A distinct structural feature, where structural segments in a protein dimer or higher oligomer were shared between two or more chains of a protein structure, characterizes 3D domain swapping. 3D domain swapping was observed as a key mediator of numerous functional mechanisms and play pathogenic role in various diseases including conformational diseases like amyloidosis, Alzheimer's disease, Parkinson's disease and prion diseases. We report the first study with a focus on identifying functional classes, pathways and diseases mediated by 3D domain swapping in the human proteome.P-value < 0.05).We used a panel of four enrichment tools with two different ontologies and two annotations database to derive biological and clinical relevant information associated with 3D domain swapping. Protein domain enrichment analysis followed by Gene Ontology (GO) term enrichment analysis revealed the functional repertoire of proteins involved in swapping. Pathway analysis using KEGG annotations revealed diverse pathway associations of human proteins involved in 3D domain swapping. Disease Ontology was used to find statistically significant associations with proteins in swapped conformation and various disease categories (We report meta-analysis results of a literature-curated dataset of human gene products involved in 3D domain swapping and discuss new insights about the functional repertoire, pathway associations and disease implications of proteins involved in 3D domain swapping.Our integrated bioinformatics pipeline comprising of four different enrichment tools, two ontologies and two annotations revealed new insights into the functional and disease correlations with 3D domain swapping. GO term enrichment were used to infer terms associated with three different GO categories. Protein domain enrichment was used to identify conserved domains enriched in swapped proteins. Pathway enrichment analysis using KEGG annotations revealed that proteins with swapped conformations are present in all six classes of KEGG BRITE hierarchy and significantly enriched KEGG pathways were observed in five classes. Five major classes of disease were found to be associated with 3D domain swapping using functional disease ontology based enrichment analysis. Five classes of human diseases: cancer, diseases of the respiratory or pulmonary system, degenerative diseases of the central nervous system, vascular disease and encephalitis were found to be significant. In conclusion, our study shows that bioinformatics based analytical approaches using curated data can enhance the understanding of functional and disease implications of 3D domain swapping. We used complete set of human proteins from 3DSwap database and examined statistically significant domains, biological process, cellular component, molecular function, biological pathways and diseases using enrichment methods. From a bioinformatics perspective, this manuscript is a case study that leverage application of robust bioinformatics methods to gain new functional and therapeutic insights from a protein structural mechanism.Computationally efficient classification, annotation and prediction algorithms are rapidly improving our understanding of protein sequence-structure-function relationships. Analysis of such relationships often helps in our understanding of novel sequence or structural features in the regulation of a particular function including molecular pathways and various disease mechanisms. Cells attain its functional integrity with the help of molecular mechanisms including protein-protein interactions -7. Prote3D domain swapping is a unique protein structural phenomenon with implications in function, form and disease Figure . Only twIrrespective of numerous biochemical and computational studies focused on the molecular basis of 3D domain swapping ,34-52, aP-value was calculated. In SEA-based approach, annotation terms of subset of genes were assessed one at a time against a list of background genes. An enrichment p-value was calculated by comparing the observed frequency of an annotation term with the frequency expected by chance and individual terms beyond the p-value cut-off . BiNGO [2 χtests [Enrichment analysis plays an important role in knowledge-based bioinformatics approaches ,55. In t). BiNGO , FunctAs). BiNGO , Onto-ex). BiNGO ,67 are e). BiNGO  and topG). BiNGO  employ t). BiNGO . For exa). BiNGO . A varie). BiNGO ,72, hype). BiNGO , binomiaχ2 tests  or combiχ2 tests  and Ontoχ2 tests ,67 for sχ2 tests ,76.Gene ontology enrichment and pathway enrichment analysis, using ontologies or annotations derived from a subset of genes characterized from an experimental or computational study, generally applied to infer new biological insights, which was otherwise impossible with candidate gene-centric approaches. Due to the generic nature of statistical methods used in enrichment analysis, current set of enrichment algorithms and related statistical methods can be used to infer enrichment from annotation databases. Enrichment calculations are currently available for various types of annotations. Annotations of protein domains , pathwtools, two ontologies and two annotation databases for inferring functional and disease insights from list of human proteins involved in 3D domain swapping. Protein domain enrichment was performed using DAVID 6.7. Protein domain annotations were derived from Pfam database, a database of evolutionarily conserved protein domain coordinates. Ontologizer 2.0, a GO term enrichment tool with command-line interface and improved statistical method for deriving GO terms enriched in a given list of proteins was used in this study. SubPathwayMiner, an R package that internally handles KEGG annotations for pathway enrichment analysis were used to derive statistically significant pathways associated with the dataset. Enriched disease ontology terms were identified using Functional Disease Ontology server that consults Disease Ontology and it's derivative disease-ontology lite for identifying significant diseases. 0 H= List of curated proteins with swapped conformations are not associated with any class of protein domains, gene ontology terms, KEGG pathways or disease ontology terms. We tested our null hypothesis individually using four different tools and associated annotations or ontologies. P-value from enrichment analyses were obtained using default statistical settings of different tools employed in this study. Protein domain enrichment P-values were derived from DAVID using a modified Fisher Exact P-value, called EASE score [P-values were derived using Ontologizer 2.0 and corrected using Bonferroni method [P-values. Disease enrichment analysis was performed using Functional Disease Ontology server and it uses a Fisher's exact test for deriving P-values.This study utilized four SE score . GO termi method . KEGG paHomo sapiens. A cursory look at 3DSwap database for the taxonomic spread would indicate that the largest fraction was from humans (25.6%)  Figure . Human pProtein domain enrichment analysis was performed using DAVID . KEGG paTo perform protein domain enrichment analysis, domains were identified in proteins involved in 3D domain swapping and a list of protein domains was obtained. This list of protein domains was compared against a reference dataset of protein domains associated with complete human proteome. Protein domain enrichment analysis was performed to understand statistically significant, conserved, functional modules associated with proteins involved in 3D domain swapping. Dataset of 45 Uniprot identifiers were used for protein domain enrichment analysis using Pfam annotations. DAVID version 6.7 with default settings was used for the analysis.x is also annotated to all parent terms of x, and this often leads to false enrichment calculations. Such relationships  were taken into account in Ontologizer 2.0 using parent-child inheritance concepts [Homo sapiens) specific GO enrichment analysis and pathway analysis. GO enrichment analysis was performed using the following parameters using Ontologizer 2.0: Gene Ontology annotations were derived from human-specific annotation data (gene_association.goa_human) [GO term enrichment analysis in this study was performed using Ontologizer 2.0, a multifunctional tool for GO term enrichment analysis. Ontologizer was selected due to the improved statistical approximation methods incorporated in it. A brief description of the method is provided here. Generic GO enrichment tools calculate the enrichment of a GO term with respect to the list of genes in the dataset and the background population using the probability of drawing the same or higher number of genes annotated to a given term. This basic concept was implemented using statistical test involving the upper tail of the hypergeometric distribution or one-tailed Fisher's exact test. Such methods do not consider relationships between the annotation terms. GO is defined as a directed acyclic graph (DAG), with various levels of relationships between the terms. Due to DAG architecture of GO, a gene or gene product annotated with a term concepts . Detaileconcepts ,84. Dataa_human) , multiplPathway enrichment analysis using KEGG pathway annotations were performed to understand the role of proteins in 3D domain swapping conformation in various biological pathways. UNIPROT Identifiers were mapped to Entrez gene identifiers using custom Perl scripts and used as the input in R package SubPathwayMiner  for pathThe disease ontology tem enrichment analysis was performed using Functional Disease Ontology server . List of3D domain swapping is a structural mechanism employed by a variety of protein structures to form oligomeric assemblies. These oligomers were often associated with aggregation diseases or proteinopathies in humans. Parkinson's diseases and Alzheimer's diseases are two major neurodegenerative diseases due to phenotypic impact of 3D domain swapping. Hitherto, no comprehensive study has been reported to analyze the impact of all proteins involved in 3D domain swapping from a whole proteome-wide or genome-wide perspective due to unavailability of a well-defined, curated dataset. We performed the initial investigation of proteins involved in 3D domain swapping in the level of protein domains, Gene Ontology, KEGG pathways and Disease Ontology. Our approach helped to understand enriched protein domains, Gene Ontology terms, biological pathways and Disease Ontology terms mediated by these proteins and their role in mediating various human diseases.Statistically significant protein domains associated with swapped proteins in the human proteome is provided Table , GO termvia 3D domain swapping. Machine learning algorithms and computational approaches may help to predict more proteins with features of 3D domain swapping [Proteins involved in 3D-domain swapping represents a large collection of proteins with a variety of functional and regulatory roles in the cell. Due to limitation in crystallizing structures in the swapped conformation, currently available repertoire of proteins in the swapped conformation may