Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening.Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined.Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed.The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone  Expressed sequence tag (EST) analysis is widely used in molecular biology. This analysis comprises the transcriptome of a given tissue at a given time. These data are deposited in a specialized resource at the National Center for Biotechnology Information (NCBI) - dbEST . The ESTThe EST database analysis requires the development of novel methods and software for data processing. The standard procedure includes processing of the biological material, production of clones, construction of libraries, and data analysis, from grouping in contigs to gene annotation and microarray design . SpecialThe strategy of bioinformatics to database analysis remains the same, variety of diverse crude sequences combined by cluster analysis in contigs should be subjected to alignment search tools and function classification by gene ontologies. It gives good results although is not always optimum. Earlier, analysis of the EST database from spider venomous glands showed  that theIdentification of short polypeptides in EST datasets is especially challenging since they may be aligned only with highly homologous proteins. They are synthesized as precursors, which are consequently processed into mature polypeptides. The enzymes involved in maturation recognize specific regulatory amino acid motifs, which help to identify precursor proteins in EST databases ,19,21.Polypeptide toxins from natural venoms are of considerable scientific and practical interest. They may be used for designing drugs of new generation . Venom oHomologous polypeptides in a combinatorial library may differ by point mutations or deletions of single amino acid residues. During contig formation such mutations may be considered as sequencing errors and can be ignored. Our method is devoid of such limitations. Instead of the whole EST dataset annotation and search for all possible homologous sequences, we suggest to consider the bank as a "black box", from which the necessary information may be recovered. The criterion for selection of necessary sequences in each particular case depends on the aim of the research and the structural characteristics of the proteins of interest.Anemonia viridis.To make queries in the EST database and to search for structural homology, we suggest to use single residue distribution analysis (SRDA) earlier developed for classification of spider toxins . In thisIn many proteins the position of certain (key) amino acid residues in the polypeptide chain is conserved. The arrangement of these residues may be described by a polypeptide pattern, in which the key residues are separated by numbers corresponding to the number of nonconserved amino acids between the key amino acids ,* - gap in the search line from 0 to any number of symbols.Since the final goal by query motifs developing was maximum retrieving of sequences from the database, we didn't try to create universal motifs with broad specificity. Conversely, many motifs were developed to ensure search specificity of key residues distribution in patterns. The first four motifs enclose the largest number of known sea anemone toxins and are the most discriminative. For motifs 5-9, we tried to achieve high identification capacity, while motifs 10-13 were made degenerative and partially overlapped earlier developed motifs.Among anemone toxins, large cysteine-free molecules exhibiting strong cytolytic activity are present. These toxins named cytolysines comprise a heterogeneous group of membrane-active molecules subdivided into several groups on the basis of primary structure homology and similarity of physical and chemical properties . For theTo check the potential of the developed pattern motifs, the efficiency of retrieval for toxin-like sequences from the reference animal toxin database was determined. Since amino acid sequences of anemone toxins were used as queries, we expected that all anemone toxins would be identified. Due to a specificity of the reference database syntax, the termination symbols in the motifs were eliminated prior to analysis. Table In the database studied with a total of 13 motifs, we were unable to identify 154 sequences of anemone toxins from 374 available, 108 of which belonged to predicted structures or sequence fragments, and the remaining 46 sequences referred to cytotoxins (motif K).As shown in Table To decrease the number of false positive results during converted database screening, the limitations on the search parameters were imposed. The identity to the screening line was searched only on long fragments started from the beginning or, after any termination symbols and ending by another termination symbol  family . A speciSRDA simplifies the database itself and the search queries, thus considerably simplify the comparing algorithm and consequently increasing the analysis rate. Thus, the search of 12 queries in the reference database of 10489 sequences on a standard desktop PC required 30 sec. We suggest that the simplicity and high rate of analysis make SRDA attractive not only for the study of polypeptide toxins but of other polypeptides as well.Since some procedures in the analyses are tedious and labor-consuming, it may be useful to combine SRDA with other progressive techniques, for example based on the Hidden Markov Model. A novel consolidated algorithm will enclose best features of all parts to aid a precise and fast technology of EST processing.A. viridis EST database showed that this method is effective for rapid retrieval of sequences from the bulk of bioinformatics data. The correct formulation of query plays the crucial role in the outcome of database screening and requires small additional study. The key residues, whose arrangement we wish to fix in the polypeptide pattern, should be selected on the basis of their structural or functional significance. The introduction of termination signals considerably decreases false positive results.The SRDA of A. viridis, we discovered 43 novel sequences. Besides toxins, we also found short peptides with regulatory neuronal function, whose role is still to be investigated, and several groups of toxin-like polypeptides.Using the procedure developed, we identified both new sequences and sequences showing high homology to already described toxins. For two known toxins, the precursor structures were determined. All retrieved sequences formed families of homologous peptides that differ by single or multiple amino acid substitutions, providing additional evidence for the combinatorial principle of natural venom formation. In addition to 23 earlier reported polypeptide toxins in sea anemone Simplification of queries and the database itself reduces the time of analysis as compared to methods based on the search for complete amino acid sequences. The procedure developed may be used for scanning newly generated databases or as a complementation to traditionally used approaches. It is suitable not only for retrieval of polypeptide toxins but for finding any type of amino acid sequences once their structural motifs have been established.SK participated in the design of the study, VBA macros creation and performed the analysis. EG conceived of the study, and participated in its design, data interpretation and coordination. All authors read and approved the final manuscript.Supplementary Excel table of reduced databank used in the analysis (read only). Use Save as command and allow macros execution to reach SRDA and complementary functions in this example.Click here for fileSupplementary listing of VBA module. General function description and how to use section. Start a MS Excel program and change security level for macros to medium. Open any existent file or create a new one . Change file type of Add_file 2 SRDA_processing.bas.txt to SRDA_processing.bas and import all functions included in batch via Microsoft Visual Basic editor (File/import file command).= function name(" and drag "fx" button located on the left from cell input line. Argument(s) required for function processing should be put in the opening window. Then copy equation to other cells.Type in necessary cell "FUNCTIONS DEFINITIONShortDo - is a main function capable to produce converted sequence, where:-Function seq - String variable enclosed amino acid sequence processed by SRDA,excpt - String variable equal to key residues (combination of any single letter coded amino acid(s) with\without termination symbol".") as a solid word.Translate - converts nucleotide to amino acids sequence in appropriated frame, where:-Function seq - String variable enclosed sequence for translation,frame - Integer variable defined translation frame, acceptable value is 1,2,3,-1,-2,-3 or 0 (by frame = 0 only reverse compliment nucleotide sequence will be created).SignalFrom - is a function for prediction of acceptable Met residue starting a signal peptide, where:-Function seq - String variable enclosed amino acid sequence for processing,limitMet - Integer variable defined a searching range (from the beginning) of Met residue,frame - Integer variable equal to frame used early by translation (frame range 1-6), this variable is important for calculation a position of first nucleotide started possible signal peptide,format - Integer variable defined output style:0 - function returns the position of the first nucleotide,1 - function returns the position of the first Met in the signal peptide,2 - function returns the position of the last nucleotide in predicted signal peptide,3 - function returns the position of the last amino acid in predicted signal peptide,other digit - function returns the best score calculated for the signal peptide.TrimSeq - is a function for partial sequence presentation, where:-Function seq - String variable enclosed nucleotide or amino acid sequence,start - Integer variable defined the first nucleotide (amino acid),finish - Integer variable defined the last nucleotide (amino acid).MatureChain - is a function for sequence termination search, where:-Function seq - String variable enclosed amino acid sequence,start - Integer variable defined a start position for termination symbol searching,frame - Integer variable equal to frame used early by translation (frame range 1-6), this variable is important for calculation a position of the last nucleotide in termination codon,format - Integer variable defined output style:0 - function returns the position of the last nucleotide in gene,1 - function returns the position of a termination symbol,start to detected terminus.other digit - function returns a polypeptide sequence from Frame6Check - prints a frame number in which analyzed sequence(s) match query, where:-Function pattern - String variable defined any text for matching,seq1 - seq6 - String variables enclosed amino acid sequences (or converted sequences) translated in 1 to 6 reading frame.Click here for fileSupplementary Table. Results of A. viridis EST database processing. Accession numbers of EST sequences in GenBank are given. Homology to known structures was estimated by UFO and PSI-BLAST.Click here for fileSupplementary Figure. Multiple sequence alignment of toxin-like, cytolysin-like and hypothetical peptides. Removable by maturation predicted domains are given in light brown. Cysteine residues are highlighted yellow, while positively charged residues Lysine and Arginine are shown in blue. (A) short toxin-like polypeptides retrieved with motifs 11 and 13; (B) long toxin-like polypeptides retrieved with motifs 11 and 13; (C) Cytolysin-like polypeptides retrieved with motif K and hemolytic toxin Equinatoxin-2 (P61914); (D) hypothetical polypeptides identified with motif K.Click here for file


