Poor sanitation could pose greater risk for enteric pathogen transmission at higher human population densities because of greater potential for pathogens to infect new hosts through environmentally mediated and person-to-person transmission. We hypothesized that incidence and prevalence of diarrhea, enteric protozoans, and soil-transmitted helminth infections would be higher in high-population-density areas compared with low-population-density areas, and that poor sanitation would pose greater risk for these enteric infections at high density compared with low density. We tested our hypotheses using 6 years of clinic-based diarrhea surveillance (2007–2013) including 4,360 geolocated diarrhea cases tested for 13 pathogens and a 2010 cross-sectional survey that measured environmental exposures from 204 households (920 people) and tested 701 stool specimens for enteric parasites. We found that population density was not a key determinant of enteric infection nor a strong effect modifier of risk posed by poor household sanitation in this setting. Most enteric pathogens are spread by fecal–oral transmission that is facilitated by the close contact of people either through direct, person-to-person transmission or through indirect, environmentally mediated transmission.12Population density may be one important source of heterogeneity, and if so, enteric infection studies designed explicitly around questions of population density and its relationship to poor sanitation will help guide future intervention programs. Yet, we are aware of few empirical studies of the relationship between population density and enteric infection risk.14The objectives of this study were to compare enteric infection rates at high- and low-population densities in a Guatemalan municipality and to determine whether poor sanitation conditions pose a greater risk at high-population density compared with low-population density. Due to the greater opportunity for individuals to come into contact with fecal pathogens through contact with their environment or with other people at higher population densities, we hypothesized that enteric infections would be more common in high-density areas compared with low-density areas and that poor sanitation would pose a greater risk for enteric pathogen infection at higher population density. Since poor sanitation theoretically contributes most to environmentally mediated enteric pathogen transmission, we would expect it to pose a greater risk for environmentally mediated pathogens than for those spread predominantly through person-to-person contact.16Nueva Santa Rosa is a municipality located southeast of Guatemala City in the department of Santa Rosa. Its population in 2011 was approximately 33,000, and 80% of people live in rural areas, whereas 20% live in urban areas. Population density ranges from extremely low  to moderate . Unlike many regions of Guatemala, just 3% of Santa Rosa's population is indigenous, and 91% speak Spanish as their first language.Vigilancia Integrada Comunitaria [VICo]) in the municipality that tested stool specimens from individuals who presented to six sentinel clinics with diarrhea symptomsThe study used two complementary data sources. The first source was an ongoing diarrhea surveillance system (2) using global positioning system coordinates identified in aerial imagery. From these 387 roofs, the final survey enrolled 204 households and 920 individuals. The reasons for 183 roofs not being enrolled were the following: structure not found by interviewers , structure not being used for living purposes , structure uninhabited at the time of the visit by field workers , no adult was present at the time of the visit to provide consent for participation , and not providing consent to participate in the study . Of the remaining 211 consenting households, seven participated in the survey but their data were removed from analysis because of data cleaning issues . Interviewers recorded observations of sanitation facility condition and use, observed feces in the household environments, and reported child feces disposal practices. Interviewers collected information about housing conditions, assets, and environmental conditions, including: water sources, water treatment practices, water quality, observations of the primary handwashing locations, and animal presence around the home.The cross-sectional survey was conducted in August–September 2010. The study drew a simple random sample of 387 roofs from all 10,770 possibly inhabited roofs , protozoans , and tapeworms  using the fecal parasite concentrator method .Interviewers asked the primary respondent, usually an adult female (76%), about diarrhea symptoms (≥ 3 loose or watery stools in 24 hours) in the past 7 days for each household member.N = 2,557) and five rural health posts  in the municipality who met the diarrhea case definition: ≥ 3 loose or liquid stools in a 24-hour period with onset in the past 7 days. We excluded diarrhea cases from the regional hospital in Cuilapa  because the hospital receives patients from the entire department. Cases originating in the Nueva Santa Rosa municipality typically present first to the municipal clinics before referral (for severe cases) to the Cuilapa hospital. Such referrals were not