represent only a small fraction of proteins that may perform its molecular role swapping ,52. HereProtein domain enrichment analysis reveals that five protein domain families were enriched in the dataset , diseases of the respiratory or pulmonary system , degenerative diseases of the central nervous system , vascular disease  and encephalitis (rabies). Neurodegenerative diseases are well-known to have strong association with 3D domain swapping, but insights into other diseases indicates that there could be more proteins with disease association and 3D domain swapping, beyond the currently well-known group of conformational diseases. Detailed table with Disease Ontology term (disease), genes associated with each disease and Domain swapping is a key pathophysiological mechanism mediating conformational disease. A detailed account of functional repertoire, molecular pathways and spectrum of diseases affected by this mechanism remains elusive. We used enrichment calculations to understand the aspects using a curated dataset of proteins involved in 3D domain swapping. Our analysis was performed using a dataset of 45 unique human proteins derived from 3DSwap knowledgebase . This daDue to oligomeric features of swapping, earlier studies have indicated that 3D domain swapping plays a crucial role in conformational diseases or deposition diseases and proteinopathies. There was limited insight on structure-function relationship of proteins involved in domain swapping due to unavailability of a large dataset to objectively analyze functional or disease implications implicated by 3D domain swapping. Proteins encoded in the human genome and reported to be involved in 3D domain swapping were analyzed in detail to understand the role of gene products in various classes of diseases, beyond conformation diseases or proteinopathies. Mapping and enrichment analysis of human proteins involved in 3D domain swapping to KEGG pathways in 'disease' class and Disease Ontology indicates that these proteins play a significant role in various other diseases categories along with well-known neurodegenerative or conformational diseases.Availability of genome-scale sequence data and annotations were considered as the ideal resource for gaining new insights from a plethora of biological data. Structural mechanisms can gain new insights about the functional aspects by mapping and database-wide enrichment analysis using annotations. In a similar way, functional mechanism may also gain new insight by using knowledge-based approaches employed in this study. In summary, the present study reports the application of knowledge-based approaches to understand new functional insights about a structural mechanism. Starting from an initial dataset of protein structures, the present study shows the importance and impact of the data integration and data mining to derive biologically relevant interpretations of global trends of a structural mechanism from sequence, functional and disease perspective. Further new insights are obtained from a translational perspective by focusing on proteins involved in 3D domain swapping in the human genome. 3D domain swapping is a unique phenomenon and may affect availability of active sites and binding sites required to impart the biological function depending on the swapped conformation. Perhaps, future drug design studies should consider these important aspects while developing therapeutics for various disease categories where 3D domain swapping is observed.In the current era of personal genomes and network medicine, clinical and therapeutic approaches are utilizing integrated approaches for the understanding of disease states and pathophysiological mechanisms. Complex disease states are often triggered by perturbations in multiple pathways by multiple genes -94. ProtInsights obtained from our disease ontology analysis indicates that 3D domain swapping is not just confined to neurodegenerative diseases, proteins in swapped conformation play a significant role in several other classes of diseases like cancer, vascular disease, pulmonary disease etc. Enrichment results discussed in this paper will be useful in such studies in the future from biochemical, functional, structural and therapeutic perspective. Our analysis also indicates that further genome-specific analysis of proteins involved in 3D domain swapping, using comparative genome analysis framework, may also add further understanding of functional, structural and pathophysiological manifestations of 3D domain swapping.3D domain swapping is an important structural mechanism associated with a diverse set of proteins involved in multitude of biological processes and molecular functions and diseases including proteinopathies. This phenomenon is often studied from the perspective of protein structure and its impact on biological pathways, correlations with biological functions and association with classes of diseases other conformational diseases were largely unknown. We performed a knowledge-based analysis of human proteins involved in 3D domain swapping to find the key functions, pathways and diseases associated with 3D domain swapping. Our study was limited to 45 unique proteins involved in 3D domain swapping. 3D domain swapping is a functionally relevant phenomenon due to its primary role in protein oligomerization; proteins with swapped oligomeric states are being identified on a regular basis using crystallography experiments. Effective algorithms that can predict swapping from structural and sequence information may also help to identify more proteins in swapped confirmation. As more proteins are being characterized in swapped conformation, performing such knowledge-based analysis using new proteins, improved annotations and enhanced ontologies may reveal additional functional classes, pathways and disease. In summary, we showed results from an initial investigation to understand conserved protein domains, functional repertoire, pathways and diseases mediated by 3D domain swapping in human proteome.The authors declare that they have no competing interests.KS curated the data, performed the analysis and compiled the first draft of the manuscript. RS conceived the project, designed the curation strategy, discussed the approaches and provided critical comments to the manuscript. All authors read and approved the final manuscript.Supplementary Table 1Click here for fileFigure S2Click here for fileFigure S3Click here for file


Our ultimate objective, however, is to discern HER2 overexpressing tissue from normal tissue in whole, nonsectioned, specimens to facilitate rapid diagnoses. Here, we use targeted nanoshells to quickly and effectively visualize HER2 receptor expression in intact ex vivo human breast tissue specimens. Punch biopsies of human breast tissue were analyzed after a brief 5-minute incubation with and without HER2-targeted silica-gold nanoshells using two-photon microscopy and stereomicroscopy. Labeling was subsequently verified using reflectance confocal microscopy, darkfield hyperspectral imaging, and immunohistochemistry to confirm levels of HER2 expression. Our results suggest that anti-HER2 nanoshells used in tandem with a near-infrared reflectance confocal microscope and a standard stereomicroscope may potentially be used to discern HER2-overexpressing cancerous tissue from normal tissue in near real time and offer a rapid supplement to current diagnostic techniques.Tumor margin detection for patients undergoing breast conservation surgery primarily occurs postoperatively. Previously, we demonstrated that gold nanoshells rapidly enhance contrast of HER2 overexpression in  Currently, breast cancer is the second leading cause of cancer-related deaths in women, and it accounts for approximately one-third of all cancers diagnosed in women in the United States . To reduPresently, intraoperative tumor margin detection occurs primarily in specialized tertiary centers, such as The University of Texas M.D. Anderson Cancer Center (MDACC). In these centers, the resected tissue receives a preliminary evaluation by a pathologist while the patient remains in the operating room; if necessary, additional tissue can be removed until the pathologist determines that the tumor margins are negative. In community hospitals, however, pathologic analysis of excised tissue only occurs postoperatively . Patient While histologic analysis remains the gold standard for tumor margin assessment, the macroscopic evaluation of whole, nonsectioned tissue specimens may also be used to provide an intraoperative estimate of tumor margin status prior to subsequent processing. This would be an invaluable tool in hospitals without onsite pathology suites. Macroscopic visualization of questionable tissue is attractive for enhancing the sensitivity and specificity of tumor margin delineation: if the number of suspicious regions that require further microscopic processing can be reduced, surgeons and pathologists can focus their attention and resources on areas that remain inconclusive. Currently, macroscopic evaluation only occurs for breast cancer specimens that involve microcalcifications or nonpalpable masses and does not occur for palpable breast masses . For non In preceding studies, we confirmed that silica-based gold nanoshells targeted to the Human Epidermal growth factor Receptor 2 (HER2) could be used for the rapid contrast enhancement of both cells  and tissex vivo human breast tissue specimens without sectioning. We first confirm the predominance of the surface targeting needed to identify the tumor margins and preferential labeling of HER2-positive tissue using two photon and hyperspectral imaging. Then, we demonstrate that anti-HER2-targeted gold nanoshells can be used as rapid diagnostic imaging agents for HER2 overexpression in intact breast tissue specimens using a standard stereomicroscope and confirm these results through reflectance confocal microscopy and immunohistochemistry. To facilitate prompt tumor margin detection intraoperatively, the ability to assess tumor margins without physical sectioning is highly desirable as sectioning may incur significant time to the surgical procedure . Thus, i∼1–3 nm diameter) was fabricated and adsorbed onto the surface of the silica cores via the amine groups to form gold nucleation sites [43H2O) was dissolved in potassium carbonate and then added with formaldehyde to help reduce the gold. After the gold layer over the silica cores was formed, the spectrum of the final nanoshell solution was visualized using a UV-VIS spectrophotometer (Varian Cary 300)  .9 particles/mL. To determine the concentration of nanoshells in solution, the absorption, scattering, and extinction coefficients were determined using Mie theory. The average nanoshell diameter, as validated by scanning electron microscopy (SEM), was 314 nm with a peak surface plasmon resonance at 840 nm. The concentration of the working nanoshell solution was approximately 2.0 × 10μL. Nanoshells were targeted to biological HER2 antigens by linking the surfaces of the nanoshells to anti-HER2 antibodies using