergone routine histology. The Institutional Review Board of Würzburg University Hospital approved all described studies and waived the need for written consent for histochemical analysis of anonymised tumor samples. Generation of the WueMel 45 melanoma cell line was done after written consent from the patient .After anonymization of tissue samples a dermatohistopathologist (C.K.) reviewed slides from all blocks, selecting representative areas of tumor tissue to be cored for generation of TMA as previously described 4 µm sections of paraffin-embedded tumors and TMA were dried at 56°C and then treated twice with xylene for 10 min at room temperature. Subsequently, sections were washed twice with absolute ethanol and twice with 70% ethanol followed by one rinse with bi-distilled water. For antigen retrieval, sections were incubated with citrate buffer pH 9.0  for 10 min at 90°C and rinsed with bi-distilled water. Next, slides were rinsed twice with phosphate-buffered saline (PBS) and thereafter incubated with Blocking Solution  for 10 min at room temperature. After two additional washing steps with PBS for 10 min at room temperature, the monoclonal α-p53 antibody  or α-TRP2 antibody  was added to the sections at a predetermined concentration in PBS, followed by an over night incubation at 4°C. After two 10 min washes in PBS, biotinylated multispecies-specific secondary antibody  was added to the sections for 30 min at room temperature. Slides were then washed twice in PBS/bovine serum albumin, and bound antibodies were visualized using streptavidin-HRP (DAKO K5003) and Vector Vip  as peroxidase substrate according to the manufacturer's guidelines. Finally, the nuclei were stained with hemalaun.The cores of specimens on the tissue microarray (TMA) slides were scored using a semi-quantitative scoring system where staining intensity and the proportion of stained tumor cells is taken into account 2) and the p value were determined applying linear regression analysis comparing p53 and TRP2 histology scores.The relationship between the p53 and TRP2 expression was statistically analyzed using the Prism Graph software 5.0 . The coefficient of determination (ROne melanoma cell line (WueMel45) was generated in our lab from a melanoma metastasis obtained from a patient for therapeutic purposes. This cell line as well as the melanoma cell lines FM88 TCAAGAGTCTGTTAGTTGTTTGAATCACATTTTTTT-3′5′-GTGATTCAAACAACTAACAGA, TCAAGAGACAGCATGAAATTGCCAACCTTTTTTTTT-3′5′-AAGGTTGGCAATTTCATGCTGT and TCAAGAGTCTGCCGAATCACTGGTGGTTTTTTT-3′5′-CCACCAGTGATTCGGCAGA, respectively (the sense strand is given), into the lentiviral vector KH1 CATGAAGCTGCCCACG-3′5′- ACAG; exchanged nucleotides displayed in bold) were introduced in the shRNA target sequence using the quick change mutagenesis kit .The shRNA vectors TRP2_shRNA #1, #2 and #3 were obtained by cloning the shRNA sequences Infectious viruses were raised by transfecting HEK293T cells CGACATGCCCGGGCATGT). Following puromycin selection flow cytometry to monitor GFP expression in pGreenFire transduced cells was performed on a FACSCanto . Mean GFP fluorescence intensities were normalized to the relative presence of the reporter constructs in the infected cells as determined by real time PCR The p53 reporter construct pGreenFire lentiviral vector codes for a puromycin resistence and for green fluorescent protein (GFP) under the control of a p53 responsive element . Antibodies to p53  TRP2  and β-tubulin  were used.Prompted by a report demonstrating an inhibitory effect of TRP2 on p53 in a melanoma cell line 2 = 0,0258) and only borderline-significant  positive correlation was observed. Moreover, of all 52 samples with very weak or no TRP2 staining (histology score of 0 or 1) 17 (33%) also displayed very weak or negative p53 staining (histology score of 0 or 1). On the other hand of all 120 samples with middle to strong TRP2 staining (histology score of 2–12) 44 (37%) displayed very weak or negative p53 staining (histology score of 0 or 1). Taken together these analyses reveal, that p53 expression is independent of the TRP2 expression level. Moreover, both proteins are detectable in the majority of samples and therefore expression is not mutually exclusive.Overall p53 expression was detectable (score ≥1) in 99 out of 139  cases of primary melanoma and in 17 out of 33  cases of metastatic melanoma . p53 immTo further scrutinize a possible role of TRP2 in the regulation of p53 we performed a series of TRP2 knockdown experiments addressing