CDKN2A (encodes p16INK4A and p14ARF) deletion, which results in both Rb and p53 inactivation, is the most common chromosomal anomaly in human cancers. To precisely map the deletion breakpoints is important to understanding the molecular mechanism of genomic rearrangement and may also be useful for clinical applications. However, current methods for determining the breakpoint are either of low resolution or require the isolation of relatively pure cancer cells, which can be difficult for clinical samples that are typically contaminated with various amounts of normal host cells. To overcome this hurdle, we have developed a novel approach, designated Primer Approximation Multiplex PCR (PAMP), for enriching breakpoint sequences followed by genomic tiling array hybridization to locate the breakpoints. In a series of proof-of-concept experiments, we were able to identify cancer-derived CDKN2A genomic breakpoints when more than 99.9% of wild type genome was present in a model system. This design can be scaled up with bioinformatics support and can be applied to validate other candidate cancer-associated loci that are revealed by other more systemic but lower throughput assays. CDKN2A (INK4A/ARF) tumor suppressor locus, which was discovered in this and other laboratories CDKN2A deletions occur early during tumor development INK4a  are reported and also through The Cancer Genome Atlas (TCGA) project Tumors evolve through the continuous accumulation and selection of randomly mutated genes. While sets of advantageous mutations are selected in tumors, neutral or even slightly detrimental mutations may also occur due to genomic instability and genetic drift. Recently, much effort has been expended to identify in primary human cancers point mutations in the exons of cancer-related genes. However, systemic mapping of genomic DNA rearrangements has lagged behind, due to technical difficulties in detecting smaller deletions, tumor heterogeneity, and the necessity to purify malignant from normal cells in situ hybridization (FISH) While point mutations and very small insertions or deletions in genomic DNA can be detected by exon re-sequencing, it can be more difficult to detect gene dosage changes of larger genomic fragments, especially deletions Here we report a novel approach, designated as Primer Approximation Multiplex PCR (PAMP), to enrich small amounts of deleted genomic DNA sequences in the presence of wild type DNA. The genomic locations of the enriched sequences are subsequently decoded by a genomic tiling array and confirmed by sequencing.CDKN2A is located on chromosome 9p21  and MTAP (methylthioadenosine phosphorylase) (not shown) are centromeric and telomeric neighboring genes respectively CDKN2A genomic fragment and was used as template to generate probes (excluding repetitive regions) for printing on the minigenomic tiling array. The frequency of repetitive sequences predicted by RepeatMasker is shown at the bottom of the diagram.The ome 9p21 . It encoCDKN2A deficient DNA. We aimed to take advantage of the fact that a shorter deleted genome sequence should be preferentially amplified compared to a much longer WT sequence using “approximated” flanking primers  and reverse (R1-10) primer group can have multiple primers. Therefore, every PCR group (for example F1-R1) pair becomes multiplex PCR. Therefore, we designated this procedure as Primer Approximation Multiplex PCR (PAMP).INK4A exons 1 and 2) deletion on chromosome 9p21 A, FB, RY and RZ) of primers were used for four PAMP reactions (CDKN2A deficient) or HEK293 (CDKN2A wild type) cell lines. Aliquots of all 4 PAMP reaction products were pooled and labeled for hybridization on an INK4A minigenomic tiling array that covers about 25 kb, including all of the exons of INK4A. As predicted in We reported previously that the Detroit 562 cell line has an approximate 20 kb (including eactions  using geB-RY (array 27) and all products pooling (array 29) produce the same result as shown in In addition, four separate arrays were used to hybridize the individual PAMP products described above. A simple plot of signal intensity ratio of mutant/WT PCR products on the tiling array revealed the genomic location of the breakpoint . This anIn order to pinpoint more precisely the area of deletion, nested PCR with pairs of specific primers was designed according to the earlier PAMP results. The PCR product was labeled for array hybridization, yielding a result very similar to that shown in 5 molecules/µg of haploid genome). The CDKN2A deleted cell line Detroit 562 was serially diluted with CDKN2A wild type HEK293 as shown in the To mimic the heterogeneous population of cancer and host cells typically found in solid tumors, various amounts of genomic DNA derived from Detroit 562 (mutant) and HEK293 (wild type) were mixed for PAMP and array hybridization. In order to test the sensitivity of our approach, we performed a titration experiment. The total genomic DNA for each assay was kept constant (100 ng). This is equivalent to about 28,000 copies of haploid genome  that rely on the absence of a detectable wild type signal, PAMP directly measures the deleted DNA. Therefore, this approach is much less vulnerable to problems associated with normal cell contamination. The experimental procedure is robust enough to detect deletions in the presence of at least 99.9% wild type sequence contamination, which could not be achieved by other procedures We have developed a general strategy that can be applied for pinpointing the genomic breakpoints in unpurified primary cancers. The amplification and tiling protocol described here allows for simple and precise C. elegansPrimer approximation PCR screening has been a useful tool for isolating deletion mutants in CDKN2A locus with a total of 500 primers in one single PCR reaction through computational simulation, which will be described elsewhere (manuscript submitted). A recent paper reported a successful multiplex PCR with more than 1000 primer pairs through the aid of computational design We used multiplex PCR to reduce the workload and cost for PAMP. We were able to multiplex 28 primers easily in a single PCR reaction. Theoretically, one can cover over 90% of the 0.5 Mb of genomic fragment around CDKN2A breakpoints are specific and unique for each cancer with this locus deleted. A highly sensitive assay, such as real-time PCR, can be designed to monitor the status of cancer progression in the blood or other body fluids. The assay should be very specific because amplification is expected to occur only from deletion-containing DNA due to very long distance between the primers in the wild type genome J recombination may be responsible for creating CDKN2A breakpoints of the two cell lines used in this study as reported by others. Second, the DNA is more stable than RNA although it is easier to map fusion transcript if it exists, such as BCR-ABL. Third, the genomic breakpoints are very likely to be different from each patient and become personalized biomarkers, thereby, reducing the risk of false positive results due to cross contamination. However, this is also the biggest hurdle to overcome. For example, many efforts to improve the PCR amplification range for detecting C-MYC/immunoglobulin translocations have had limited success because the breakpoints are scattered across a more than 300 kb region Using breakpoint sequences as cancer-specific biomarkers to monitor minimal residual diseases has been explored CDKN2A with 1 kb resolution. We are currently working on methods to increase multiplexing and reduce the volume of each PAMP reaction for broader applications.Our approach aims to identify breakpoints within a 1 Mb genomic fragment as FISH or other cytogenetic techniques are available for larger genomic rearrangements and the cost and labor significantly increase when the target region expands. We are able to inexpensively produce a tiling array covering a genomic fragment of 0.5 Mb around the The cell lines described in the paper were obtained from the American Type Culture Collection  and cultured as recommended. The genomic DNA was extracted with DNAzol  following the instructions from the manufacturer.INK4A minigenomic tiling array covering a 25 kb fragment in the CDKN2A locus for proving the concept of our approach. DNA probes were generated by PCR with BAC clone RP11-149I2  as template and avoiding the repetitive genomic sequences that were predicted by RepeatMasker. The PCR products were purified with DNA Clean-up and Concentrator-5 , resuspended in 3×SSC and printed on poly-L-lysine slides at 0.1 mg/ml along with Human Cot-1 DNA , which is enriched for repetitive sequences, and herring sperm DNA , which was used as nonspecific control. The printing procedure has been described and essentially followed the manual of the DeRisi arrayer with silicon microcontact printing pins  http://derisilab.ucsf.edu/microarray/protocols.html).We created an INK4A exons 1-2 along the CDKN2A locus were synthesized by Integrated DNA Technologies . Groups of forward and reverse primers  were used to generate amplicons from 0.1 µg of genomic DNA templates in a total of 10 µl of solution mixing with 10 µl of Taq 2×Master Mix . The reaction was assembled at 4°C in a PCR workstation and transferred to a thermocycler with the block preheated to 94°C. The cycling conditions were a 3-minute denaturation step at 94°C followed by 35 cycles at 92°C for 30 sec, 55°C for 30 sec and 68°C for 2.5 minutes with a final extension step at 68°C for 5 minutes. One µl of unpurified product was subsequently used as templates for another round of amplification to label the amplicons with the same PCR protocol except that dTTP was replaced by a 4∶1 mixture of aminoallyl dUTP  and dTTP for probe labeling. The labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns, eluted in 9 µl of sodium bicarbonate (pH 9.0) and coupled with 1 µl of DMSO dissolved Cy3 or Cy5 NHS esters  for 30 to 60 minutes. The Cy3 and Cy5 labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns and eluted with 10 µl of 10 mM Tris-HCl (pH 8.0). Paired Cy3 and Cy5 labeled amplicons were combined with 3.6 µl of 20×SSC, 0.5 µl of Hepes (pH 7.0) and finally 0.5 µl of 10% SDS. The mixed solution was heated for 2 minutes at 95°C, cooled to room temperature and hybridized to the minigenomic tiling arrays at 63°C overnight essentially as previously described A simplified PAMP scheme is shown in 50%-HSI) were used to divide the intensity (GI) of each feature representing genomic probes. Each (GI):(50%-HSI) ratio, the normalized genomic probe signal, was plotted at the Y-axis against the corresponding probe's genomic location at the X-axis to ease data interpretation  were used for 35 cycles of PCR . The two sets of internal primers  were used for the second PCR reaction under the same conditions. A single band about 1 kb in size was excised and extracted with Qiaquick gel extraction kit . The purified product was directly sequenced with internal primers.ment see  and 4. EHs578T cell line: Four sets of uniplex PCR reactions were performed by pairing 2 single forward (FA1 and FA2) and 2 single reverse (RX3 and RX4) primers. The PCR program was the same as that for PAMP. The products were analyzed by agarose gel electrophoresis. A single band from the shortest distance pair (FA2-RX3) was excised and sequenced with an internal primer.