previously described methods . Prior tNormal and cancerous (HER2-negative and HER2-positive) breast tissue specimens were supplied by the Cooperative Human Tissue Network (CHTN) through a protocol approved by the Institutional Review Board (IRB). Tissues were designated as normal or cancerous by pathologists at the medical centers where the tissue samples were obtained. Additionally, HER2 status was previously determined by pathologists at the respective medical centers prior to the patients undergoing any form of medical treatment.Before use, samples were thawed briefly in a 37°C water bath and cut on a disposable cutting board using a 5 mm punch biopsy to maintain size consistency. At least two punch biopsies were taken from each specimen for control and experimental conditions. Each cut specimen used was 5 mm in diameter with an average thickness of 1 mm. Tissue samples were subsequently incubated in prewarmed antibody diluent for 1 minute at room temperature with gentle agitation in a 24-well plate. After prerinsing, the samples were incubated in either antibody diluent or the aforementioned targeted-nanoshell cocktail in polyethylene sample vials (Sigma Aldrich). The vials were placed on a nutator in an incubator at 37°C for 5 minutes. After incubation, the tissue samples were removed from the vials and rinsed 3 times in 1x PBS briefly in a 24-well plate. Samples were moved to a clean well of 1x PBS prior to imaging. z-stack (depth) increment of 5 μm. In order to calculate the percentage of area covered by nanoshells, ImageJ imaging software was implemented after image acquisition. Recent research has shown that ImageJ can be used to analyze signal intensity of silica-gold nanoshells under different imaging systems [Both HER2-positive and HER2-negative cancerous samples were evaluated for surface labeling of HER2-targeted nanoshells by employing two-photon imaging of intact breast tissue specimens. Samples were placed directly on a glass coverslip (Fisher Scientific), and an additional coverslip was placed on top of the tissue in order to facilitate moderate tissue compression. For image acquisition, a Zeiss multi-photon confocal microscope (LSM 510 META NLO) was used in tandem with a Coherent Chameleon femtosecond-pulsed, mode-locked Ti: sapphire laser. This system was set to operate as formerly described . Specifi systems , 24. Eacμm using a Leica CM1850 UV cryostat. Cancerous specimens were sectioned at −20°C and normal specimens at −30°C. The different temperatures were used to maintain optimal tissue morphology as recommended by Leica. Additionally, Magalhães et al. reported on the use of different temperatures to slice normal and cancerous tissue [ To confirm the presence of nanoshells on the surface of the tissues, HER2-positive cancerous, HER2-negative cancerous and normal tissue samples were incubated with nanoshells as previously described. A thin layer of pathological ink was placed on the tissue surface for orientation. The tissues were embedded in OCT media  and frozen rapidly over dry ice. The specimens were cut at a section thickness of 8 s tissue . The sec Spectral data of each field of view (FOV) was used to determine if nanoshells were present on each slice of tissue. Comparisons were made between tissue surfaces and tissue beyond the surfaces to determine the presence of nanoshells; spectral data from tissue that was not incubated with nanoshells was also used as a negative control. Normal and HER2-positive cancerous breast tissue specimens (from patients who had and had not received neoadjuvant chemotherapy) were imaged using a Zeiss Discovery. V8 stereomicroscope equipped with a VisiLED MC1000 light source. For macroscopic imaging of breast tissue specimens, a thin plastic black stage was placed beneath a glass coverslip to enable ease of tissue placement and to provide a consistent black background among all samples. The specimens (controls and respective nanoshell-labeled counterparts) were placed alongside each other on top of the coverslip. Images were taken at both 1x and 2x magnification under the same lighting conditions. Following widefield imaging, the aforementioned samples were prepared for microscopic analysis under reflectance confocal microscopy. For this component of the study, a Lucid VivaScope 2500 inverted confocal microscope was used. Samples were placed directly on glass slides that were modified by the addition of an adhesive 1 mm deep, 20 mm diameter silicon isolator (Invitrogen). To compress the tissue slightly and consistently among samples, an adhesive tissue cassette  was placed directly on top of the silicone isolators above the tissue specimens. Multiple images were taken at a power of 0.4 mW and at the same distance from the glass surface for both samples and controls. After reflectance imaging, the samples were prepared for histological processing. Additionally, reflectance intensity measurements were recorded using ImageJ processing software as formerly described .μm. Multiple sections from each specimen were prepared for either immunohistochemistry (IHC) or hematoxylin and eosin (H&E) staining. IHC for the HER2 antigen was executed using the Histostain Plus AEC Broad Spectrum Kit (Invitrogen) per manufacturer's instructions. H&E staining was also performed per manufacturer's instructions (Sigma Aldrich) for the alcoholic Eosin Y solution. For image acquisition, a standard brightfield microscope (Zeiss Axioskop 2 equipped with a Zeiss Axiocam MRc5 color camera) was used at a magnification of 20x.Once images were collected under both stereomicroscopy and RCM imaging systems, normal and HER2-positive cancerous samples (with and without previous neoadjuvant chemotherapy) were embedded in OCT media and sectioned to a thickness of 5 z-direction represents 5 μm into the tissue. Qualitatively, the first image (taken at the surface or at 0 μm) in The goal of this study was to evaluate the distribution of anti-HER2-conjugated gold nanoshells on resected intact tissue specimens. For comparison, the nanoshell labeling between HER2-positive and HER2-negative tissue samples was evaluated using a two-photon imaging system. As previously reported, this imaging system is capable of enhancing and capturing the luminescence signature of the gold nanoshells  while alμm beyond the surface of the same tissue. Spectra from multiple (n = 3) specimens that were incubated with anti-HER2 nanoshells were acquired, and analysis showed that tissues without nanoshells had very similar spectra across different patients. μm beyond the surface. In fact, the spectra of the tissue beyond the surface of the nanoshell-labeled specimen are very similar to the spectra of the surface of the control. These results support our findings that the targeted nanoshells primarily localized to the surface of the tissue.To further validate the surface binding of the nanoshells, hyperspectral images of different tissue sections were also acquired. ex vivo HER2-positive tissue specimens, we assessed the potential of using a standard stereomicroscope to visualize this enhanced contrast. For this component of the study, human breast tissue specimens that overexpressed HER2 receptors at the time of patient diagnosis were evaluated and compared to normal breast tissue. Due to the ultimate goal of utilizing gold nanoshells to rapidly label tumor margins intraoperatively in diverse patient populations, we examined tissue from patients who had and had not undergone neoadjuvant chemotherapy. All tissue samples were incubated with either antibody diluent buffer or the anti-HER2-targeted nanoshells for 5 minutes at 37°C. As shown in Based on previous results demonstrating the preferential labeling of HER2-targeted nanoshells on the surface of intact While the degree of nanoshell labeling can be visualized without image adjustments under a standard stereomicroscope, the superior extent of this labeling can be seen more clearly after a simple contrast enhancement using imaging software (ImageJ). As seen in ∼2.5 to 3 times greater for both the HER2-positive tissue sample receiving chemotherapy and for the HER2-positive tissue not receiving chemotherapy when compared to the normal tissue sample. To validate the enhanced nanoshell labeling seen by macroscopic imaging, the surfaces of the same tissue samples were also imaged using reflectance confocal microscopy . ConcurrSubsequent histological analysis shown in ex vivo human breast tissue and confirmed binding location via confocal and darkfield hyperspectral microscopy. By utilizing silica-gold nanoshells designed as rapid diagnostic imaging agents, surgeons and pathologists may be able to realize tumor margin status directly in the operating room after both macroscopic and microscopic assessment. While multiple methods of intraoperative tumor margin detection are currently under investigation [ex vivo specimens based on the desire to enhance current methodologies without delay in clinical translation due to regulatory concerns associated with in vivo systems.In this study we demonstrated the ability to use targeted gold nanoshells to rapidly improve visualization of a specific biomarker associated with disease aggression and progression (HER2) in intact tigation –31, we aex vivo intraoperative tumor margin detection without the need for lengthy incubation times or the use of optical clearing agents. The ability to enhance contrast of malignancy using topically applied agents has previously been demonstrated for oral and breast tissue using fluorescently labeled deoxy-glucose and epidermal growth factor (EGF) conjugates –34 as weex vivo tissue from normal tissue within the same incubation time, and we demonstrate that this difference can be observed macroscopically. These results are supported by microscopic imaging and immunohistochemistry against HER2.Recently, we verified that silica-based gold nanoshells could be used to enhance contrast of both HER2-overexpressing cells and tissue sections within 5 minutes of incubation time , 11. HowBy employing macroscopic imaging intraoperatively, clinicians may be better able to distinguish cancerous and normal breast tissue prior to further microscopic analysis and subsequent histological processing. Ultimately, this system could also be used for other diagnostic applications, for other anatomical locations, and for other biomarkers associated with disease. By facilitating fast and accurate tumor margin results intraoperatively as a supplement to current diagnostic methods, we expect to reduce the amount of time spent in surgery due to inadequate tissue removal. To translate these findings more readily to the clinic, we are presently developing a low-cost widefield imaging system that can be used to detect the overexpression of HER2 (and other extracellular biomarkers) through contrast enhancement provided by gold nanoshells. In addition, we plan to collect data from diverse patient populations and assess results with fresh tissue samples. In this way, the use of gold nanoshells may demonstrate widespread efficacy or be limited only to specific patient subsets.