the expression and transcriptional activity of p53. Initially, we analyzed the expression of p53 and TRP2 in the melanoma cell lines FM88, M26, MelU, MelJuso, and WueMel45. p53 was present in all five melanoma lines, whereas TRP2 expression was detected by western blot in all cell lines except WueMel45. Three short hairpin RNAs were engineered to suppress TRP2 expression. The efficacy of TRP2 knockdown was confirmed by immunoblot; all three shRNAs strongly reduced TRP2 protein expression in melanoma cell lines and the knockdown was most prominent for TRP2-shRNA_#2 . To thisTo test whether the transcriptional activity of p53 may be altered by TRP2 knockdown, the set of melanoma lines were stably transduced with a reporter gene construct (pGreenFire) coding for GFP under the control of a p53 response element. The mean GFP expression was measured by flow cytometry and normalized for the relative number of incorporated pGreenFire copies as determined by real time PCR. As we have shown previously expression levels of p53 do not necessarily correlate with its transcriptional activity suggesting posttranslational inactivation of p53 Interestingly, TRP2 knockdown by two of the three shRNAs (#1 and #3) showed no impact on p53 reporter gene activity. However, infection with the TRP2-shRNA_#2 led to increased p53 reporter gene activity in FM88, Mel-U and MelJuso but not in M26 and WueMel 45 melanoma cells . TherefoInactivation or at least partial repression of the p53 tumor suppressor pathway is thought to occur in almost all human cancers, and inactivating p53 mutations are the genetic alterations most frequently observed in malignancies Caenorhabditis elegans the TRP2 homolog TYR2 as a protein secreted by neurons and acting paracrine in neighboring germ cells to suppress CEP-1 (p53 homolog in the worm) dependent apoptosis In our study we investigated the role of the TRP2 in p53 regulation prompted by a report of Sendoel and colleagues who identified in


Permanent pacemaker implantation is available in Nigeria. There is however no national registry or framework for pacemaker data collection. A pacemaker database has been developed in our institution and the results are analyzed in this study.The study period was between January 2008 and December 2012. Patient data was extracted from a prospectively maintained database which was designed to include the fields of the European pacemaker patient identification code.Of the 51 pacemaker implants done, there were 29 males (56.9%) and 22 females (43.1%). Mean age was 68.2±12.7 years. Clinical indications were syncopal attacks in 25 patients (49%), dizzy spells in 15 patients (29.4%), bradycardia with no symptoms in 10 patients (17.7%) and dyspnoea in 2 patients (3.9%). The ECG diagnosis was complete heart block in 27 patients (53%), second degree heart block in 19 patients (37.2%) and sick sinus syndrome with bradycardia in 5 patients (9.8%). Pacemaker modes used were ventricular pacing in 29 patients (56.9%) and dual chamber pacing in 22 patients (43.1%). Files have been closed in 20 patients (39.2%) and 31 patients (60.8%) are still being followed up with median follow up of 26 months, median of 5 visits and 282 pacemaker checks done. Complications seen during follow up were 3 lead displacements (5.9%), 3 pacemaker infections (5.9%), 2 pacemaker pocket erosions (3.9%), and 1 pacemaker related death (2%). There were 5 non-pacemaker related deaths (9.8%).Pacemaker data has been maintained for 5 years. We urge other implanting institutions in Nigeria to maintain similar databases and work towards establishment of a national pacemaker registry. Bradyarrhythmias are a cause of sudden death in Nigeria, though the precise incidence is unknown. Pacemaker implantation is an accepted intervention which has been shown to improve the quality of life and reduce mortality in patients with bradyarrhythmias. Published experience in Nigeria has shown that implantation rates are low, the main indication for implantation is complete heart block (CHB) and most patients receive ventricular implants . PacemakA pacemaker implantation and follow up service was established in our institution in 2008. The aim of this study was to review our experience by analysis of our pacemaker database.Following patient referral the clinical indication for pacemaker therapy is established from the history and the diagnosis confirmed with a 12 lead ECG (and 24 hour holter if necessary). Cardiac function is assessed with a transthoracic