GcvB inhibits yifK mRNA translation by pairing with a sequence immediately upstream from the Shine-Dalgarno motif. Surprisingly, we found that some target sequence mutations that disrupt pairing, and thus were expected to relieve repression, actually lower yifK expression and cause it not to respond to GcvB variants carrying the corresponding compensatory changes. Work prompted by these observations revealed that the GcvB target sequence in yifK mRNA includes elements that stimulate translation initiation. Replacing each base of an ACA trinucleotide near the center of the target sequence, by any other base, caused yifK expression to decrease. Effects were additive, with some triple replacements causing up to a 90% reduction. The enhancer activity did not require the ACA motif to be strictly positioned relative to the Shine-Dalgarno sequence, nor did it depend on a particular spacing between the latter and the initiating AUG. The dppA mRNA, another GcvB target, contains four ACA motifs at the target site. Quite strikingly, replacement of all four ACAs by random trinucleotide sequences yielded variants showing over 100-fold reduction in expression, virtually inactivating the gene. Altogether, these data identify the ACA motif as a translation-enhancing module and show that GcvB's ability to antagonize the enhancer function in target mRNAs is quintessential to the regulatory effectiveness of this sRNA.GcvB is an archetypal multi-target small RNA regulator of genes involved in amino acid uptake or metabolism in enteric bacteria. Included in the GcvB regulon is the  Salmonella, we discovered that the sequence base-pairing with GcvB in the target mRNA functions as a translational enhancer. Replacing an ACA motif near the center of the sequence with unrelated trinucleotide sequences leads to a decrease in translational initiation efficiency that can be as severe as more than 90%. Interestingly, some of these replacements concomitantly render the mRNA insensitive to GcvB variants carrying the appropriate compensatory changes, suggesting that targeting the enhancer element is paramount for GcvB regulatory effectiveness. Overall the data presented in the paper unveil the role of the ACA motif in the translation initiation process and lay the grounds for further analysis of the mechanism involved.The majority of small RNA (sRNA) regulators in bacteria act by inhibiting translation initiation in target messenger RNAs. The study of this regulatory mechanism not only allows a better understanding of sRNA function but it can also provide new insight into aspects of the translation initiation process that remain incompletely characterized. This was the case in the work described here. Analyzing the mechanism by which GcvB, a multi-target sRNA, downregulates a putative amino acid transporter in  E. coli and SalmonellaA relevant chapter in the expanding field of RNA-mediated gene regulation is devoted to the activities of multi-target trans-encoded small RNAs in bacteria. Acting in concert with chaperon protein Hfq, these RNA regulators function by base-pairing with short, often imperfectly complementary sequences in the 5′ untranslated regions (UTR) of target messenger RNAs. They can affect translation and turnover of several mRNAs simultaneously thus reprogramming gene expression of whole gene families in a coordinate manner in response to environmental cues  upstream from the translation initiation codon. Removal of this sequence (as part of a 27 nt deletion), besides causing the loss GcvB regulation, affected gltI translation, suggesting that the CA-rich element acts as a translational enhancer. Consistent with this interpretation, crafting the 27 nt segment at the corresponding position of an unrelated mRNA conferred simultaneously GcvB control and increased translational efficiency GcvB uses a specific sequence region to pair with most, although not all lac-based genetic screen aimed at identifying genes controlled by trans-encoded small RNAs in Salmonella. A random library of lacZ fusions to chromosomal genes was generated using a phage Mu-derived transposon (MudK) and screened for isolates whose LacZ levels changed (either increased or decreased) upon inactivating Hfq hfq mutant background, carried the lacZ insert translationally fused to the yifK gene yifK might be a GcvB target. We thus proceeded to test this hypothesis and characterize yifK regulation. While this work was underway, Sharma and coworkers identified yifK mRNA as a member of the gcvB regulon by microarray analysis; however, these authors could not confirm direct regulation by GcvB due to low reporter fluorescence of the yifK-gfp fusion used in the study yifK expression might be indirect.Some years ago, our laboratory performed a in vivo and in vitro evidence that GcvB downregulates yifK directly by pairing with a sequence immediately preceding the Shine-Dalgarno (SD) motif in yifK mRNA. A surprising observation in the course of this study was that some target sequence mutations that disrupted pairing did not cause yifK expression to increase – as expected for the relief of GcvB repression – but had the opposite effect. The drop in expression was not suppressed by deleting gcvB nor was it accentuated in a GcvB mutant carrying the appropriate compensatory changes. Closer analysis revealed that the GcvB target sequence includes elements that stimulate yifK mRNA translation. In the absence of such elements, the role of GcvB pairing in regulation becomes marginal.Here we present lac) transposon in the yifK gene; one predicted to produce a LacZ protein fusion to the 48th amino acid (aa) of the 461 aa YifK (yifK87::MudK); the other with LacZ inserted after the 95th aa of YifK (yifK88::MudK) yifK87::MudK produced significantly higher ß-galactosidase activity and was chosen for the present study. A survey of protein sequence databases showed YifK to be a highly conserved protein with the characteristic signature of amino acid transporters. The known role of GcvB in the regulation of some members of this family made this small RNA the likeliest candidate to control yifK expression. This was confirmed by deleting the gcvB gene and testing the effects of the deletion on the expression of the yifK87::MudK fusion (hereafter referred to as yifK-lacZY). As shown in gcvB deletion causes a nearly 5-fold increase of ß-galactosidase activity in exponentially growing cells, while effects decline in stationary phase. Somewhat surprisingly, LacZ levels in the gcvB-deleted strain are not as high as the levels measured in a strain deleted for hfq  under error-prone conditions and introduced into the chromosome of a strain harboring the yifK-lacZY reporter fusion via lambda red recombination. Most of the isolates originating from the gcvB mutagenesis carried changes in the gcvB promoter or in the promoter of the adjacent gcvA gene  that might constitute a site for Hfq binding hfq background , yifK mRNA being otherwise undetectable when expressed from the wild-type promoter (data not shown). The analysis identified two yifK mRNA species, a 1.4 kilobase (Kb) mRNA covering just the yifK coding portion and a longer, 2.0 Kb RNA extending into the adjacent argX-hisR-leuT-proM tRNA operon. As shown in yifK translation in ΔgcvB or Δhfq cells protects the 1.4 Kb RNA against RNase E cleavage. To confirm this interpretation, the analysis was repeated with strains that, besides the promoter “up” mutation, carried a mutation in the Shine-Dalgarno sequence . As shown in gcvB or Δhfq strains but not in the rne ts mutant, consistent with the idea that reduced translation renders yifK mRNA susceptible to RNAse E degradation.Northern blot analysis was used to assess the effects of GcvB regulation on yifK and the tRNA operon suggests that the 1.4 Kb RNA originates from processing of the longer form. Likely, under normal conditions  yifK transcription contributes only to a small fraction of the four tRNAs, as most the tRNA operon transcription results from a strong promoter located immediately upstream from the argX gene E.coli, showed that this tRNA precursor is processed by the concerted actions of RNase E and RNase P in a pathway that, intriguingly, also sees the participation of Hfq Absence of any obvious transcription termination signals in the intercistronic region between yifK expression was exquisitely sensitive to the growth medium and virtually silenced in minimal medium. As a result, a strain with the yifK-lacZY fusion is phenotypically Lac− when plated in minimal medium. We exploited this phenotype to positively select for spontaneous Lac+ mutants. The selection yielded two classes of mutations, one genetically linked to the yifK-lacZY locus, the second mapping elsewhere. All of the linked mutants that were analyzed were found to harbor the -33 C:G to A:T promoter change obtained previously (see above). The unlinked mutations mapped in a chromosomal interval encompassing the gene for leucine response regulator, Lrp. Prompted by this observation, we introduced an lrp insertion mutation into the yifK-lacZY-containing strain. The resulting strain acquired a Lac+ phenotype . Trying to clarify this observation, portions of the region of interest were mutagenized separately. As shown in yifK-lacZY repression. Repression is restored upon combining the compensatory alleles. Thus, this portion of the target sequence behaves as expected, and the behavior of the compensatory mutant strongly suggests that GcvB represses yifK through a base-pair interaction. In vitro toeprint experiments, showing that GcvB inhibits the binding of ribosomal 30S subunit to yifK translation initiation site, specifically and in a dose-dependent manner  between the most conserved base of the SD motif yifK-lacZ expression drops sharply in both constructs . Similar effects were observed in a separate construct where the segment between SD sequence and the initiating AUG was replaced by the sequence found at the corresponding position in the chiP gene ifK expression to be less sensitive to repression by GcvB.A peculiarity of the dppA gene, a major GcvB target dppA mRNA includes four ACA motifs clustered within a 15 nt segment near the SD sequence . In the next experiment, we randomly mutagenized all four ACA repeats (in the same lacZ fusion background) and screened the mutants on MacConkey lactose indicator plates. Out of 43 mutants analyzed, 18 formed white colonies and had ß-galactosidase activities ranging between 0.5 and 5% of the wild-type levels. Four representative isolates from this group are shown in − mutants. 