Laboratory-induced dyspnea (breathing discomfort) in healthy subjects is widely used to study perceptual mechanisms, yet the relationship between laboratory-induced dyspnea in healthy volunteers and spontaneous dyspnea in patients with chronic lung disease is not well established. We compared affective responses to dyspnea 1) in COPD patients vs. healthy volunteers (HV) undergoing the same laboratory stimulus; 2) in COPD during laboratory dyspnea vs. during activities of daily living (ADL).2 (PETCO2) during restricted ventilation, evoking air hunger. We used the multidimensional dyspnea profile (MDP) to measure intensity of sensory qualities  and work/effort (W/E)) as well as immediate discomfort (A1) and secondary emotions (A2). Ten of the COPD subjects also completed the MDP outside the laboratory following dyspnea evoked by ADL.We induced moderate and high dyspnea levels in 13 COPD patients and 12 HV by increasing end-tidal COCOPD patients and HV reported similar levels of immediate discomfort relative to sensory intensity. COPD patients and HV reported anxiety and frustration during laboratory-induced dyspnea; variation among individuals far outweighed the small differences between subject groups. COPD patients reported similar intensities of sensory qualities, discomfort, and emotions during ADL vs. during moderate laboratory dyspnea. Patients with COPD described limiting ADL to avoid greater dyspnea.In this pilot study, we found no evidence that a history of COPD alters the affective response to laboratory-induced dyspnea, and no difference in affective response between dyspnea evoked by this laboratory model and dyspnea evoked by ADL. How well do laboratory models contribute to our understanding of clinical dyspnea? Dyspnea is defined by the American Thoracic Society as “… a subjective experience of breathing discomfort that consists of qualitatively distinct sensations that vary in intensity. The experience … may induce secondary physiological and behavioral responses” . This shAs is the case with pain models, a multidimensional perspective on dyspnea may also be useful for addressing question about the transferability of laboratory models to practice. A customary single-dimension measure of dyspnea, such as use of a modified Borg scale to rate sensory intensity (SI), may not adequately assess the differences seen between clinical and laboratory dyspnea. Although SI may predict affective response, the relationships among SI, A1 and A2 are likely to be influenced by factors such as sensory quality (SQ), pathophysiology, and environment. Thus, while laboratory models have been valuable in defining neural mechanisms of dyspnea, and in testing potential palliative interventions, they are prone to the criticism that clinical dyspnea is fundamentally different than laboratory dyspnea and that the experience of patients greatly alters their perceptual and emotional responses -14. For In this study, we tested the extent to which one laboratory model of dyspnea (air hunger) evokes different affective responses between COPD patients and HV. We also assessed differences in affective responses of COPD patients to the lab dyspnea model and dyspnea associated with activities of daily living (ADL). Using a multidimensional measurement instrument that incorporates qualitative and affective measurements, we first compared dyspnea perceptions of Healthy and COPD volunteers in the laboratory . We, the1) and the intensity of specific sensory qualities (SI) does not differ between healthy and COPD volunteers.H0_1 During experimentally induced dyspnea, the relationship between immediate affective response  does not differ between healthy and COPD volunteers.H0_2 During experimentally induced dyspnea, the relationship between immediate unpleasantness, discomfort and emotional affective response  and experimentally induced dyspnea.Subjects at least 45 years old were recruited through advertisement and referral by pulmonary physicians. We screened subjects for self-reported cardiopulmonary or neurological conditions, and administered the Baseline Symptom Inventory BSI-18) to identify subjects prone to panic disorder . No subj to ident2 (PETCO2) was sampled in one nostril through an unobtrusive tube. During experiments, subjects breathed through a mouthpiece; tidal PCO2 and pressure at the airway opening were sampled in the common line, and we monitored pulse rate, arterial oxygen saturation and blood pressure throughout the experiment. Data were digitized and recorded for later analysis .Subjects began each session by sitting comfortably and reading a standard text while end-tidal PCO2 value <0.49 (R < 0.7) for the regression of on-line discomfort ratings  on PETCO2.Using a standardized script  .We employed a hypercapnic stimulus with minute ventilation constrained to 0.13 liters/min/kg , by limiting flow to an inspiratory reservoir bag  The Multidimensional Dyspnea Profile (MDP) was completed immediately after each test, and subjects were instructed to focus on the final 30 to 60 seconds of the stimulus exposure (corresponding to the targeted end-point). Physiologic data associated with MDP ratings were averaged over the same 30 to 60 seconds.a The experimenters immediately reduced the stimulus if the subject rated 100%. The BDVAS was intended as a continuous measure of A1 (immediate discomfort), and was used to target comparable levels of A1 among subjects prior to completion of the MDP , or unpleasantness’ using an online ‘Visual Analog Scale’ (BDVAS) anchored with “No Discomfort” at 0 and “Stop Now” at 100% of full scale (%FS). 1); the intensity of specific sensory qualities (SQ) such as a sense of “air hunger” and “breathing work and effort” [2). Respondents also chose a single SQ that most aptly described the dyspnea sensation.  ,15. Resp effort” -23 and eAfter completing all laboratory experiments, COPD patients took home copies of the MDP to complete daily for 2 weeks following episodes of dyspnea associated with normal Activities of Daily Living (ADL). Patients also recorded the events that had provoked the reported dyspnea. MDP responses were averaged within subject to provide equal weighting, and were then compared to responses obtained from the same subjects following laboratory dyspnea.Descriptive statistics by subject type and stimulus intensity are reported as means and standard deviations. The statistical significance of between group  item response differences was evaluated by the Mann–Whitney U test, while the Wilcoxon test was used to evaluate within group (by stimulus intensity) differences. Repeated measures ANOVA was used to assess group by stimulus intensity interactions. Additional analyses to assess internal consistency and test-retest reliability of the MDP in a laboratory setting are provided in Additional file Analyses were performed on 12 HV and 13 COPD patients whose characteristics are shown in Table 1 . There was no significant difference by subject type .As would be expected from the similarity in underlying constructs, on-line BDVAS corresponded closely to subsequent MDP ratings of AETCO2 above resting) that evoked moderate  and high  ratings were not significantly different between subject groups (P = 0.18), and mean BDVAS and A1 were equivalent at both stimulus levels . The descriptor phrase “I feel that my breathing requires work or effort” (WE) was chosen as the best descriptor by 35% of subjects following moderate and 22% of subjects following high laboratory stimulus presentations. All other descriptor phrases, such as “my chest or lungs feel tight”, or “I am breathing a lot” were chosen as best descriptor by only 13% of subjects following laboratory stimulus presentations. Best descriptor choice did not differ significantly between COPD and healthy subjects.Despite discrimination of best descriptor choice at both stimulus levels, subjects provided roughly equal scale ratings of both sensory qualities; this apparent contradiction is discussed below. Following stimulus exposure at the moderate level, subjects rated AH at 46% full scale compared with 57% for WE (p = 0.035). Following exposure at the high level, corresponding ratings were 83% versus 86% (p = 0.223). Sensory quality ratings did not differ significantly by subject type.1 differed significantly between levels of perceived sensory intensity, (p < 0.001). We compared A1 ratings between groups at roughly matched levels of air hunger. There were no significant differences in mean ratings of A1 between healthy and COPD patients . To account for small differences in sensory intensity within level, we plotted mean A1 versus mean ratings of AH by stimulus level and subject type  COPD patients and HV report similar unpleasantness for a given intensity of sensation (AH and WE) in laboratory experiments; 2) COPD patients and HV reported similar emotional responses during laboratory dyspnea; 3) COPD patients reported similar frustration and anxiety with ADL as they did with laboratory dyspnea. Given the modest sample size, we consider below whether any important differences are likely to have gone undetected by this study (‘Type II error’).We showed that COPD patients and HV undergoing the same laboratory dyspnea experience report the same quality of sensation and the same immediate unpleasantness. This implies that long experience to clinical dyspnea has not greatly altered the immediate processing of respiratory discomfort. This is consistent with findings in competitive breath-hold divers whose perception of laboratory air hunger is not different from normal subjects .We observed that the emotional component of dyspnea differed widely among individuals at both moderate and high strength laboratory stimulus levels  is virtually nil at moderate stimulus, and at most 14% FS at high stimulus for anxious  were older than healthy volunteers (54.8 ± 4.8). We found no reports of age associated differences in dyspnea perception during well-controlled stimulus exposure among adult subjects who differ in age by 10 to 15 years. Indirect evidence of age associated differences comes from studies of resistive load threshold detection among older asthmatics ,29. As wThere was a clear favorite choice of descriptor that best characterized the sensation during laboratory dyspnea, yet there was considerable overlap in the ratings of descriptors on a scale from 0–10. Our interpretation of this is that when subjects are presented with several parallel rating scales, many tend not to greatly separate ratings. On the other hand, when forced to make a choice about the most prominent sensation, the subject makes a judgment. As expected from results in earlier studies that exposed subjects to a similar dyspnea stimulus , air hunETCO2) associated with the BDVAS ratings differed somewhat between COPD patients and HV, although the difference was not statistically significant. In COPD, PETCO2 is not a reliable indicator of arterial blood gasses due to heterogeneous ventilation/perfusion ratio, the effect of which causes PETCO2-PaCO2 difference to vary with tidal volume, TE, and PICO2-PaCO2 difference. Furthermore, ratings of SI for a given ∆PCO2 is known to vary widely among individuals [2. Because the range of ∆PCO2 was modest, approximately 2–10 mmHg, symptoms and signs attributable to elevated PaCO2, such as headache, elevated blood pressure and flushing, did not appear to affect discomfort ratings.The average changes from resting  in multi multidimensional models of pain, ‘Discomfort’ is employed in the ATS dyspnea statement.Authors O’Donnell, Schwartzstein, Lansing, Gulifoyle, Elkin, and Banzett declare that they have no competing interests to report.CRO participated in study design, study conduct, data collection and analysis, and manuscript preparation. RMS participated in study design, data analysis, and manuscript preparation. RWL participated in study design, data analysis, and manuscript preparation. TG participated in data management and analysis and in manuscript preparation. DE participated in study design, data collection, data management, and in manuscript preparation. RBB participated in study design, study conduct, data collection and analysis, and manuscript preparation. All authors have read and approved this manuscript in its final form.