echocardiogram. Pacemaker implantation is performed in a dedicated theatre suite equipped with a fluoroscopic C arm. The implantation team is composed of a surgeon who performs the implantation, a cardiac physiologist who performs the checks of pacemaker parameters, a pacemaker technician to operate the fluoroscope for imaging and a scrub nurse. Monitored parameters are the heart rhythm, heart rate, non-invasive blood pressure and peripheral oxygen saturation. A standard subclavian approach is used after infiltration with local anaesthesia in all cases. Prior to lead fixation the R wave  and pacing threshold are checked. Target values are R wave greater than 6 millivolts (mV), P wave greater than 2 mV, lead impedance less than 1200 Ohms and pacing threshold less than 1 volt (V). Diaphragmatic pacing is checked at 10V. The pacemaker pocket is irrigated with 1g of ceftriaxone, the pacemaker lead connected to the pulse generator and the wound closed in layers. An arm sling is used in all cases to restrict movement of the arm on the operation side (to reduce the risk of lead displacement) and the patient is transferred to the ward.Patients are monitored on the ward for 48 hours to exclude lead displacement. After 48 hours a pacemaker check is done and the patient is given a copy of both the pacemaker implantation report  and paceA Microsoft Access database was designed and has been maintained prospectively since the inception of the programme in January 2008. Data storage covers the fields recommended by the European pacemaker patient identification codes . Sample Of the 51 patients implanted there were 29 males (56.9%) and 22 females (43.1%). Ages ranged from 22-92 years with a mean age of 68.2±12.7 years. Age distribution is as shown in The distribution of clinical indications for pacemaker therapy was syncopal attacks in 25 patients (49%), dizzy spells in 15 patients (29.4%), documented bradycardia with no symptoms in 10 patients (17.7%) and dyspnoea/heart failure in 2 patients (3.9%). The ECG diagnosis was Complete Heart Block (CHB) in 27 patients (53%), second degree heart block (SDHB) in 19 patients (27.2%) and Sick Sinus Syndrome with bradycardia (SSS) in 5 patients (9.8%). There was no patient with atrial fibrillation.The distribution by pacemaker manufacturer was Medtronic (Minneapolis Minnesota USA) in 33 patients (66.7%), Pacetronix (Kolkata India) in 10 patients (19.6%) and St Jude (St Paul Minnesota USA) in 7 patients (13.7%). Pacing modes used were VVI (Ventricular Demand Pacing) in 9 patients (17.6%), VVIR (Rate Responsive Ventricular Demand Pacing) in 20 patients (39.2%), DDD  in 2 patients (3.9%) and DDDR  in 20 patients (39.2%). Overall single chamber ventricular pacing (VVI(R)) was used in 29 patients (56.9%) and dual chamber pacing (DDD(R)) in 22 patients (43.1%) with a progressive annual reduction in the use of ventricular pacing and an increased use of dual chamber pacing over the study period . DistribAt implantation the average R wave obtained was 11.8±4.6 mV while the average P wave obtained was 2.9 + 1.8 mV. The average atrial pacing threshold was 0.65±0.54 V and the average ventricular pacing threshold was 0.59±0.39 V. Average impedance for the atrial leads was 650.2±191.7 Ohms and was 780.5±230.2 Ohms for the ventricular leads. Complications seen postoperatively and during follow up were 3 lead displacements (5.9%), 3 pacemaker infections (5.9%), 2 pacemaker pocket erosions (3.9%), and 1 pacemaker related death (2%). Details of complications seen and time interval from implantation when they occurred are shown in At follow up in the pacemaker clinic, 282 pacemaker checks have been done on the 51 patients implanted in our institution. Of these 51 patients, 31 patients (60.8%) still remain under follow up in the pacemaker clinic. For these patients, the follow up period has ranged from 1-60 months with a median follow up of 26 months. The number of clinic visits ranged from 114 with a median of 5 visits.File closure has been done in 20 patients (39.2%). The reasons for file closure were pacemaker removal in 5 patients (25%), non-pacemaker related death in 5 patients (25%), transfer to another hospital in 5 patients (25%), lost to follow up in 2 patients (10%), inability to attend from transportation problems in 2 patients (10%) and pacemaker related death in 1 patient (5%). Of the 5 non-pacemaker related deaths, 4 patients were reported to have suffered myocardial infarctions while 1 patient died of complications of