4 of the initial 43 mutants formed red colonies and expressed significant levels of ß-galactosidase  previously showed that GcvB's target sequence in the gltI gene of Salmonella acts as transferable translation enhancer  at the chromosomal site to be modified and: 2) replacing the tetAR module by a DNA fragment carrying the desired changed through positive selection tetracycline-sensitive recombinants Scarless modification of chromosomal DNA sequences at the single base-pair level was achieved with a two-step recombineering procedure as previously described 600 of 0.35) as previously described 32P-labeled primer ppF49. The same DNA primer was used for the sequencing reactions. Reactions were performed with the fmol DNA Cycle Sequencing System from Promega, according to the manufacturer's protocol. Reaction products were fractionated on a 10% polyacrylamide-8 M urea gel. For Northern blot analysis, RNA was fractionated on a 1% agarose-formaldehyde gel, blotted onto a nylon membrane, and hybridized to the appropriate 32P-labeled DNA oligonucleotide probes.RNA was prepared by the acid-hot-phenol method from exponentially growing cells (ODIn vitro coupled transcription/translation was performed using New England Biolabs' PURExpress In vitro Protein Synthesis kit (NEB #E6800) according to the manufacturer instructions. Genes to be analyzed were cloned under T7 promoter control in the DFRH plasmid provided with the kit. The hybrid genes carried yifK wt or mutant 5′ UTR sequences fused to the cat-3×FLAG coding sequence (chloramphenicol acetyl transferase in-frame fusion to the 3×FLAG epitope). Final volume of the transcription/translation reaction was 25 µl in all cases. In addition to kit solutions A and B, reaction mix contained, 10 U of RNase inhibitor SUPERase (Ambion) and template plasmid DNA added to either 0.5 or 5 pM final concentration. Incubation times at 37°C varied from 15 to 90 min. Reactions were stopped by addition of equal volume of 2× Laemmli buffer and immediate freezing. Aliquots were loaded on 12.5% Acrylamide gels and Western analysis performed as previously described et alyifK mRNA were synthesized in vitro from T7 DNA templates generated by PCR amplification of chromosomal DNA (from strains MA8020 or MA11793) with primers ppI22 and ppI23. 2 pmol of RNA were annealed with 5′end-labeled primer ppI23 (1 pmol) in 10 mM Tris-acetate [pH 7.6], 0.1 M potassium acetate, and 1 mM DTT for 1 min at 90°C and chilled in ice for 5 min. Then, all dNTPs , Mg Acetate  were added; this was followed by preincubation with 2 pmol of 30S ribosomal subunit (a gift of Dominique Fourmy and Satoko Yoshizawa) at 37°C for 5 min. In experiments involving GcvB, 5, 1 or 0.5 pmol of sRNA were added prior to both, addition of the 30S ribosomal subunit and the preincubation step. After the 5-min period, 2 pmol of tRNAfMet were added and preincubation at 37°C continued for 15 additional min. Finally, Reverse Transcriptase  was added and samples incubated for 15 min at 37°C. Following phenol chloroform extraction and ethanol precipitation, resuspended samples were loaded onto a 10% polyacrylamide-8 M urea gel along with the sequencing reaction samples generated with the same primer.Toeprinting reactions were carried out as described by Darfeuille 600≈0.7). All experiments included parental or reference strains as normalization controls. Standard deviations were generally less than 5% of the mean.β-galactosidase activity was assayed in toluene-permeabilized cells as described in Figure S1gcvB-linked mutations relieving yifK repression. A DNA fragment spanning the gcvB gene and a linked cat marker  was amplified by PCR under error-prone conditions using oligonucleotides ppF17 and ppF18 as primers (yifK-lacZY/pKD46) and recombinants were selected on MacConkey lactose plates supplemented with chloramphenicol as described in the text. Red-colored colonies were picked and the region of the gcvB locus analyzed by DNA sequencing. Most of the isolates were found to harbor DNA sequence changes in the gcvA-gcvB intergenic region, which affected either the -35 or -10 box of the gcvB promoter, or the -10 box of the gcvA promoter gcvA coding sequence and a mutation affecting the CG-rich stem of gcvB's Rho-independent terminator were also identified (data not shown). primers  and chro primers  as templ(TIF)Click here for additional data file.Figure S2lrp insertion mutant as a function of the growth medium. Bacteria were grown in minimal medium . The probes used were ppI67 (GcvB) and ppB10 (5S)(Comparing GcvB sRNA levels in wild-type and in an dium NCE  supplemepB10 (5S).(TIF)Click here for additional data file.Figure S3dppA-lacZ fusion by GcvB variants. GcvB alleles GA84,85 and ACA86–88 fall outside the portion of GcvB that pairs with dppAWT in down-regulating dppA-lacZ. This shows that the sequence changes do not affect the functioning of the GcvB. The Δ symbol denotes a complete deletion of the gcvB gene.Regulation of (TIF)Click here for additional data file.Figure S4yifK translation start site in members of the Enterobacteriaceae family. Sequences (from NCBI database) are 100% identical among isolates from the same species. Green shading denotes conservation of the ACA motifs analyzed in in this study.Alignment of sequences preceding (TIF)Click here for additional data file.Figure S5yifK variant with optimal spacing between the SD and the initiator AUG still depends on the upstream ACA for optimal expression. Replacing the upstream ACA with GGG lowers expression as well as the response to GcvB repression (see main text and A (TIF)Click here for additional data file.Table S1Salmonella enterica serovar Typhimurium strain MA3409 dppA::pCE40 (lacZY) fusion was constructed as described Relevant strains used in this work. Strains are derived from (DOCX)Click here for additional data file.Table S2DNA oligonucleotides used in this work. Changes from the wild-type sequence are in red. Nucleotide insertions are in bold and underlined. “N” denotes an equimolar mixture of all four nucleotides, “V” denotes an equimolar mixture of A, C and G; “H” denotes an equimolar mixture of A, T and C.(DOCX)Click here for additional data file.Table S3Salmonella strains used as source of DNA; described in the text) or from primers annealing to each other (“self”). Further details on DNA oligonucleotide primers are given in Relevant alleles constructed in this work. Template DNA was from plasmids pSEB5  or chrom(DOCX)Click here for additional data file.


RET oncogene have been directly associated with multiple endocrine neoplasia type 2 and hereditary medullary thyroid carcinoma, other mutations are classified as variants of uncertain significance (VUS) until the associated clinical phenotype is made clear. Currently, some 46 non-synonymous VUS entries exist in curated archives. In the absence of a gold standard method for predicting phenotype outcomes, this follow up study applies feature selected amino acid physical and chemical properties feeding a Bayes classifier to predict disease association of uncertain gene variants into categories of benign and pathogenic. Algorithm performance and VUS predictions were compared to established phylogenetic based mutation prediction algorithms. Curated outcomes and unpublished RET gene variants with known disease association were used to benchmark predictor performance. Reliable classification of RET uncertain gene variants will augment current clinical information of RET mutations and assist in improving prediction algorithms as knowledge increases.Although reported gene variants in the  Medical genetics involves diagnosis, management, and determining risk of hereditary disorders The outlay of time and labor to validate the disease association concerning a variant of uncertain significance (VUS) within the coding portion of a gene can be daunting and cost prohibitive There are established methods for predicting mutation severity based on amino acid substitution penalties, structural disruption, sequence homology (ortholog conservation) or neural nets, such as PolyPhen RET gene (REarranged during Transfection) is located on chromosome 10q.11 codes for 20 exons. The transcript length is 5,659 bps and translates to the 1,114 amino acid residue protein  as shown in RET plays a critical role in neural crest development. It can also undergo oncogenic activation in vivo and in vitro by cytogenetic rearrangement. It can be further classified by Gene Ontology (GO) categories (www.geneontology.org) of biological process of homophilic cell adhesion, posterior midgut development, and protein amino acid phosphorylation. Its GO annotated cellular location is component integral to membrane and the GO category of molecular functions lists ATP binding, calcium ion binding and transmembrane receptor protein tyrosine kinase activity. Functional domains of the RET protein are also summarized in The human RET is essential for the development of the sympathetic, parasympathetic and enteric nervous systems. Disruption of function by germline mutations in RET have been associated with several diseases in humans including three related inherited cancers: multiple endocrine neoplasia type IIA (MEN2A), multiple endocrine neoplasia type IIB (MEN2B), and familial medullary thyroid carcinoma (FMTC). RET gene variants may vary in functional severity, where some are reported as benign, some pathogenic, and some of uncertain significance. Curated RET oncogene mutations have been recently reported by Margraf et al. RET gene variants has been curated as benign (6%), pathogenic (52%) and VUS (42%), meaning unknown or uncertain association with disease or phenotype outcome. This archive currently hosts 146 RET variants, including 62 VUS entries that can be accessed at http://www.arup.utah.edu/database/.Although well understood codon changes often guide patient therapy or surgical options Accurate prediction of disease association for novel mutations and uncertain gene variants is of great importance to medicine and biology. Informatics tools for predicting disease severity of uncertain gene variants will aid in the improvement of genetically-informed patient care. With a rapidly growing number of on-line resources for gene variants collections, the opportunity to apply machine learning algorithms to well curated disease causing gene sets becomes increasingly desirable.RET proto-oncogene into categories of benign or pathogenic. The PSAAP algorithm performance has also been compared to four well-established prediction tools available on-line and agreement between algorithms summarized.The absence of any gold standard for predicting phenotype severity in uncertain gene variants prompts two questions. Are algorithms trained specific to a gene/disease setting more appropriate to use than generalized on-line prediction tools? Does agreement between several and varying algorithms influence clinician decision-making? This study expands a recently reported algorithm, we here term Primary Sequence Amino Acid Properties (PSAAP), which uses feature selected amino acid physicochemical properties of primary amino acid sequence RET curated mutations was used to evaluate performance of different categories of classifier algorithms. The best performing algorithm (using Weka) was Naïve Bayes. Algorithm metrics for this novel Bayes classifier of RET disease outcome were calculated using the above test set data. Evaluation of the classifier yielded a sensitivity of 0.938, specificity of 0.867 and positive predictive value (precision) of 0.883. Performance for our Primary Sequence Amino Acid Properties (PSAAP) classifier is summarized in RET gene variants with known disease outcomes. Following the 88% of the PSAAP classifier, MutPred was next closest to predicting the correct disease outcomes for the known RET variants with 84% precision. PolyPhen yielded the highest specificity for RET variant disease association of 92%, yet had the lowest precision at 54%. PMut correctly predicted gene variant disease outcomes with 72% precision but had the lowest specificity at 59%. RET mutations using the four established prediction algorithms.The independent test set of RET non-synonymous VUS mutations (n = 46) was performed using our recently reported algorithm Next, evaluation of RET uncertain gene variants (VUS) using the established on-line prediction tools are also summarized in Results from analysis of the RET benign variants, 7 of 24 agreed across all algorithms, while only 6 of 22 predicted pathogenic RET variants showed agreement across the different methods. Although only 13 