Fast, specific identification and surveillance of pathogens is the cornerstone of any outbreak response system, especially in the case of emerging infectious diseases and viral epidemics. This process is generally tedious and time-consuming thus making it ineffective in traditional settings. The added complexity in these situations is the non-availability of pure isolates of pathogens as they are present as mixed genomes or hologenomes. Next-generation sequencing approaches offer an attractive solution in this scenario as it provides adequate depth of sequencing at fast and affordable costs, apart from making it possible to decipher complex interactions between genomes at a scale that was not possible before. The widespread application of next-generation sequencing in this field has been limited by the non-availability of an efficient computational pipeline to systematically analyze data to delineate pathogen genomes from mixed population of genomes or hologenomes.de-novo assembly. In depth analysis of the data generated revealed the presence of sequences corresponding to Japanese encephalitis virus. The genome of the virus was also independently de-novo assembled. The presence of the virus was in addition, verified using standard molecular biology techniques.We applied next-generation sequencing on a sample containing mixed population of genomes from an epidemic with appropriate processing and enrichment. The data was analyzed using an extensive computational pipeline involving mapping to reference genome sets and Our approach can accurately identify causative pathogens from cell culture hologenome samples containing mixed population of genomes and in principle can be applied to patient hologenome samples without any background information. This methodology could be widely applied to identify and isolate pathogen genomes and understand their genomic variability during outbreaks. Viral pathogens have been a major cause of epidemics worldwide -3. The rThe availability of next-generation sequencing (NGS) technology has enabled the scale and ease of addressing biological questions on a genomics perspective ,18. The Hologenome, a term borrowed from evolutionary biology is defined as the sum of the genetic information of the host and its microbiota . HologenCultured population of viruses co-exists and interacts intricately with their host genomes . Althougde novo sequence assembly to accurately identify viral pathogens from mixed populations of genomes. The pipeline relies on the specificity of sequence mappings and the differential distribution of the mapped reads across genomes. As a proof of concept, we applied the methodology on a cell culture hologenome consisting of human, bacterial and viral genomes, and could specifically identify the viral pathogen. This methodology could potentially be applied for rapid and specific identification of viral pathogens during epidemic outbreaks.Here we report the sequencing and analysis methodology, involving computational algorithms for reference mapping and QiaAMP viral RNA minikit) kit as per manufacturer's instructions. The RNA was eluted in 60 μlitre of AVE buffer.The sample was collected and isolated during an epidemic of acute viral encephalitis from an anonymous patient suffering from fever and acute encephalitis from Baba Raghav Das (BRD) Medical College and Nehru Hospital, Gorakhpur, India. Sample was procured and processed as per ethical procedures laid down by BRD Medical College, Gorakhpur, India and National Institute of Virology, Pune, India. Using standard virus isolation protocols samples were inoculated in human Rhabdosarcoma (RD) and Baby Hamster kidney (BHK) cell lines for virus isolation ,26. CellThe RNA library was prepared according to the manufacturer's instructions using RNA Sample-prep kit  for sequencing on Illumina sequencing platform. Two microgram of total RNA was fragmented using divalent cation. Cleaved RNA was converted to cDNA using reverse transcriptase (SSRT-II Invitrogen) and random primers. The fragments were further subjected to second strand cDNA synthesis using DNA polymerase as per manufacturer's instructions. End-repairing process followed by A-base addition and adapter ligation was further performed on the cDNA fragments. Approximately 350 base pair products were separated by gel excision and enriched with PCR to create the final library.Clusters were generated on the flow cell using cBot Paired end cluster generation kit  as per manufacturer's instructions. The sequencing runs were performed on Illumina Inc, USA) using 76 × 2 base reads. The sequence-quality files generated was transformed to Sanger quality scores using custom scripts.The paired end reads were mapped to the reference datasets using Mapping and Assembly with Qualities (MAQ) software . The datde novo assembly algorithm based on de Bruijn graphs was used for the de novo assembly. The entire read data was partitioned into smaller subsets for analysis. De-novo assembly was attempted on the subsets with different k-mers. The data was compiled and compared using in-house scripts.Velvet , a populJapanese encephalitis virus were used. RNA was initially reverse transcribed using JEV PM1R at 50°C using Superscript II reverse transcriptase . The cDNA was amplified using forward primer JEV C1F and reverse primer JEV PM1R. PCR amplification was carried out by denaturing 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec 58°C for 30 sec, 72°C for 1 min and final extension of 3 min using Taq DNA polymerase (Fermentas).Experimental validation of the virus was performed using reverse transcription (RT)-polymerase chain reaction. Specific primer JEV PM1R (reverse primer): 5'-CGGARTCTCCTGCTTCGCTTGG-3' and JEV C1F (forward primer): 5'-GGCAGAAAGCAAAACAAAAGA-3' specific for Japanese encephalitis Virus genome . JE specific primers mapping in the C-prM region amplified approximately 400 bp amplicon as expected [Reference mapping is an attractive strategy, as it is fast and frugal on compute and memory requirements, however is limited by the availability of the genome under consideration in the reference dataset. This could limit the widespread application of this methodology in identifying pathogens that have not been sequenced before, as in the case of new and emerging infectious diseases. This limitation could be potentially overcome using a expected ,26, subsJapanese encephalitis genome were analyzed in depth to understand the genome organization and genomic variations of the isolate. We identified a total of 209 genomic variations in the genome with high confidence. The complete list of variations, their genomic loci, their type (i.e. synonymous and non-synonymous) and the genes that harbor them are summarized in Additional file Furthermore the reads and alignments in the reference assembly that mapped to de novo assembly to identify the pathogen as Japanese encephalitis virus. Furthermore we also validated the identity of the virus using RT-PCR techniques. In summary we successfully demonstrate the utility of such approaches in identification of viral pathogens from mixed population of genomes. As cheaper and faster sequencing technology becomes widely available in research labs, this approach would open up immense opportunities in identifying viral pathogens in outbreaks, apart from being used in regular bio-surveillance of infectious diseases and screening for agents used in bioterrorism. This methodology could also find application in special situations like food processing, beverage, and water quality monitoring for identification of food borne and waterborne pathogens respectively.Here we have used next-generation sequencing approach and generated over 22 million sequence reads from a cell culture hologenome sample consisting of human, bacteria and virus. We also successfully applied a pipeline of computational algorithms including reference mapping and JN644310.Raw sequence data is available at the NCBI Short Read Archive with ID: SRX099040. Sequence generated using de novo genome assembly tools is available at Genbank with ID: The authors declare that they have no competing interests.KKP, GNS, VPB & MMG-Collected the samples; GNS, VPB & MMG-Isolated and inoculated the virus and maintained the cell culture; AP, RK, MS, SKV & SSB-Generated the nucleic acid sequences and conducted the molecular studies; AP, VP & VS-Developed the algorithms and conducted the computational analysis; MMG, SSB & VS-Designed the study, analyzed the results and drafted the manuscript.Read Statistics. Excel file containing detailed information on various read statistics.Click here for fileRT-PCR validations. JPG image depicting reverse transcriptase validation for Japanese encephalitis.Click here for fileList of Variations. Microsoft DOC file containing table on variations identified in Japanese Encephalitis genome (NC_001437).Click here for file


A sample of 55 TMD patients were selected. Each was examined with standard clinical procedures and genotyping techniques.Specimens from the TMJ of ank mutant mice was joint space narrowing. Within TMD patients, closed lock was more prevalent among ANKH-OR homozygotes  and the elder .The major histological finding in ank mutant mice. In the human sample, ANKH-OR polymorphism was found to be a genetic marker associated with TMJ closed lock. Future investigations correlating genetic polymorphism to TMD are indicated.Fibrous ankylosis was identified in the TMJ of  Temporomandibular disorders (TMD) are a developing issue in public health with clinical signs being observed in 50% of the population Internal derangement has been associated with 90% of cases suffering from TMJ closed lock ank mutations and ANKH polymorphisms, respectively, as determinants for arthritis in mice ankThe over-expression of certain genes such as lumican ank mutant as well as wild-type mice, and (2) to conduct a case-control research study to identify polymorphisms of the ANKH gene, using a sample of TMD patients in Japan. A special interest was to establish a relationship between the types of TMJ internal derangement and ANKH polymorphisms.Since genetic effects on TMJ internal derangement have not been fully clarified and the progressive ankylosis gene was associated with joint disorders, an investigation of this gene may help to gain insights into the pathogenesis of TMD. Therefore, this study aimed (1) to carry out a histological examination of the TMJ collected from ank/ank mutant and wild-type mice  were purchased for the animal experiment. The mice were separately euthanised with carbon dioxide gas at 3, 4 and 5 months of age, since the life span of ank/ank mutant mice was less than 6 months Prior to commencement, animal and