prostate cancer.There are currently 10 centres in Nigeria known to implant pacemakers. There are 3 centres in Lagos and 1 each in Enugu, Ibadan, Abuja, Port Harcourt, Calabar, Ife and Ilorin . To date a centre in Lagos  have pubth World survey of cardiac pacing and implantable cardioverter-defibrillators [th world survey showed that there are slightly more males receiving implants than females  which is similar to our experience. The use of dual chamber pacing in 43.1% of patients in our series is considerably higher than reported in other West African series and reflects the worldwide trend of increased use of dual chamber pacing seen in the 11thth World survey. Similar to the findings of other West African series, most patients in our environment are diagnosed with CHB and about 50% present having had syncopal attacks. This is unlike the pattern in the Western World where 30% or less of patient present with CHB and Sinus Node Dysfunction (SND) is the predominant indication for cardiac pacing [The mean age in our series was 68 years with 56.9% being male and 43.1% female. This is within the mean age range of 65 to 75 years reported in the 11Maintaining a stock of pacemakers locally makes it easier to proceed directly to permanent pacemaker implantation. In this series of 51 patients, only one temporary pacemaker was implanted. This is unlike the earlier experience from Lagos  and thisIn our practice there has been a progressive decrease in the use of single chamber ventricular pacing and an increase in dual chamber pacing over the last 5 years of our experience. In the 1990s initial recommendations urged more use of dual chamber pacing as it was thought that the hemodynamic benefits of AV synchrony would translate into improved longevity, improved quality of life and reduction in strokes . The firIt has been suggested that dual chamber pacing though more expensive may be offset by reduced replacement for pacemaker syndrome and improved quality of life . None ofWe noted that all the pacemaker infections occurred in dual chamber implants. It has been shown that there can be a higher complication rate with dual chamber implants . We willWe have considered the re-use of pacemakers as there is some evidence that it is safe and could reduce costs for patients . AverageOnly 1 pacemaker related death occurred in this series. This occurred in an elderly patient who received a Pacetronix implant (VVI) with a tined, non-steroid eluting endocardial lead. Over a period of 2 years there was a gradual rise in pacing threshold from implantation level of 0.8V to 2.5V. Pacing amplitude had been increased to 5V and the patient advised on a change of implant. She however declined and died suddenly, presumably from sudden failure to capture. This singular experience informed our current practice where we no longer use Pacetronix implants and all pacemaker leads used are steroid eluting and active fixation.Of the 5 non-pacemaker related deaths, 4 were reported as being secondary to myocardial infarctions. Diagnosis of myocardial infarction was made by cardiac enzymes and electrocardiogram changes in 1 patient, and purely from ECG changes in the other 3 patients. The incidence of Ischaemic heart disease (IHD) is known to be on the increase in Nigeria  so the cThere is great variability in implantation rates between different countries as shown in the 11th World survey of cardiac pacing and implantable cardioverter-defibrillators . The larIn the midst of this great variability in the number of pacing centres and implants done per country, it is of great concern that Nigeria was not included in this survey. This stresses the urgency in establishment of a framework for a national registry so that the efforts of various implanting institutions in Nigeria can be captured.A pacemaker implantation and follow up service has been established in our institution and a robust database has been developed and maintained. Early results show that the main indications for implantation are complete heart block and second degree heart block. Use of dual chamber pacing is higher than has been reported from other West African Centres. Continued patient follow up may be able to address questions of pacemaker therapy unique to our environment. Complications rates have been low. Complete follow up information is available for 49 patients (96%). We urge other implanting institutions in Nigeria to maintain similar databases and work towards establishment of a national pacemaker registry.