DEFA1 and DEFA3 genes has been reported. Moreover, the DEFA3 gene has been found to be absent in a significant proportion of control population subjects. CNVs involving immune genes, such as α-defensins, are possibly contributing to innate immunity differences observed between individuals and influence predisposition and susceptibility to disease.Copy number variants (CNVs) account for a significant proportion of normal phenotypic variation and may have an important role in human pathological variation. The α-defensin cluster on human chromosome 8p23.1 is one of the better-characterized CNVs, in which high copy number variability affecting the DEFA3 absence in 697 samples from different human populations. The proportion of subjects lacking DEFA3 has been found to vary from 10% to 37%, depending on the population tested, suggesting differences in innate immune function between populations. Absence of DEFA3 was correlated with the region's haplotype block structure. African samples showed a higher intra-populational variability together with the highest proportion of subjects without DEFA3 (37%). Association analysis of DEFA3 absence with 136 SNPs from a 100-kb region identified a conserved haplotype in the Caucasian population, extending for the whole region.We have tested the DEFA3 gene may be suggestive of population-specific selective pressures with potential impact on human health.Complexity and variability are essential genomic features of the α-defensin cluster at the 8p23.1 region. The identification of population differences in subjects lacking the  DEFB1, DEFA6, DEFA4, DEFA1, DEFT1, DEFA3 and DEFA5) and at least two centromeric clusters of β-defensin genes  [Defensin genes encode a family of small cationic peptides that act as antimicrobial mediators of the innate immune system . DefensiDEFA1 and DEFA3) and β-defensin  genes in chromosome 8p23.1 has been well detected and characterized [DEFA1 and DEFA3 gene copies has been reported to range from 4 to 11 in a sample of 111 subjects, the DEFA3 allele being completely absent in 10% of them [DEFA1, DEFT1 and DEFA3 has been replaced by DEFA1A3, following recommendations of Aldred et al, since these genes have been considered as being part of a copy number variant (CNV) region [DEFA1 and DEFA3 alleles in 27 subjects and found between 5 and 14 copies per diploid genome, with DEFA3 being absent in 26% of them [Chromosome band 8p23.1 is known to be a frequent site of chromosomal rearrangements mediated by low copy repeats (LCRs) or segmental duplications (SDs). It has been described that as many as one in four individuals from the general population carry a 4.7 Megabase (Mb) inversion of the region . In addicterized -14. The  of them . Gene noDEFA1 and DEFA3 being considered as members of the same CNV (DEFA1A3), they encode different peptides, HNP-1 and HNP-3, respectively. The mature HNP-1 and HNP-3 peptides differ only in their N-terminal amino acid, due to a single nucleotide difference, C3400A, between the DEFA1 and the DEFA3 genes [DEFA1A3 CNV cluster encode the HNP-2 peptide. The three peptides are constitutively produced by neutrophil cell precursors and packaged in granules before mature neutrophils are released into the blood. During phagocytosis, the defensin-containing granules fuse to phagocytic vacuoles where defensins act as antimicrobial agents [Despite A3 genes . This C3l agents .DEFA3 in samples from different human populations. For this purpose, we used the International Haplotype Map (HapMap) Project collection and a cohort of Spanish healthy individuals.Recent work has shown that CNVs are a major source of genetic variation . IndividWe have analyzed 786 samples from four populations with ancestry in Europe, Africa or Asia (the HapMap collection), including Spanish healthy individuals. The source used for this study was the HapMap collection of 269 samples utilized by the International HapMap Consortium for the study of human genomic variation, initially through the investigation of SNPs and their associated haplotypes , and 180DEFA1 and DEFA3 differs only by a single nucleotide (C3400A), which allows distinguishing between DEFA1 and DEFA3 by HaeIII digestion, since a restriction site for this enzyme is absent in the DEFA3 sequence. All samples had at least one DEFA1 copy, but DEFA3 was absent in several subjects of all populations. DEFA3 was absent in different proportions depending on the population tested, ranging from 10% in the Chinese/Japanese dataset to 37% in the Yoruba samples .The coding sequence of DEFA6, DEFA4, DEFA1, DEFA3 and DEFA5), six α-defensin pseudogenes  and one θ-defensin pseudogene (DEFT1P)  Figure . Such cl3 Figure .DEFA1A3 CNV, previously reported to be variable in copy number between individuals , but only in four cases where a gain or loss was detected, DEFA3 is absent. Copy number variation in the DEFA1A3 region is reported to be much more common than the variation identified by Redon et al [DEFA1A3 CNV makes it undetectable with BAC arrays. Moreover, the presence of segmental duplications in the region entails a bad SNP coverage of the region by the Affymetrix SNP array, which does not allow an accurate detection of the CNV. Thus, the study of this CNV for association purposes has to be performed by quantitative methods or by the analysis of paralogous sequence variants.HapMap samples have been tested for the presence of CNVs by two different techniques Affymetrix SNP array and BAC array . DEFA1A3on et al . HoweverDEFA1A3 cluster and the single copy gene DEFA5 was chosen for the linkage disequilibrium analysis (based on human genome assembly hg17) . However, only 136 of the SNPs had genotype data in all four populations. Interestingly, almost all genotyped SNPs are located outside the DEFA1A3 cluster  Figure . The Hapr Figure . The absOf the 136 SNPs analyzed in all four populations, 55 were monomorphic in at least one of them . Monomorphic SNPs can be used to measure genetic variability, by analyzing their distribution in the different populations. The Chinese and Japanese groups had the highest proportion of monomorphic SNPs (34%) which was very similar to that observed for Caucasian samples (31%), whereas the Yoruba samples had the smallest number of monomorphic SNPs (24%). This indicates that genetic variability is higher within Yoruba samples, while Chinese/Japanese and Caucasian populations show similar proportions of genetic variability. This higher variability for Yoruba samples is similar to that detected in the HapMap analysis for the whole genome . InteresDEFA3 locus absence results; the Yoruba samples showing highest LD variability and also having the highest proportion of DEFA3 absence.The patterns of linkage disequilibrium (LD) in each population are summarized in Figure DEFA3 is inherited together with neighbor SNPs, an association study was performed using the HapMap data for the 100-kb region including the DEFA1A3 cluster. All the SNPs of the region genotyped in the HapMap project were tested for association with the C3400A PSV, which defines the presence or absence of DEFA3 gene, respectively. No association for any of the genotyped SNPs was found in the Yoruba or Japanese/Chinese populations. However, a significant association was found between absence of DEFA3 and 18 SNPs in the Caucasian samples, under a recessive mode of inheritance .To assess whether e Figure . Associae Figure . Moreovek Figure . This esSeveral studies have recently reported a previously unknown high prevalence of copy number variation in humans . A recenDEFA1A3 and DEFB4/DEFB103A) in the 8p23.1 region have been extensively characterized [DEFA3 allele. In the present work, we have tested the absence of the DEFA3 allele in different human populations, finding significant differences between them, which could be indicative of differences in innate immune function between populations. This is not surprising since the different human population groups have been exposed to different environments regarding infectious agents and other factors. One obvious way by which CNVs result in human phenotypic diversity is by altering the transcriptional levels of the genes which vary in copy number [DEFA1A3 in which variation in DEFA1 and DEFA3 copy number, and DEFA3 absence could underlie variable resistance to infection among individuals. Different selective pressures acting in each geographic region could likely explain population differences in DEFA3 absence.CNVs involving α- and β-defensin genes  and DEFA3 (GenBank accession number L12691) genes differing by a single nucleotide. A fragment of 304 bp around C3400A SNP was PCR amplified with fluorescently labelled primers  and digested with HaeIII enzyme. In order to accomplish complete digestion, we used saturating conditions (2.5 U/25 μl reaction) of the enzyme to digest a short DNA fragment containing only one cutting site. In addition, in all the runs, a DEFA3 negative sample was included, as a positive control of the assay. About 2 μl of digestion product was added to 10 μl HiDi formamide containing ROX500 marker (Applied Biosystems) and run on an ABI 3100 capillary system (Applied Biosystems). Peaks were analysed using Genemapper software (Applied Biosystems).A PCR amplification assay followed by restriction enzyme digestion (PCR-RFLP) has been used to discriminate The UCSC Genome Browser  served aDEFA3 absence in different human populations. Genotyping data from HapMap public database [DEFA3 absence using logistic regression models. Odds ratios (OR)and 95% confidence intervals (95% CI) were calculated for eachgenotype compared with the homozygous for the major allele . Analyses were initially done under a codominant inheritance model (three genotypes separated). Then, simplified models were fitted: a dominant model , a recessive model , an overdominant model (homozygous grouped) and a log-additive model . The model with lowest Akaike information criteria was the recessive one (minus twice the log likelihood of the model plus the number of variables in the model) and it was selected for an easy summary of the results. P values were derived from likelihood ratio tests, and a significance level of 5% (two sided) was used for the analyses. All these analyses were performed using the SNPassoc R package [Between groups chi-square test was performed to compare the proportion of database  was used package .Haploblocks were constructed using Haploview program . HaplotyEB carried out the genetic molecular studies, the bioinformatics work and drafted the manuscript. JRG carried out the statistical analysis. NB participated in the bioinformatics work and design of the study. XE conceived the study and participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Results of the association study for the three population groups. The p-values under all inheritance modes tested are shown.Click here for file


This study sought to produce monoclonal antibodies (MAbs) which reacted with the MUC2 core protein. Two MAbs [3A2 (IgG1) and 4F1 (IgM)] were produced by immunising female BALB/c mice with gel-formed mucin from the LS174T colon cancer cell line followed by a KLH conjugate of a 29 amino acid synthetic peptide whose sequence was derived from the variable number of tandem repeats (VNTR) region of a MUC2 cDNA clone. The MAbs reacted with synthetic MUC2 VNTR peptides but not synthetic MUC1 or MUC3 VNTR peptides, and showed specific reactivity in Western blotting with a high molecular weight protein produced by the LS174T colon carcinoma cell line. The use of shorter peptides indicated that the minimum peptide epitopes for these MAbs were different. Mab 3A2 reacted with amino acids 5-19 of the MUC2 VNTR by inhibition ELISA but not by direct ELISA, while 4F1 reacted with this peptide in both assays. Furthermore, 4F1 reacted in direct ELISA when a larger (29 amino acid) MUC2-derived peptide was coated onto the assay plate by incubating in carbonate buffer or by drying the peptide onto the assay plate, while 3A2 only reacted when this peptide was coated in carbonate buffer. The different specificity of the MAbs was also illustrated by the reactivity of 4F1 but not 3A2 with partially deglycosylated cystic fibrosis mucin. Immunohistochemical analysis with these MAbs revealed a strong reactivity with lung, gastric and colon tumours relative to normal tissue, with some breast and ovarian tumours also reacting. Both MAbs stained some normal goblet cells in the perinuclear region but not the mucin droplet or secreted mucin, indicating a reaction with immature (poorly glycosylated) mucin in the endoplasmic reticulum and/or golgi, but not with mature (fully glycosylated) mucin. In contrast, tumours showed strong diffuse cytoplasmic staining. 4F1 also showed weak apical cytoplasmic staining in some goblet cells and stained some tumours which showed no reactivity with 3A2. These antibodies should prove useful in the study of MUC2 structure and function, and in the diagnosis of some tumours.