tracked in the surveillance system and excluding cases from the Cuilapa hospital ensured that we did not double count cases originating in Nueva Santa Rosa. Each stool sample  included case demographic characteristics and socioeconomic characteristics; VICo collected few environmental and household exposure characteristics related to enteric infections, except for drinking water source and floor material.The VICo health facility–based surveillance system collected stool specimens between September 2007 and December 2013 to monitor infectious causes of diarrhea.Supplemental Text includes additional details. Samples were tested for STH , protozoan parasites , and tapeworms  by direct smear microscopic examinationSalmonella spp., Shigella spp., Campylobacter spp.) by direct cultureEscherichia coli pathotypes  using conventional polymerase chain reactionSupplemental Table 1 summarizes the number of VICo samples and pathogen-specific test results.The details of VICo specimen collection and testing have been previously describedSupplemental Text). The cut point corresponded to 5,348 persons/km2, which was the 74th percentile of the distribution.For each household in the cross-sectional survey, we identified the number of roofs within a 50-m radius. We chose a 50-m radius to be consistent with recent enteric infection sanitation studies conducted in urban and rural environments,2, maximum = 5 km2; median location density = 754 persons/km2, maximum = 2,579 persons/km2). We converted the continuous population density measures into dichotomous groups by classifying households above the 75th percentile of the distribution  as “high density.” We used a 75th percentile cutoff to maintain consistency with our high-density definition from the cross-sectional survey.Each diarrhea case identified through VICo surveillance had a recorded home village or populated place . We matched each case to the official list of populated places from Guatemala's Instituto Nacional de Estadística (INE), and we excluded four cases that we could not match to a location. The remaining 4,360 cases originated from 31 locations in the municipality. We calculated village- and age-specific population estimates for 2010 (midpoint of the 2007–2013 surveillance) by increasing location-specific 2002 census populations by INE municipal-level growth rates through 2010.Supplemental Information).In the cross-sectional survey we defined a household's sanitation as “improved” if: 1) it had a toilet that met the World Health Organization/United Nations Children's Fund Joint Monitoring Program (WHO/UNICEF JMP) criteriaShigella spp., Campylobacter spp., pathogenic E. coli, rotavirus, norovirus).Using the surveillance data, we calculated crude incidence rates by dividing incident cases by the person-time at risk in the municipality, which we estimated using the surveillance dates and 2010 population estimates described above.Supplemental Text includes minimum detectable effect calculations, statistical analysis details, and some exploratory analyses of the geographic distribution of infection.We compared the prevalence of each enteric infection between high-density areas and low-density areas in the cross-sectional survey. We examined whether household sanitation was differentially associated with enteric infection prevalence by estimating the association between poor sanitation conditions and enteric infection prevalence separately in low- and high-density households, controlling for possible confounding factors informed by our causal model  and 2,710 cases (high density). In the cross-sectional survey, the classification of households into low- and high-density areas resulted in 57 high-density households (292 people) and 147 low-density households (628 people). Characteristics of the cross-sectional survey households were similar to households of diarrhea cases detected through VICo surveillance, except for participant age—young children accounted for the majority of VICo cases .Supplemental Table 2 includes stratified IRRs). Pathogen-specific adjusted incidence rates were higher in high-density areas for Shigella spp.  and rotavirus .Distance to the surveillance facility was a strong confounder of the association between population density and diarrhea because it was strongly, positively associated with both population density and the probability of reporting to a surveillance site. For this reason, our analysis focused on adjusted IRR estimates that stratified by distance and age. Diarrhea incidence was lower in high-density areas versus low-density areas after adjusting for distance to surveillance site and age  . Although not statistically different, protozoan parasite prevalence tended to be lower in high-density households: G. lamblia prevalence was 4.8% in high-density households compared with 7.8% in low-density households , and E. coli prevalence was 24.5% in high-density households compared with 33.3% in low-density households .We detected no statistically significant differences in prevalence between high- and low-density households in the cross-sectional survey . DiarrheG. lamblia infection  compared with low-density areas , with some evidence for effect modification . However, after adjusting for potentially confounding covariates, there