Healthcare workers in many countries are recommended to receive influenza vaccine to protect themselves as well as patients. A monovalent H1N1 vaccine became available in Hong Kong in December 2009 and around 10% of local healthcare workers had received the vaccine by February 2010.We conducted a cross-sectional study of the prevalence of antibody to pandemic (H1N1) 2009 among HCWs in Hong Kong in February–March 2010 following the first pandemic wave and the pH1N1 vaccination campaign. In this study we focus on the subset of healthcare workers who reported receipt of non-adjuvanted monovalent 2009 H1N1 vaccine . Sera collected from HCWs were tested for antibody against the pH1N1 virus by hemagglutination inhibition (HI) and viral neutralization (VN) assays.We enrolled 703 HCWs. Among 104 HCWs who reported receipt of pH1N1 vaccine, 54% : 44%–63%) had antibody titer ≥1∶40 by HI and 42% (95% CI: 33%–52%) had antibody titer ≥1∶40 by VN. The proportion of HCWs with antibody titer ≥1∶40 by HI and VN significantly decreased with age, and the proportion with antibody titer ≥1∶40 by VN was marginally significantly lower among HCWs who reported prior receipt of 2007–08 seasonal influenza vaccine . After adjustment for age, the effect of prior seasonal vaccine receipt was not statistically significant.Our findings suggest that monovalent H1N1 vaccine may have had suboptimal immunogenicity in HCWs in Hong Kong. Larger studies are required to confirm whether influenza vaccine maintains high efficacy and effectiveness in HCWs. In a separate study we investigated antibody seroprevalence in HCWs who reported that they had not received pH1N1 vaccine We recruited HCWs between February 11 and March 31, 2010 in 6 public hospitals comprising the Hong Kong West cluster of the local Hospital Authority, with a total workforce of around 7,000 HCWs in one acute care teaching hospital and five non-acute hospitals Serum samples were stored in a refrigerated container at 2–8°C immediately after collection and delivered to the laboratory at the end of each working day for storage at −70°C prior to testing. Serum specimens were tested for antibody responses to A/California/04/2009 (H1N1) by hemagglutination inhibition (HI) and viral microneutralization (VN) assays using standard methods as previously described The conventional neutralization test for the A/California/04/2009 was carried out in micro-titre plates using neutralization of virus cytopathogenic effect (CPE) in Madin-Darby Canine Kidney (MDCK) cells. Serial serum dilutions in quadruplicate were mixed with 100 tissue culture infectious dose 50 (TCID50) for 2 hours and added to MDCK cells. One hour after infection, serum-virus mixtures were removed and serum free MEM with 2 ug/ml trypsin was added to each well. The plates were incubated and cytopathic effect was observed to determine the highest serum dilution that neutralized ≥50% of the wells. A virus back titration and positive and negative control sera were included in each assay.We compared the differences in the proportion of HCWs with pH1N1 antibody titer ≥1∶40 both by HI and VN between groups with chi-squared tests or Fisher's exact test and used the phi coefficient to compare results between the two assays. We compared antibody seroprevalence over time in our study with the date of administration of pH1N1 vaccines to HCWs in Hong Kong We recruited 703 HCWs during the study period, and seroprevalence data for the 599 HCWs who reported that they had not received pH1N1 vaccine have been reported elsewhere Around 10% of HCWs in Hong Kong had received pH1N1 vaccine by January 2010 . We founAmong the 101 HCWs without confirmed pH1N1 infection, the proportion of HCWs with antibody titer ≥1∶40 by VN significantly decreased with older age , and was significantly lower among HCWs who reported receipt of 2007–08 seasonal influenza vaccine . In the Influenza vaccination is recommended as the primary prevention measure against infection, and HCWs are often one of the groups targeted to receive vaccine not only for their direct protection but also to indirectly protect vulnerable patients against nosocomial infection While only 42% of HCWs had antibody