human sub-projects of this study have both received appropriate ethics approval from the Institutional Review Board of Kyoto University (approved ID Number: G86). Commercially bred 5′-ATCAAGAAGTTCACCTTCGTC-3′) and AnkRTR4 (5′-CTTTTTCTGCTTCCGGTAGAC-3′) as the primers to perform the RT-PCR technique.To identify the expression of ANKH in TMJ, an mRNA expression analysis was conducted. Synovial cells used for this purpose were collected from a patient undergoing TMJ arthroscopy due to closed lock, with appropriate written informed consent received. Procedures for preparation of the primary culture and the reverse transcriptase polymerase chain reaction (RT-PCR), as suggested in literature This study hypothesised that closed lock patients were more likely to carry a homozygous ANKH polymorphic genotype. The calculation of a minimal sample size based on the statistical power to hypothesis testing was unattainable, since the homozygosity/heterozygosity distribution of the ANKH polymorphisms studied has never been reported. The percentage of controls carrying a homozygous ANKH polymorphism would be required for estimating the sample size of such an unmatched case-control study. To achieve a satisfactory sample size for an adequate statistical power, sample collection was carried out from January 2003 to December 2006. A sample was recruited for the study at the outpatient clinic of oral and maxillofacial surgery, Kyoto University Hospital. Inclusion criteria for subjects of the study were: (1) having a Japanese ethnicity background; (2) being older than 15 years of age; (3) presenting signs and symptoms that were characteristic of a diagnosis of TMJ clicking (disc displacement with reduction) or closed lock (disc displacement without reduction) The genotyping techniques reported by a previous study were used to identify the ANKH-OR polymorphic site and the ANKH-TR polymorphic site in the 5′-noncoding region and the promoter region of the ANKH gene, respectively Data entry and statistical analysis were implemented with JMP 8.0 . Data analysis included descriptive statistics (frequency distribution and cross tabulation). A univariate logistic regression method was used to assess the individual (unadjusted) contribution of explanatory variables including genotypes of ANKH polymorphisms, gender and/or age ank/ank mutant and the wild-type mice, six 3-month-olds, two 4-month-olds and two 5-month-olds were examined. Mutant individuals of all age groups showed similarly narrower and/or ankylosed superior and inferior synovial cavities filled with fibrous connective tissue throughout the entire joint space  were female. The participants' age ranged from 15.5 to 69.7 years and the mean age of female and male subjects was 39.7±18.1 years and 30.8±12.1 years, individually. Thirty-nine  and 16  cases were separately diagnosed with closed lock and TMJ clicking .TCGCCCCG (Allele-1) and TCGCCCCGTCGCCCCG (Allele-2) located in the putative 5′-noncoding region , ANKH-TR , gender  and age . In the multivariate regression model, participants that were ANKH-OR homozygotes (Genotype 1/1 or Genotype 2/2) showed a higher risk of TMJ closed lock than those who were ANKH-OR heterozygotes (Genotype 1/2)  . With thank/ank mutant mice. Joint space narrowing found in this group represents a relevant pathological trait of fibrous ankylosis ank/ank mutant mice ank function has been associated with calcified debris, excessive calcification and cartilage erosion in the joints of ank/ank mutant mice ank gene and fibrous ankylosis of the TMJ.Fibrous ankylosis was notably found in the TMJ of ank mutant mice than their heterozygous littermates, based on an autosomal dominant trait of the disorder This study has demonstrated for the first time an enhancing effect of homozygous ANKH-OR polymorphisms on closed lock of the human TMJ. ANKH-OR homozygotes were approximately 8 times more likely to develop closed lock than their control group counterparts. A past study has reported a more serious phenotype of craniometaphyseal dysplasia amongst homozygous Age was related to the type of TMJ internal derangement in this sample and this agreed with a previous study ank gene This study identified ANKH expression in TMJ synovial cells. It was found that mutations and polymorphisms of the ANKH gene predisposed closed lock in humans and fibrous ankylosis in mice. Although this study did not indicate an equivalence between fibrous ankylosis in mice and closed lock in humans, past studies have reported relationships and similarities between the two disorders.. Fibrous adhesion, known as Type I TMJ ankylosis ank mutant mice has been identified by this study.This study has manifested that the ANKH-OR polymorphism is a genetic marker associated with TMJ closed lock. ANKH expression in the TMJ has been confirmed. In addition, fibrous ankylosis in the TMJ of TMD has become an important issue in public health and dentistry. A genetic approach to the pathogenesis adds to current understanding of aetiology of TMD. Succeeding investigations into the influence of the ANKH gene on mechanisms of TMD development are indicated.


Lung tumors showing ground-glass opacities on high-resolution computed tomography indicate the presence of inflammation, atypical adenomatous hyperplasia, or localized bronchioloalveolar carcinoma. We adopted a two-staged video-assisted thoracoscopic lobectomy strategy involving completion lobectomy for localized bronchioloalveolar carcinoma with an invasive component according to postoperative pathological examination by permanent section after partial resection.Forty-one patients with undiagnosed small peripheral ground-glass opacity lesions underwent partial resection from 2001 to 2007 in Hokkaido University Hospital. Localized bronchioloalveolar carcinoma was classified according to the Noguchi classification for adenocarcinoma. Malignant lesions other than Noguchi types A and B were considered for completion lobectomy and systemic mediastinal lymphadenectomy. Perioperative data of completion video-assisted thoracoscopic lobectomies were compared with data of 67 upfront video-assisted thoracoscopic lobectomies for clinical stage IA adenocarcinoma performed during the same period.Postoperative pathological examination revealed 35 malignant and 6 non-malignant diseases. Histologically, all of the malignant diseases were adenocarcinomas of Noguchi type A (n = 7), B (n = 9), C (n = 18), and F (n = 1). Eleven of 19 patients (58%) with Noguchi type C or F underwent two-staged video-assisted thoracoscopic lobectomy. Three patients refused a second surgery. There was no cancer recurrence. The two-staged lobectomy group had a significantly longer operative time and more blood loss than the upfront lobectomy group. There was no surgical mortality or cancer recurrence.Two-staged lobectomy for undiagnosed small peripheral ground-glass opacity lesions showed satisfactory oncological results. However, low compliance for and invasiveness of the second surgery are concerns associated with this strategy. In 1995, Noguchi et al. proposed the classification of adenocarcinoma into six pathologic types, A to F, based on growth patterns -4. Most Given that tumors demonstrating pure GGO or mixed GGO lesions with a slight solid part represent early-stage cancer without invasiveness, they might be good candidates for limited surgery with curative intent. The outcome of limited surgery with curative intent for patients with pure GGO lesions is reportedly satisfactory -7. AlthoHowever, there was a limitation in this therapeutic strategy: intraoperative pathological examination sometimes led to a misdiagnosis of Noguchi type or cancer invasiveness, because the Noguchi type of adenocarcinoma was originally diagnosed based on postoperative pathological examination by permanent section ,10,13. MTo avoid these intraoperative pathologic diagnostic errors and resultant inappropriate surgeries, we adopted the following therapeutic strategy for patients with undiagnosed small peripheral GGO lesions for 7 years (2001–2007). According to the pathological diagnosis by permanent section after partial resection, patients with Noguchi type A or B adenocarcinoma underwent postoperative follow-up without two-staged lobectomy. For other Noguchi type adenocarcinomas (including type C) and cancers of other histological types, two-staged lobectomy was considered. There have been no reports of a therapeutic strategy similar to ours. We herein report the perioperative and survival outcomes of this two-staged lobectomy strategy based on pathological examination by permanent section in patients with undiagnosed small peripheral GGO lesions..The therapeutic strategy for patients with undiagnosed small peripheral GGO lesions in our institute is as following. First, video-assisted thoracoscopic (VATS) partial resection was performed after obtaining informed consent for the possibility of two-staged lobectomy. Based on postoperative pathological examination by permanent section, patients who were diagnosed with non-malignant disease or Noguchi type A or B adenocarcinoma did not undergo two-staged lobectomy. On the other hand, patients who were diagnosed with adenocarcinoma other than Noguchi type A or B or with cancers of other histologic types underwent two-staged lobectomy if the patient met the medical criteria for lobectomy and agreed to the second surgery. The institutional review board of Hokkaido university hospital approved this retrospective study and waived the requirement for informed consentThe subjects were 41 patients with undiagnosed small peripheral GGO lesions who underwent pulmonary resection in Hokkaido University Hospital from January 2001 to December 2007. The clinicopathological data were obtained from the medical charts. GGO was defined as a localized or focal misty increase in lung attenuation that did not obscure the underlying vascular markings. Typical CT findings of GGO lesions were shown in Figure The usefulness and safety of our therapeutic strategy for patients with undiagnosed small peripheral GGO lesions were statistically analyzed. To evaluate usefulness, overall survival (OS) and DFS after resection were analyzed. OS was the period between the date of operation and the date of death or the last date of confirmed survival. DFS was the period between the date of operation and the date of recurrence or the last date of confirmed recurrence-free survival. For patients who underwent two-staged lobectomy, OS and DFS were the period between the date of first operation and the last date.To evaluate safety, perioperative data of patients who underwent two-staged VATS lobectomy (two-staged lobectomy group), such as operative time, blood loss, ratio of conversion to open thoracotomy from VATS lobectomy, postoperative complications, period of postoperative drainage, length of postoperative hospital stay, and surgery-related death, were compared with data of 67 patients with adenocarcinoma who were diagnosed with clinical stage IA disease and underwent VATS lobectomy during the same period (upfront lobectomy group). The following patients were excluded: patients who underwent partial resection of another lobe at the same time and patients with a past history of lung resection. Conversion to