out of 46 (28%) were concordant, these variants may count as having a higher degree of confidence in prediction due to the varied methodologies and basis of classification. Importantly, the focus of molecular research and clinical efforts could therefore be directed to this prioritized listing of RET uncertain variants. Curated variants are shown mapped across the length of the protein in Of special interest, for predicted RET gene variants with known pathological (MEN2) outcomes (n = 5) were identified during routine genetic testing at ARUP Laboratories. To further benchmark a gold standard of truth for RET mutation prediction, all five algorithms were used to classify this set of not yet seen variants. Our novel Bayes trained PSAAP classifier correctly identified all five variants as pathogenic. PMut called 3 disease causing variants correctly, but classified two others as “neutral” mutations, when in fact these changes were known to be associated with disease. PolyPhen also correctly identified 3 as probably damaging (pathogenic), but missed classified the same 2 variants as PMut. SIFT predicted 4 of these variants would affect function (pathogenic), but called one of the same variants “tolerated.” MutPred correctly predicted all 5 as pathogenic.Finally, several unpublished RET proto-oncogene have been directly associated with MEN2 and hereditary medullary thyroid carcinoma, and provide guidance for patient care. Accurate classification of phenotype severity for novel mutations and uncertain variants as relating to disease is of great importance to proper patient care. Although correlation of genotype-phenotype offers therapy options that would otherwise remain hidden and may lead to disease specific mutation-guided management strategies, appropriate caution is justified when clinicians are asked to trust computational outcomes for determining patient care Mutations in the RET gene variants using primary amino acid sequence properties and Naïve Bayes gene-specific and clinically curated disease outcomes. Comparison of this recent PSAAP algorithm with established on-line prediction tools may improve our understanding of predicting mutation status in the RET proto-oncogene.On-line mutation prediction tools have been available for many years. Prediction tools such as PolyPhen Sorting Intolerant From Tolerant (SIFT) was first published in 2003 by Ng and Heinikoff from work done at the Fred Hutchinson Cancer Research Center in Seattle Polymorphism Phenotyping (PolyPhen) is an EMBL based tool from 2002 from Ramensky et al. MutPred is a recently developed prediction algorithm by Li, Mooney and Radivojac PMut was first published in 2005 by the Molecular Modeling Unit at the Institut de Recerca Biomédica, Parc Científic de Barcelona, Spain RET variants and associated MEN2 disease demonstrates our PSAAP classifier model compares very well to other established prediction tools. A distinguishing feature of the PSAAP model herein reported is the algorithm was trained specifically to curated RET disease outcomes, as summarized in RET variants as compared to generalized prediction tools available on-line.Benchmarking the established prediction algorithms with curated RET variants warrant closer investigation by traditional and molecular techniques. Furthermore, algorithm agreement in a clinical setting may be just as important for “benign” as it might be for “pathogenic.”Ranking agreement of predicted phenotype severity across several complimentary algorithms may provide an additional level of clinical confidence in computational classifiers. At a minimum, these five all-in-agreement “predicted pathogenic” what was found, what does it mean and what to do about it can be answered for each individual patient and genetic test result. Among the key features critical for a decision support framework in clinical genetic testing is a reliable phenotype classification tool and scoring metric to predict consequences of a variation that alters protein structure. For these uncertain gene variants, the in-house algorithm trained specifically on available RET curated outcomes seems to outperform well-established and generalized prediction tools available on-line. More importantly, agreement between several predictors may provide research priority for novel and uncertain gene variants.Personalized treatment in genomic medicine cannot advance until questions such as RET proto-oncogene into categories of benign or pathogenic. This novel application of classification algorithms for computational prediction of phenotype severity in uncertain gene variants could be generally applied to any gene-disease setting where a corpus of curated gene variants are trusted and where reported mutations impact clinical care.The use of machine learning algorithms to classify uncertain gene variants in disease is a promising tool to strengthen our underlying knowledge of disease pathogenesis. Software algorithms to better classify gene variants of uncertain significance are necessary to move translational research forward. This follow up study used the PSAAP algorithm to “reclassify” 46 variants of uncertain significance within the RET variants were characterized by physicochemical differences in primary amino acid sequence resulting from the mutation. Attributes of mutation status were characterized using values of 544 physical, chemical, conformational, or energetic properties (AAindex v9.4) RET variant, matrices of delta values for each biochemical property of the substituted amino acid were calculated by Python scripting and the resulting mutation described by an array of variables archived using SQL - where each matrix corresponds to the absolute value of the difference between the value of the property in the amino acid present in the wild type and the one in the mutant.Non-synonymous RET mutations with known pathogenicity was used to train and test machine learning classification algorithms. Although training and test sets included different disease subtypes such as MEN2A (n = 40), MEN2B (n = 3), FMTC (n = 5), MEN2A and FMTC (n = 36) - class labels of “pathogenic” and “benign” were used to describe all curated disease association. Random selection was used to build a 2/3 training set (n = 56) and 1/3 test set (n = 28). Attribute selection (feature selection) was performed during classification training/testing. Machine classification algorithms were implemented using the Weka software package (v3.6) As previously described, representative algorithms from different categories of classification  were evaluated for their ability to correctly predict mutation status in the training set RET uncertain variants (n = 46) was then analyzed using our PSAAP  classification algorithm. The workflow of our PSAAP algorithm is summarized in The PSAAP algorithm performance was evaluated using the test set, with sensitivity (true positive rate), specificity (true negative rate), and positive predictive value (precision) calculated. A data set of non-synonymous RET mutations (known disease association) and RET uncertain variants (VUS data) were analyzed and compared using four existing mutation prediction algorithms. These established prediction tools are mainly based on phylogenetic properties such as sequence homology, amino acid substitution penalties or structural disruption. MutPred (mutdb.org/mutpred) RET variants and RET VUS entries were evaluated using respective default settings.Next, both curated RET disease variants (n = 5) with known pathogenic outcomes  were identified during routine genetic testing at ARUP Laboratories. This nascent set of RET variants was also analyzed and compared by all prediction algorithms to further benchmark some standard of performance and precision. Data and methods used for this study were approved by the Institutional Review Board of the University of Utah.Finally, several unpublished


Drosophila embryo resulted in localized alterations in nuclear morphology and cellular movement. In addition, global defects in germ-band (GB) extension and retraction are observed when external force is applied during morphogenetic movements, suggesting a long-range physical coupling within the GB layer of cells. Further local application of force resulted in redistribution of non muscle myosin-II in the GB layer. Finally these perturbations lead to altered segmental gene (engrailed) expression patterns later during the development. Our observations suggest that there exists a tight regulation between nuclear morphology and cellular adhesive connections during morphogenetic movement of cells in the embryo. The observed spatial changes in patterning genes, with perturbation, highlight the importance of nuclear integrity to cellular movement in establishing gene expression program in a developmental system.The development of an organism is accompanied by various cellular morphogenetic movements, changes in cellular as well as nuclear morphology and transcription programs. Recent evidence suggests that intra and inter-cellular connections mediated by various adhesion proteins contribute to defining nuclear morphology. In addition, three dimensional organization of the cell nucleus regulate the transcription programs. However the link between cellular morphogenetic movements and its coupling to nuclear function in a developmental context is poorly understood. In this paper we use a point perturbation by tissue level laser ablation and sheet perturbation by application of force using magnetic tweezers to alter cellular morphogenetic movements and probe its impact on nuclear morphology and segmental gene expression patterns. Mechanical perturbations during blastoderm stage in a developing  Drosophila embryogenesis, cells within an embryo are repositioned to different spatial regions in three dimensions and thus experience varied chemical gradients, established during cellular blastoderm stage by maternal proteins Drosophila melanogaster, GB invagination is one of the major morphogenetic movements for gastrulation, during which cells segregate into three germ layers - endoderm, mesoderm and ectoderm. At this stage, movement of cells is highly coordinated in both space and time and segmental gene expression patterns emerge During early Drosophila embryo to elucidate the coupling between nuclear morphology, its position and global gene expression program. For this we have employed single point perturbation by tissue level laser ablation technique In this work, we use micromanipulation methods in a live Drosophila embryo was carried out. rd egg length  Tissue level laser induced perturbation and, (2) Application of external force, using a custom built magnetic tweezers, on cells within an embryo, injected with 100 nm paramagnetic beads  of 15–20 cells leads to large scale differential changes in GBE; depending on the ablation position . rd and posterior end ablation. Nuclear trajectories were determined during GBE in control and 1/3rd ablated embryos (control- rd ablated- rd egg length) do not move further towards the anterior region and thus get repositioned due to the perturbation. While ablation at 2/3rd egg length does not have any effect on movement of cells inside the embryo. However ablation at posterior end stalls GBE and is lethal to the embryo, like that of 1/3rd ablation. Further for 1/3rd egg length ablation, nuclei of GB and early amnioserosa cells above the ablation point spread and increased in size, compared to those in a control embryo. rd ablated embryo and quantification of nuclear area is shown in To probe the robustness of