was no evidence for an association between poor sanitation conditions and A. lumbricoides infection and no evidence for effect modification by population density .Poor household sanitation was not associated with diarrhea at either low density  or high density , nor was it consistently associated with nfection . In unad density . Due to Campylobacter spp., pathogenic E. coli), but we did observe some evidence for higher adjusted incidence rates of medically attended diarrhea attributed to Shigella spp. and rotavirus in higher density areas—both pathogens are most likely dominated by person-to-person transmission.G. lamblia and E. coli infection within high-density households, but the differences were not statistically significant , but the range of density we studied was lower than in large city centers and urban slum environments—for example, the population density of slums in Dhaka, Bangladesh, is 205,415 people/km237Population density is a complex exposure and readers should interpret our findings in the context of the study's methods and setting. There are three main points to consider. First, the study included households across a wide range of population densities , but in larger studies it would be useful to look for associations and effect modification across a finer gradient of population density. When we stratified the medically attended diarrhea rates by quartiles of population density, we saw no clear dose–response relationship ; the cross-sectional survey was sufficiently small that we felt we could not conduct a similar analysis.Second, we reduced the continuous range of population densities into dichotomous groups of high and low density that corresponded to the 74th percentile of the density distribution. We dichotomized population density due to the relatively small study sample. We chose the cut point using a prespecified and repeatable method designed to reflect local conditions (2), but were larger than areas used to calculate 50-m point densities in the cross-sectional survey (area = 0.008 km2). Population densities calculated over larger areas will be lower because larger areas include more open space,Finally, we used two different approaches to estimate population density that arose from our different data sources. For the surveillance data, we estimated population density for populated places using their estimated boundaries and estimated population. The areas were small (median = 0.9 kmN = 701 stool specimens). The study had power to detect stratified PRs associated with poor sanitation of between 2.13 and 2.81, and a RERI of ≥ 2.25, but many of the associations estimated in this study were smaller than this . Third, the cross-sectional analysis estimated population densities by applying the number of residents per roof in the study households to all roofs in a 50-m radius—a complete census would be more accurate. Fourth, we used a single exposure definition of “poor sanitation” in low- and high-density areas, which was consistent with estimating effects under a counterfactual causal model. Yet, if the features of household or neighborhood sanitation conditions that drive enteric pathogen transmission differ depending on population density, a single definition of poor sanitation across different densities could potentially lead to misclassification of exposure, and in turn, mask some heterogeneity between enteric infection risk associated with poor sanitation in low- versus high-density areas. Finally, the cross-sectional analysis focused on household-level sanitation conditions and not on neighborhood sanitation conditions. This approach implicitly assumes that household sanitation conditions are most relevant for enteric infection risk or that household conditions reflect the broader sanitation conditions in the relevant transmission area around a household. We could not test this assumption because we found that predictions of neighborhood sanitation conditions using spatial location were unreliable . A measurement approach that characterized sanitation conditions over the same area used to calculate population density would be the best approach for future studies because it would better characterize the joint exposures of poor sanitation and population density within a spatially relevant transmission area.This study had some limitations owing to the use of previously collected data to test our hypotheses. These limitations would not obtain in a prospective study designed de novo to test these hypotheses, and we hope that the limitations encountered in this analysis help guide future research. First, our stratified estimates using the surveillance data corrected for age and distance to the surveillance site, but it remains possible that there was residual confounding between high- and low-density areas that we could not adjust for in the analysis. Second, the cross-sectional survey that we used to test the joint relationship between population density and poor sanitation was relatively small (han this . This liIn the rural and small urban areas of Nueva Santa Rosa, Guatemala, population density was not a major determinant of enteric pathogen transmission, nor did it act as a strong effect modifier of risk posed by poor household sanitation.Supplemental Datas.