open thoracotomy from VATS lobectomy was performed if there was severe adhesion of lymph nodes to the pulmonary artery, severe adhesion of the pleura, or uncontrolled vascular injury. Postoperative pulmonary fistula was defined as air leakage that continued for more than 7 postoperative days or necessitated pleurodesis or reoperation.The survival rate was calculated using the Kaplan-Meier method. Comparison between the two-staged and upfront lobectomy groups was performed using the Mann–Whitney U test and chi-square test. All statistical analyses were performed using StatView 5.0 software . A p-value of <0.05 indicated a significant difference.The characteristics of 41 patients with preoperatively undiagnosed small peripheral GGO lesions are shown in Table The median postoperative follow-up period was 67 months  in 41 patients with GGO lesions and 67 months  in 35 patients with malignant diseases. Although two patients with Noguchi type C adenocarcinoma died of other diseases, no patents had recurrence of lung cancer. In patients with Noguchi type A or B adenocarcinoma, the 5-year OS and DFS were 100%  and upfront lobectomy (n = 67) groups are shown in Table In this study, we reported the results of limited resection for Noguchi type A and B BAC and two-staged lobectomy for other primary lung cancers. In patients with Noguchi type A or B adenocarcinoma n = 16), the 5-year OS and DFS were 100%. In patients with Noguchi type C adenocarcinoma (n = 18), the 5-year OS and DFS were 87.2% and 100%, respectively. Patient survival was satisfactory and comparable with that of reports from other institutes. The postoperative follow-up period in the present study might not be long enough, because adenocarcinoma often recurs after 5 postoperative years ,16., the 5-yThere have been several reports on the therapeutic strategy in which partial resection is initially performed for tumors comprising a GGO component, and whether lobectomy was added was determined based on the intraoperative Noguchi type diagnosis on pathological examination by frozen section -12. LobeThere is a limitation in Noguchi type diagnosis based on intraoperative pathological examination by frozen section. Determination of the Noguchi type of adenocarcinoma was originally based on postoperative pathological examination by permanent section . YoshidaWe adopted a two-staged lobectomy strategy to avoid misdiagnosis due to intraoperative pathological examination by frozen section for undiagnosed small peripheral GGO lesions. That is, two-staged lobectomy was added based on the results of postoperative pathological examination by permanent section after partial resection. No patients who underwent this protocol were subjected to surgical overtreatment. However, of 19 patients with a diagnosis other than Noguchi type A or B disease, 8 (42%) did not undergo two-staged lobectomy. Although five of them did not undergo this procedure for medical reasons, three refused. There seems to be a problem with compliance to undergo a second surgery. It might be psychologically and financially overwhelming. The ratio of undertreatment was higher than the ratio of misdiagnosis based on intraoperative pathological examination in past reports .Eleven patients underwent two-staged VATS lobectomy. The perioperative outcomes of these surgeries were compared with those of upfront VATS lobectomy for c-stage IA adenocarcinoma. Although there was no mortality in either group and no significant differences in morbidity, the time of operation and estimated blood loss were significantly greater in the two-staged group than in the upfront lobectomy group. The rate of conversion to open thoracotomy was as high as 18% in the two-staged group, although there was no significant difference with the upfront lobectomy group . It is speculated that two-staged lobectomy requires dissection of adhesions, and thus results in a longer operative time, more blood loss, and sometimes conversion to open thoracotomy. Two-staged lobectomy seems more invasive than upfront lobectomy. Recently, to avoid adverse effects of two-staged surgery, we performed limited resection according to the GGO ratio and standardized uptake value of PET-CT regardless of Noguchi classification with permanent section.The therapeutic strategy for undiagnosed small peripheral GGO lesions  had satisfactory oncological results. However, there are concerns in terms of the invasiveness of the procedure and low compliance for two-staged lobectomy.The authors declare that they have no competing interests.KO and YH wrote the manuscript. Other authors collected data. All authors read and approved the final manuscript.


Analysis of signals by means of symbolic methods consists in calculating a measure of signal complexity, for example informational entropy or Lempel-Ziv algorithmic complexity. For construction of these entropic measures one uses distributions of symbols representing the analyzed signal.sequential spectrum that is suitable for analysis of the wide group of signals, including biosignals.We introduce a new signal characteristic named The paper contains a brief review of analyses of artificial signals showing features similar to those of biosignals. An example of using sequential spectrum for analyzing EEG signals registered during different stages of sleep is also presented.Sequential spectrum is an effective tool for general description of nonstationary signals and it its advantage over Fourier spectrum. Sequential spectrum enables assessment of pathological changes in EEG-signals recorded in persons with epilepsy. There are many ways of using symbolic dynamics for time series analysis and all of them need coding i.e. conversing of the analyzed time series into symbols series. The differences between different symbolic methods are in coding procedure and/or in calculated complexity measure, such as entropy -3 or Lemseq-spectrum) in analogy to frequency spectrum. Sequential spectrum like Lempel-Ziv complexity, belongs to methods applying short ordered sequences of symbols (tuples). The important difference is that in the case of seq-spectrum it is the values of first derivative of the signal that is encoded. Moreover, in seq-spectrum only mono-sequences i.e. tuples containing only one kind of symbol are considered and lengths of mono-sequences correspond to frequencies.We introduce a new symbolic measure that is similar to frequency characteristics and we call it a sequential spectrum  we calculate the first range differences and represent them by the symbols from the two-elements set {0,1}:For a signal represented by the time series P, for example .As a result of signal's encoding we obtain binary symbol series, Nxs], i.e. mono-sequences of length N consisting only one type of symbol (s≡0 or s≡1), are counted in series P. As the result we obtain the cardinality, L[Nxs], and we repeat counting procedure for all possible values of N (limited by the length of symbols series P), so obtaining the distribution of cardinalities. Knowing cardinalities L[Nxs] we calculate binary occupancy, O[Nxs], for mono-sequences, [Nxs], in the binary symbol series P,Next, tuples  . But it is not always the case, because when sampling frequency increases additional local maxima and minima appear in the time series representing the analyzed signal so mono-sequences might become shorter.N is a positive integer number.The small resolution of seq-spectrum in the range of short mono-sequences is a consequence of the fact that the length The shape of seq-spectrum and its width contains information about general properties of the analyzed signal.relative seq-spectrum:To compare the seq-spectrum appointed for the state A with the seq-spectrum for the state B we define Before using seq-spectrum for analysis of biosignals, the features of seq-spectrum were tested on well defined artificial signals. Knowing the results of analysis of artificial signals with different characteristics we could start to analyze complex natural signal like EEG.Nx0] and [Nx1]  which is the example of signals with frequency changing regularly with time. Such a behavior is observed in some biosignals. For our simulation of chirp signals we used formula:a = 1 and b = 0.where Nx0] and [Nx1] are different from each other . Figure As opposed to the Fourier spectrum, the seq-spectrum Figure  of the cThe signals produced by complex nonlinear biological systems are usually nonstationary and processes generating these signals can be described by deterministic chaos. Our last example concerns analysis of artificial signal generated by chaotic system with logistic map dynamicsN = 1 and N = 2, contribute to the seq-spectrum , i.e. we calculate the differences between pairs of seq-spectrum values in all sleep stages and the respective value for the chosen stage. Figure In order to highlight the difference between sleep stages we apply the relative seq-spectrum (Eq.4) OIn most cases in the spectrum it is also possible to distinguish in the relative seq-spectrum two ranges but for REM stage the values are either only positive or only negative. However, it is not the rule - the curve of the relative spectrum for REM is sometimes similar to that for stage S1.For stage S1 situation is more complicated. It is possible to distinguish three or four ranges in which the difference of binary occupancies between stage S1 and the state of vigilance is positive or negative. Border of these ranges are not usually sharp - for short mono-sequences  the value of the relative cover is positive. The range in which the values are negative usually comes after this range (within the limits from 3-4 to 9). The next range contains again positive values.The question arises whether universal ranges of the values of the binary occupancy do exist. This would make it a tool for automatic sleep staging. But it is doubtful since EEG signals show strong individual diversification.Seq-spectra calculated for EEG-signals from polysomnographic recordings make it possible to distinguish normal sleep from certain sorts of sleep disorders. In pathological sleep short mono-sequences shows greater contribution than in the case of normal sleep Figure . CompariAnother example concerns using sequential spectrum for analysis of EEG-signals in epilepsy. Comparison of seq-spectra of EEG-signals of persons suffering from epilepsy and those of healthy persons shows that seq-spectra in pathological cases are much broader than those of healthy persons. All mono-sequences longer than 7 have a bigger contribution to seq-spectra in pathological cases than that for healthy persons Figure . HoweverNx0]) and O after the fashion time-frequency methods.Nx0]) and O([Nx1]) of stationary random signals high congruity exists. It decreases when autocorrelation of the signal grows or the signal is non-stationary, or chaotic. This requires further research because asymmetry between spectra O([Nx0]) and O([Nx1]) appears also for signals in which epochs appear about the constant amplitude. It results from the way of coding the signal (Eq.1); when the signal is constant there is an assigned symbol "1".Sequential spectrum can also be used to categorize processes generating signals. Between seq-spectra O([The author declares that they have no competing interests.


Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt) and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg) were examined using the novel Mouse Drawer System (MDS) aboard the International Space Station (ISS) over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2) and transforming growth factor-beta1 (TGF-β1) were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1 relative to ground controls. Total colonic TGF-β1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-β1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-β1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice. These effects include bone loss, cardiovascular alteration (cardiac atrophy and change in heart rhythm), muscle loss, and immune dysfunction, among others While numerous effects of spaceflight on the immune system have been reported, the potential contribution of spaceflight-associated effects on the gut microbiota to such changes has received little attention to date. Increasing evidence points to numerous interactions of the gut microbiota with the immune system + T cells into T helper 1 cells and promotion of CD8+ T cell proliferation. However, it plays other roles, such as increasing antibody secretion from plasma cells + and CD8+ T cells have been observed +/CD4+) was reduced in comparison to ground controls TGF-β1 is a multifunctional cytokine that plays an integral role in adaptive immunity, mucosal immunoregulation as well as bone formation, osteoblast proliferation and differentiation, and is influenced by the gut microbiota In this component of the MDS Experiment on the ISS, we examined effects on selected mucosal and systemic immune parameters and also attempted to assess the effects on the gut microbiota by examining the colonic community in flight mice, in the corresponding ground and vivarium controls, and in transgenic versus Wt mice. The lack of gut community diversity made inferences into interactions of gut microbiota and the immune system difficult, as did the limited number of mice surviving the flight. However, due to the unique nature of the MDS experiment and inability to readily conduct additional replicates, we carried out analysis of the TGF-β1 and IL-2 levels in the available tissue and analysis of the colonic microbiota community, and provide here an observational analysis of the findings. These results may provide further insight not only into effects of spaceflight on the immune system in systemic and mucosal compartments, but also into design issues for future studies aiming to examine effects of long term space flight on the gut microbiota, and associated effects on host parameters.et al., 2012 et al., 2012 have previously outlined details concerning consequences and timing of rodent death for both flight mice and ground controls Full details regarding the MDS Experiment and flight opportunity have been previously described in Cancedda Both TGF-β1  and IL-2 were quantified within the colonic tissue of PTN-Tg and Wt mice, in order to observe the effects of long-term space flight on cytokine production at the mucosal level . PTN-Tg Wt mice appeared to be affected by flight more so than their transgenic counterparts with respect to inguinal node cytokine production, but additional testing would be required to confirm this observation. As shown in Levels of TGF-β1 in the inguinal lymph nodes of Wt mice compared to PTN-Tg mice were different under flight conditions. The percentage of active TGF-β1 was higher in the Wt flight mouse compared to the PTN-Tg flight mice . The Wt The brachial lymph node homogenate was investigated for effects of the PTN-Tg mutation and of spaceflight on peripheral cytokine production by quantifying the levels of IL-2 in addition to total and active TGF-β1 . IL-2 coTotal TGF-β1 levels were lower in brachial nodes of PTN-Tg mice than the Wt mouse in ground control conditions, but the lowest total brachial TGF-β1 levels were observed in vivarium-housed mice. The flown Wt mouse exhibited decreased levels of active and total TGF-β1 in flight conditions when compared to levels in the Wt ground control. Conversely, the brachial nodes of PTN-Tg mice had higher levels of both active and total TGF-β1 in the flight condition when compared to levels in ground controls. Notably, the brachial lymph node concentrations of active TGF-β1 of the PTN-Tg mice were higher in flown conditions than in the Wt counterpart, as was the percent active TGF-β1. Under flight conditions, the percent active TGF-β1 was lower in the Wt mouse and higher in the PTN-Tg mice compared to levels in ground controls, although statistical analysis was not possible due to the limited sample size.Statistical analysis was only possible for mice housed under vivarium conditions; however the difference in TGF-β1  in the brachial lymph nodes between PTN-Tg and Wt mice was not statistically significant.Lactobacillus reuteri, which encompassed 84–99% of the total phylotypes within each of these samples. Colonic samples from the vivarium-housed mice were richer and mostly clustered further away, but were also dominated by this phylotype, although to a lesser degree . A common feature was the uneven diversity within each sample.Colonic contents collected from mice subjected to spaceflight, the corresponding MDS ground control, and from vivarium-housed mice were examined in order to determine whether spaceflight affected the gut community. All of these samples were liquid and contained no visible particulate material. Analysis of pyrosequencing data yielded a total of 137 phylotypes shared among these samples. Cluster analysis comparing the colonic community among each sample indicated little difference in the microbiota of mice exposed to spaceflight versus those in the corresponding ground control MDS system . In all The present study investigated the effects of long-duration spaceflight on selected immune system parameters in both Wt and PTN-Tg mice. While considerable evidence points to changes in the efficacy of the immune system during spaceflight reg) phenotype, which are in turn essential for maintaining intestinal immune homeostasis and controlling excessive inflammatory responses at the mucosal interface TGF-β1 is a highly conserved, pleotropic cytokine with varied effects on numerous cell types, including regulation of B cell differentiation and class switching, attenuation and regulation of Natural Killer (NK) cell activity and regulation of dendritic cells (DC) maturation and antigen-presenting activity In addition to its integral role in the immune system, TGF-β1 also plays a role in wound healing and fibrosis, and bone formation reg induction and development In this component of the MDS study, the effect of spaceflight on mucosal immunity was examined by quantifying IL-2 and TGF-β1 levels within colonic tissue of flight and ground control mice, as well as in vivarium controls. Our results suggest a spaceflight associated decrease in total TGF-β1 levels in colonic tissue of PTN-Tg mice and the Wt mouse. Given the role of TGF-β1 in Tet al. (2009) when splenocytes from flight mice were activated by anti-CD3 stimulation Activation of T cells requires extensive cell-cell communication and signal transduction, effected through appropriate ligand-receptor recognition. An important cytokine which activates resting T cells is IL-2, secreted by activated TH cells. In this study, it was observed that the Wt mouse under flight conditions produced lower IL-2 levels in systemic tissues  compared to its ground control counterpart. Similar results with respect to IL-2 production were observed by Gridley The percentage of active TGF-β1 in the inguinal nodes was somewhat higher in the Wt flown mouse compared to the ground control, but an opposite pattern emerged in the brachial lymph nodes. Microgravity shifts the normal head-to-foot hydrostatic gradient, leading to fluid retention in the upper extremities and limiting pressure to the cervical areas Exposure to low gravity plays a role in the inhibition of lymphocyte proliferation due to its impact on cytokine secretion, T cell locomotion and differential structuring of lymphocyte cytoskeletal elements The PTN-Tg mice in this study were over-expressing pleiotrophin under control of the human bone-specific osteocalcin promoter The effect of over-expressing PTN under the osteocalcin promoter on immune parameters has received little attention to date. In this study, it was observed that the levels of IL-2 in both lymph node tissues of Wt and PTN-Tg were lower in vivarium conditions when compared to ground control counterparts. These differences in vivarium in relation to ground control conditions may be due to the nature of the housing environment, or to decreased stressors. Reduced production of TGF-β1 in PTN knockout mice in response to CG treatment has been previously reported L. reuteri strain made it impossible to assess the potential impacts of spaceflight on the gut community in any of the mice. Gut transit time in mice is very fast, having been estimated at approximately 4.5 hours The observation that the collected colonic contents contained no visible particulate material and that the communities consisted primarily of a single In summary, while spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1, direct correlations with effects on the gut microbiota could not be made, due to the scant colonic content. These immune alterations could result from a variety of factors including impeded movement of lymphatic fluids, effects of microgravity on T cell populations, or from spaceflight-associated effects on the gut microbiota.Future studies could potentially address these elements, as well as investigate the site of cytokine production. It was not possible to perform histopathology or immunohistochemistry experiments in this study since the limited sample number and diminutive proportions only provided enough tissue for homogenate cytokine analysis. Such experiments could provide insight as to whether immunological adaptations to space flight took place in the lymphoid tissues. However, it must be noted that changes in immune parameters, and potentially even in the gut microbiota may not be due to microgravity itself, but instead due to other elements such as changes in diet, exposure to radiation or even psychological stressors and the resulting neuro-immune interactions PLoS Collectionsth, 2009. The surviving 1 Wt and 2 PTN-Tg mice were returned to earth after 91 days, via the Space Shuttle Atlantis (STS-129 mission). The authors participated in a tissue sharing process, receiving specific samples  at the end of the flight period. During the tissue collection, a Biospecimen Reporting for Improved Study Quality (BRISQ) checklist was employed to ensure specimen uniformity. Animal care protocols were approved by the University of Ontario Institute of Technology (UOIT) Animal Care Committee, the American Institutional Animal Care and Use Committee (IACUC), Ethics