titers ≥1∶40 by VN, which is consistent with the findings of another study of 409 HCWs in Japan who were provided with one dose of pH1N1 vaccine, and just 38% achieved antibody titers ≥1∶40 by HI within 21 days of vaccination Another possible reason for lower antibody response is imperfect effectiveness of vaccine in field condition for the general population. Various controlled trials have shown good immunogenicity of the pH1N1 vaccine Although we did not record time of receipt of pH1N1 vaccine in participants in our study, the majority of HCWs in Hong Kong had received pH1N1 vaccine before mid-January 2010, i.e. at least one month before the start of our study . AntibodIt is important to note several limitations of our study in addition to the relatively small sample size. First, we conducted a cross-sectional seroprevalence study following the first pH1N1 wave, and we did not have baseline (pre-pandemic) data to enable us to compare the pre-pandemic antibody titer with the post-pandemic antibody titer among HCWs Vaccination is the most effective way to protect HCWs and the patients around them from influenza, but uptake of influenza vaccines tends to be low in many countries including Hong Kong. Our study indicated lower antibody response than expected among HCWs after receipt of pH1N1 vaccine than antibody results of other two clinical studies using the same pH1N1 vaccine


Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.In the yeast  S. cerevisiae, three chromosomal regions are epigenetically silenced with respect to transcription: the telomeres, the silent mating loci (HMR and HML), and the ribosomal DNA (rDNA) repeats  complex SAS-I and the chromatin-condensing activities of NAD+-dependent histone deacetylase (KDAC) Sir2 (silent information regulator-2) In a eukaryotic cell, selective transcriptional repression or silencing dictates the accessibility of specific chromatin domains by the transcriptional machinery; this results in varying degrees of transcriptional competency across the eukaryotic genome. Euchromatin refers to the chromatin regions at which transcription is generally active, whereas heterochromatin refers to those that are largely devoid of transcriptional activity   has been identified as the only type I PRMT in HMT1 in conjunction with the synthetic genetic array (SGA) methodology to systematically and comprehensively screen for all non-essential yeast genes that interact with HMT1, and generated an HMT1 genetic interaction network based on our findings. Gene ontology (GO) analysis of our SGA data showed that HMT1 interacts with genes encoding various components of the Rpd3L (large) complex. In the Hmt1 loss-of-function mutants, recruitment of Rpd3 to the telomeric boundary region is increased. In mutants carrying both Rpd3 and Hmt1 deletions, increased silencing at the telomere and increased Sir2 recruitment at telomeric boundary region is observed compared to Δhmt1 mutants. The Hmt1 loss-of-function mutants display a decrease in the levels of H4K5 acetylation (a known Rpd3 substrate) and an increase in the levels of acetylated H4K16 (a known Sir2 substrate) at the telomeric boundary regions. Finally, mutants lacking either Sir2 or Rpd3 display a decrease in the levels of dimethylated H4R3 at the telomeric boundary regions, albeit to a different degree. Overall, our results indicate that Hmt1 has the potential to influence the recruitment and actions of KDACs in order to promote the maintenance of silent chromatin.In the current study, we used a null allele of HMT1 and other genes, we conducted a synthetic genetic array (SGA) analysis using the Δhmt1 mutant as the query strain. This approach allowed us to construct and analyze double mutants in which the Δhmt1 mutation was combined with deletions in most of the non-essential genes of S. cerevisiae. Such information was expected to enable us to uncover any role Hmt1 may play within a broad biological network. The sensitivity of our screen was maximized by constructing the Δhmt1 query strain from a parental strain (15578-1.2b) developed by the Hartman lab To obtain further insight into the interactions between hmt1  and compiled the known physical