morphogenetic movement and its effect on Drosophila embryo  is blocked as shown by comparing the GB front position in case of control and perturbed case . Cells near the electromagnet and further away did not show notably different displacement as shown in The coordinated cellular movements during morphogenesis, evident from the cellular displacement versus time traces, show that cells are intercalated and cells migrate as a sheet during various morphogenetic movements, in our case GBE and GBR. Perturbations to this sheet, either due to ablation or application of external force, evidence differential changes in the coordinated cellular movement. Defects in GBE or GBR upon perturbation suggest that the developing embryos are highly sensitive mechanical systems. In addition, our results show that nuclear morphology is intimately linked with cellular movements; transiting from circular nuclear shapes to emergence of heterogeneity in nuclear architecture and chromatin compaction. This may be a result of cytoskeletal reorganization dynamics and due to changing inter-cellular adhesions with development. Our results further show that morphogenetic movements require localization of non-muscle myosin II at the inter-cellular junction and any perturbation to this localization, in terms of cytoskeletal integrity, leads to alterations in cellular movements within the embryo. From the force versus distance calibration plot shown in Drosophila embryo. The observed changes in segmental gene expression patterns, obtained when the force was applied from the two sides- left and right side of the embryo near the posterior end reveal a handedness in patterning genes or cellular movement. These results may have some bearing on earlier results, which showed that for correct handedness of embryonic hindgut in Drosophila, type-I myosin; Myo31DF was required Drosophila development respectively. Pouille et alsnail homozygous mutant where there is no mesoderm invagination, local mechanical deformation could rescue the cell movement by promoting Fog-dependent signaling leading to cortical sub-membranal myosin-II accumulation. Further, Fernandez-Gonzales et alDrosophila development. Our results further shows that application of force on posterior cells when GBE has just commenced leads to myosin II fluidification or formation of small punctas, suggesting that myosin reorganization was sensitive to mechanical tension. Taken together our results suggest that developing embryos comprise of a collective and dynamic mechanical network that exploits robust morphogen signaling gradients to elicit precise segmental genetic patterns. While the signaling gradients may be robust, our results highlight the importance of the mechanical sensitivity in physical cellular networks and their associated morphogenetic movements. Local perturbations to these morphogenetic movements are amplified to global defects, resulting in altered segmental gene expression patterns and thus developmental changes in the Drosophila embryo. However, quantitative links between physical modulation of cellular morphogenetic movements, acto-myosin reorganization and segmental gene expression patterns requires further investigation.Changes in global expression patterns of segment polarity gene- engrailed, on application of force, may be both due to alterations in prestressed nuclear architecture and global repositioning of cells within the embryo. Further, application of force could also mechanically activate signaling pathways upstream of the engrailed, such as the Armadillo pathway, and lead to observed changes in gene expression Transgenic fly with EGFP (Enhanced Green Fluorescent Protein) fused to one of the core Histone protein, H2B, (H2B-EGFP) is used for labeling the nuclei in embryo. To see the global effect of perturbation on pattern formation en-Gal4 UAS myr mRFP recombinant was made by:(♀) X ;UAS myr mRFP; (♂)Step 1: ;en-Gal4; (♀) X ;Cyo/Tft; (♂)Step 2: ;en-Gal4/UAS myr mRFP; (♀) X ;en-Gal4 UAS myr mRFP/Cyo; (♂)Step 3: Screen larvae, grow till adult & collect Cyo ;en-Gal4 UAS myr mRFP/Cyo; (♀) X ;en-Gal4 UAS myr mRFP; (♂)Step 4: Cross used for experiment: H2B-EGFP In these flies, H2B-EGFP marked the nucleus in green while the cells expressing the segment polarity gene- engrailed, had mRFP (monomeric Red Fluorescent Protein) at the plasma membrane. As a marker for cytoskeletal reorganization during embryo development (morphogenesis), transgenic fly with EGFP tagged to non-muscle myosin II regulatory light chain (MRLC) (spaghetti squash (Squash EGFP on chromosome II)) was used.To collect freshly laid embryos, flies  were kept in a cut bottle with sucrose plate at the bottom for an hour at 25°C in an incubator. Collected embryos were washed with water and aligned on #1 coverslip along a double sided tape with the dorsal side facing downwards and covered with Halocarbon oil 700 .rd distance or 2/3rd distance from the posterior end. In each case, ablation was carried out at blastoderm stage during the embryogenesis  or Olympus FV1000 confocal microscope using 60×, 1.4 NA oil objective and 20×, 0.70 NA objective . Ablation experiments were performed using Titanium-sapphire 80 MHz pulsed femtosecond lasers  mode locked at 835 nm. Embryos were aligned facing dorsal side down and ablation was carried out in one of the three regions at the dorsal side of the embryo: at the posterior end, 1/3th to 13th mitotic cycle embryos) or before, were aligned on the coverslip as described above. 100 nm paramagnetic beads  were injected in these embryos from the posterior side using Femtotips II (outer diameter 0.7 micron) on Eppendorf Femtojet microinjection set up. Embryos were allowed to develop till the required experimental stage at room temperature in a moist chamber. Force was applied using a custom made long pointed electromagnet mounted on a XYZ stage at 30° angle. Further details of electromagnet calibration and microinjection are provided in supplementary To apply mechanical force on cells in the embryo, early stage embryo, Bowne's stage 4 . All the experiments were acquired at 5 second per frame. For efficient bleaching, tornado bleach option was used after second frame with 100% 488 nm line of Argon ion laser  for 50 iterations with pixel time 8 microsecond in a circular region of interest (ROI) with diameter 90 pixel (approximately 3.7 micron). Post bleaching 23 additional frames was acquired. Background subtraction was done during analysis to correct for photobleaching and nuclear movements were also corrected.http://rsbweb.nih.gov/ij/index.html), Zeiss LSM image examiner and Olympus FV10 ASW. Nuclear tracks were determined by using ImageJ plugin- MtrackJ (http://www.imagescience.org/meijering/software/mtrackj/). All the graphs and quantifications were done using OriginPro 7.5 .Acquired images were processed and analysed using ImageJ software  XY tracks of the centroid position of individual nuclei (marked by H2B-EGFP) are determined by tracking them using ImageJ plugin (MtrackJ) from the time lapse images acquired (B) Displacement versus time plot shows highly coordinated movement of nuclei during the morphogenetic movement . Nuclei from posterior end were tracked. Direction of movement of cells is bottom to top (A-P axis).(TIF)Click here for additional data file.Figure S2The evolution of nuclear shape in a Drosophila embryo. Note the spherical nuclei at the earlier time points: more asymmetric shapes emerge with cellularization, and shapes become more variegated and region specific with the onset of germ band extension. The representative z-stack projected images shown here start from just before 13th mitotic cycle (0′) and continue into germ band extension, 112′ later at room temperature. The time points in minutes are indicated. Dorsal side of the embryo is imaged. Scalebar = 20 µm.(TIF)Click here for additional data file.Figure S3Schematic details of laser induced perturbation experiment. Image shows the dorsal view of a live embryo expressing H2B-EGFP. Markings on arrow indicate different positions of ablation – posterior end, 1/3rd and 2/3rd from the posterior. Ablation is performed at one of the three regions using Titanium sapphire multiphoton laser mode locked at 835 nm optimized to give 190 mW at the focal plane of 40×, 1.3 NA objectives.(TIF)Click here for additional data file.Figure S4Effect of tissue level ablation on GBE. Each row shows the movement of cells and GBE front in control, 2/3rd ablation, 1/3rd ablation and posterior end ablation respectively at different time points  after ablation. 0′ corresponds to cellular blastoderm stage. The plane in which most nuclei are observed was chosen to be the ablation plane . For spot ablation, 835 nm laser was parked at a diffraction-limited region for ∼750 msec at one of the three positions as described. The images shown above are 3D reconstruction image using Zeiss LSM 510 Meta software. Scalebar = 50 µm.(TIF)Click here for additional data file.Figure S5Arrest of GBE by tissue level ablation of Drosophila embryo. Nuclear XY trajectories for the embryo ablated at 1/3rd position from the posterior end obtained from the time lapse images. (A) XY tracks of 70 nuclei determined using ImageJ plugin- MtrackJ. 2D tracking is done till the nuclei went out of the plane. Ablation perturbs the movement of nuclei as seen from the tracks and also stops the GBE below the ablation point. (B) Movement of cells below and above the ablations spot.(TIF)Click here for additional data file.Figure S6Impact of spatial position on nuclear size. (A) Representative images  and Nuclear area of the cells in front of GB front (early amnioserosa cells) is plotted for the control and 1/3rd ablated after 2.5 hr of cellularisation. N = 4. .(TIF)Click here for additional data file.Figure S7Engrailed pattern in embryos at different stages of development. First column shows the nuclei image as marked by H2B-EGFP, second column shows engrailed pattern and third is the merge. Embryos are imaged post GBE (first row), post GBR (second row) and 20 hour after egg laying (third row).(TIF)Click here for additional data file.Figure S8Engrailed patterning in embryo injected with 100 nm paramagnetic beads from the posterior and without force & with force, anterior region without force application and with irreversible force application protocol (as described in main manuscript).(TIF)Click here for additional data file.Materials and Methods S1Calibration of electromagnet to estimate the force exerted.(DOC)Click here for additional data file.Movie S1Movie of morphogenetic movement namely germ band extension (GBE) and retraction (GBR) in a live embryo. Nucleus labeled with H2B-EGFP.(AVI)Click here for additional data file.Movie S2Movie of tracks of individual nuclei in a live embryo during cell movement at the posterior region.(AVI)Click here for additional data file.Movie S3Movie for control case showing zoomed in view of nucleus labeled using H2B-EGFP, when force was applied during the blastoderm stage in embryos injected with 100 nm paramagnetic beads.(AVI)Click here for additional data file.Movie S4Movie for irreversible case showing zoomed in view of nucleus labeled using H2B-EGFP, when force was applied during the blastoderm stage in embryos injected with 100 nm paramagnetic beads.(AVI)Click here for additional data file.Movie S5Movie for reversible case showing zoomed in view of nucleus labeled using H2B-EGFP, when force was applied during the blastoderm stage in embryos injected with 100 nm paramagnetic beads.(AVI)Click here for additional data file.


Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to “low pulmonary reserve”, ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction.Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages.Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology. A stereotyped pattern of structural changes which occur in the human lung as it ages, termed “senile lung”, is characterized by airspace enlargement that is similar but not identical to acquired emphysema A limitation of previous surveys of organ-specific aging programs is the use of binary constructs of the aging phenotype, focusing on “young” versus “old”. Since the young organ is not necessarily the “control” for the old organ, we sought to develop an alternative approach to describe tissue aging. By performing a genome-wide transcriptional time course survey of the aging murine lung (over six time points), we were able to extract genes that not only displayed more complex patterns of expression with aging but also reflected known histologic events that could not be replicated by simple pair-wise comparisons. In this study, we focus on the gene cluster which corresponds to the transcriptional transition attending the onset of airspace enlargement, e.g. 8–12 months of age.Previous genomic surveys of murine lung aging showed that 1) the terminal structural changes seen in the aged lung are associated with an altered transcriptome and 2) that the aging lung harbors both tissue-specific and aging specific molecular signatures. Misra and colleagues found that airspace enlargement in senescent DBA/2 mice is associated with the down-regulation of elastin and several collagen genes despite increased collagen content compared with the young adult controls In this study, we show that airspace enlargement develops during the mid-range of the murine life-span and progresses through the late, preterminal time points and is accompanied by early oxidative stress, cell death and elastase activation. We also show that several genes are transiently induced during the onset of this structural change, and may possibly be the first signature of a tissue-specific aging program. This period, we further demonstrate, is punctuated by a marked, transient induction of immunoglobulin genes, accompanied by B lymphocyte (B-cell) activation/expansion, immunoglobulin deposition and macrophage infiltration. Taken together, our strategy has shown that time course data informed by structural surveys can reveal relevant pathways involved in tissue-specific aging that might be overlooked with conventional young-old pairwise analyses.In order to delineate the critical signaling events which attend the onset of airspace enlargement during the aging of DBA/2 mice, we performed a detailed histologic and molecular analysis of the lungs of the mice at six different time points during adult life: 2 months, 4 months, 8 months, 12 months, 16 months and 20 months. These time points loosely correspond to specific maturation stages in humans: early adulthood (2–4 months), middle-age (8–12 months) and old age (16–20 months) . At each2 of 0.28. Since weight has a bimodal curve morphology with age, we considered whether analysis of mice ≥8 months of age might show an independent association between MLI and weight. The observed linear association between MLI and weight in this group, however, was eliminated with adjustment for age (p = 0.68). A significant association existed between MLI and age (R2 0.89) with no evidence of improved association when weight was included in the model. Taken together, these data show that even when corrected for weight, age remains an independent factor contributing to MLI.We found a nonlinear pattern of airspace enlargement, denoted by MLI (mean linear intercept), that commenced at 12 months of age and progressed thereafter . The airAirspace enlargement frequently accompanies various forms of lung injury that result in oxidative stress, cell death, reduced proliferation and/or local inflammation. Increased oxidative stress is a signature of systemic aging and likely contributes to the higher incidence of malignancy, fibrosis and low-grade inflammation in elderly persons Nine clustering profiles of the time course transcriptome were generated using a K-means strategy. Of the nine cluster patterns, one was selected for further interrogation based on the timing of induction consistent with the onset of the airspace phenotype . This clAlthough immunosenescence involves overall dampened immune responses with aging, autoimmunity is an observed accompanying pathology Flow cytometry on whole lung preparations and BAL  specimens from 2 months, 8 months, 12 month and 20 month old lungs showed an increase in B-cell content between 8 and 12 months . B-cell Autoimmunity has been proposed as a component of immunosenescence, however to date no culprit immunogenic lung proteins have been identified and the lung pathology accompanying most rheumatologic illnesses is fibrosis rather than airspace enlargement. We examined whether selected autoimmune cytokines  were induced in the aging lung Taken together, these data show that an induction of B-cell expansion/activation and immunoglobulin production in the aging lung accompanies the earliest stage of airspace enlargement. Further, the immunoglobulin deposition is associated with an influx of macrophages.Our finding of early oxidative stress followed by immune cell infiltration and immunoglobulin deposition prompted us to determine whether the enhanced oxidative stress might contribute to matrix turnover that could trigger an immune response. Recent reports show that both aging and non-aging related redox changes can alter matrix homeostasis Airspace enlargement is a well-recognized pathological signature of respiratory aging Despite the fact that airspace enlargement is a known feature of the aged lung, whether the lesion develops from injury or simply reflects reduced matrix abundance in tissue is a subject of debate. Recent observations, including those reported here, support the former paradigm. The oxidant injury, elastase activation and cell death preceding the onset of airspace enlargement in our studies strongly implicate age-associated tissue stressors. Consistent with this paradigm, Sato reported that SMP30-deficient mice, a proposed model for the “senile lung”, not only develop accelerated age-associated airspace enlargement but also display increased oxidative stress, cell death and susceptibility to cigarette smoke induced pulmonary emphysema Several investigators have shown or postulated reduced immune function in aged persons and in murine models of aging We propose three possible mechanisms connecting the immune signature with the airspace lesion  were obtained from the specific pathogen-free Charles River-National Institute of Aging (NIA) facility. These mice were temporarily housed in a Johns Hopkins Medical Institution mouse facility accredited by the American Association of Laboratory Animal Care until time of euthanization. The animal studies were reviewed and approved by the institutional animal care and use committee of Johns Hopkins School of Medicine.Total RNA was extracted from murine lung using the Trizol Reagent method . The six RNA samples from each time point were pooled into two groups comprised of three murine specimens. RNA samples were labeled and hybridized to Illumina Sentrix MouseRef-8 Expression Beadchips  according to manufacturer's protocol.Affy, a Bioconductor package (http://www.bioconductor.org), utilizing the quantile normalization method to reduce the variation between microarrays that can develop during the processes of sample preparation, manufacture, fluorescence labeling and hybridization R to assess sample variability.The microarray data was normalized with Differentially-expressed genes were categorized using modified Best K-Means clustering (Spotfire 9.1.1). The number of clusters was chosen empirically based on visual inspection as differing from evenly spaced profiles. To reduce the background and ensure the clustering quality, only genes with detectable hybridization signals in the arrays from all aging groups were included. Nine profiles were generated using Spotfire cluster initialization with a data centroid based sea. One cluster was selected for further analysis based upon a peak transcriptional induction coincident with the onset of airspace enlargement. Genes within this cluster were ranked based on similarity to exemplar and examined by Gene Ontology Definition for pathway assignment. The top 200 genes, ranked by similarity to exemplar, are shown in Differentially expressed genes were classified into functional categories based on the Gene Ontology (GO) definition using publicly available web-based tools Onto-express and David . For each level of annotation, the calculated p-value represents the probability that the specific gene-function was randomly distributed between groups Total RNA isolated from lung tissues was treated with DNase and reverse-transcribed using a first-strand DNA sysnthesis kit from Invitrogen. The PCR was performed on an ABI Fast 7500 System . TaqMan probes for the respective genes were custom-generated by Applied Biosystems based on the sequences in the Illumina array and used per manufacturer's instructions. The expression levels of target genes were determined in triplicate from the standard curve and normalized to Gapdh mRNA level.Western blot analysis was performed using standard methods. Primary antibodies and dilutions were as follows: IgG1 , IgA , IgM , CTGF (Abcam 1∶5000), psmad2 , iNOS (Abcam 1∶200), beta-actin , MMP12 .Histologic, morphometric and immunohistochemical methods were as described previously TUNEL staining was performed using the Calbiochem TdT-FragEL DNA Fragmentation Detection Kit per standard protocol as published Serum samples and lung lysates from mice at designated ages were subjected to Mouse IgG ELISA analysis per Roche protocol. IL17 and IL12/23 ELISA assays were performed per Invitrogen and R&D protocols, respectively.Lungs were minced and incubated at 37°C and single cell suspensions were prepared. Lymphocytes were gated with characteristic low forward scatter/side scatter, using a FACSAria instrument and FACSDiva for data acquisition (Becton Dickinson), and Flowjo for analysis (Tree Star Inc) as previously published Lung tissue lysates were prepared in a cold room at 4C. Tissue was homogenized in 50 µL PBS and centrifuged at 14000 RPM for 20 min. The supernatant was removed and used as sample lysates. Fifty µg of lung lysates were loaded on a10% Criterion Zymography Precast Gel (Biorad) and run at 120 V. Twenty-five µL of recombinant mouse MMP9 protein  was loaded as a positive control. The gel was soaked in 1× Renaturing Buffer (Biorad) twice for 30 minutes each at room temperature and incubated in 1× Development Buffer (Biorad) overnight at 37C. The gels were stained with Coomassie Brilliant Blue R-250 Staining Solution (Biorad), followed by 1× Destain Coomassie R-250 Solution (Biorad) until a clear band appeared against a blue background.Results are expressed as means ± SEM unless otherwise stated. Screening comparisons across multiple time points were performed by one-way ANOVA. These were followed by pairwise-comparisons using the two-sample t-tests or Mann-Whitney rank sum tests. All statistical analyses were performed with Sigmastat . A p<0.05 was considered significant.Figure S1Strategy for analysis of lung phenotype at different ages.(TIF)Click here for additional data file.Figure S2Caspase activity in the aging lung. A. Caspase activity in lung lysates from mice at designated ages. N = 4–6 mice per time point. *p<0.01.(TIF)Click here for additional data file.Figure S3Immunoglobulin expression in the aging lung. Western blotting for immunoglobulins in lung lysates from mice at designated ages. Top-IgM blot, Bottom-IgG blot.(TIF)Click here for additional data file.Figure S4T cell subsets in the aging lung. A. Relative Proportion of CD4+ and CD8+ cells in CD3+ lymphocyte subset quantified by flow cytometry in lung mononuclear cells isolated from mice at designated ages. N = 4–6 mice per time point. B. Relative proportion of FoxP3+ cells in CD4+ lymphocyte subset from mice at designated ages. N = 4–6 mice per time point. Asterisk designates p<0.05 compared with 2 month time point.(TIF)Click here for additional data file.Figure S5MMP12 expression in the aging lung. Densitometric analysis of MMP12 protein expression in lung lysates from mice at designated ages normalized to actin. N = 4–6 mice.(TIF)Click here for additional data file.Table S1Top 200 genes in airspace peak.(PDF)Click here for additional data file.Table S2Real-time PCR validation of genes in airspace peak.(PDF)Click here for additional data file.