Committee of the Animal Facility of the National Institute for Cancer Research  and by the Public Veterinary Health Department of the Italian Ministry of Health.Details regarding the mouse drawer system and exposure of C57BL/10J Wt and PTN-Tg mice to zero gravity on the ISS have been previously described in “The Mice Drawer System Experiment and the Space Endurance Record-Breaking Mice” (2012) g, 30 min). The supernatants were collected and the centrifugation process was repeated until tissue and fat were no longer prevalent in the supernatant. The final products were stored at -80°C for future assay.The inguinal and brachial lymph nodes and colonic tissue were removed by dissection; flash frozen, shipped immediately to UOIT and stored at -80°C. To prepare the samples, tissues were weighed and suspended in phosphate buffered saline with 0.5% protease inhibitor cocktail and DMSO . Tissues were homogenized using a Tissuemiser homogenizer  and centrifuged (16 100×Contents contained within the colon of each mouse (∼100 mg/mouse) were recovered during dissection and frozen at −80°C. DNA was recovered from each sample by first grinding in liquid nitrogen then purified using the QIAamp DNA stool mini kit . Bacterial tag-encoded FLX amplicon pyrosequencing was performed as previously described Both TGF-β1 and IL-2 were quantified by enzyme linked immunosorbent assay (ELISA), using kits DY240 and DY402 respectively,  and high affinity protein binding ELISA plates (Greiner Bio-One). Microcentrifuge tubes were siliconized with Sigmacote®  prior to preparation of dilutions and activation of samples for TGF-β1 quantification. Levels of both active and total TGF-β1 were determined. Activation of TGF-β1 samples to remove the Latency Associated Protein (LAP) was carried out using standard acidification protocols as outlined by R&D Systems. The optical density was determined using a Synergy HT plate reader  at 405 nm.Statistical analyses were performed in InStat 3  using unpaired two-tailed T-tests. However, statistical analysis was not possible for all ground controls and space flight mice due to the small sample size which survived flight. For cluster analysis, phylotype frequency distributions were first calculated for each colonic sample with abundance normalized by expressing the contribution of each individual phylotype as a percent of the total sample. This data was pooled then subjected to cluster analysis using Statistica  using city block distances


Down syndrome  is the most common genetic cause of mental retardation in the human population and key molecular networks dysregulated in DS are still unknown. Many different experimental techniques have been applied to analyse the effects of dosage imbalance at the molecular and phenotypical level, however, currently no integrative approach exists that attempts to extract the common information.SOD1, APP, RUNX1 and DYRK1A as well as a large proportion of novel genes (N = 62). Furthermore, we characterized these genes using gene ontology, molecular interactions and promoter sequence analysis. In order to judge relevance of the 324 genes for more general cerebral pathologies we used independent publicly available microarry data from brain studies not related with DS and identified a subset of 79 genes with potential impact for neurocognitive processes. All results have been made available through a web server under http://ds-geneminer.molgen.mpg.de/.We have performed a statistical meta-analysis from 45 heterogeneous publicly available DS data sets in order to identify consistent dosage effects from these studies. We identified 324 genes with significant genome-wide dosage effects, including well investigated genes like Our study represents a comprehensive integrative analysis of heterogeneous data including genome-wide transcript levels in the domain of trisomy 21. The detected dosage effects build a resource for further studies of DS pathology and the development of new therapies. Down syndrome (DS) is the most frequent genomic aneuploidy with an incidence of approximately 1 in 700 live-newborn ,3. DS haSOD1 and DYRK1A ..VSNL1), http://ds-geneminer.molgen.mpg.de/. Examples of possible views and information are shown in Figure These examples show that the meta-analysis approach identified multiple additional genes that might be involved in DS pathology. In order to enable the community to check any particular gene of interest for DS relevance in the studies under analysis, we have agglomerated all information of the meta-analysis into a WEB-interface per se interesting that, in spite of such heterogeneity, common dosage effects could be identified at all and it should be highlighted that whole-genome data was fairly robust across experiments. Additional file The statistical meta-analysis approach was described previously by Rasche et al. . The scoThe score used in this analysis allows detecting genes that could be either up- or down-regulated in different studies. An overview of the fold-changes for the genes across the different experiments is given in Additional file We studied 255 HSA21 genes matched with the probe sets from the microarrays. Of these only 77 showed consistent dosage effects  TJP1 (Tight junction protein ZO-1) interacts with two HSA21 genes, JAM2 and CLDN8, (ii) FOS  interacts with HSA21 genes ETS2, SUMO3, RUNX1 and indirectly with ERG, (iii) RHOQ  interacts directly with ITSN1 and TIAM1 and indirectly with SYNJ, and (iv) PIK3R1 interacts directly with IFNAR1 and indirectly with IFNAR2. It should be emphasized that current information on molecular interactions is far from complete, thus we either might miss important interactions not yet detected and/or we might count false positive interactions due to the high error rates of current annotations of interactions.In this study we monitored molecular interactions of HSA21 genes that might function as drivers of dosage effects Figure . For exa(i) differential gene expression in the cerebellum region versus whole "brain" or cerebrum areas which has been reported in other studies  different patterns of gene expression associated to particular developmental stages ; these changes were reported before by Dauphinot el al. [(iii) differences in ES studies.Several of the DS genes (N = 79) extrapolated to more general neurological phenotypes Figure . The denh et al. ), (ii) dt el al. , and  and different developmental stages  as the backbone annotation for all studies. IDs were mapped to human Ensembl gene IDs. Mapping and merging of the information was done within R and the BioConductor package. In total, information on 19,388 ENSEMBL genes was mapped.ftp://ftp.ncbi.nlm.nih.gov/pub/sage/mappings.Differential expressed tags were extracted from additional files of the studies. Identifiers (based on sequences) were cross-tagged with the information displayed in the updating tables (SAGEmap_Hs and SAGEmap_Mn) from the SAGE site (i) the fold-change (DS vs. controls), (ii) the standard error of the fold-change from the replicated experiments in that study and (iii) the expression p-value  that indicates whether or not the gene is expressed in the target samples under study. For RT-PCR and MALDI experiments we computed the fold-change of the mean expression (DS vs. controls) as well as the reported standard error of the ratio. When mean and standard variation for each group (DS and controls) was provided we calculated the ratios as well as their associated standard errors.We incorporated only case-control studies in the meta-analysis in order to derive expression fold-changes. Affymetrix gene chip annotations were adapted from the latest genome annotation (version 12). Affymetrix data were normalized with GC RMA. For transcriptome case-control studies three pieces of information were stored for each gene; i-th gene in the j-th study, sij, was computed as described in Rasche et al. [In order to score the different categories of information such as binary counts and quantitative gene expression values, we summarized the scores of the individual experiments for each category. For microarray studies, the score of the e et al. :ij is the fold-change, pij is the average detection p-value and eij is the standard error of the ratio derived from the experimental replicates of the study. Thus, the fold-change is weighted with its reproducibility across the experimental replicates and with the likelihood of the gene being expressed in the study's case or control samples.Here rFor RT-PCR and MALDI studies we applied the following equation:ij is the fold-change and eij is the standard error of the ratio.Here rThe total score of the gene was computed as the sum across all individual study scores.In order to assess the significance of the overall gene scores we generated random scores by re-sampling the scores 50,000 times with replacement within the same study. Using the random distribution as background we assigned as significant those genes that were above the 99.9 percentile of the background distribution.ij be the score of the ith gene in the jth study, then Ei is a measure for the uniformity of the score distribution over the individual experiments:For each gene, entropy of the score distribution was computed in order to quantify the relevance of the gene across many experiments. Let sHigh entropy is assigned to a gene if many experiments contribute to the overall score whereas low entropy is assigned if only a few experiments contribute to the overall score.http://cpdb.molgen.mpg.de. Over-representation analysis of TF target sets was performed with Fisher's test based on annotation from TRANSFAC [Gene Set Enrichment Analysis  of the TRANSFAC . Motif eTRANSFAC  with sigIn order to proof general brain relevance of the 324 genes, we collected DS-independent gene expression studies to decipher brain features, performed with Affymetrix technology and, with experiments deposited in GEO or ArrayExpress , the significant non-HSA21 genes (green bars) and the non-significant genes (blue bars).Click here for fileFigure S2. Genomic location of DS dosage effects in A) human B) mouse. Significant genes are marked in red, non-significant genes in white.Click here for fileFigure S3. A) Categorization of the 35 qualitative studies, B) Categorization of the 34 quantitative studies. C) Venn diagram of dosage effects detected with mouse and human data alone and with the combination of all data, D) correlation between average PCR and microarray values for the detected 324 dosage effects.Click here for fileFigure S4. Cross-validation with DS dosage effects detected with an HSA21 microarray [croarray . Box-ploClick here for file