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/27/prepubText of standardized script.Click here for fileSchematic of hypercapnic stimulus apparatus and on-line rating device.Click here for fileMultidimensional Dyspnea Profile.Click here for fileMDP Test-Retest Measures.Click here for fileSubject Characteristics.Click here for filePlot of MDP A1 ratings versus on-line BDVAS ratings showing close correspondence among both types of subjects. (DOC 36 kb)Click here for fileInterindividual variability of anxiety ratings (A2) following moderate and high level stimulus exposure by subject type . Note that the range of ratings at matched A1 is similar between subject type. Click here for file


In birds and feathered non-avian dinosaurs, within-feather pigmentation patterns range from discrete spots and stripes to more subtle patterns, but the latter remain largely unstudied. A ∼55 million year old fossil contour feather with a dark distal tip grading into a lighter base was recovered from the Fur Formation in Denmark. SEM and synchrotron-based trace metal mapping confirmed that this gradient was caused by differential concentration of melanin. To assess the potential ecological and phylogenetic prevalence of this pattern, we evaluated 321 modern samples from 18 orders within Aves. We observed that the pattern was found most frequently in distantly related groups that share aquatic ecologies , suggesting a potential adaptive function with ancient origins. Complex pigmentation patterns like spots and stripes are common features of avian plumage A fossil feather with a distinct gradient of coloration along the proximo-distal axis was recovered from the Fur formation in Denmark 2S , which dissolves feather keratin by breaking disulfide bridges of cystine 2S was performed to ensure keratin degradation to the point where melanosomes, but not feather keratin, were observed. After a maximum of 6 hours of incubation, the Na2S solution was carefully rinsed away by slowly injecting distilled water through the slides as before. After drying, the two slides were separated, leaving prints of melanosomes on both slides , and melanosome concentration was quantified on both slides using ImageJ’s ‘count particles’ function from light micrographs of the dissolved feathers.Based on relationships between melanin concentration and darkness in human hair h slides , which wWe then tested if the relationship between melanosome density and color held true in the fossil feather  using two methods. First, we placed a copper wire mesh on top of the feather (the part), dividing it longitudinally into five equal-width 6.2 mm bins. Tin foil was wrapped around the sides and bottom of the fossil to reduce charging in the scanning electron microscope. Twenty images were taken within each bin at randomized locations along the proximal end of barb rami using a Philips XL 30 ESEM . MelanosSecond, we used the trace metal mapping techniques developed and described in −2) were calibrated from the dead time corrected fluorescence counts in each channel by using thin film standards deposited on mylar from Micromatter , and collected under the same conditions of beam intensity and sample to detector distance. Data processing consisted of taking the median intensity over a 3×3 pixel area which improved signal to noise ratios over areas with lower count rates. We calculated mean metal concentrations in fifteen different regions of interest (ROIs) throughout the feather and in the surrounding matrix  using beam line 10–2. The incident x-ray energy was set to 11.0 keV using a Si (111) double crystal monochromator with the storage ring Stanford Positron Electron Accelerating Ring (SPEAR) containing 350 mA at 3.0 GeV. The fluorescence lines of the elements of interest, as well as the intensity of the total scattered X-rays, were monitored using a silicon drift Vortex detector (SII NanoTechnology USA Inc.). The microfocused beam of ∼50×50 µm was provided by a focusing x-ray polycapillary optic (XOS). The incident and transmitted x-ray intensities were measured with nitrogen-filled ion chambers. Samples were mounted at 45° to the incident x-ray beam and were spatially rastered in the microbeam while data were collected continuously during stage motion. Beam exposure was 30 ms per pixel. Metal concentrations , from the United States National Museum collection. Two independent observers (authors MDS and LD) graded feathers on a scale of zero to three, with zero indicating absence of the gradient, 1 indicating presence of the gradient, 2 indicating a binary pigment deposition with a uniformly dark pennaceous feather and an unpigmented afterfeather , and 3 indicating an inverse gradient . Examples of these gradient categories are depicted in Spearman’s rank analysis  showed a2 = 5.12, p = 0.02). A bimodal pigmentation pattern  was found in an additional 15% of samples. No pattern was observed in the remaining 44% of samples. Interestingly, the occurrence of within-feather pigmentation gradients seems to be associated with ecology, as gradients are most commonly observed in waterbirds that are distantly related to one another  is observed in 3%. The standard gradient was found in contour feathers with higher frequency than in flight feathers , corroborating the assumption that melanin expression may be costly et al. found no evidence for condition dependence of melanin-based ornamentation in an identical study of Barn Owls  The question of why a feather is colored a certain way can be answered at both a proximate and ultimate level Alternatively, the high frequency of pigmentation gradients and their conservation in numerous distantly related waterbird clades suggest that these gradients may serve an adaptive ecological function. Such convergent evolution in animals with shared ecologies typically indicates adaptation and/or constraint ArchaeopteryxThese data suggest that within-feather pigmentation gradients are relatively common amongst birds, and are found most frequently in waterbirds. Additionally, we demonstrate that melanosome concentration reflects feather brightness, and thus may help improve the discriminant power of fossil feather color reconstructions. The gradients seen in dark feathers lacking other within-feather pigmentation patterns Figure S1Part (left) and counterpart (right) of a fossil feather recovered from the Fur Formation of Denmark. Identical darkness gradients can be observed in both, ruling out uneven splitting as the cause of the gradient observed in the part.(TIFF)Click here for additional data file.Figure S22S feather degradation, and subsequent melanosome density analysis.The process of Na An intact feather is placed between two glass microscope slides, and wetted with Na2S. After an incubation period and rinsing, the slides are separated to reveal a melanin print on both slides. Light micrographs of the melanin print are then analyzed to quantify differential melanosome densities in various regions of the feather. A) Intact contour feather from Himantopus mexicanus, B) Same feather after treatment with Na2S, removing most of the keratin surrounding the melanin, C) and D) close ups of dark (C) and light (D) regions of the feather showing higher concentrations of melanosomes in the dark region. Scale bars = 10 µm(PDF)Click here for additional data file.Figure S3A) Locations of 15 regions of interest (ROIs), where mean metal concentrations were analyzed. B) Representative image of matrix surrounding the fossil feather, demonstrating that no melanosomes are present.(PDF)Click here for additional data file.Figure S4Representative pictures of feathers exhibiting the four pigment concentration gradient categories. (0) absence of gradient (Ramphastos tucanus), (1) presence of gradient , (2) binary gradient (Sarcoramphus papa), (3) inverse gradient (Fluvicola nengeta).(PDF)Click here for additional data file.Table S1Average trace metal concentration for the 15 regions of interest (ROIs) shown in (XLSX)Click here for additional data file.


Rapamycin, an immunosuppressant agent used in renal transplantation with antitumoral properties, has been reported to impair longitudinal growth in young individuals. As growth hormone (GH) can be used to treat growth retardation in transplanted children, we aimed this study to find out the effect of GH therapy in a model of young rat with growth retardation induced by rapamycin administration. Three groups of 4-week-old rats treated with vehicle (C), daily injections of rapamycin alone (RAPA) or in combination with GH (RGH) at pharmacological doses for 1 week were compared. GH treatment caused a 20% increase in both growth velocity and body length in RGH animals when compared with RAPA group. GH treatment did not increase circulating levels of insulin-like growth factor I, a systemic mediator of GH actions. Instead, GH promoted the maturation and hypertrophy of growth plate chondrocytes, an effect likely related to AKT and ERK1/2 mediated inactivation of GSK3β, increase of glycogen deposits and stabilization of β-catenin. Interestingly, GH did not interfere with the antiproliferative and antiangiogenic activities of rapamycin in the growth plate and did not cause changes in chondrocyte autophagy markers. In summary, these findings indicate that GH administration improves longitudinal growth in rapamycin-treated rats by specifically acting on the process of growth plate chondrocyte hypertrophy but not by counteracting the effects of rapamycin on proliferation and angiogenesis. Rapamycin or sirolimus  is used as immunosuppressant in transplanted patients. Rapamycin directly inhibits mammalian target of rapamycin (mTOR), a ubiquitous kinase that controls different pathways related with cell proliferation, growth and metabolism In pediatric renal transplantation, rapamycin is mainly used as a substitute for calcineurin inhibitors when nephrotoxicity or proliferative diseases occur Over the past two decades, introduction of recombinant human growth hormone (hereafter referred as GH) treatment has been used to improve growth retardation in children with chronic renal disease, including transplanted patients The study presented