SAP also promotes the formation of immuno-regulatory Mreg macrophages. To elucidate the endogenous function of SAP, we used bleomycin aspiration to induce pulmonary inflammation and fibrosis in mice lacking SAP. Compared to wildtype C57BL/6 mice, we find that in -/-Apcs “SAP knock-out” mice, bleomycin induces a more persistent inflammatory response and increased fibrosis. In both C57BL/6 and -/-Apcs mice, injections of exogenous SAP reduce the accumulation of inflammatory macrophages and prevent fibrosis. The types of inflammatory cells present in the lungs following bleomycin-aspiration appear similar between C57BL/6 and Apcs-/- mice, suggesting that the initial immune response is normal in the -/-Apcs mice, and that a key endogenous function of SAP is to promote the resolution of inflammation and fibrosis.Fibrosing diseases, such as pulmonary fibrosis, cardiac fibrosis, myelofibrosis, liver fibrosis, and renal fibrosis are chronic and debilitating conditions and are an increasing burden for the healthcare system. Fibrosis involves the accumulation and differentiation of many immune cells, including macrophages and fibroblast-like cells called fibrocytes. The plasma protein serum amyloid P component  inhibits fibrocyte differentiation  Fibrosing diseases, such as pulmonary fibrosis, cardiac fibrosis associated with hypertensive heart disease, primary myelofibrosis of the bone marrow, liver fibrosis, and renal fibrosis are chronic and debilitating conditions and are an increasing burden for the healthcare system Following their recruitment from the blood into a tissue, monocytes can differentiate into macrophages, dendritic cells, or fibrocytes + monocytes There are multiple sources of fibroblast-like cells present in fibrotic lesions We previously found that the plasma protein Serum Amyloid P component  inhibits human and murine fibrocyte differentiation Pentraxins including SAP bind to the receptors for the Fc portion of immunoglobulin G (FcγR) to initiate signaling cascades consistent with FcγR ligation, and SAP has been crystallized bound to FcγRIIa -/-Apcs  “SAP knockout mice” appear to be normal and have normal viability -/-Apcs mice are more susceptible to experimental bacterial lung infection In tissue culture, human and mouse monocytes spontaneously differentiate into fibrocytes when there is no SAP present -/-Apcs mice. We find that the induced lung inflammation and fibrosis in the -/-Apcs mice is persistent compared to C57BL/6 wild type mice. However, the inflammatory cells present in the lungs after bleomycin aspiration appear similar between C57BL/6 and -/-Apcs mice, suggesting that the initial response to the insult is independent of SAP, and that a key function of SAP is to promote the resolution of inflammation and fibrosis.To elucidate the endogenous function of SAP, in this report we used bleomycin to induce inflammation and fibrosis in the lungs of -/-Apcs “SAP-knock-out” mice This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Texas A&M University Animal Use and Care Committee (TAMU AUP #2009-0265 and #2013-0007). All procedures were performed under anesthesia, and all efforts were made to minimize suffering. 4-6 week old C57BL/6 mice  and 5 cells/well in 200 μl with human SAP at the indicated concentrations. On day 3 of the incubation, wells were supplemented with IL-13 and M-CSF, as described previously Mouse spleen cells were isolated as described previously, except that the spleens were digested with Accutase  for 30 minutes at room temperature according to the manufacturers' instructions 2 anti-rat IgG  used at 2.5 μg/ml. After blocking with PBS/20% rat serum  for 30 minutes at 4°C, cells were incubated with 2.5 μg/ml biotinylated anti-CD45 . After a 30 minute incubation on ice, cells were washed and then incubated with streptavidin-APC (BioLegend). After a 30-minute incubation on ice, cells were washed twice, resuspended in 100 μl ice-cold PBS/2% BSA, and analyzed using a C6 flow cytometer .Spleen cells were analyzed by flow cytometry, as described previously BAL cytospins and frozen lung tissue sections (5 μm) were prepared and immunochemistry performed as described previously Lungs of naïve mice were purged of blood, excised, weighed, snap-frozen in liquid nitrogen, and stored at −80°C, as described previously Blood from untreated mice, and mice euthanized at day 7 and 21 after bleomycin aspiration was collected and serum stored at −20°C. Exogenous human SAP was measured by ELISA, as described previously Statistical analysis was performed using Prism . Statistical significance between two groups was determined by t test, or between multiple groups using analysis of variance (ANOVA), and significance was defined as p<0.05.-/-Apcs mice were originally generated on a 129 background and then backcrossed to C57BL/6 mice -/-Apcs mice develop glomerulonephritis in adults >6 months old. This hybrid genetic background (129×C57BL/6) has since been shown to generate autoantibodies and glomerulonephritis independent of the -/-Apcs locus -/-Apcs mice were re-derived and then backcrossed for an additional two generations to C57BL/6 mice. Mice were checked for -/-Apcs gene deletion by PCR, and the sera were screened for absence of SAP protein by ELISA and/or western blotting (data not shown). To further reduce the possible effects of autoimmunity found in >6 months old mice, all experiments were performed on 4–6 week old mice. However, as young -/-Apcs mice may have altered lung and kidney characteristics due to the presence of autoantibodies, low grade fibrosis, and/or underlying infections, we compared the organ weights of 4–6 week old naïve C57BL/6 and -/-Apcs mice, using organ weight increase as an indicator of edema associated with inflammation -/-Apcs and C57BL/6 mice, but the liver and lung weights were lower in -/-Apcs mice  and -/-Apcs mice . Compared to C57BL/6 mice, -/-Apcs mice have increased numbers of CD3+ T cells and reduced numbers of CD11b+ monocytes and macrophages in the spleen  . However, -/-Apcs spleen cells had two discrete populations of spleen cells staining for a common epitope of FcγRII and FcγRIII, whereas spleen cells from C57BL/6 contain a single population  , whereaspulation . Rat IgGen cells . These d-/-Apcs mice had an altered response to bleomycin aspiration, we measured heart rate, pulse distension, and arterial oxygen saturation (pulse Ox) at 7 days after aspiration, when there is acute inflammation and alveolar cell death, and at 21 days when there is peak fibrosis and the acute toxic effects of bleomycin have receded -/-Apcs mice, bleomycin caused a significant reduction in heart rate compared to mice receiving saline  and 21 days (to measure the fibrotic response) -/-Apcs mice.SAP injections inhibit neutrophil, macrophage, and fibrocyte accumulation in the lungs of bleomycin-treated C57BL/6 mice -/-Apcs mice given bleomycin aspiration without SAP treatment contained significantly more cells than the saline control  or newly recruited inflammatory macrophages (CD11b+ CD11c+) Apcs-/- . SAP treleomycin . These d-/-Apcs mice .Exogenous SAP injections reduce inflammation and fibrosis in many organ systems -/- mice , althoug-/- mice . In addi-/-Apcs mice had lower protein levels compared to C57BL/6 mice (-/-Apcs mice (To determine if exogenous or endogenous SAP levels could also regulate bleomycin-induced lung damage, we measured total lung BAL protein levels, as a measure of lung tissue damage following bleomycin aspiration L/6 mice . The bleL/6 mice . SAP inj-/- mice . SAP inj-/-Apcs mice have a more severe inflammatory response and impaired lung function following bleomycin aspiration. To determine if -/-Apcs mice also have a more severe fibrotic response, lung sections were stained with picrosirius red to detect collagen. Compared to C57BL/6 mice, at 21 days after bleomycin treatment, -/-Apcs mice had increased numbers and size of fibrotic lesions and more extensive deposition of collagen (-/-Apcs mice, SAP injections significantly reduced picrosirius red staining (-/-Apcs mice is an increased fibrotic response to bleomycin, that this phenotype can be rescued by SAP injections, and that treatment with exogenous SAP can reduce fibrosis.The data above suggest that collagen . In addistaining . These d-/-Apcs mice had a persistent inflammatory response and increased fibrosis in the lungs after bleomycin aspiration. However, the inflammatory cells present during the initial phases of inflammation were similar between C57BL/6 and Apcs-/- mice, suggesting that the initial response to the insult is independent of SAP. These data suggest that a key function of SAP is to promote the resolution of inflammation and fibrosis.We found that compared to C57BL/6 mice, -/-Apcs mice, although we did observe that SAP injections reduced the BAL cell numbers in both strains of mice. These data suggest that the initial inflammatory response to bleomycin is similar in C57BL/6 and -/-Apcs mice, and that the levels of endogenous SAP are incapable of moderating the inflammatory response. These data may also help explain the strain variation in response to bleomycin. C57BL/6 mice are sensitive to bleomycin-induced lung fibrosis, but Balb/c and DBA/2 strains are resistant Many groups have shown that SAP injections can reduce inflammation and fibrosis induced by multiple different stimuli -/-Apcs mice had significantly more cells in the BAL, and these cells contained a significant number of inflammatory CD11b+ macrophages. The cells present in the lung tissue of -/-Apcs mice also contained increased numbers of CD11b+ inflammatory macrophages, and CD206+ M2a pro-fibrotic macrophages. Compared to the C57BL/6 mice, the overall inflammatory and fibrotic response was elevated and persistent in the -/-Apcs mice, although the composition of the cells found was not significantly different. These data suggest that the inflammatory response is not aberrant in -/-Apcs mice, but that the absence of endogenous SAP leads to the persistence of signals that drive inflammation. As SAP binds to apoptotic cells and other debris formed during inflammation, and promotes phagocytosis of this material, the absence of SAP in -/-Apcs mice, or reduced levels of SAP in susceptible individuals, may be an additional contributing factor to persistent inflammation and fibrosis following inflammation At day 21 following bleomycin aspiration, compared to C57BL/6 mice, -/-Apcs mice following bleomycin aspiration suggests that SAP has no protective effect on lung epithelial cells. However, following bleomycin exposure the apoptotic lung epithelial cells, when coated with SAP, are likely to be removed by phagocytosis without generating a pro-inflammatory response The observation that there was no significant difference in the BAL protein composition of C57BL/6 and -/-Apcs spleen cells had an increased numbers of IgG binding cells, altered FcγR expression, and increased inhibition of fibrocyte differentiation by SAP, indicating that endogenous SAP alters the expression of FcγR, and/or binding of SAP to the FcγR. These data also indicate that an additional function of endogenous SAP is to modulate FcγR expression and/or IgG binding, possibly to modulate the inflammatory response.Compared to C57BL/6 mice, -/-Apcs mice have significantly smaller lungs, with increased collagen content. This correlates with reduced pulse Ox and increased pulse distension in the -/-Apcs mice. These data suggest that the lungs of -/-Apcs mice are less efficient at oxygenating the blood, and that the heart is compensating to overcome this problem. These findings may be related to the reduced number and/or altered functionality of fibrocytes detected in the spleens of -/-Apcs mice. Together, our results suggest that endogenous and exogenous SAP levels are important in the regulation of bleomycin-induced lung inflammation and fibrosis, and that SAP promotes the resolution of inflammation and reduces fibrosis by multiple mechanisms.Compared to C57BL/6 mice, naïve