Using three different assays, we examined 103 serum samples collected from different civet farms and a market in China in June 2003 and January 2004. While civets on farms were largely free from SARS-CoV infection, ≈80% of the animals from one animal market in Guangzhou contained significant levels of antibody to SARS-CoV, which suggests no widespread infection among civets resident on farms, and the infection of civets in the market might be associated with trading activities under the conditions of overcrowding and mixing of various animal species. In IFA, monolayers of Vero cells infected with SARS-CoV at a multiplicity of infection of 0.02 TCID50/cell were methanol-fixed 24 h postinfection, exposed to a range of serum dilutions, and bound antibody detected by using fluorescein isothiocyanate–conjugated protein A . Groups of samples that reacted positively in either VNT or IFA were also subjected to Western blot analysis with a recombinant SARS-CoV nucleocapsid (N) protein expressed in Escherichia coli. Bound antibodies were detected by using alkaline phosphatase–conjugated protein A/G .All serum samples were inactivated at 56°C for 30 min, transferred to the Australian Animal Health Laboratory, and inactivated by gamma irradiation before analysis. Anti–SARS-CoV antibody in serum was detected by using immunofluoresence antibody assay (IFA) and quantified in a microtiter virus neutralization test (VNT). The SARS-CoV (strain HKU-39849) used in both VNT and IFA was plaque purified three times in Vero cells, and stock virus . Total RNA was extracted from these samples by using the Trizol method , followed by first-strand cDNA synthesis using the Superscript II RNase H reverse transcriptase  and random hexamer primers. PCR amplification was conducted by using Ex Taq polymerase (TaKaRa). Three pairs of SARS-CoV–specific primers were used to amplify regions in the N gene , the M gene  and the S gene , respectively.Of 103 civet serum samples tested, 18 were positive on at least one of the three assays used, for ≈17% overall seroprevalence. However, when seroprevalence among civets from farms and the market was compared, differences were observed. For samples taken in January 2004, 14 of 18 obtained from the Xinyuan Live Animal Market in Guangzhou tested positive by all three assays , for a sIntestinal tissues collected from the 56 civets were tested by RT-PCR using N-gene primers; none of the samples were positive. Negative results were confirmed by RT-PCR with M- and S-gene primers. Therefore, virus isolation from these tissues was abandoned. The other 47 serum samples taken in June 2003 from Henan and Hunan provinces were negative by VNT or IFA . WesternWhile civet selection was derived from a convenience sample and limited because of time constraints imposed by the slaughter campaign, this study showed a marked difference in SARS-CoV antibody prevalence between animals from the market and those selected from the farms. Animals selected from one market in Guangzhou in January 2004 had a much higher prevalence of SARS-CoV antibodies than those selected from farms in the same period or from farms in two other provinces in June 2003. These results raise the possibility that civets, rather than being the natural animal reservoir of SARS-CoV, are infected mainly in markets or during other trade-related activities. Our results suggest that mass slaughter of civets on farms might not be necessary to control SARS-CoV spread. A more effective approach might be to implement testing in live animal markets and farms for susceptible animals and to apply quarantine regulation and targeted slaughter for markets or farms with infected animals.While Guan et al.  and one Chinese ferret-badger  from the same market at the same time, which demonstrates possible interspecies SARS-CoV transmission during trading. Sequence analysis of the S genes showed that one civet isolate (SZ16) was more closely related to the raccoon dog isolate (SZ13) than the other two civet isolates (SZ1 and SZ3), which further supports interspecies transmission in the market , group 2 , and group 3 (infectious bronchitis virus) and found no cross-reactivity (data not shown). We cannot rule out the possibility that an unknown coronavirus can infect civets, which may give low levels of cross-reactivity in the assays used in this study. However, such cross-reactive antibodies are not likely to positively react in all three of the assays used in this study.The most basic limitation of our study was the nonrandom sampling, which limits the generalization of our results. However, this study is a first step in investigating the role of civets in transmitting SARS-CoV. Much remains to be done, including studies on the prevalence of infection with SARS-CoV and related coronaviruses that use more robust methods to sample susceptible animals in markets, farms, and the wild. Improved serologic tests should be developed that can detect SARS-CoV–specific antibodies from different animal species, without relying on live SARS-CoV. Other issues that remain to be resolved include the rate of new infections in susceptible animal species, the characteristics of the animals that become infected, and the nature of the exposures that lead to interspecies transmission.


Surprisingly, despite a contrary belief in the community, the common driving motif is an unreliable means of establishing zero-lag synchrony. Although dynamical relaying has been validated in empirical and computational studies, the deeper dynamical mechanisms and comparison to dynamics on other motifs is lacking. By systematically comparing synchronization on a variety of small motifs, we establish that the presence of a single reciprocally connected pair – a “resonance pair” – plays a crucial role in disambiguating those motifs that foster zero-lag synchrony in the presence of conduction delays  from those that do not (such as the common driving triad). Remarkably, minor structural changes to the common driving motif that incorporate a reciprocal pair recover robust zero-lag synchrony. The findings are observed in computational models of spiking neurons, populations of spiking neurons and neural mass models, and arise whether the oscillatory systems are periodic, chaotic, noise-free or driven by stochastic inputs. The influence of the resonance pair is also robust to parameter mismatch and asymmetrical time delays amongst the elements of the motif. We call this manner of facilitating zero-lag synchrony resonance-induced synchronization, outline the conditions for its occurrence, and propose that it may be a general mechanism to promote zero-lag synchrony in the brain.Zero-lag synchronization between distant cortical areas has been observed in a diversity of experimental data sets and between many different regions of the brain. Several computational mechanisms have been proposed to account for such  Understanding large-scale neuronal dynamics – and how they relate to the cortical anatomy – is one of the key areas of neuroscience research. Despite a wealth of recent research, the key principles of this relationship have yet to be established. Here we employ computational modeling to study neuronal dynamics on small subgraphs – or motifs – across a hierarchy of spatial scales. We establish a novel organizing principle that we term a “resonance pair” , which promotes stable, zero-lag synchrony amongst motif nodes. The bidirectional coupling between a resonance pair acts to mutually adjust their dynamics onto a common and relatively stable synchronized regime, which then propagates and stabilizes the synchronization of other nodes within the motif. Remarkably, we find that this effect can propagate along chains of coupled nodes and hence holds the potential to promote stable zero-lag synchrony in larger sub-networks of cortical systems. Our findings hence suggest a potential unifying account of the existence of zero-lag synchrony, an important phenomenon that may underlie crucial cognitive processes in the brain. Moreover, such pairs of mutually coupled oscillators are found in a wide variety of physical and biological systems suggesting a new, broadly relevant and unifying principle. Since first reported in cat visual cortex Complex dynamics in spatially embedded systems arise in a broad variety of physical and biological contexts. Arrays of coupled semiconductor lasers are a prominent example. Because of their extraordinary internal speed, even small time delays due to the finite speed of light are usually nonnegligible in arrays of coupled lasers resonance-induced synchrony – arises consistently in candidate computational models at the neuronal, population and mesoscopic spatial scales and is robust to mismatches in system parameters and even time delays. Remarkably, we show that the resonance effects of a synchronized pair are not necessarily localized, but may instead propagate throughout the network. We hence propose resonance-induced synchrony as a general and unifying mechanism of facilitating zero-lag synchrony in the brain.The presence of a node that drives two common-driven nodes that reach zero-lag synchrony between them due to the driver's influence is intuitively appealing and finds anatomical support, for example, by shared input through bifurcating axons We studied zero-lag synchronization – quantified as the average zero-lag cross-correlation between two nodes A and B (The notation we adopt for the motifs of three nodes follows the notation of Sporns and Kötter (2004) We first focus on the four motifs depicted in For the smallest-scale system we consider, each node comprises a single excitatory Hodgkin-Huxley neuron resonance pair: Its presence dramatically alters the dynamics and synchronization properties of the driven nodes. Supplementary The structural variations introduced in motifs M6, M9 and M3+1 over the common driving M3 share an essential feature: The driver node 2 is mutually connected and synchronized with at least one other node. We denote this mutual connection The emergence of zero-lag synchrony in motifs of coupled Hodgkin-Huxley neurons shows a strong dependence on the time delay, consistent with prior work  , where S , increasThe bottom panels of The delay in a resonance pair can either enhance or reduce the synchronization. In motif M6 the driven nodes (1 and 3) synchronize whenever the resonance pair (1 and 2) synchronizes, whether this is in-phase or anti-phase synchrony. This corresponds to a drop in the incoherence R of the driving node. Hence, the synchronization between nodes 1 and 3 depends on the time delay in the resonance pairs because the synchronization between the resonance pair (1 and 2) – and thus the incoherence – also depends on the time delay. It therefore appears that synchronization between the reciprocally connected nodes leads to a more regular (less incoherent) output from the master node which then facilitates synchronization between this node and the other slave node.To investigate whether these results translate to neuronal activity at the next spatial scale, we exploited the computational parsimony of the neural model of Izhikevich Similar to motifs of Hodgkin-Huxley neurons, synchronization of populations of spiking neurons also shows a dependence on the delay time between