here was designed to find out the effect of GH therapy on longitudinal growth in a model of young rat with growth retardation induced by rapamycin administration.vs RGH: 18.1±3.8 ng/ml).As shown in As shown in As resumed in vs C); RGH: 31±2 positive cells/100 cells (p≤0.05 vs C)].Finally, chondrocyte proliferation was assessed in growth cartilage by immunodetection of bromodeoxyuridine (BrdU). BrdU-labelled chondrocytes were localized in the proliferative zone in all groups . AlthougPicrosirius red/alcian blue staining showed morphological anomalies in the primary spongiosa of both groups treated with rapamycin . In cont2 between both groups treated with rapamycin and C animals .Tartrate-resistant acid phosphatase (TRAP) positive cells were found in the primary spongiosa and along the vascular invasion front in all groups . In the vs C); RGH: 68±3 positive cells/100 terminal chondrocytes (p≤0.05 vs C)].VEGF immunohistochemical signal was localized within the cytoplasm of hypertrophic chondrocytes from the early hypertrophic zone to the distal end of the cartilage in all groups . This paAutophagy was studied in growth cartilage of rapamycin treated rats using two different approaches. First, fluorescent immunodetection of total microtubule-associated protein light chain 3 (LC3) revealed a strong signal mostly in the cytoplasm of early hypertrophic chondrocytes in both groups . This siPeriodic acid-Schiff (PAS) stain was used to identify glycogen deposits, as an index of the energetic reserves of growth plate chondrocytes. As shown in Expression and phosphorylation of several proteins related to glycogen biosynthesis and chondrocyte hypertrophy were studied. IGF-I expression, assessed by immunohistochemistry and in situ hybridization, was confined to the hypertrophic zone of growth cartilage without differences between groups (data not shown). As shown in This study shows for the first time that GH administration increases longitudinal growth in young individuals treated with rapamycin at therapeutic doses. We in situ hybridization and immunostaining for IGF-I showed no differences between groups. As we have previously shown The present work also shows that GH acts locally in the growth plate to promote linear growth in rapamycin treated animals. GH is known to bind to specific receptors located in the epiphyseal chondrocytes to increase longitudinal growth An important finding of this study was that GH enhanced chondrocyte hypertrophy in rapamycin treated animals, as disclosed by a significant increase in the height of the terminal chondrocyte. Similar effects on chondrocyte hypertrophy have also been found in uremic young rats receiving GH Chondrocyte hypertrophy is a complex process regulated by largely unknown molecular mechanisms. We have previously shown that mTOR inhibition by rapamycin decreased chondrocyte hypertrophy in the growth plate et al. showed that cartilage-specific inactivation of GSK3β in mice does not affect bone growth A recent report has showed that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3β It is worth to mention that rapamycin antiproliferative and antiangiogenic activities remained in the growth plate of RGH animals. Despite being a well known mitotic agent, GH did not increase cell proliferation in the growth cartilage of RGH animals. In addition, alterations in vascular invasion seen in rapamycin treated animals, such as abnormal arrangement of septa and blood vessels in primary spongiosa, decreased VEGF expression in terminal chondrocytes, and reduction of TRAP positive cells near chondro-osseous junction, were also found in RGH group. Antitumoral effects of rapamycin are thought to be related to inhibition of cell proliferation and angiogenesis. Our study importantly indicates that GH treatment can promote growth without interfering with these effects, at least at the growth plate level. Further studies are needed to clarify whether the potential of GH to increase the risk of malignancy in kidney transplanted children In summary, our study reports that GH administration improves longitudinal growth in young rats treated with rapamycin. This GH promoting effect is not related to antagonism of the antiproliferative and antiangiogenic actions of rapamycin, but to an increase in the growth cartilage chondrocyte hypertrophy which involves GSK3β inactivation and β-catenin stabilization.ad libitum and RAPA and RGH were pair fed with C group. The study complied with the Directive 86/609/EEC on the protection of Animals used for Experimental and other scientific purposes in the European Union. The protocol was approved by the Ethics Committee on Animal Research of the University of Oviedo, Spain.Female Sprague–Dawley rats aged 4 weeks and weighing 70±5 g were housed in individual cages under controlled conditions of light (12 h light/dark cycle) and temperature (21–23°C). All animals received standard rat chow  and tap water. Control animals were fed in situ hybridization studies.After 3 days of adaptation to the experimental area, 1 mg/kg/day of rapamycin diluted in 5% dimethyl sulfoxide  was administered intraperitoneally at 10.00 am for seven days to 20 animals whereas control  rats were injected with 5% DMSO. On day 0 of the protocol, rapamycin treated animals were classified into two groups of 10 individuals each: RAPA and RGH. From day 0 to 6 of the protocol, in addition to rapamycin injection, RGH animals received also 10 IU/kg/day of intraperitoneal recombinant human GH  at 09.00 and 17.00 h, whereas C and RAPA received vehicle. Animals were killed under anesthesia on day 7 of the protocol. Three days before sacrifice, each animal received 20 mg/kg of calcein (Sigma) by intraperitoneal route for calculation of osseous front advance as an index of longitudinal bone growth rate Concentrations of rapamycin were measured in whole-blood samples using an Abbott Imx analyzer  as previously described Nose to tail-tip length was measured under anesthesia on days 0 and 7. Longitudinal growth rate was measured in 10 µm-thick frontal sections of the proximal end of tibiae as described elsewhere 2.Frontal sections (5 mm thick) of proximal tibiae fixed in formalin were stained by the following methods: alcian blue/safranine  for morphometric analysis, alkaline phosphatase stain  for chondrocyte maturation, TRAP stain (Sigma) for osteoclast identification, periodic acid-Schiff (PAS) reaction to stain glycogen deposits (Merck), and picrosirius red/alcian blue/hematoxylin (Merck) for analysis of bone and cartilage extracellular matrix. Heights of the growth cartilage and its hypertrophic zone were identified following morphological criteria and measured at regular intervals using an image analysis system previously described Immunodetection of BrdU, VEGF, IGF-I and collagen X were carried out as described elsewhere In situ hybridization was used to define the mRNA distribution pattern of VEGF and IGF-I in the growth cartilage following a methodology previously described A total of 30 µg of protein, extracted from tibial frozen samples as previously described Data are given as mean ± SEM. Differences between groups were assessed by ANOVA. Comparisons between two groups were performed by Student t test. A P value ≤0.05 was considered significant. All data sets were analyzed using SPSS 15.0 software package .Figure S1GH effects on chondrocyte expression of collagen type X and alkaline phosphatase activity. (A) Representative images of immunohistochemistry experiments showing collagen type X expression in growth plates of control rats (C), rats treated with rapamycin (RAPA) or rapamycin and GH (RGH). (B) Representative sections of proximal tibial growth plates showing alkaline phosphatase activity in hypertrophic chondrocytes of C, RAPA and RGH animals.(TIF)Click here for additional data file.


To test whether the canonical nonhomologous end joining (NHEJ) pathway of double-strand break (DSB) repair is involved in the formation of this class of CNVs, chromosome integrity was monitored in NHEJ–deficient Xrcc4−/− mouse embryonic stem (ES) cells following treatment with low doses of aphidicolin, a DNA replicative polymerase inhibitor. Mouse ES cells exhibited replication stress-induced CNV formation in the same manner as human fibroblasts, including the existence of syntenic hotspot regions, such as in the Auts2 and Wwox loci. The frequency and location of spontaneous and aphidicolin-induced CNV formation were not altered by loss of Xrcc4, as would be expected if canonical NHEJ were the predominant pathway of CNV formation. Moreover, de novo CNV junctions displayed a typical pattern of microhomology and blunt end use that did not change in the absence of Xrcc4. A number of complex CNVs were detected in both wild-type and Xrcc4−/− cells, including an example of a catastrophic, chromothripsis event. These results establish that nonrecurrent CNVs can be, and frequently are, formed by mechanisms other than Xrcc4-dependent NHEJ.Spontaneous copy number variant (CNV) mutations are an important factor in genomic structural variation, genomic disorders, and cancer. A major class of CNVs, termed nonrecurrent CNVs, is thought to arise by nonhomologous DNA repair mechanisms due to the presence of short microhomologies, blunt ends, or short insertions at junctions of normal and  in vivo, arise by a mutagenic mechanism other than canonical NHEJ, a pattern similar to that reported for model translocation events. Potential roles of alternative end joining and template switching are discussed.Copy number variants (CNVs) are a major factor in genetic variation and are a common and important class of mutation in genomic disorders, yet there is limited understanding of how many CNVs arise and the risk factors involved. One DNA damage response pathway implicated in CNV formation is nonhomologous end joining (NHEJ), which repairs broken DNA ends by Xrcc4-dependent direct ligation. We examined the effects of loss of Xrcc4 and NHEJ on CNV formation following replication stress in mouse cells. Cells lacking NHEJ displayed unaltered CNV frequencies, locations, and breakpoint structures compared to normal cells. These results establish that CNV mutations in a cell model system, and likely  Recurrent CNVs arise during meiosis by nonallelic homologous recombination (NAHR) in regions flanked by large segmental duplications Canonical NHEJ, along with homologous recombination (HR), is one of the two major mechanisms used to repair DNA double-strand breaks (DSBs) in eukaryotic cells. NHEJ directly joins two DSB ends without using extensive sequence homology to guide repair through the action of a well-defined set of proteins, including the Xrcc4-ligase IV complex, which is dedicated to and essential for this pathway Xrcc4-ligase IV-independent forms of DSB end joining also exist, variably called alt-EJ or microhomology-mediated end joining. Alt-EJ is ordinarily less efficient than and/or suppressed by NHEJ such that its activity is often revealed principally in the absence of NHEJ proteins. For example, in the absence of Xrcc4-ligase IV, alt-EJ becomes important in class-switch recombination C. elegansIn contrast to end joining mechanisms, which obligatorily proceed through DSB