Short tandem repeats (STRs) are abundant in human genomes. Numerous STRs have been shown to be associated with genetic diseases and gene regulatory functions, and have been selected as genetic markers for evolutionary and forensic analyses. High-throughput next generation sequencers have fostered new cutting-edge computing techniques for genome-scale analyses, and cross-genome comparisons have facilitated the efficient identification of polymorphic STR markers for various applications.An automated and efficient system for detecting human polymorphic STRs at the genome scale is proposed in this study. Assembled contigs from next generation sequencing data were aligned and calibrated according to selected reference sequences. To verify identified polymorphic STRs, human genomes from the 1000 Genomes Project were employed for comprehensive analyses, and STR markers from the Combined DNA Index System (CODIS) and disease-related STR motifs were also applied as cases for evaluation. In addition, we analyzed STR variations for highly conserved homologous genes and human-unique genes. In total 477 polymorphic STRs were identified from 492 human-unique genes, among which 26 STRs were retrieved and clustered into three different groups for efficient comparison.We have developed an online system that efficiently identifies polymorphic STRs and provides novel distinguishable STR biomarkers for different levels of specificity. Candidate polymorphic STRs within a personal genome could be easily retrieved and compared to the constructed STR profile through query keywords, gene names, or assembled contigs. Short tandem repeats (STRs), also known as short sequence repeats or microsatellites, are genome segments composed of short repeating sequences. The length of the fundamental repeat unit varies from one to six nucleotides . STRs arin silico methods and verified by PCR [in silico tools are available for detecting both perfect and imperfect STRs within a single species [in silico STR marker screening [in silico STR marker discovery across individual NGS samples. The lobSTR program was developed by Gymrek et.al., who constructed a comprehensive survey of STR variations from NGS-derived personal genomes [In traditional approaches, genomic STR markers have typically been discovered by analyzing DNA sequences through d by PCR . Various species ,12. In r species . Traditi species . Recent  species . The lat species . Hoffmancreening . Their e genomes ,19. An a genomes . HoweverWe have performed a statistical analysis of the STR distributions in several datasets including chromosomal genes, combined DNA index system (CODIS) genes, disease-related genes, cross-species homologous genes, and genes that are unique to humans. The two major reasons for performing statistical analyses on different gene sets were: (1) to determine the most frequently appearing lengths of polymorphic/conserved STR patterns, and (2) the most frequently occurring regions of polymorphic STR motifs. To understand the extent of variation in the identified STRs, the distribution scale ranged from 1 to 84 bp. In addition, we selected the interval from 20 to 84 bp to analyze conserved degrees of identified STRs. Retrieved STRs from the different datasets are shown and discussed in the following sub-sections.vWA (ENSG00000110799) defined by CODIS contain multiple short repeat patterns, and the adopted CGSSR program could successfully identify the three STR markers "ACAG", "AGAT", and "TCCA" for vWA.CODIS is a collection of investigated and verified DNA markers provided by the U.S. Federal Bureau of Investigation (FBI) to criminal justice services. Thirteen STR markers (within ten defined genes and three intergenic segments) were examined in this program. From the verification results, our proposed system could successfully detect and list all 13 STR markers from 7 genomes, including six individual genomes from the 1000 Genomes Trio Project and one from the Ensembl reference genome. All retrieved STR markers are listed in Table It was also observed that most polymorphic STRs within a family group agreed with inherited characteristics, i.e., the daughter's alleles were inherited from either one of her parents. Based on CODIS STR markers for comparing these two families from the Trio Project, the results show that 7 of 13 STR loci displaying identical repeat patterns/numbers among all selected individuals, and only one or two STR patterns possessing minor differences in length could be found between parents and daughter in both families. These results strongly suggest that distinguishability at the individual level in the post-NGS era would likely improve if more distinct STR markers were added to support CODIS.To verify the accuracy and efficiency of the proposed system in detecting STR markers, we selected 13 well-known genes containing disease-related STRs. All identified STRs occurred in different genetic locations including protein coding regions, 5′ UTRs, 3′ UTRs and introns; large variations in repeat number might be causally related to serious genetic diseases according to previous medical reports. Table DMPK and AR genes were not consistent with heredity principles. This phenomenon might be a result of mixed sequencing data from heterozygous alleles. More