interactions into a list. These physical interactions were then superimposed on the HMT1 genetic interaction dataset, and the overlap was graphically displayed  as one of the most enriched among the 123 genetic interactions identified in our SGA screen (Saccharomyces Genome Database (SGD) as ones encoding components of the Rpd3L complex, six displayed negative synthetic genetic interactions  with Δhmt1, two displayed positive synthetic genetic interaction (Δrpd3 and Δume6) with Δhmt1, and the other seven genes fell outside our significance criteria . For Δsds3, and Δume6, our follow-up screens indicated that they were false negatives (hmt1 to HMT1 spots for Δsds3 and Δume6) as they had the same interaction type in the original SGA screen, but their p-values from the original SGA screen did not pass the cutoff criteria  complex, and they did not display any genetic interactions with Δhmt1 (HMT1 and Δhmt1 rows for Δeaf3 and Δrco1). Although Δsin3 resulted in a synthetic genetic interaction with Δhmt1 in the initial screen, this interaction could not be confirmed by our subsequent SGA analysis (hmt1 suggests that Hmt1 has an influence on the activity of the Rpd3L complex.Using software that identifies the enrichment of specific gene function groups based on annotated gene ontology (GO) terms A screen . Both poA screen  and by sA screen . Of the criteria . In our criteria . Two neganalysis . NeverthHMT1. The catalytic subunit of the Rpd3L complex, Rpd3, has a well-known role in the maintenance of silent chromatin at the S. cerevisiae telomere; mutants lacking RPD3 display enhanced silencing in this region Our SGA screen revealed interactions between genes encoding components of the Rpd3L complex and in vivoPreviously, a complex consisting of Rpd3 and Sin3 was shown to be specifically recruited to create a repressive chromatin domain hmt1 mutant backgrounds. Directed chromatin immunoprecipitation (ChIP) experiments were then performed to determine the effects Hmt1 has on the occupancy levels of Rpd3 at distances of 0.35, 0.6, 1.4, 2.8, and 5 Kb from the telomeric repeats  and loss of silencing in the Hmt1 loss-of-function strains (hmt1 and hmt1(G68R)) as previously reported rpd3 strain (hmt1/Δrpd3 and Δrpd3/hmt1(G68R). Thus, our results indicate that the Δrpd3 phenotype is dominant to the silencing defect caused by mutations in Hmt1.Since Hmt1 and Rpd3 affect the dynamics of silent chromatin formation in opposing manners hmt1, Δrpd3, and Δhmt1/Δrpd3 double mutant (hmt1/Δrpd3 double mutant when compared to the Δhmt1 mutant (hmt1 (black bars) to Δhmt1/Δrpd3 (striped bars)). As previously seen, Sir2 recruitment increased minimally within 1 Kb of the telomeric end hmt1 (black bars) to Δhmt1/Δrpd3 (striped bars), and Δhmt1 to Δrpd3 (gray bars), regions A and B) and this trend continues in both the Δhmt1/Δrpd3 double mutant even at a distance greater than 1 Kb from the telomeric end (hmt1 (black bars) to Δhmt1/Δrpd3 (striped bars) and Δhmt1 to Δrpd3 (gray bars), regions C, D, and E). Thus, our ChIP data support the results from the silencing assay in which we observed more silencing in the Δhmt1/Δrpd3 double mutant strains when compared to the single Hmt1 mutant strain.To examine this relationship further at the molecular level, we used directed ChIP to compare the level of Sir2 occupancy in Δe mutant . We obserpd3-null mutants display enhanced acetylation of H4K5 hmt1 mutants , not that of Δhmt1 (less silenced than the wild-type). This is also supported by the change in Sir2 occupancy level in these Δrpd3/Δhmt1 double mutants, in which Sir2 recruitment at the telomeric region displays a trend more similar to that of Δrpd3 than Δhmt1. We note that these results do not distinguish whether such an increase in Rpd3 recruitment is simply a consequence of decreased Sir2 occupancy in the Hmt1 loss-of-function mutants, or the decrease in Sir2 occupancy seen in these mutants are due to an increase in Rpd3 recruitment. While we have not tested the role of Hmt1 in controlling the boundaries of the silenced domains at HMR and HML silent mating type loci, our previous work has demonstrated that Sir2 recruitment in Hmt1 loss-of-function mutants is decreased