intermediates and could occur throughout the cell cycle, mechanisms based on replication template switching have also been proposed to explain the presence of microhomologies at CNV junctions. Lee et al. de novo CNVs that closely mimic the nonrecurrent class of human CNVs de novo CNVs that resemble nonrecurrent CNVs in vivo in size and structure. In particular, both human nonrecurrent CNVs and those induced in our experimental system have breakpoint junctions primarily characterized by microhomologies, blunt ends, or small insertions. These observations led to the hypothesis that NHEJ participates in the formation of nonrecurrent CNVs, although their induction by replication stress and the occurrence of complex events also raised the possibility of template switching mechanisms We previously reported an experimental approach for inducing in vivo, the proposed mechanisms have only been inferred from junction sequences; direct experimental tests have been lacking. Because of the strong functional implications of the potential alternative mechanisms of nonrecurrent CNV formation, we sought to definitively explore the role of the well-defined, canonical NHEJ pathway. We report studies of CNV formation using Xrcc4−/− mouse embryonic stem (ES) cells and the DNA polymerase inhibitor aphidicolin (APH). Xrcc4 is an essential component of DNA ligase IV that is absolutely required for NHEJ de novo CNVs in mouse ES cells as it does in human fibroblasts, but that there is no difference in the frequency or structure of spontaneous or induced CNVs between wild-type and −/−Xrcc4 cells. CNV breakpoint junctions were characterized by blunt ends and microhomologies regardless of genotype, with no observed shift in microhomology lengths. We conclude that replication-associated CNVs in mouse ES cells are created through mechanism(s) other than canonical NHEJ and discuss the potential roles of alt-EJ and template switching in the context of both simple and complex CNVs observed in the presence and absence of Xrcc4.For both experimentally-induced CNVs and those occurring in humans Xrcc4 deletion mutation in the −/−Xrcc4 mouse ES cells used in these studies. Supporting this, these cells also demonstrated a large decrease in survival after exposure to ionizing radiation   , demonst−/−Xrcc4 cells, de novo CNVs were identified in untreated and APH-treated clones at a frequency of 1.31 and 7.34 de novo CNVs per clone, respectively (p<10−14)  . When aleriments . Thus, tde novo CNVs in wild-type and −/−Xrcc4 cells. While CNVs consisted of a mix of both deletions and duplications, there was a clear overrepresentation of deletions in both wild-type and −/−Xrcc4 cells. 130 of 143 (90.9%) CNVs from wild-type cells and 195/234 (83.3%) CNVs from −/−Xrcc4 cells were of the deletion type. The abundance of deletions compared to duplications is consistent with results seen in normal human fibroblasts after replication stress, in which 65–82% of CNVs were deletions in vivoBecause NHEJ deficiency might affect CNV structure independently of frequency, we compared numerous features of de novo CNV size between wild-type and −/−Xrcc4 cells . These sizes are similar to de novo CNVs seen in −/−Xrcc4 cells, which had a median size of 63 kb (7.7 kb to 26.2 Mb). We did note that CNVs arising in mouse ES cells (median = 62 kb) were 2.2-fold smaller than de novo CNVs seen in similar experiments with human fibroblasts (median = 138 kb) There was no difference in overall /− cells . De novo−/−Xrcc4 mouse ES cells were distributed throughout the genome, with most arising in distinct, nonoverlapping regions  were deletions (p<0.0001). Most importantly, there was no apparent difference in the spatial distribution of CNVs between wild-type and −/−Xrcc4 cells, including that hotspots accounted for 35.0% (50/143) and 35.5% (83/234) of all de novo CNVs, respectively.Consistent with previous observations in human fibroblasts, spontaneous and APH-induced CNVs in both wild-type and  regions . SuperimAuts2 gene at chromosome 5G2. One or more CNVs in Auts2 were seen in 28/55 APH-treated clones, and accounted for 8.5% of all de novo CNVs. In addition, a hotspot was seen in the Wwox gene at 8E1, with CNVs found in 12/55 APH-treated clones. Both of these hotspots corresponded to hotspots seen in human fibroblasts , reinforcing that there is no apparent increase in sequence homology at breakpoint regions in Xrcc4-deficient cells.Similarly, none of the sequenced junctions had long stretches of homology that would suggest a shift toward HR in NHEJ-deficient cells. To explore this further, we examined the breakpoint regions of unsequenced deletions in vivo−/−Xrcc4 clones, again suggesting no Xrcc4-dependent structural difference. These complex CNVs were initially scored as simple deletions based on aCGH data, but sequencing revealed the presence of small retained sequences, as well as duplications and inversions that were below the resolution limit of the array  of nonhomologous junction formation.The experiments reported here demonstrate that APH-induced replication stress creates In many ways, the results of this CNV study are similar to observations made using a two-DSB translocation model system Importantly, alt-EJ and template switching models of CNV formation are not mutually exclusive, and indeed might be considered more similar than different . The dem−/−Xrcc4 clone suggests that these catastrophic rearrangements can occur via an NHEJ-independent pathway, in contrast to chromosome shattering followed by religation via NHEJ The results from this study also relate to the nature of chromothripsis events and CNV hotspots. Chromothripsis is a recently-described, catastrophic chromosome rearrangement seen in 2–3% of cancers de novo CNVs in mouse cells are distributed across the genome, but include hotspots. Only some of these hotspots correspond to the syntenic regions of hotspots in human fibroblasts, indicating that hotspot conservation can vary between cell types and perhaps species. As seen in human fibroblasts, some CNV hotspots in mouse ES cells correspond with molecularly-characterized common fragile sites or in cytogenetic bands that contain fragile sites As with human cells, Xenopus extracts and mammalian cells The observation that hotspot CNVs are almost all deletions and tend to be almost twice as large as non-hotspots, coupled with the possible fragile site connection, raises interesting possibilities for their formation. APH and hydroxyurea are known to activate the firing of dormant replication origins in in vitro. Evidence from breakpoint junction structures further demonstrates that the CNVs did not form via HR. Instead, the data implicate a replication-dependent alt-EJ and/or template switching mechanism. Because of the strong similarity of the observed CNVs to the major classes of nonrecurrent normal and pathogenic CNVs seen in humans, we argue that these conclusions are generalizable to most de novo, nonrecurrent CNV formation in both germline and somatic human cell lineages, with the simple difference that event rates are higher in our model system because replication is exogenously stressed. Although not mutually exclusive, important features distinguish the remaining alt-EJ and template switching mechanisms, specifically the manner in which the strands are stably resolved. Also enigmatic is precisely which DNA intermediate is the substrate for template switching and which proteins are involved in executing the transfer when little or no microhomology is present. Major efforts moving forward should thus be to delineate the precise strand intermediates and protein mechanisms involved in mediating nonrecurrent CNV formation.In summary, the experiments described here demonstrate that canonical, Xrcc4-dependent NHEJ is not involved in CNV formation in somatic cells cultured −/−Xrcc4) was homozygous for a targeted inactivation of Xrcc4All experiments were performed with two isogenic male mouse ES cell lines. The first (TC1) was wild-type, while the second  unique sequence oligonucleotides (Roche Applied Science). Arrays were prepared according to the manufacturer's protocol. Arrays were scanned on an Axon 4000B scanner (Molecular Devices) with GenePix software at 532 and 635 wavelengths. Data extraction, normalization, and visualization were achieved by using manufacturer-provided software . Arrays were analyzed for copy number differences using SegMNT, part of Nimblegen's NimbleScan software package, as well as our software platform, VAMP, as previously described CNV breakpoint junctions were amplified using the Expand Long Template PCR System (Roche Applied Science). For deletions, PCR primer pairs were generated that flanked deletion breakpoints, whereas for duplications, primers were designed within the duplicated region, directed outward, as described previously CNVs in our model system are relatively rare events and therefore the numbers of CNVs per clone are expected to fit a Poisson distribution determined by the mean frequency of CNVs in all clones. Therefore, p values of treated vs. untreated samples were determined using the one-sided E-test of Krishnamoorthy and Thomson for comparing two Poisson mean rates −/− CNV sets. Regions with 5 or more overlapping CNVs were very rarely observed by random placement  PCR confirmation of mutant Xrcc4 allele with deletion of exon 3 GCTGAGTACTTAGATTTGAGTAC; XRev1 ACCTGGGTGACCCTTACACG. (B) IR sensitivity of Xrcc4−/− ES cells. Wild-type and Xrcc4−/− cells were irradiated with indicated doses of X irradiation, cultured for 7 days, and surviving colonies were stained and counted. IR sensitivity is expressed as the percentages of surviving colonies over unirradiated controls.Confirmation of (TIF)Click here for additional data file.Figure S2Examples of APH-induced CNVs showing Nimblegen aCGH intensity data (log2R). Each dot represents a single probe on the array. (A) A 107.0 kb deletion at 8E1 in clone X6-21 is easily detected by a reduction in the log2R intensity. (B) A 486.6 kb duplication at 9C–D in clone X6-7 can be identified by an increase in the log2R values.(TIF)Click here for additional data file.Figure S3Xrcc4−/− (red) cells, in each of three experiments. It is evident that wild-type cells from Experiment 1 formed unusually low numbers of de novo CNVs compared to all other experimental groups. As a result, when data are combined, there is an apparent increase in CNV formation in Xrcc4−/− cells  and /− cells .(TIF)Click here for additional data file.Figure S4CNV coverage at all hotspots in mouse ES cells. The x-axis shows the position along the chromosome, while the y-axis indicates that fraction of hotspot CNVs that crossed a particular 10 kb genomic window.(TIF)Click here for additional data file.Figure S5Xrcc4−/− cells. Each of these CNVs was called as a deletion based on aCGH data. Breakpoint junction sequencing revealed small duplications (blue), interrupted deletions (red), and inversions (gray).Demonstration of complex CNV rearrangements in wild-type and (TIF)Click here for additional data file.Table S1Monte Carlo simulation to identify CNV hotspots.(DOCX)Click here for additional data file.Table S2de novo CNVs.List of (XLSX)Click here for additional data file.