The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three  transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant P < 0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGF beta 1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study.


Tumour tissue oxygenation has been measured in man during carbogen breathing  using a commercially available polarographic electrode system (Eppendorf p02 histograph). At least 200 tumour measurements in each of 17 patients with accessible tumours were taken before, and subsequently continuously after the commencement of carbogen breathing for periods of 10 to 30 min. In 12 out of 17 patients studied there was a significant increase in median tumour p02 during the first 10 min of carbogen breathing (range 9 to 1800%). There was an initial rapid increase in tumour p02 which was maintained until 8 to 12 min, but then decreased throughout the subsequent treatment period. Although there was a reduction in the proportion of point measurements < or = 10 mmHg in 11 out of 13 patients, during carbogen breathing, measured points of < or = 2.5 mmHg were only eliminated in three out of 11 tumours. The time course has implications for the planning of clinical trials utilising radiotherapy with carbogen breathing.


Cdk2/Cdk4 double knockouts has indicated that these two Cdks are required to phosphorylate Rb during late embryogenesis. The lack of Rb phosphorylation is progressive and associated with reduced E2F-inducible gene expression. Cdk2 and Cdk4 share the essential function of coupling the G1/S transition with mitosis. However, proliferation in early embryogenesis appears to be independent of Cdk2 and Cdk4. We discuss these observations and propose molecular mechanisms that establish the requirement for Cdk2 and Cdk4 at the G1/S transition. We are considering that the balance between proliferation and differentiation is disturbed, which affects especially heart development and leads to embryonic lethality in Cdk2-/-Cdk4-/- mutants. We also discuss the specific functions of Cdk4 and Cdk6, which ironically do not compensate for each other.Progression through the mammalian cell cycle is associated with the activity of four cyclin dependent kinases . Knockout mouse models have provided insight into the interplay of these Cdks. Most of these models do not exhibit major cell cycle defects revealing redundancies, and suggesting that a single Cdk might be sufficient to drive the cell cycle, similar as in yeast. Recent work on  Cdk2 knockout mice, though Cdk2 was considered to be a unique kinase bound to cyclin E, regulating S phase initiation and progression. This perplexing observation has been quickly addressed by further in vivo analysis demonstrating that Cdc2, which was previously demonstrated to control G2/M, is also able to bind cyclin E and compensates for Cdk2 in S phase [Cdk4 and Cdk6 does not affect cell cycle initiation and progression, suggesting that Cdk2 compensates for the lack of cyclinD dependent kinases [Cdk2-/-Cdk6-/- mice display similar phenotypes as Cdk6 or Cdk2 single knockout mice [Cdk2-/-Cdk4-/- mice and provide new models of mammalian cell cycle regulation.Cell cycle regulation plays an essential role in cellular homeostasis and contributes to determine the fate of cells. Most factors influencing the decision, whether to start a new round of division or not, act at the G1/S transition. Mitogenic factors induce expression of cyclin D and therefore stimulate the activities of Cdk4 and Cdk6. The activation of the cyclin D/Cdk complexes is the first step leading to cell cycle entry and is followed by several waves of cyclin expression . Each family of cyclins binds to a specific Cdk, which is active at a specific phase of the cell cycle and contributes to the activation of the next cyclin/Cdk complex. Recent studies in different Cdk knockout mice have challenged this common model of mammalian cell cycle regulation. Single loss of Cdk2, Cdk4, or Cdk6 did not significantly affect cell proliferation in vivo or in vitro [ S phase . SimilarCdk2/Cdk4 double knockout (DKO) mice and for the first time we observed reduced Rb phosphorylation in vivo and in vitro [Cdc2 and cyclin A2. On the other hand, HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes and cell proliferation. This result suggests that Cdk6 and Cdc2 can regulate cell proliferation, but these two kinases might not phosphorylate Rb to full extent, leading to decreased Cdc2 expression. The declining Cdc2 expression acts as a negative loop leading to proliferation defects. The fact that more Cdk6 is bound to cyclin D1, in the absence of Cdk4, is apparently not sufficient to compensate the lack of Cdk2 and Cdk4 (see below). From our experiments, we conclude that Cdk2 or Cdk4 is required, at a certain point, to phosphorylate Rb thereby maintaining higher levels of Cdc2 protein expression. These two G1/S kinases contribute to the activation of G2/M cyclin/Cdk complexes, and doing so, couple the G1/S transition with mitosis. Characterization of the double knockout mice of Cdk2 and Cdk4 revise the picture of the cell cycle, combining features from the classic mammalian model and features from the yeast model. Nevertheless, though we have uncovered the dynamics of this molecular mechanism, we still need to understand why Rb phosphorylation starts to decline only at midgestation.We recently generated in vitro . The decCdk4 or Cdk2 induces relocation of free p27 to Cdk2 or Cdc2 complexes respectively, which affects their activity. Studies with p27 knockout mice have shown that loss of p27 can rescue a normal entry of S phase in Cdk4-null [Cdk2-null MEFs (unpublished data). In Cdk2-/-Cdk4-/- DKO, these inhibitors might be relocated to Cdc2 complexes and therefore, through their inhibition, contribute to the lack of Rb phosphorylation. We tested this hypothesis by knocking out p27 in Cdk2-/-Cdk4-/- mice, but did not observe a rescue of embryonic lethality or proliferation of MEFs [Cdk2-/-Cdk4-/- MEFs but Cdc2 did not restore normal proliferation (unpublished data).The late embryonic lethality of DKOs suggest that Cdk2 and Cdk4 are required to regulate the Rb/E2F pathway only late in development. In agreement with this, cell proliferation in early embryogenesis is comparable in DKO and wild type embryos. Our analysis has shown that Rb phosphorylation decreases progressively after E13.5, and we can only speculate how Rb phosphorylation is maintained in early development. Therefore, we propose four possible models that are not mutually exclusive and can possibly explain our findings Figure  could beCdk2-/-Cdk4-/- embryos is probably linked to the progressive loss of Rb phosphorylation observed at midgestation. So far, we do not know if the heart defect is related to hypophosphorylation of Rb. A similar cardiac phenotype was observed in cyclin D1-/-D2-/-D3-/- triple knockouts [cyclin D-null and Cdk2-/-Cdk4-/- mice. Cyclin D-null mice do not display an Rb defect, however, it cannot be excluded that the Rb/E2F pathway is deregulated in cyclin D-null cardiomyocytes. This pathway plays a major role in cardiogenesis and the levels of free activated E2F is critical for normal cardiac function [Cdk2-/-Cdk4-/- mutants, Rb represses E2Fs, probably interacting with repressors like Jumonji, which then inhibits cardiomyocyte growth. Further characterization of this pathway might help to better understand the complexity of heart development and the relation to the cell cycle players. Studies of Cdk2-/-Cdk4-/- cardiomyocytes will be a good experimental model to determine how the combined loss of Cdk2 and Cdk4 affects the balance between differentiation and proliferation. We need to determine why the cardiogenesis is more sensitive to inactivation of Cdk2 and Cdk4 than differentiation of other cell types.The embryonic lethality in DKOs is most likely associated with cardiac failure. The small size of the nockouts . It is lfunction . Anotherfunction . The inafunction . From E1Cdk2-/-Cdk4-/- mutants are embryonic lethal, whereas Cdk2-/-Cdk6-/- mice develop normally. Focusing on animal growth and control of cell proliferation, several observations suggest that Cdk4 and Cdk6 do not completely compensate for each other in vivo. Cdk4 single knockout males and females display reduced animal size [Cdk6 reduces the size of the females but to a lesser extend than Cdk4 mutation [Cdk4-null but not in Cdk6-null MEFs [Cdk4-/- and Cdk2-/-Cdk4-/- mutants compared to wild type or Cdk2-/-Cdk6-/- mutants. The complete inactivation of Cdk4 and Cdk2 leads to more pronounced lack of proliferation (at least in the hematopoietic linage and in MEFs) and affects cardiac development, thereby inducing embryonic lethality [Studies with double knockout mouse models have pointed out some differences between Cdk4 and Cdk6. Indeed mal size ,4, whileethality .Cdk2-/-Cdk4-/- mice. Other substrates might also be involved, such as Smad3, phosphorylated by Cdk4 and Cdk2 but not yet tested for Cdk6 [At the biochemical level, few differences have been described between Cdk4 and Cdk6 . In vivfor Cdk6 . Smad3 mfor Cdk6 ). All thCdk2-/-Cdk4-/- mouse model will teach us more details about tumorigenesis.Our knowledge of cell cycle regulation has greatly improved through the characterization of knockout mouse models. The overlap of Cdk functions adds more complexity to the in vitro model of the cell cycle (specific Cdk/cyclin complexes for each cell cycle phase). On the other hand, we can consider that all Cdks are redundant, which would result in a model similar to the yeast cell cycle. Our recent results show that the redundancy is not complete and each Cdk might have its own niche. This specificity could be essential for small subpopulations of cells  or affect cell cycle regulation globally. Indeed, Cdk2 and Cdk4 share a common role in the G1/S transition, which couples this phase with mitosis through E2F-inducible gene expression. Embryonic stem cells might proliferate independently of this coupling, and we presented four models to describe how this coupling can take place during embryogenesis. Among E2F-inducible genes, we focused on the role that Cdc2 can play as a kinase to phosphorylate Rb, however we cannot exclude that other E2F-targets are also important. Moreover, these four molecular mechanisms act probably in concert to establish the G1/S checkpoint. This role might be important with regards to cancer cells. Could the combined targeting of Cdk2 and Cdk4 be a valuable approach for cancer therapy? To answer this question, we have to determine if Rb wild type cancer cells require Cdk2 and Cdk4 activities throughout tumor progression. Future experiments with the Cdk: cyclin dependent kinaseDKO: double knockoutES cell: embryonic stem cellsMEF: mouse embryonic fibroblastPK is co-Editor-in-Chief of Cell Division but was not involved in the editorial and peer review process of this manuscript.