at the E silencer and immediately upstream of the α2 gene in HML and I silencer of HMRHML locus HM silent mating loci, more experiments must be done in order to elucidate the precise molecular mechanisms underlying this phenomenon at the silent mating loci.Interestingly, it has previously been shown that spreading of the SIR complex at telomeric boundary regions is antagonized by the presence of Rpd3 hmt1 loss-of-function mutants. This line of evidence supports our observation of increased Rpd3 recruitment at these regions in these mutants, as Rpd3 deacetylates acetyl-H4K5 Based on our Rpd3 ChIP data, it is likely that an increase in Rpd3 recruitment to these regions would impact the acetylation state of histones, since Rpd3 substrates include a wide range of acetylation sites within histones H4 and H3 in vitro study of the four acetylatable lysine residues in the N-terminal tail of histone H4 reveals that each acetylated histone H4 has a different ability to act as a substrate for Hmt1/PRMT1 Previous studies in mammalian systems have established that KATs and PRMTs have synergistic activities In conclusion, our data indicate that loss of either Hmt1 or its catalytic activity influences Rpd3 occupancy at the telomeric boundary region, and, as a consequence, the levels of H4K5 acetylation in this region. Given the proposed mechanism for how Rpd3 antagonizes Sir2 action at these regions All yeast strains used in this study are listed in hmt1 query strain. 2) images of each plate were taken with a CCD camera (Bio-Rad) to assess the degree of cell growth for each double mutant. These images were then processed using customized MATLAB code adopted from studies done by Collins et al. Three independent screens were carried out to identify all of the genes that interact Δhmt1. These interactions were then scored based on the percentage difference in the colony size when compared to a control query strain containing only deletion library mutation. Based on the results from all three screens, a p-value was assigned to each genetic interaction tested. The following criteria were used to score and filter the raw data for inclusion in the final dataset: 1) the double mutant had a corresponding control mating present on the final selection medium; and 2) any interactions with dubious or putative ORFs were removed; and 3) the double mutant exhibited a growth difference of 50% or more relative to the control, and the p-value was < or  = 0.001. The genetic interaction network was created using Cytoscape The SGA methodology was performed essentially as described previously ChIP procedures were performed as described previously HMT1 or RPD3 were constructed in a published yeast strain with ADE2 integrated at the right telomere of chromosome 5 Null mutations of Figure S1HMT1 interactors from the SGA analysis: A) HMT1 interactors found in the Boone lab study only; B) HMT1 interactors identified in both the Boone lab study and in this study; C) HMT1 interactors found in this study that had negative synthetic interactions; and D) HMT1 interactors found in this study that had positive synthetic interactions. Colonies represents non-query single mutant is marked by a white square and the double mutant (both query and non-query) is marked by a white circle.Tetrad analysis was used to confirm (TIF)Click here for additional data file.Table S1HMT1 as described by the current study. The growth difference ranges from +1 (increasing colony size) to -1 (decreasing colony size) and the p-value for each interaction is also shown. Only genes that passed the p-value criteria are included in this table.The list of all genes that display a genetic interaction with (PDF)Click here for additional data file.Table S2hmt1 query mutants. Query S.D.: standard deviation of Δhmt1 query colonies. No. Sets: number of replicates scored. Visual Significance: binary assessment of replicate quality; 1 = good, 0 = poor.The unfiltered list of SGA data from three independent screens. Control Average: average colony size for control query mutants; Control S.D.: standard deviation of control query colonies, Query Average: average colony size for Δ(XLSX)Click here for additional data file.