Young children played a major role in pneumococcal nasopharyngeal carriage, acquisition, and transmission in the era before pneumococcal conjugate vaccine (PCV) use. Few studies document pneumococcal household dynamics in the routine-PCV7 era.We investigated age-specific acquisition, household introduction, carriage clearance, and intra-household transmission in a prospective, longitudinal, observational cohort study of pneumococcal nasopharyngeal carriage in 300 American Indian households comprising 1,072 participants between March 2006 and March 2008.clearance 1.53 ; 5–8 years, HRclearance 1.71 ) and adults . Exposure to serotype-specific carriage in older children (2–8 years) most consistently increased the odds of subsequently acquiring that serotype for other household members.Pneumococcal acquisition rates were 2–6 times higher in children than adults. More household introductions of new pneumococcal strains were attributable to children <9 years than adults ≥17 years (p<0.001), and older children (2–8 years) than younger children (<2 years) (p<0.008). Compared to children <2 years, carriage clearance was more rapid in older children  drive intra-household pneumococcal transmission: first, by acquiring, introducing, and harboring pneumococcus within the household, and then by transmitting acquired serotypes more efficiently than household members of other ages. Pneumococcal conjugate vaccines are being introduced into routine childhood immunization schedules around the world, including in low-resource countries. Routine infant vaccination with heptavalent pneumococcal conjugate vaccine  has substantially reduced pneumococcal disease in the United States Streptococcus pneumoniae plays a major epidemiologic role both as a disease precursor Nasopharyngeal (NP) colonization with Longitudinal studies of intra-household pneumococcal NP transmission have been conducted prior to PCV7 introduction This longitudinal study aimed to characterize pneumococcal NP acquisition, carriage duration, and transmission in American Indian households during the routine PCV7 era. PCV7 was first introduced to these communities in 1997 during a group-randomized efficacy trial The study was approved by the Navajo Nation, the White Mountain Apache tribe and the institutional review boards of the Johns Hopkins Bloomberg School of Public Health, the Navajo Nation, and the Phoenix Area Indian Health Service. Written informed consent was obtained from all adult participants and parents or guardians of enrolled children.The study protocol has been previously described Daycare attendance was defined as ≥4 hours per week outside of the home with ≥2 members of another household. PCV7 vaccination was defined as ≥3 doses. 23-valent pneumococcal polysaccharide  vaccination was defined as any previous receipt of PS23.specimen refers to a single sample from an individual; an isolate refers to a pneumococcal strain, specified by its serotype identified from a swab. A carriage episode refers to the isolation of a serotype from an individual along with as any subsequent isolation of that same serotype at consecutive visits. Carriage episodes were incident if preceded by ≥1 swab negative for that serotype and prevalent otherwise. Serotype acquisition occurred when an isolated serotype was immediately preceded by ≥1 swab negative for that serotype. Acquisitions were individual new acquisitions if the individual had never previously carried the serotype in the study, and individual reacquisitions otherwise. Acquisition of a serotype never previously observed among participating household members was a household introduction. Household introductions were unique if a single household member acquired the new serotype and concurrent if simultaneously acquired by >1 member. In order to investigate serotype-specific differences in household introduction, unadjusted categorical binomial regression was used to analyze the serotype-specific proportion of concurrent versus unique household introductions for the ten most-commonly-introduced serotypes .A NP To analyze carriage duration, incident carriage episodes were aligned for survival analysis on a time metric of “days from first observed carriage”. Clearance was defined by two consecutive swabs negative for a carried serotype and assumed to occur at the midpoint between the last swab positive and the first swab negative for that serotype. Carriage episodes concluding with a missed swab or the last study visit were right-censored. Unadjusted median time to clearance was calculated using survival analysis. Cox regression with cluster robust standard errors was used to analyze the effect of age on clearance of a carried serotype, using only the first incident observation per individual and adjusting for time-varying  and time-fixed  covariates. To examine whether clearance rate was affected by serotype in children, the above Cox regression was repeated for children <9 years, limiting to the first acquisition of one of the five most commonly carried serotypes. Serotype was included in the regression as a categorical variable and its inclusion tested for significance using a Wald test. Serotype values were carried forward for missed visits only if isolation of that same serotype both preceded and followed the missed visit.Individual acquisition and household introduction rates were compared using Poisson regression with cluster robust standard errors. Exposure was defined as [number of swab specimens obtained–1] for all Poisson models, since acquisition could not occur at first visit by definition. All analyses were conducted in Stata 12 Multilevel analysis was used to investigate age-specific intra-household transmission patterns for the four serotypes with the greatest number of putative transmission events . For each serotype, separate three-level  mixed logistic regression models for children and adults were fitted A total of 1072 individuals in 300 households were enrolled in the study. Of the 7504 NP specimens anticipated from the 1072 participants, 6541 (87%) were successfully collected. Most (61%) participants provided all seven swabs, and 88% provided ≥5. A comprehensive description of prevalence and serotype distribution has been published The 2412 pneumococcal isolates represented 1655 distinct carriage episodes; 400 (24.2%) were prevalent and 1255 (75.8%) incident. Per-swab carriage probabilities are summarized in Concurrent serotype-specific carriage within households was also common: for 41.7% of isolates, ≥1 other household member simultaneously carried the same serotype. Among children <9 years, there was no difference between females and males in the proportion of isolates concurrently carried by other household members . Among adults ≥17 years, however, concurrent household carriage was significantly more common among adult females than males .Most carriage episodes  were observed at a single visit. Four individuals <5 years carried the same serotype at all seven study visits (two 6C and one each 19A and 33F). Compared to children <2 years, older children and adults cleared carried serotypes at a greater rate . Of the five most-commonly carried serotypes, serotype did not significantly affect the hazard of clearance among children <9 years (p = 0.13) or adults ≥17 years (p = 0.37).Figure S1). Of other risk factors, only the association between rate of acquisition and reported number of children <6 years in the household reached significance .Five hundred fifty-one (551) household introductions were observed; 78.8% (434/551) were unique and 21.2% (117/551) were concurrent. Of the concurrent introductions, two household members simultaneously acquired the serotype on 105 occasions, three on 11 occasions, and four on one occasion. Of individuals with ≥5 swabs, 48.3% (457/945) had at least one household introduction. For the ten most commonly introduced serotypes in this study, there was no difference in the proportion of concurrent versus unique household introductions by serotype – except for serotype 15A, which was more commonly concurrently introduced  than other serotypes.; Figure S1). No other risk factor reached statistical significance.In multivariable Poisson analysis, the rate of new household introduction was 3–5 times higher in children <9 years compared to adults 17–39 years and varied significantly with month of study entry , while the impact of gender among children was non-significant .Considering serotypes 6C, 19A, 35B, and 22F, exposure to carriage in a child 2–8 years was most consistently associated with a significant increase in the odds of other household members acquiring that serotype at the next visit Table 4.This study is the largest longitudinal household analysis of pneumococcal carriage and transmission to date and the first conducted in the setting of routine PCV use. Concordant with pre-PCV7 household studies, our findings support a major role for children in pneumococcal acquisition and transmission in the household, but we have now shown that this holds true even for settings of PCV use and high pneumococcal transmission.Numerous studies indicate that duration of carriage decreases with increasing age Children have previously been observed to be the major route of introduction of pneumococcus into the household, both in developing Among adults in this community, men may introduce serotypes into the household at a higher rate than women; conversely, women may be more involved in intra-household transmission (given their higher rates of concurrent carriage). These findings may reflect higher rates of community exposure in men and effective household contact in women or a bias in the subset of men who participated in the study compared with women.By isolating the effect of age on transmission – independent of age-specific carriage prevalence – multilevel analysis further confirmed the importance of older children in pneumococcal household dynamics, especially as transmitters to adults. Interestingly, for serotype 6C, exposure to carriage in children <2 years was significantly associated with adult acquisition, while exposure to carriage in older children was not. This observation may be due to imprecision alone or a true difference in serotype-specific transmissibility patterns. In a recent pre-PCV7 Kenyan study, mothers and siblings of all ages transmitted to neonates with similar efficiency Our study has several limitations. Risk factor data was collected via interview, potentially introducing reporting bias. Most households had members who declined to participate, especially older adults and especially men. Our analyses adjusted for reported household composition and accounted for the number of swabs obtained by age category. Still, we cannot exclude the possibility that observed acquisitions may have been transmitted from missing household members. Our inferences assume that consecutively carried or transmitted serotypes represented the same pneumococcal strain. Multi-locus sequence typing or whole genome sequencing may provide stronger strain-specific inferences than serotyping alone This study provides evidence that, in the setting of PCV, children remain the major route of pneumococcal entry into the household, a reservoir for maintaining carriage within households, and both the source and recipient of the majority of intra-household transmission. The 13-valent conjugate vaccine  now in routine use in the United States contains capsular polysaccharide to serotype 6A, which will likely confer cross-protection to serotype 6C Figure S1Seasonal trends in individual acquisition (⧫) and household introduction (•). Incidence rate ratios and 95% confidence intervals obtained from Poisson regression analysis (with January as reference month of study entry).(TIF)Click here for additional data file.