Allergic inflammation is commonly observed in a number of conditions that are associated with atopy including asthma, eczema and rhinitis. However, the genetic, environmental or epigenetic factors involved in these conditions are likely to be different. Epigenetic modifications, such as DNA methylation, can be influenced by the environment and result in changes to gene expression.To characterize the DNA methylation pattern of airway epithelial cells (AECs) compared to peripheral blood mononuclear cells (PBMCs) and to discern differences in methylation within each cell type amongst healthy, atopic and asthmatic subjects.PBMCs and AECs from bronchial brushings were obtained from children undergoing elective surgery for non-respiratory conditions. The children were categorized as atopic, atopic asthmatic, non-atopic asthmatic or healthy controls. Extracted DNA was bisulfite treated and 1505 CpG loci across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I. Gene expression for a subset of genes was performed using RT-PCR.STAT5A and CRIP1. We demonstrate STAT5A gene expression is decreased whereas CRIP1 gene expression is elevated in the AECs from asthmatic compared to both healthy and atopic subjects.We demonstrate a signature set of CpG sites that are differentially methylated in AECs as compared to PBMCs regardless of disease phenotype. Of these, 13 CpG sites were specific to healthy controls, 8 sites were only found in atopics, and 6 CpGs were unique to asthmatics. We found no differences in the methylation status of PBMCs between disease phenotypes. In AECs derived from asthmatics compared to atopics, 8 differentially methylated sites were identified including CpGs in We characterized a cell specific DNA methylation signature for AECs compared to PBMCs regardless of asthmatic or atopic status. Our data highlight the importance of understanding DNA methylation in the epithelium when studying the epithelial contribution to asthma. Asthma and atopy are two of the most common chronic inflammatory conditions in the western world Epigenetic control of gene expression has two general mechanisms: either the DNA itself is chemically altered by the addition of a methyl group to a cytosine base in a cytosine-guanine (CpG) dinucleotide by a DNA methyltransferase (DNMT), or the histone proteins that package DNA into chromatin are chemically modified. DNA methylation generally results in gene silencing or repression by either changing the conformation of chromatin (facilitated by methyl CpG binding domain proteins (MBDs) and histone deacetylases), or by simply blocking transcriptional machinery from accessing the DNA. Thus, although the genetic make-up of an individual is identical throughout different tissues of the body, DNA methylation is important for the regulation of unique gene expression profiles in each tissue or cell type et al., have demonstrated that epigenetic modifications can also be inherited by using a murine model in which a maternal diet, rich in methyl donors, was shown to increase the severity of allergic airway disease in offspring in utero) also had increased severity of allergic airway disease Epigenetic modifications to DNA have been described as the potential link between environmental effects and clinical phenotypes The tracheo-bronchial epithelium is the interface between the environment and the submucosa and represents the first line of defense against inhaled exogenous agents. There is now substantial evidence that the airway epithelium of subjects who have asthma is abnormal, including increased expression of epithelial growth factor receptor (EGFR) at denuded and damaged sites The objectives of this study were first to identify the DNA methylation signature of AECs compared to PBMCs. The second objective was to identify epigenetic differences within AECs and PBMCs from non-atopic asthmatic, atopic asthmatic, atopic and healthy children.This study is part of ongoing research investigating the phenotype of AECs from mild asthmatic and atopic children 2 atmosphere culture and collected at passage 2 and 3. PBMCs were isolated from 10 mL of whole blood using ficoll gradient centrifugation as previously described 6 cells using the Flexi Gene DNA Kit and QIAamp columns according to the manufacturer’s instructions . Total RNA was extracted from AECs using the RNeasy mini kit (Qiagen).AECs were obtained by either trans-laryngeal, non bronchoscope brushing Bisulfite treatment of 250 ng of genomic DNA was performed using the EZ DNA Methylation-Gold Kit . DNA methylation of 1505 CpG sites across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I, according to the manufacturer’s protocol . The GoldenGate Methylation Cancer Panel I has previously been validated with other technologies such as pyrosequencing P-values >0.05 in more than 10% of samples (n = 122) were also removed leaving 1027 CpG sites across 671 genes. Remaining loci with detection P-values >0.05 were imputed using the K-nearest neighbour algorithm (0.5% of data points) methylumi package 2 ((Cy5+1)/(Cy3+1)) was calculated, where Cy5 and Cy3 were the methylated and unmethylated sequences, respectively t-test http://www.R-project.org) A Kruskal-Wallis test with a Dunn’s Multiple Comparison test between disease phenotype groups for sex (p = 0.22) and age (p = 0.84) and found no significant differences thus we did not adjust for these parameters in subsequent analyses. To avoid gender bias, all CpG sites on the X chromosome (n = 84) were removed from the analysis as well as probes containing single nucleotide polymorphisms (n = 272). Probes with detection www.ingenuity.com) using the biological functions application. As the Illumina GoldenGate Cancer Panel I is an array based on cancer related genes, we used our dataset as the reference set for pathways analysis to avoid bias towards cancer associated pathways. A right-tailed Fisher’s exact test was used to calculate a P-value which determined if the association between the genes identified and the biological pathway was a result of chance alone.The genes containing CpG sites which showed differential DNA methylation between AECs and PBMCs were classified by IPA , STAT5A  and PPIA  were obtained from GeneWorks . Expression was normalized to PPIA. A Kruskal-Wallis and a post hoc Dunn’s multiple comparison test was performed using GraphPad Prism Version 4.0 for Windows . A p-value of less than 0.05 was deemed significant.Gene expression of cysteine-rich protein 1 (CRIP1) and signal transducer and activator of transcription 5A (STAT5A) was determined via two-step reverse transcriptase polymerase chain reactions (RT-PCR). Briefly, cDNA was synthesized using radom hexamanucleotide primers and Multiscribe Reverse Transcriptase (Applied Biosystems) and used in a PCR reaction containing 2X Sybrgreen Master Mix (Applied Biosystems) under standard cycling conditions. Primers for CRIP1 . We were able to construct a cell specific signature consisting of 80 CpG sites across 67 genes which had differential DNA methylation in AECs compared to PBMCs . Table SWe then evaluated the DNA methylation profiles of PBMCs and AECs for each disease phenotype and found 96, 71, and 89 differentially methylated CpG sites across 83, 58, and 77 genes in the healthy, atopic and asthmatic groups respectively . A full We next sought to determine if there were differences in the DNA methylation profiles of AECs from asthmatics compared to healthy controls. We found no differentially methylated CpG sites in this comparison . We alsoWhen the DNA methylation signatures of PBMCs from asthmatic, atopic and healthy individuals were compared, we found no CpG sites that were differentially methylated between the subject groups .CRIP1 as it had the most significant q-value and z-score difference for DNA methylation between atopic and asthmatic AECs , a cytoskeletal protein, is less methylated in AECs compared to PBMCs  ADAM33 in epithelium and hypomethylation in fibroblasts which strictly regulated gene expression in utero supplementation with methyl donors and both allergic airways disease as well as differential methylation of the runt-related transcription factor 3 (Runx3) gene Runx3 gene at three separate CpG loci when we compared AECs to PBMCs from healthy subjects (apoA-1) gene; we found decreased methylation in AECs as compared to PBMCs from all of our subjects , Asthma is classically characterized by chronic airway inflammation, variable airway obstruction, airway remodeling and hyperresponsiveness This study has several limitations. While a small sample size for each disease phenotype studied is likely to influence our ability to detect differential CpG methylation, we were able to determine a number of differentially methylated CpG sites between asthmatic and atopic AECs. Additionally, we used the Illumina GoldenGate Methylation Cancer Panel I which was designed to screen candidate genes originally identified in cancers and thus focuses on cancer related pathways. Even so, we were able to identify pathways within this cancer-centric structure that were relevant to our study. The inability to determine the entire epigenetic profile of the airway epithelium may result in potentially missing many disease specific epigenetic modifications. With regards to disease, our study also focused on pediatric subjects with asthma and atopy. Therefore we cannot determine the epigenetic component of atopy and asthma with increasing age, which may identify a stronger epigenetic fingerprint. Also, all subjects included in the study displayed mild disease therefore it is difficult to generalize our results to all subtypes and severities of asthma. However our findings do indicate that epigenetic changes are present early in disease pathogenesis and highlight the importance of understanding these mechanisms of gene regulation further. Although the individuals within the study were well characterized for asthma and atopy we did not have enough power in the study cohort to stratify the groups by specific allergens, which may also influence the epigenetic profile of the airway epithelium.In summary, we have characterized a cell specific DNA methylation signature for AECs and PBMCs that is maintained regardless of the presence of asthma or atopy. Our data also highlights the importance of determining the effects of DNA methylation in the airway epithelium in discriminating atopy and asthma rather than using PBMCs. We conclude that future studies are required to determine the complete epigenetic signature of the airway epithelium in individuals with atopy and asthma to elucidate candidate genes which may be involved in the pathogenesis of disease and amenable to targeted therapies.Table S1Differentially Methylated CpG Sites in AECs Compared to PBMCs. We identified 80 CpG sites which are differentially methylated between AECs and PBMCs. Z-score difference is presented as AEC relative to PBMC.(DOCX)Click here for additional data file.Table S2Biological Functions in Differentially Methylated Genes in AECs Compared to PBMCs. We identified 67 genes which contain the 80 differentially methylated CpG sites between AECs and PBMCs, these genes are classified into 19 overrepresented biological functions.(DOCX)Click here for additional data file.Table S3Differentially Methylated Sites in Healthy AECs Compared to PBMCs. We identified 96 CpG sites which are differentially methylated between healthy AECs and PBMCs. Z-score difference is presented as AECs relative to PBMCs.(DOCX)Click here for additional data file.Table S4Differentially Methylated Sites in Atopic AECs Compared to PBMCs. We identified 71 CpG sites which are differentially methylated between atopic AECs and PBMCs. Z-score difference is presented as AECs relative to PBMCs.(DOCX)Click here for additional data file.Table S5Differentially Methylated Sites in Asthmatic AECs Compared to PBMCs. We identified 89 CpG sites which are differentially methylated between asthmatic AECs and PBMCs. Z-score difference is presented as AECs relative to PBMCs.(DOCX)Click here for additional data file.Table S6Biological Functions in Differentially Methylated Core Genes in AECs Compared to PBMCs. We identified 47 core genes which contain 57 differentially methylated CpG sites between AECs and PBMCs, these genes are classified into 19 overrepresented biological functions.(DOCX)Click here for additional data file.