The cytophotometric DNA content and the argyrophilic nucleolar organiser regions (AgNORs) of biopsy specimens taken before undergoing any treatment were examined in 91 surgically treated oesophageal carcinoma cases. There was a significant linear dependence between the mean DNA content and the number of AgNOR per nucleus (AgNOR number) . The DNA distribution pattern and the range of the AgNOR number also showed a significant correlation (P < 0.01). Twenty three of 28 cases with a low AgNOR number (< 4) were then determined to have a diploid pattern (type II), while 17 out of 22 cases with a high AgNOR number (> or = 6) had high ploidy values (type IV). The patients with a type II DNA distribution pattern and a low AgNOR number thus showed a good post-operative course with a 5 year survival rate of 55.2%, whereas no patients survived over 4 years among the 17 cases with both a type IV DNA pattern and a high AgNOR number (P < 0.001). These data thus demonstrate the close relationship between cytophotometric DNA content and AgNOR number and suggest that the combined detection of these two parameters, using biopsy specimens, should be of benefit in making an accurate preoperative evaluation of prognosis for patients with oesophageal carcinoma.


Fourteen patients with advanced breast cancer were treated with the ribonucleotide reductase inhibitor didox 6 g m-2 given by intravenous infusion over 36 h every 3 weeks. None responded and toxicity was minimal. Possibilities for the more effective use of this agent are discussed.


The data presented in this article shows the longitudinal analysis of tear fluid cytokine profiles, blood CD4 and CD8 counts and HIV viral load in 34 dry eye patients with HIV infection during the HAART therapy. Clinical samples were collected from HIV patients with dry eye disease at the time of presentation to the clinic (visit 1), three months (visit 2) and 6 months (visit 3) after the presentation. At each time point tear samples were evaluated for 41 cytokines using Luminex bead based multiplex assay and blood samples were tested for HIV viral load and CD4 and CD8 counts. Specifications TableValue of the data•In our earlier reports cytokines IFN-gamma-inducible protein 10 , epidermal growth factor (EGF) and growth-regulated oncogene (GRO) levels were significantly altered in dry eye patients with human immunodeficiency virus (HIV) infection compared to immunocompetent dry eye patients at the time of presentation (visit 1/baseline) to the clinic •Here we report the longitudinal tear cytokine profiling data of HIV patients with dry eye disease over the period of 6 months with two follow ups (3 months each) and its association with the systemic CD4 (cluster of differentiation 4), CD8 counts and HIV viral loads.•This longitudinal data will help researchers to understand the changes in tear cytokines in HIV patients with dry eyes over the time and its association with systemic and viral factors and study further on HIV associated ocular inflammation and its pathogenesis.1The data presented herein was obtained from longitudinal analysis of tear cytokine profiles in 34 dry eye patients with HIV infection at 3 time points . Systemi2The data herein was obtained from HIV patients who had complaints of symptoms related to dry eyes  with a prior informed consent and were evaluated following the guidelines of the International Dry Eye Workshop (DEWS), 2007


CYP2C9*30 (A477T) is associated to diminished response to the antihypertensive effects of the prodrug losartan and affected metabolism of other drugs. Here, we investigated molecular mechanisms involved in the functional consequences of this amino-acid substitution. Molecular dynamics (MD) simulations performed for the active species of the enzyme (heme in the Compound I state), in the apo or substrate-bound state, and binding energy analyses gave insights into altered protein structure and dynamics involved in the defective drug metabolism of human CYP2C9.30. Our data revealed an increased rigidity of the key Substrate Recognition Sites SRS1 and SRS5 and shifting of the β turn 4 of SRS6 toward the helix F in CYP2C9.30. Channel and binding substrate dynamics analyses showed altered substrate channel access and active site accommodation. These conformational and dynamic changes are believed to be involved in the governing mechanism of the reduced catalytic activity. An ensemble of representative conformations of the WT and A477T mutant properly accommodating drug substrates were identified, those structures can be used for prediction of new CYP2C9 and CYP2C9.30 substrates and drug-drug interactions.Cytochrome P450 2C9 (CYP2C9) metabolizes about 15% of clinically administrated drugs. The allelic variant  CYP2C9 can affect the clinical response of drugs metabolized by CYP2C9, especially those with a narrow therapeutic index and can induce adverse drug reactions (ADR). The rare allelic variant CYP2C9*30 found in Japanese subjects has been associated to diminished response to the antihypertensive effects of the prodrug losartan [Cytochrome P450 2C9 (CYP2C9) is the most expressed member of the human CYP2C family and metabolizes more than 15% of clinically administrated drugs including hypoglycemic agents, anticonvulsants, anticoagulants, nonsteroidal anti-inflammatory drugs (NSAIDs), antihypertensives, diuretic drugs  and sevelosartan , 11. It losartan . Recentllosartan . HoweverCYP2C9*30. Our results highlighted conformational and dynamic changes believed to be involved in the governing mechanism of the catalytic activity reduction and confirmed that protein dynamics affected by missense mutations could play a crucial role in affected regulation of substrate entry, recognition and metabolism of CYP2C9.Here we provide insights into the dysfunctional metabolism mechanism in human CYP2C9.30 based on molecular dynamics (MD), quantum mechanics and docking simulations. Molecular dynamics (MD) simulations combined with energetic analyses have been demonstrated to be a pertinent approach to understand molecular mechanisms of amino-acid substitutions affecting drug metabolism or regioselectivity of CYP –18. In tThe A477T mutation present in CYP2C9.30 variant is located in the Substrate Recognition Site 6 (SRS6) (Supplementary The Root Mean Square Fluctuations (RMSF) results confirmed the stability of the WT and mutant A477T structures during the MD simulations. The mean RMSF values of the merged MD trajectories for each of the six systems varied between 1.08 Å and 1.18 Å. However, the comparison of the atomic fluctuations per residue see  revealedvs 5.6% in the apo mutant MD simulations) and F476 (a HB conserved in 26.3% in the apo WT vs 2.1% in the apo mutant MD simulations). Changes of the HB interactions of Q214 due to the A477 mutation were also observed in the case of bound losartan to CYP2C9 [In order to find noticeable conformational differences of the substrate binding pocket of CYP2C9 between the WT and the A477T mutant, we performed structural clustering of the five merged MD trajectories for each of the studied systems based on the binding site residues see . The stro CYP2C9 . Therefoo CYP2C9 ) and mayHydrophobic interactions between F476 and other hydrophobic residues of the CYP2C9 binding pocket were monitored within a distance of 5 Å during the MD simulations see . We obseSubstrate positions are crucial for the oxidation reaction. On the contrary, the losartan reached an equilibrium position , remaining in the same subpocket in contact with the heme similarly to its initial docking position in both WT and A477T mutant proteins see . Along tThe RMSD of Cpd I along the MD simulations  showed tWe explored the structure and dynamics of the substrate access channels for the WT and the A477T mutant. Significant changes of the substrate access channels were found to occur in the presence of the mutation A477T. The open channels detected along all MD simulations for the WT and the mutant proteins are summarized in We observed that several channels were open in more than 20% of the MD simulations of the diclofenac-bound state . This observation is in accordance with the large movements of diclofenac observed during the MD simulations suggesting a dynamic behavior of the channels. Similar results have been reported in a previous study  for CYP2The comparison between the apo WT and mutant proteins showed several important changes due to the mutation. Most of the substrate and solvent access channels were more closed in the A477T mutant as compared to the WT. More precisely, the channels 2b, 2f, 5 and S1 were more closed in the apo state of the mutant than of the WT see . The chaWe carried out molecular docking calculations to identify different conformations of the WT and A477T mutant of CYP2C9 properly accommodating the substrates for the catalytic reaction. In order to explore the binding spectrum of CYP2C9, we studied five drugs metabolized by CYP2C9 by molecular docking: diclofenac, flurbiprofen, warfarin, losartan and glimepiride. It has been shown that the metabolism of diclofenac, losartan and glimepiride is strongly affected in the A477T mutant . To takeTen independent docking calculations were performed for each protein centroid