nodes in the presence of a resonance pair. Supplementary To further study the robustness of the relationship between motifs and synchronization with respect to the underlying dynamical systems, we next utilized a neural mass model, which represents a reduced model of cortical dynamics. A neural mass model is a parsimonious representation of the dynamics of a very high-dimensional system, and replaces thousands of equations for each population of neurons with a small number (here only three) of nonlinear equations per node. These represent the dynamical behavior of the essential summary system statistics (here mean firing rate) and hence a reduced representation of spontaneous cortical dynamics. Here we employ a population representation of conductance-based model neurons The dynamics of these delayed-coupled neural masses shows chaotic oscillations fast dynamics (and coupling strength: For weak coupling strength (c = 0.01), phase synchronization is not reached. However, as illustrated in supplementary Despite the dissimilarities between the neuronal systems at different scales, synchronization and incoherence of the neural mass model also exhibits a dependence on the time delay in the presence of a resonance pair  The simple common-driving motif (M3) where synchronization at zero lag is not achieved in the weak-coupling regime, independent of the time delay; (ii) A ring of three mutually coupled systems (M13) or a common-driving motif that also contain direct coupling between the driven nodes (M8) require a relative strong coupling and negligible delay in order to promote synchronization , becauseThe preceding analyses show that the effect of the resonance pair can influence the common driving motif even when it is placed outside the motif itself (e.g. M3+1). Here we further investigate the propagation of the resonance pair effect by considering larger structures in which the resonance pair is distant from the driver node (2). This procedure is schematically shown in We observe that zero-lag synchronization between the driven nodes 1 and 3 is virtually independent of the distance along a polysynaptic chain from the resonance pair . For a fFinally, to highlight the influence of the resonance pair in the dynamics, we removed the feedback connection to node N . That is, the emergence of synchronization between the resonance pair then stabilizes synchrony amongst unidirectionally coupled nodes. The same phenomenon underlies the propagation down a polysynaptic chain . InteresConnectivity also plays a role on the onset of synchronization. We studied the temporal onset of zero-lag synchronization in neural mass models for different motifs by (1) examining the transient dynamics following random initial conditions, and (2) studying the response to a transient perturbation. An example is shown in us state . In contSynchronization hence arises quickly in the presence of a resonance pair. Is it possible to adjust the dynamics of the driver node without such reciprocal coupling to induce synchronization? We next studied this possibility by fine-tuning the input current , a scenario that is broadly assumed to underlie zero-lag synchrony, fails completely in the weak-coupling regime and is sensitive to parameter mismatch. However, the addition of one or more mutually coupled pairs fosters the emergence of zero-lag synchrony in the outer nodes of triplet motifs, and beyond. We find that this effect is robust to many of the particular details of the system, the spatial scale and parameter asymmetry, and can propagate through a multi-synaptic relay chain. In stark contrast, the further addition of a reciprocal connection between the driven nodes introduces frustration for delays that favor out-of-phase synchrony and fails to promote zero-lag synchronization. The disruptive effect of adding new edges that close the motif reinforces the observation that it is the topology  that determines the zero-lag synchrony. This is also evident by the fact that an increase in the coupling over two orders of magnitude in the unidirectional motif (M3) is less effective than adding a single feedback connection (where the effective coupling within that pair is simply doubled).resonance pair because it can induce zero-lag synchronization between outer nodes. We find that an entire family of three- and four-node motifs exhibits zero-lag synchronization in the presence of such a resonance pair. Perhaps the archetypal motif in this family is M9  and scales: motifs of neurons and motifs of cortical regions. The results are also robust with respect to the delay, the coupling strength, the oscillatory frequency band, and arise in autonomous, chaotic systems as well as noise-driven excitable dynamics. It seems reasonable to propose that resonance-induced synchronization will prove important for other neuronal systems, such as dendritic oscillations in single-neuron dynamics Despite the robustness of the present effect in different classes of models and dynamical regimes, the universality and extent of the phenomenon remains to be clarified. Phase-resetting curves (PRCs) can be useful to predict whether phase or out-of-phase synchronization will arise Computational studies of anatomically derived brain networks have shown that motifs M9 and M6 are the first and second most abundant of all three-node motifs in the macaque visual cortex The influence of a resonance pair is not limited to local synchronization dynamics but also, through propagation, to larger networks, decaying only slowly with the polysynaptic distance see  and 12. The notion of motifs as fundamental building blocks of complex networks has yielded considerable prior success We simulated neuronal motif dynamics at different scales, and for different dynamical scenarios. First, representing the microscopic scale, each node was taken as a single neuron. For this endeavor we utilized the Hodgkin-Huxley model. Second, at the circuit scale, we took each node as a large population of spiking neurons. Third, at the mesoscopic scale, we considered a simplified coarse-grained version in which each population was taken as a neural mass model.Each node was modeled by the well-known Hodgkin-Huxley equations nization . The extFor this large-scale circuit model, each node represented populations of 500 randomly connected neurons described by the Izhikevich model Each neuron receives input from 80 neurons of the same population and from 25 excitatory neurons of each afferent population. The synaptic current is given byThe equations were integrated using a fixed-step first-order Euler method with time steps of 0.05 ms, starting with random initial conditions. To avoid spurious synchronization at the onset of simulations, neural populations were activated with random noise in 600 ms sequential windows (with a 500 ms overlap). The first transients of 1 s were discarded before further analysis.The preceding large-scale circuit model is a high dimensional system. Whilst the dynamics are instructive, the large number of parameters and equations preclude an intuitive perspective of the system. We therefore additionally studied a reduced system Each node represents the mean dynamics of an ensemble of neurons, with spontaneous dynamics arising from the interaction between excitatory and the inhibitory sub-populations. The model is derived from the biophysical Morris-Lecar model This neural mass model comprises three state variables: The mean membrane potential of the excitatory pyramidal neurons, neural-activation function, whose shape reflects a sigmoidal-saturating grow with The fraction of channels open dde23.Our simulations employ the previously published parameter values: Figure S1Dynamics of pairs of neural mass models. (a), (d) and (g) show the time traces of the average membrane potential of the excitatory pyramidal neurons; (b), (e) and (h) show the auto-correlation function of node A; (c), (f) and (i) show the cross-correlation function between nodes A and B; respectively for a pair bidirectionally connected, unidirectionally connected, and disconnected nodes. Parameters are c = 0.01, and (TIFF)Click here for additional data file.Figure S2Example dynamics of Hodgkin-Huxley neurons coupled on motif M6 for different time delays. From left to right, panels show anti-phase synchronization ((TIFF)Click here for additional data file.Figure S3Synchronization dynamics and incoherence in populations of Izhikevich neurons. Panels (a–d) as per (TIFF)Click here for additional data file.Figure S4Example dynamics of populations of Izhikevich neurons coupled as motif M6 for different time delays. From left to right, panels show phase synchronization ((TIFF)Click here for additional data file.Figure S5Synchronization dynamics and incoherence in weakly coupled neural mass models. Panels (a–d) as per (TIFF)Click here for additional data file.Figure S6Example dynamics of neural mass models coupled on motif M6. From left to right, panels show anti-phase synchrony at the slow rhythm (no time delay), weak synchrony ((TIFF)Click here for additional data file.Figure S7Cross-correlation for strongly coupled neural mass models. Top panels show the crosscorrelations between nodes 1 and 2 (a–b), and nodes 1 and 3 (c–d) as a function of the delay for coupling strength c = 0.05. Bottom panels panels show the cross-correlations between nodes 1 and 2 (e–f), and nodes 1 and 3 (g–h) as a function of the delay for coupling strength c = 0.15. First and third columns correspond to a zoom of second and forth columns respectively. Black (blue) lines represent results for motif M3 (M6), and continuous (dashed) lines represent the crosscorrelation at zero lag . Phase synchrony, and complex synchronous states can be found in motif M6 depending on the time delay (TIFF)Click here for additional data file.Figure S8Example dynamics of neural mass models strongly coupled as motif M6 for different time delays. From left to right, panels show phase synchrony ((TIFF)Click here for additional data file.Figure S9Incoherence and synchronization dependence on the time-delay mismatch in Hodgkin-Huxley neurons. (a–c) Top panels show incoherence: Colors represent different nodes. Bottom panels show cross-correlations for motif M6′. Continuous lines indicate the cross-correlation coefficients at zero time lag, and dashed lines indicate the maximum cross-correlation coefficients across all time lags. Panels a, b and c represent pairs of nodes: 1–2, 1–3, and 2–3 respectively. Results are averaged over 40 trials.(TIFF)Click here for additional data file.Figure S10Zero-lag synchronization dependence on the delay  in motifs of neural mass models. The curves are color coded as in (TIFF)Click here for additional data file.Figure S11Kernel test in motifs of Hodgkin-Huxley neurons. Nearly identical cross-correlation functions are obtained when the external driving is considered identical to the spikes within the motifs (see (TIFF)Click here for additional data file.