The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP ACPP; EC 3.1.3.2) has been known for more than 70 years The association between prostate cancer and serum prostatic acid phosphatase  is essential for the maintenance of the spermatozoa in vitro against β-glycerophosphate In vivo, the ecto-5′-nucleotidase activity of PAP is responsible of dephosphorylating adenosine monophosphate (AMP) to adenosine 2], an essential regulator of vesicular traffic PAP exerts its phosphatase activity SNARE proteins comprise a large family found in yeast and mammalian cells, with the primary function to mediate docking and fusion of vesicles with the cell membranes The mouse prostate consists of three different lobes: anterior (AP), dorsolateral (DLP) and ventral prostate (VP); and it does not show spontaneous development of neoplasia −/−) −/− mouse prostate showed disturbed vesicular trafficking, loss of cell polarity and development of slow-growing non-metastatic prostate adenocarcinoma. Here we report the interaction of TMPAP with snapin; and suggest that TMPAP regulates endo-/exocytosis and the disruption of these processesis a hallmark of prostate adenocarcinoma.To understand the physiological function of PAP, we studied the prostate of our PAP-deficient mouse model  of the prostatic acid phosphatase gene  with the neo gene as described earlier −/− mice was not affected by the gene modification. PAP−/− mice were backcrossed to the C57BL/6J strain (Harlan Laboratories Inc.) for 16 generations to obtain homogenous background. Age-matched C57BL/6J male mice were used as controls in all the experiments.Mice deficient in PAP were generated by replacing exon 3  and analyzed at the Biocenter Oulu EM core facility using Philips 100 CM Transmission Electron Microscope with CCD camera.DLP samples from age-matched PAPSaccharomyces cerevisiae Mav 203 strain according to the Clontech's two-hybrid protocols. Inserts of positive clones were amplified by PCR, and the DNA was automatically sequenced.To screen for interacting partners of human TMPAP, yeast two-hybrid screening was performed using the Matchmaker Gal4 two-hybrid System 3 (Clontech) in accordance with the manufacturer's instructions. The bait construct consisted of the coding region of human TMPAP  cloned in frame into NcoI/SmaI sites of pGBKT7 using PCR generated linkers. A human thymus cDNA library cloned in pACT2 (Clontech) was used as the prey. The bait and prey plasmids were co-transformed into The FRET variant acceptor photobleaching was used. In this technique, the efficiency of energy transfer between two molecules (and consequently the interaction between them) is measured by comparing the fluorescence of the donor molecule before and after the selective photobleaching of the acceptor moleclule Cultured cells were mounted 24 hours after transfection and epifluorescent images were acquired using an Olympus CellR imaging system with 60× oil immersion NA 1.45 objective. Images were collected with a CCD camera . The system was equipped with automated filter wheels for excitation filters and emission beam-splitter/emission-filter cubes for epifluorescence imaging. GFP fluorescence was excited at 450 nm and collected at 510/40 nm. DsRed fluorescence was excited at 575 nm and collected at 640/50 nm. Acceptor fluorescence was bleached for 5 minutes with maximal burner power.http://rsb.info.nih.gov/ij/) and ImagePro 5.1 (Media Cybernetics). Background fluorescence was subtracted prior to calculations. The FRET efficacy defined as the percentage of donor fluorescence increase was calculated with the following equation: E = 1-(Ib/Ia), where ‘Ib’ is the fluorescence intensity of the donor before photobleaching and ‘Ia’ is the post-bleach fluorescence intensity.Images were quantified and processed using Olympus Biosystems AnalySIS software, ImageJ (freely available at The detailed methodology can be found in the Supplementary Methodology in Gene expression files containing microarray raw-data can be accessed from ArrayExpress repository database (accession number E-MTAB-1191).−/−mice examined (n = 8), detecting a hyperplastic growth already at the age of 3 months, followed by mouse prostatic intraepithelial neoplasia (mPIN) at 6 months and prostate adenocarcinoma at 12 months  staining . Bulging−/− mice lost the regular structure of uniform columnar monolayer transforming to a cuboidal multilayer epithelium with hyperchromatic nuclei and presence of pseudo-lumens as a sign of loss in cell polarity. Further analysis of prostate ultrastructural changes showed an increased number of electron-lucent enlarged vacuoles (−/− mice DLP cells and their contents secreted into the lumen (Important changes observed by transmission electron microscopy (TEM) included irregularities and invaginations of the basement membrane into the epithelium  in additvacuoles , and burvacuoles . Exosomevacuoles . Lamellahe lumen .−3, n = 4), six-  and 12 month-old  PAP−/− mice DLP, but the apoptosis status was not different between genotypes at the same time points   .Snap25, Syt1, Syt4, Nrxn1, Cart, Abpa, Cplx1) and synaptic vesicle traffic  .To determine the effects of PAP deficiency at gene expression level, we performed microarray experiments of mouse prostatic tissue. The Gene Ontology analyses of differentially expressed genes showed, among the most significant groups, those genes associated with regulated secretory pathway, neurotransmitter secretion  . Compara−/− prostates and the differential expression of genes related to vesicle fusion, such as Snap25, Syt1, Syt4 and Cplx1, led us to search for proteins interacting with TMPAP. The yeast two-hybridization screening of human thymus library detected seven out of 15 clones expressing snapin (NM_012437.3), as a clear candidate for interaction with TMPAP.The disturbed exocytosis observed in the ultrastructural studies of PAPin vivo proof of the physical protein-protein interaction, was used to determine the interaction between TMPAP and snapin. Data analysis revealed significant FRET between TMPAP and snapin  compared to experiments with negative control  while in experiments with positive control FRET efficacy reached a level of 37.7±6.5% , but absent in androgen insensitive prostate cancer cells (PC3) −/− mice, progressive changes were observed in the prostatic tissue leading to the development of prostate adenocarcinoma at the age of 12 months. Despite the presence of prostate cancer, we have not detected any metastatic lesions. Histologically, the mouse DLP has been considered to be the analogous area to the peripheral zone of the human prostate, where the majorities of adenocarcinomas reside et al. suggested that the mouse models carrying genetic modifications that affect the tumor development in the DLP are more significant for the studies on those pathologies associated with the peripheral zone of the human prostate In all the PAP−/− mice.In humans, it has been observed that PTEN (phosphatase and tensin homolog deleted on chromosome 10) is downregulated in prostate cancer tissue specimens −/− mouse prostates showed a high amount of nanovesicles, compatible in size with exosomes. According to this finding, other authors have reported a significant increment of exosomes in plasma of prostate cancer patients compared to healthy donors or benign hyperplasia et al. showed that augmented levels of hypoxia inside solid tumors increased the release of exosomes −/− mice. The interaction between TMPAP and snapin detected in LNCaP cellsisanindication that disturbed exocytosis is involved in the phenotype we observed. The vesicular traffic is an intrinsic factor in the regulation of cell polarity, which is gaining attention as a determinant of tumor development The ultrastructural studies of PAP−/− mice.Our results in addition to our previous knowledge of the presence of PAP in the endosomal-lysosomal pathway et al., 70% of snapin is found in the cytosol In −/− mouse prostate. This could establish a significant starting point for uncontrolled cell proliferation and the development of prostate adenocarcinoma. Interestingly, in DRG of PAP−/− mice the levels of PI  P2 are increased when compared to wild-type mice 2 is the main regulator for clathrin-based endocytosis −/− mice requires a concomitant increased endocytosis mechanism to keep the cell membrane homeostasis.According to this model, the lack of TMPAP would lead to the observed dysregulation of vesicular traffic, exocytosisand release of exosomes in PAP−/− mouse model shows that TMPAP is required for the normal function of prostate in mice, and its deficiency leads to prostate adenocarcinoma. This suggests that TMPAP acts as a regulator of endo-/exocytosis mechanism.In summary, this PAPFile S1Supplementary fileincludes: Figure S1: Pan cytokeratin immunohistochemistry of DLP from 12 month-old animals. Tables S1: Proliferative cell count statistics. Tables S2: Proliferative and non-proliferative cell counts. Tables S3: Apoptotic cell count statistics. Tables S4: Apoptotic and non-apoptotic cell counts. Tables S5: Significant ontological groups in the cellular component category obtained with Genomatix Bibliosphere software from two-color microarrays experiments. Tables S6: Significant ontological groups in the biological process category obtained with Genomatix Bibliosphere software from two-color microarrays experiments. Supplementarymethodology.(DOCX)Click here for additional data file.