structure. The three top-scored substrate poses were taken for the analysis. This resulted in 360 substrate poses in MD generated WT CYP2C9 conformations and 360 poses in MD generated A477T mutant conformations for each of the five drugs. The two small NSAID flurbiprofen and diclofenac were found to bind in different positions in the CYP2C9 binding pocket, while the other ligands showed preferable binding zones. The comparison of the WT and the mutant showed differences for the diclofenac and flurbiprofen docking in the so called "flurbiprofen-binding" subpocket corresponding to the catalytic site subpocket close to the heme . These tThen, we focused on the SOM positions regarding the Cpd I oxygen atom, which was included in the docking calculations. We considered that the docking poses showing a distance between the SOM  and the In order to analyze other possible positions of the losartan in the CYP2C9 active site we compared the docking poses of losartan with the recently reported co-crystalized structures, PDB ID 5XXI (WT–losartan bound) and 5X23 (A477T mutant–losartan bound). The most similar positions were found when the losartan was docked into the active site of the centroid number 4 of CYP2C9 WT apo representing 5.76% of the entire MD simulations for WT apo (RMSD = 5.41 Å) and of the centroid number 0 of A477T mutant–losartan bound representing 6.59% of the entire MD simulations for the A477T apo mutant–losartan bound (RMSD = 4.36 Å). These docking poses suggest that losartan can also adopt positions similar to the co-crystalized ones in CYP2C9 WT and CYP2C9 A477T mutant–losartan bound. Although RMSD are not very low, the losartan is similarly oriented in the docked and experimental structures see . HoweverIn the three best WT apo MD derived centroids the residues F476 and L362 adopted different conformations . The resCYP2C9*30. Our data demonstrated that the A477T mutation present in CYP2C9.30 led to increased rigidity of the SRS1 and SRS5 regions and to shifting of the β turn 4 of SRS6 toward helix F thus decreasing the substrate access to some protein channels. We observed different behaviors of the substrates diclofenac and losartan during the MD simulations for the WT and the A477T mutant, suggesting that A477T can alter differently the metabolism of various substrates. The overall stabilization of the WT and the mutant structures in the cases of a bound substrate observed for the performed here MD simulations as well as for the available crystal structures demonstrated the importance to study structural and dynamics properties of the apo protein in order to detect molecular mechanisms due to the mutation without screening effects of a bound ligand. Molecular docking of five drug substrates of CYP2C9 into WT and A477T mutant structures generated by MD simulations indicated that some conformations of the A477T mutant were able to correctly accommodate the substrates, however, their number was very low compared to the WT MD conformations. All together these conformational and dynamic changes are believed to be involved in the governing mechanism of the catalytic activity dysfunction. Finally, an ensemble of representative conformations of the WT and A477T mutant properly accommodating the substrates were identified, these structures can be used for future prediction of unknown CYP2C9.1 and CYP2C9.30 substrates and drug-drug interactions.We investigated molecular mechanisms implicated in the altered substrate metabolism in CYP2C9.30 exhibiting < 50% of the WT enzyme activity. MD simulations performed for the active species of the enzyme, Compound I, in apo or substrate-bound states, and binding energy analyses for several drugs revealed molecular mechanisms involved in the defective drug metabolism in carriers of Six crystallographic structures of CYP2C9 were taken from the Protein Data Bank (PDB) and analysed: the native sequence flurbiprofen–bound one (PDB ID 1R9O) , losartaThe initial models of diclofenac-bound and losartan-bound WT and A477T mutant structures were generated using docking with Autodock 4.2 program . The besThe six initial CYP2C9 models  were fully solvated with explicit TIP3P water molecules  and neutThe NAMD2.6 program  was usedTo find the most representative structures of the substrate binding pocket for each of the six systems we merged the five MD trajectories. For the structural clustering, we took 25,000 protein conformations (1 conformation every 10 ps) for each system and used the quality threshold algorithm  of the Vwww.chemaxon.com). Interestingly, ChemAxon predicted two different pKa values for losartan, 4.29 or 8.0, starting from two different smile structures downloaded from BindingDB and DrugBank, respectively. We analyzed possible protonation of the losartan molecules co-crystallized with CYP2C9 in the PDB structures ID 5X23 and 5XXI and decided to consider the protonated state of the losartan, which corresponds better to the losartan environment in the binding sites. Gasteiger atom charges were added using the AutoDockTools package. Molecular dockings were carried out with the Autodock 4.2 program [Five drugs known to be CYP2C9 substrates were docked into the crystal CYP2C9 structures PDB IDs: 1OG5 and 4NZ2 with restored native residues, 1R9O, 5XXI, 5X23, as well as into the initial models of the WT or the mutant A477T and into 240 representative conformations selected after the MD simulations for the six studied systems. The heme was kept in the intermediate Cpd I state. Drug protonation states were calculated at pH 7.4 with the major microspecies option of the ChemAxon package Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Sarcophilus harrisii) predominantly affecting their facial regions. DFT1’s cellular origin is that of Schwann cell lineage where lesions are evident macroscopically late in the disease. Conversely, the pre-clinical timeframe from cellular transmission to appearance of DFT1 remains uncertain demonstrating the importance of an effective pre-clinical biomarker. We show that ERBB3, a marker expressed normally by the developing neural crest and Schwann cells, is immunohistohemically expressed by DFT1, therefore the potential of ERBB3 as a biomarker was explored. Under the hypothesis that serum ERBB3 levels may increase as DFT1 invades local and distant tissues our pilot study determined serum ERBB3 levels in normal Tasmanian devils and Tasmanian devils with DFT1. Compared to the baseline serum ERBB3 levels in unaffected Tasmanian devils, Tasmanian devils with DFT1 showed significant elevation of serum ERBB3 levels. Interestingly Tasmanian devils with cutaneous lymphoma (CL) also showed elevation of serum ERBB3 levels when compared to the baseline serum levels of Tasmanian devils without DFT1. Thus, elevated serum ERBB3 levels in otherwise healthy looking devils could predict possible DFT1 or CL in captive or wild devil populations and would have implications on the management, welfare and survival of Tasmanian devils. ERBB3 is also a therapeutic target and therefore the potential exists to consider modes of administration that may eradicate DFT1 from the wild.Devil Facial Tumour 1 (DFT1) is one of two transmissible neoplasms of Tasmanian devils ( Sarcophilus harrisii) belongs to the Dasyuridae family, it is a carnivorous marsupial that is extinct on mainland Australia and now found only on the island of Tasmania. Superficial dermal cutaneous lesions of wild Tasmanian devils can be found commonly in the form of skin sores [The Tasmanian devil  A pilot study of thirty-five Tasmanian devils differing in age, sex and geographic location were selected  to compa°C. Serum samples were retrieved from the frozen archive and thawed at room temperature immediately before analysis.Blood samples from Tasmanian devils  were col°C. Sections were deparaffinised, rehydrated and stained using Jung autostainer XL (Leica) for Haematoxylin  and Eosin, dehydrated cleared and mounted in CV Mount .Tasmanian Devil tissues were fixed in 10% Neutral Buffered Formaldehyde  for 24 hours and selected tissues were cassetted and processed overnight using a standard 15 hour overnight procedure in the TP1050 tissue processor . Tissues were orientated on the EG1160 (Leica), embedded in paraffin wax  and sectioned at 3 microns using Leica RM2245 microtome and adhered to microscope slides  for 20 minutes at 60°C for 8 minutes using a Pascal pressure chamber  then cooled to 20°C. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide  in methanol  for 30 minutes. Detection of primary antibodies was achieved using Mach1 Universal HRP-Polymer detection kit . Protein block (Background Sniper BS966L10) was applied for 20 minutes prior to application of primary antibodies. Monoclonal rabbit anti-human ERBB3  was diluted 1:50 with antibody diluent  and applied to both devil tumour and normal devil control tissues at room temperature for 30 minutes. Negative control was omission of primary antibody with buffer substitution. Universal HRP-polymer was applied for 30 minutes (MRH538L10) followed by 1 drop of Betazoid DAB Chromogen 3,3 Diaminobenzidine (BDB900G) in 1ml of substrate buffer (DB900) applied for 4 minutes. Tris buffered saline  was used to rinse between all steps. Slides were rinsed, stained with Carazzi’s Haematoxylin for 5 minutes, washed for 3 minute in tap water, dehydrated, cleared and mounted in CV mount. Sections were viewed under light microscopy using Olympus BX41  and selected areas were photographed using an Olympus digital camera (DP20).Archival Tasmanian devil tissues and tumours were sectioned at 3 microns, floated onto Superfrost plus slides (Menzel Glaser) and subjected to standard deparaffinisation and rehydration techniques using an automated stainer (Leica). Antigen retrieval in tissue sections was conducted in citrate buffer at pH 6.0  at 120°C. The supernatant was removed and wells were washed 4 times with 300 uL of 1X wash solution using an Immunowash 1575 . One hundred microliters of prepared biotinylated anti-ERBB3 was added to each well and the assay plate incubated for 1 hour at room temperature. The assay plate was washed as described after which 100 uL of prepared HRP-streptavadin conjugate was added to each well and the assay plate incubated for 45 minutes at RT. The assay plate was again washed as described and 100 uL of TMP substrate was added and the plate incubated for 30 minutes at room temperature in the dark, after which 50 uL of stop reagent was added to each well. The absorbance of each well was measured at 450 nm using a Tecan Infinite M200 microplate reader .Serum ERBB3 levels were measured using the RayBio anti-human ERBB3 ELISA Kit  according to manufacturer’s instructions. Briefly, serum samples were diluted 1/5 in Assay Diluent A and 100 uL of standard or diluted sample were added in duplicate to wells of a 96 well assay plate and incubated for 24 hrs at 4The ELISA standard curve was plotted using Prism v5  and results for each serum interpolated and corrected for dilution. The significance of differences in serum ERBB3 between groups was determined using a Kruskal-Wallis test with Dunn’s Multiple Comparison utilizing Prism v5 .DFT1 histology  and HaemSerum ERBB3 levels are shown in Fifteen Tasmanian devils without neoplasia  were studied with an average serum ERBB3 of 32 pg/ml. Collectively, CHD Tasmanian devils serum ERBB3 levels ranged from <30–663 pg/ml which could be considered representative of the reference range for Tasmanian devils. Wild caught devils 6 and 7 were unremarkable and had serum ERBB3 levels <30 pg/ml however devil 9 (220 pg/ml) and devil 10 (92 pg/ml) both recorded skin abscesses. The ERBB3 levels in the CHDD group  ranged from <30–220 pg/ml all had a small isolated dermatopathy such as abscess (devil 9), pyogranuloma (devil 10), skin tag with associated inflammation (devil 11) and small focus of dermatitis (devil 12) all recorded a low serum ERBB3 levels of <92 pg/ml. The CHJD  approximately 10 months old had an unremarkable clinical history that indicated serum was collected for a health check only, reflected in the low serum ERBB3 level of <30 pg/ml.2 containing capped at one devil per 100 m2.Further assessment of data and clinical history  revealedWe noted that skin injuries were commonly recorded although no abnormality was noted for devil 1, alopecia bilaterally around the hind limbs and flank was present on one mother due to her 3 pouch young (devil 2) and multiple puncture wounds were present on the remainder . Given that these devils were otherwise clinically healthy it would suggest that skin wounds caused by biting may contribute to some elevation in the serum ERBB3 of Tasmanian devils. There is also the possibility that simply being a Tasmanian devil living in a free range enclosure as opposed to wild populations may in itself be contributory to elevation in serum ERBB3 due to more frequent devil-devil engagement. Our results indicate that Tasmanian devils without injuries or an isolated skin lesion have serum ERBB3 levels <30 pg/ml whereas Tasmanian devils with multiple injuries or large abscesses have serum ERBB3 levels ranging from 92–663 pg/ml. Together, these results suggest that cancer-free Tasmanian devils have a serum ERBB3 range of <30–663 pg/ml.All devils with DFT1 were wild caught and all subjected to field autopsy with most serum samples reaching the laboratory within one to three days. We assessed the available clinical history  includinWe included Tasmanian devils with cutaneous lymphoma (CL) in the study for two reasons. Firstly, they were non-DFT1 devils with a severe skin condition that can affect the facial regions and secondly, the disease presentation of alopecia, excoriation and thickened plaques is distinct from DFT1 . Our resThe capture and detection of antibody in our ELISA assay is selective for the extracellular domain (ECD) of transmembrane ERBB3 in serum or plasma, thus ERBB3’s ECD is cleaved and shed from the plasma membrane would be a natural assumption. In contrast the ERBB3 receptor is internalised, although very slowly, for negative regulation and inactivation –71 utiliDefective internalisation, recycling and degradation of cell surface proteins and ligands is an emerging feature of cancer . It is tAs well as functional transmembrane forms, secreted soluble forms of Epidermal Growth Factor Receptors have been well documented for ERBB1 –84, ERBBThe antigenic peptide used for this assay is located within the N-terminal domain of the full length ERBB3 protein. Full length ERBB3 translates into a 180 kDa protein whereas ERBB3 transcripts, created by intron read through and alternative polyadenylation signals result in serum ERBB3 isoforms translating into various proteins ranging in size from 22–75 kDa . SecreteThe correlation of serum levels with disease severity and progression would be the foundation of a good biomarker  as well;ERBB3 is crucial to the sequential transition from precursor to immature and finally mature Schwann cells where ERBB3 is down-regulated as myelination proceeds . The aduDespite evidence for multiple resistance mechanisms for existing therapeutic targeting of ERBB1/2 –141 numehttp://www.utas.edu.au/news/2015/10/16/19-world-first-trial-of-tasmanian-devil-vaccine-begins-in-the-wild/). Confidence that immunisation can be successful stems from research showing that Tasmanian devils have a competent immune system [TM.However, managing wildlife disease is considerably more difficult than human disease because of limited data, the effect of the disease on the host and the transmission of disease within a dynamic population makes it difficult to model . Previoue system , 198–200e system , 201. Ane system . Develope system  however,e system , 204, ERe system –207 or be system  includine system  all showe system . Veterine system  the onlyRecent investigations reveal that the tumour microenvironment of metastatic DFT1 expressed B7-H1 and DFT1 cell lines could upregulate B7-H1. Immune-Our research has highlighted ERBB3 as a potential therapeutic target however treatment of Tasmanian devils with DFT1 with therapeutic regimes such as chemotherapy and radiotherapy are impractical. However, a combinatorial approach using therapeutic cancer vaccines including inactivated allogenic DFT1 cancer vaccine, ERBB3 monoclonal antibody, ERBB3 Peptide or xenogeneic vaccine in combination with anti-immune checkpoint blockade therapy would be easier to implement in the field as well as providing a sustained immunological response against DFT1.ERBB3 had previously avoided scrutiny due to its kinase inactivity; however, ERBB3 has now been the subject of intense investigation over the past decade and is now recognised as a potent partner of the epidermal growth receptor family. ERBB3 upregulation during developmental, dedifferentiation and regenerative processes encapsulates the Schwann cell’s inherent plasticity and imparts certain characteristics of malignant transformation advantageous to transmission of DFT1. Our pilot study has shown for the first time that ERBB3 is consistently expressed immunohistochemically and that ERBB3 is also elevated in the serum of Tasmanian devils with advanced DFT1 and cutaneous lymphoma. Therefore, our research indicates that serum ERBB3 has the potential to be employed as a biomarker of DFT1 or CL in Tasmanian devils to assist conservationists in the management and welfare of Tasmanian devils and species survival. The simplicity of the ELISA Serum ERBB3 methodology is easily incorporated into routine laboratory batch testing and equally applied to include rapid turnaround of results for urgent cases. Extension of this research is necessary to include greater numbers of healthy Tasmanian devils both with and without visible injuries, devils with large and small DFT1 lesions as well as pre-clinical DFT1. This will firmly establish the normal reference range for serum ERBB3 from which potential pre-clinical DFT1 may be identified. In addition, ERBB3 is now recognised as a therapeutic target and therefore the potential exists to consider modes of administration in addition to existing whole cell vaccination such as ERBB3 monoclonal antibody, peptide or xenogeneic vaccines including checkpoint inhibitors. A combinatorial immunotherapeutic approach will enhance cytotoxic destruction, provide long term immunity from DFT1 and therefore eradicate this transmissible tumour from the wild.S1 Fig(DOCX)Click here for additional data file.


The transcription factors GATA4, GATA5 and GATA6 play important roles in heart muscle differentiation. The data presented in this article are related to the research article entitled “Genome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis”   Specifications TableValue of the data•GATA4, 5 and 6 are important regulators of heart muscle differentiation (cardiomyogenesis).•These data identify genes differentially regulated by each of these GATA factors during cardiomyogenesis.•These data also identify genes that are regulated by these factors in common during this process.•These data will help us understand the molecular mechanisms that govern the function of these factors and therefore improve our knowledge about the gene regulatory network involved in cardiomyogenesis.•The analysis of gene ontology (GO) terms associated with cardiogenic GATA-regulated genes provides the different biological processes in which these factors are involved in during normal development and homeostasis.1The data represents RNA-Seq performed on cardiogenic samples, single GATA4, 5 and 6 depleted samples as well as samples with GATA4, 5 and 6 triple depleted, described in the related research article gata4, gata5 or gata6 . Sheet 2: The 361 genes that are exclusively reduced by gata4 knockdown (see sox7 and sox18 (highlighted in red). Sheet 3: represents GO biological process terms associated with all the 1480 genes that are reduced in gata4 knockdown  according to the manufacturer's instruction. 50fg of RNA for Activin were injected. Xenopus gata4 splice morpholino, gata5 splice morpholino Activin β B DNA constructs for mRNA synthesis have been described previously 2.2Xenopus laevis embryos were obtained as previously described 2.3Xenopus laevis genome (version 9.1) using HiSat2 At least 30 explants were used for RNA preparation with a previously described protocol


This study clearly shows the potential that oxygen sensors possess in terms of assisting in the optimised development of commercial RTE salad products.Optical oxygen sensors were used to ascertain the level of oxygen consumed by individual salad leaves for optimised packaging of ready-to-eat (RTE) Italian salad mixes during refrigerated storage. Seven commonly found leaves in Italian salad mixes were individually assessed for oxygen utilisation in packs. Each leaf showed varying levels of respiration throughout storage. Using the information obtained, an experimental salad mix was formulated (termed Mix 3) which consisted of the four slowest respiring salad leaves—Escarole, Frisee, Red Batavia, Lollo Rosso. Mix 3 was then compared against two commercially available Italian salads; Mix 1  and Mix 2 . Optical sensors were used to non-destructively monitor oxygen usage in all mixes throughout storage. In addition to oxygen consumption, all three salad mixes were quality assessed in terms of microbial load and sensorial acceptability. In conclusion, Mix 3 was found to consume the least amount of oxygen over time, had the lowest microbial load and was most sensorially preferred ( More recently, the use of high O2 atmospheres  have been used as an alternative technique to low O2 equilibrium modified atmosphere (3% O2) [2, as well as CO2, within the pack relates to the metabolic state of the produce [The growth in the ready-to-use vegetable market ~10% p.a) has been largely due to increasing demand consumers for fresh, healthy and convenient foods 0% p.a ha. Consumes i.e., >0% O2 hav2 sensors have been shown to provide valuable information in food packaging applications [2 detection to monitor O2 levels in different forms of food packaging provides ample information as to the package environment conditions of a food product throughout storage and shelf life. Problems associated with packaging and containment failures can be instantly observed post packaging using this technology [2 sensors in specific food applications, such as: MAP cheese [sous vide products [Optical Oications ,9,10. Thchnology . ResearcP cheese , vacuum P cheese , MAP andP cheese , cooked P cheese , MAP andP cheese , as wellproducts . 2 sensors have not been employed to monitor O2 levels in retail packs of fresh produce and have never been evaluated as a technology to assist in the optimised development of commercial RTE salad products. Therefore, the objective of this study was to employ the use of non-destructive O2 sensing technology to ascertain the level of O2 consumed by individual salad leaves so that the optimised packaging of RTE Italian salad mixes might be determined. An increase in the O2 gas fill of such MAP salads is also used as an alternative to traditional 3%–5% O2.To date, the use of O2, 5%–10% CO2 and 85%–90% N2) and monitored for O2 levels over time. In order to appropriate the correct level of O2 to a mixed salad product, it was necessary to understand the individual O2 requirement for each salad leaf within the product mix. Individual salad leaves were packaged using a higher O2 level  to assess the true O2 levels utilised by each leaf and subsequent salad mixes during the shelf life period. Mixes of salad leaves were also prepared as described; totalling 170 g, with approximately 25% of each leaf and contents are described in 2, 5%–10% CO2 and 85%–90% N2) without salad fill and monitored for oxygen over time. This was undertaken to assess the level of permeation of gases using these commercial packaging materials over a 14 day period. The level of permeation was negligible as sample free packs were seen to hold initial gas fill within <0.5% O2, showing poor permeability.Salad leaves used in this study were grown in Ireland (and in close proximity to the processing facility where product packaging was undertaken) and consisted of the following salad leaf varieties: Escarole, Frisee, Radicchio, Lollo Rosso, Cos, Iceberg and Red Batavia. Salad leaves undergo treatments such as washing and shredding by manufacturer prior to packaging. Approximately 170 g of each salad leaf was packaged individually for assessment, using an Ishida SE multi head weigher  and stored at 4 °C. A Sandiacre Novus 350  was used to form, fill and seal samples in orientated polypropylene/low density polyethylene laminate films (220 × 290 mm) of 45 micron thickness with modified atmospheres. Control RTE salad mixes were produced commercially with normal packaging conditions  was used in conjunction with O2 sensors allowing for regular non-destructive monitoring of oxygen within packs. The Mocon OpTech-O2 device allows for reliable and accurate O2 readings from 0.001% to 25% O2 in 0.5 s. The device causes excitation of the phosphorescent dye embedded in the optical oxygen sensor and is quenched by molecular oxygen. Calibration of instrumentation was carried out using a Platinum CalCard allowing for instant calibration before each set of measurements were taken. O2 readings were taken at day 0, 3, 5, 7 and 10 for commercially prepared MAP RTE salads, individual salad leaves and modified salad mixes. The final measurement was taken on day 10 of storage as no further O2 drop was observed and samples had exceeded microbial limits.Oxygen sensors were prepared using Pt-OEPK  spotted on Durapore  paper using Gilson P100 pipette  and cut to a size of 5 mm in diameter. These sensor materials were then placed on 3 cm Avery price marking stickers  for adhesion to packaging films. A Mocon OpTech-OMicrobial testing was carried out during refrigerated shelf-life assessment of individual salad leaves and RTE Italian salad Mixes 1, 2 and 3 on sampling days 1, 3, 5, 7 and 10. Total viable counts (TVC) were carried out to determine microbial numbers (colony forming units (cfu)/g samples) in accordance with ISO standard method (ISO 4833:2003)  using toA sensory panel made up of 26 members was recruited in University College Cork, Ireland. Panellists were selected based on their availability on test days and were regular consumers of RTE salad products. All panellists had previous experience in carrying out sensory analysis. A list of descriptors for sensory analysis was selected and these are presented in versus PC 2 is presented; other PC’s did not yield additional information. To derive significance indications for the relationships determined in the quantitative APLSR; regression coefficients were analysed by jack-knifing  was used to process the mean data accumulated from the 26 panellists during the sensory evaluation. Principal component (PC) 1 -knifing .2 modified atmosphere conditions consisting of <5% O2. This practice concurs with similar reported practices [2 levels were monitored over time. From 2 provided in such packs may be insufficient for optimised product respiration, as by day 3 of storage, less than 0.5% O2 remained within packs. This move towards an anoxic environment leads to quality-linked deteriorative processes within the pack [In commercial Irish applications, Italian mixed RTE salad leaf products are packaged in low Oractices . In thesthe pack ,22.2 requirement for each salad leaf within the product mix. Consequently, individual salad leaves typically used within Italian-styled RTE mixed salad leaf products  were packaged individually within MAP formats  to ensure adequate O2 was available for respiration) containing a pre-fitted optical O2 sensor. The individual salad leaf packs were monitored over seven days for O2 levels to ascertain O2 consumption by each salad leaf type. 2 consumed by each salad leaf type during storage . The O2 differential between leaves showed that after seven days, samples ranged in oxygen utilisation by up 3.2% O2. For example, the Radicchio salad leaf was found to consume the greatest amount of O2 over the seven days of storage (15.79%), whereas the Lollo Rosso leaf was determined to consume the least amount of O2 (12.47%) over the same period of time. The consumption of O2 in packs appears to slow after day 7, at which the product is spoilt.It was necessary to understand the individual O2 required by individual salad leaves to respire adequately over a seven day storage period was far greater than the typical <5% O2 provided in typical commercial MA packaging applications. This insufficient level of O2 utilised in MAP could lead to anaerobic respiration with the production of undesirable metabolites and associated physiological disorders [The data generated in this study showed that the amount of Oisorders . 2 utilisation can be categorised in a table representing each salad leaf in the order of increasing respiratory levels. 2 consumption by individual salad leaves in order of increasing O2 utilisation on storage days 3, 5 and 7. Escarole, Lollo Rosso and Frisee were consistently the slowest respiring salad leaves while Cos, Radicchio and Iceberg were consistently found to utilise the greatest amount of in-pack O2. Individually packed leaves underwent microbial (TVC) testing.It is apparent that each leaf respires at different rates under the same conditions. The level of OEach individually packed salad leaf was measured by total viable count (TVC) assay. Testing was carried out on day 1, 3, 5, 7 and 10 to establish the microbial load in colony forming units per gram of sample. 2 over 10 days and had the second lowest log TVC value. Results can also be ranked in terms of which salad leaf has the lowest microbial load in order to understand the difference in microbial quality across all samples. This can be seen in In the case of Radicchio, a correlation between highest level of oxygen utilisation of all leafs and microbial counts at day 7 are observed. The inverse of this can be seen with Lollo Rosso, where it was found to consume the lowest level of O2 utilised by each salad leaf , an experimental salad mix was formulated using the four lowest respiring leaves determined by the O2 sensor previously. This Italian-styled formulation (Mix 3) and two similar commercially available formulations (Mixes 1 and 2) were modified atmosphere packaged using the same packaging conditions, in the same manufacturing plant, using salad materials grown and processed in the same manner. All three salad mixes were produced in volume and a shelf-life evaluation study undertaken. Oxygen measurements were taken throughout product storage and optical O2 sensor readings showed that Italian salad Mix 3 had the lowest O2 consumption of all three salad mixes  of all seven leaf types assessed previously and commonly found in Italian-style mix leaf salads. Salad Mix 2 was also comprised of the Cos leaf, which was also found to be amongst the highest O2 consumers (13.33%) of all leaves assessed. Consequently, the use of Radicchio and other high respiring leaves in mixes ultimately results in a final product that consumes greater amounts of O2. Therefore, it was essential to provide a modified atmosphere with enough O2 to allow sufficient O2 for respiration irrespective of what leaves selected. It is important to recognize that extremely low O2 levels or excessively high CO2 levels can result in the generation of off-flavours or visible tissue damage [Based on the results obtained by monitoring the level of Oad mixes . After sns Mixes  and 2 wee damage . 2 over time. By day 7, Mix 3 was found to still maintain acceptable limits of <log 6 [2 over time appears to result in a product that has a lower microbial count than that of one which consumes more.All three salad leaf mixes were assessed for microbial quality during refrigerated storage . Microbif <log 6 ,23, wherp ≤ 0.01), flavour (p ≤ 0.05) and acceptability (p ≤ 0.01) were significantly and positively correlated. Day 7 data contrasted significantly with salad samples found to be negatively correlated with overall appearance, flavour and acceptability, along with a positive correlation for wilting appearance (p ≤ 0.01) and non-crispy texture (p ≤ 0.01). As for the individual salads, it was apparent that salad Mix 3 was favoured amongst panellists over salad Mixes 1 and 2, where overall appearance and acceptability were significantly correlated (p ≤ 0.01) and descriptors such as wilting appearance and non-crispy texture were found to be negatively correlated (p ≤ 0.05) showing a true preference for salad Mix 3. This selection appears to be attained by the use of Red Batavia instead of Radicchio. Panellists were asked if any individual leaf was found to be particularly undesirable and in this case 16 of the 26 panellists (61.5%) found the Radicchio leaf to be undesirable within the salad mix. The Radicchio leaf is understood to be a spicier and a more bitter intense leaf [2 (21%) in packaging did not appear to have an adverse affect on browning of the salad leaves. Data derived by Jack-Knifing uncertainty testing for individual salad leaves find no significance in LSB or LEB. Salad Mixes 1, 2 and 3 were assessed for sensory acceptability over the seven day storage period . Panellists were made aware of relevant descriptors  and ratinse leaf  which mo2 sensor in determining the oxygen respiration levels shows a novel and effective tool in understanding the O2 levels used in respiring salads leaves packaged in modified atmosphere conditions. This valuable information let the development of a product with a longer, more stable shelf life and a product more acceptable to consumers.With the understanding that Mix 3 was found to be the most desirable in terms of sensory analysis, the formulation of a mix with a lower microbial load and slower respiration rate compared well to common commercially produced salad mixes. The use of an optical O2 utilised by individual salad leaves was successfully achieved using optical O2 sensors. Information gathered using O2 sensors showed that an O2 level in excess of 10% is required to provide salad leaves with enough O2 to comfortably respire within a typical shelf-life period of seven days. The apparent lack of O2 within such packs has been previously shown to cause many deleterious quality processes. The use of optical O2 sensors showed that a salad mix can be optimised by understanding specific product requirements, like that for oxygen, and subsequently, careful product selection; thereby assisting in extending product shelf-life and promoting quality.The ability to non-destructively assess the level of O


Systemic therapy for cutaneous squamous cell carcinoma is typically reserved for rare cases of metastatic disease or lesions which cannot be adequately managed with local therapies. The relevant published literature is comprised only of case series and reports. Herein the authors present two cases of neglected locally advanced cutaneous squamous cell carcinoma not initially amenable to local therapies, which achieved dramatic partial responses to palliative Cisplatin and Docetaxel, facilitating sequential palliative radiotherapy. These reports add to the limited literature supporting the use of platinum-based therapy in locally advanced and metastatic cutaneous squamous cell carcinoma. They are the first describing the use of Cisplatin and Docetaxel in this setting. Non-melanomatous skin cancers are the most common forms of human malignancy, with more than 1 million cases expected to be newly diagnosed in the United States in 2009 . The majThe authors here aim to contribute to the literature by reporting two cases of locally advanced and metastatic cutaneous SCC treated with sequential palliative chemotherapy and radiotherapy.2 and Docetaxel 55 mg/m2 every 3 weeks. He tolerated 12 cycles extremely well with only mild myelosuppression, oral mucositis and tinnitus. The chest lesion showed an early and sustained dramatic response to therapy  due to myalgias, oral mucositis and 2 episodes of febrile neutropenia requiring hospitalization and G-CSF support. The visible lesion showed an early and sustained significant response to therapy : 1 of these then had surgery alone, 2 had radiotherapy alone and 1 had both, resulting in 2 patients having no evidence of disease (NED) at 13 and 22 months post-chemotherapy respectively, 1 having NED after further surgery and radiotherapy required for a local recurrence at 8 months post-chemotherapy, and 1 dying of disease at 19 months post-chemotherapy. Seven of the 13 patients achieved a partial response (PR): 2 of these then received no local therapy, 3 had radiotherapy alone, and 2 had surgery and radiotherapy, resulting in 4 patients dying of their disease at 12, 11, 8 and 4 months post-chemotherapy respectively, and 3 having NED after 12, 11 and 10 months post-chemotherapy respectively. One of the 13 patients had no response to chemotherapy, received subsequent interferon and died of disease 10 months post-chemotherapy. The last of the 13 patients progressed through chemotherapy, received subsequent retinoids and died of disease 4 months post-chemotherapy. Overall then, of 13 evaluable patients, 6 had NED and 7 had died of their disease at the time of manuscript preparation.Sadek et al.  reported2/d for 4 days and Bleomycin 20 mg/d for 4 days via continuous infusion. The first patient received a reduced dose of Bleomycin (15 mg/d). Cycles were repeated every 3 weeks. The first patient achieved a CR, the second a PR, and the third progressed through chemotherapy. All underwent subsequent surgery within 3 weeks. No recurrences had been recorded during short follow-up periods of between 2 - 9 months.Denic  reported2 and Doxorubicin 50 mg/m2 every 3 weeks for up to 4 cycles. Seven of the 12 patients received chemotherapy alone; 2 of these 7 achieved a CR and had NED at 12 and 4 months from the initiation of chemotherapy respectively, 2 achieved a PR (1 for 3 months before dying of progressive disease and 1 for 6 months before developing progressive disease), and 3 patients had no response to chemotherapy . One of the 12 patients received chemotherapy followed by surgery which resulted in a PR after chemotherapy and a CR of 11 months’ duration following surgery. The remaining 4 of 12 patients received chemotherapy followed by radiotherapy: 2 had no response to chemotherapy but achieved CRs of 32 and 30 months post-radiotherapy respectively, 1 achieved a CR after chemotherapy that persisted for 13 months post-radiotherapy and 1 achieved a CR after chemotherapy that persisted for 11 months post-radiotherapy until that patient died of comorbid lung cancer.Guthrie et al.  also rep2 on day 1 and 5-Fluorouracil 1 g/m2/d via 4-day continuous infusion. Cycles were repeated every 3 weeks. Patients received between 2 - 6 cycles. The first patient with metastatic disease achieved a CR and had NED 24 months following chemotherapy. The second patient achieved a PR and developed progressive disease 3 months following chemotherapy. Two patients with locally advanced disease achieved CRs; one was lost to follow-up 3 months following chemotherapy, and the other died of a myocardial infarction but had NED 13 months following chemotherapy. Two patients with locally advanced disease achieved PRs; one achieved a subsequent CR following consolidative surgery and the other refused chemotherapy after 2 cycles and outcome data was not provided. One patient with locally advanced disease had no response to 6 cycles of chemotherapy and died of extensive locoregional disease and a CVA.Khansur and Kennedy  reported2/week). The patient recurred one month later, but achieved a second CR after 7 additional cycles before developing progressive disease requiring 2 cycles of palliative 5-Fluorouracil (600 mg/m2/d) for 5 days every 2 weeks. The patient progressed and died of disease 1 month later. Alternatively, Wollina et al. [2 on days 1 - 13 and subcutaneous interferon-α-2a 3 x 3 million units 3 times weekly for 3 cycles).Other options for systemic treatment include retinoids and interferon-alpha. Lippman et al.  reporteda et al.  describeTo the authors’ knowledge the present reports are the first describing the use of Cisplatin and Docetaxel in the management of locally advanced or metastatic cutaneous SCC. The rationale for their use was similar to that employed within other reported series; the agents have proven efficacy for SCC in other disease sites , they ar


Plasmodium falciparum protein, Pfs25, produced from the baculovirus expression system. Pfs25 is a malaria transmission-blocking vaccine candidate, with a compact and complex structure including 22 cysteines. This supplementary data is related to the research “Disulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate”  [1]. In brief, Pfs25 was digested with trypsin/Lys-C and derived peptides separated by High Performance Liquid Chromatography (HPLC) and analyzed by mass spectrometry (MS) by MSE fragmentation. The theoretical peptides and their respective masses along with disulfide bond locations with linked peptides are presented here alongside the mass spectrometry analysis. The raw mass spectrometry data is made available through the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.This article contains the peptide data obtained while performing disulfide bond mapping of the recombinant  Specifications TableValue of the data•The derived peptides and mass spectrometry data is provided here for further details from the disulfide bond analysis of Pfs25.•The disulfide bond locations and linked peptides are discussed alongside the mass spectrometry analysis here and data made accessible to the scientific community.•Pfs25 is a compact and complex 17.9 kDa protein with 22 cysteines (11 disulfide bonds) that has presented difficulty in prior disulfide bond analysis•A method was developed to map the disulfide bonds of a complex and compact protein, which may be applicable to other proteins, an important step in recombinant protein development for vaccines.1The Pfs25 disulfide bond mapping peptides are discussed in further detail in this manuscript to further support the elucidation of disulfide bonds of Pfs25 as discussed in Utilizing Biopharmalynx 1.3 the mass spectral data was analyzed and compared to the theoretical peptides to obtain the localization of the 11 disulfide bonds present in the recombinant Pfs25. The disulfide bond locations and linked peptides  masses are presented in 210 and Cys24. The remaining ten fragment ions were consistent with constituent peptides  were consistent with an internal disulfide bond linkage between Cyspeptides , Fig. 5.795 to Cys113 linkage. Four fragment ions  were specific to the linkage between T14 and T16 and confirmed the linkage of Cys115 to Cys129. A further 21 fragment ions were consistent with the linkage of T12, T14, and T16 and remaining 11 fragments consistent with constituent peptides  were specific to the linkage between T12 and T14 and confirmed the Cyspeptides , Fig. 6.8137 and Cys148. Ten fragments were consistent with constituent peptides  and a 2489 UV/Vis Detector  set at 214 nm. An XBridge  BEH 300 C18  was used at a column temperature of 37 °C and gradient with 0.1% Triflouroacetic acid (TFA) in purified water (Mobile Phase A) and 0.1% TFA in acetonitrile (Mobile Phase B) as described in 10.3E mode using MassLynx v4.1 . RAW MS files have been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.MS analysis was done with a QTOF Premier mass spectrometer  equipped with an electrospray source as described in 10.4The mass spectral data was analyzed using BiopharmaLynx 1.3  as described in