recently developed assembly and reference mapping methods might be capable of distinguishing heterozygous alleles and overcoming such problems [The polymorphisms of disease-related STRs within all individuals were detected and compared, as shown in Table problems .To discover and distinguish important features of polymorphic STRs extracted from orthologous genes and human-unique genes within a human genome, we performed a statistical analysis of STR distributions from previously collected gene sets. In Table Max. Dev.) and average deviation (Avg. Dev.) in base pairs. The definitions of Max. Dev. and Avg. Dev. are denoted in Eq (1) and Eq (2), respectively. Max. Dev. represents the largest number of repeat differences (in bp) of a specified STR within the identical genes from any two individuals, and Avg. Dev. is obtained by taking an average-of-length difference (in bp) between all corresponding STRs within the identical genes from all possible pair combinations among 7 individuals.To observe the levels of STR marker variation within homologous genes, we calculated maximum deviation  in Figure Polymorphic STRs identified from each chromosome were analyzed and compared for 7 individuals. The total number of genes is 56,852 in this study, of which 617 were not successfully detected due to serious sequence variations and/or query genes located at defined boundaries. Moreover, a successful rate of 98.91% was achieved for polymorphic STR analyses in this study. In addition, we did not consider STR motifs in the Y chromosome since it belongs exclusively to males. Hence, we only performed a statistical analysis of the STR distribution of both polymorphic and conserved STRs among all acquired genomes. Figures Furthermore, we also evaluated the total length of STRs (TLSTR), total length of selected genes (TLgene), total number of genes (TNgene), total number of STRs (TNSTR), total number of polymorphic STRs (TNpSTR), density of polymorphic STRs, and occurrence ratio of polymorphic STRs in each chromosome. These data are summarized in Table Alternatively, highly variable STR patterns among 7 human genomes can be determined by assessing the extent of STR variations using a Manhattan-like scatter plot for all human chromosomes. The quality setting for all identified STR patterns is defined as 1.0 for this plot. Through the Manhattan plot Figure , severalWe designed a comprehensive web-based system called ISP for efficiently identifying polymorphic STRs among different individuals. Several useful functions were designed for users to retrieve and verify all potentially important STR biomarkers and compare personal STRs to 7 published human genomes. Users can enter an Ensembl gene ID, gene descriptions, gene names, or any related keywords, and the system immediately responds with query results for the appropriate gene selection. Users can then select an interesting gene and a pop-up dialog for STR quality and STR variation settings is displayed on the web page. For real-time analysis, only two quality values of 1.0 and 0.9 are currently available, and variation degrees are automatically decided and unlocked for selection depending on the selected genes. A quality of 1.0 indicates that all identified STRs are perfect STRs, while a quality of 0.9 indicates that an identified STR contains less than 10% noise including mutations, insertions, and deletions. Variation degree is calculated as the true difference in base pairs between any two polymorphic STRs.In the proposed system, users can provide customized sequences for STR polymorphism analysis. Once the query sequences are uploaded, the system will apply BLAST+ to align the query sequence against the reference human genome. Once the query sequence is successfully aligned to one of the collected human genes, the newly identified STRs within the query sequence are compared to all 7 human genomes for polymorphic STR analyses. The query results are exactly the same as described above. Here, the threshold for identity in BLAST+ was set at 99%. Such a relatively high threshold value avoids ambiguous situations caused by non-human sequences. Finally, the compared results are displayed via a tabulated interface and sent via email. For security reasons the URL was designed with embedded encryption.The system also includes four test gene sets including disease-related genes, CODIS genes, homologous genes, and genes related to a GO term of GO:0001501. Corresponding statistical reports stored in Microsoft Excel files are provided in the developed system. For online queries for interesting genes, users can click on the folder "ISP Datasets", and four different gene sets and their corresponding identified polymorphic STRs are available for each individual gene. To comprehensively analyze polymorphic STRs for all human genes, the folder "Chromosome Statistics" provides 23 Excel files, each of which contains the total number of STRs, total number of polymorphic STRs, total length of selected genes, total length of STRs, percentages of exact genetic locations of all detected STRs, percentages of different variation degrees for all polymorphic STRs, and two different degrees of STR quality (perfect STR and imperfect STR with less than 10% noise content). All these statistics can be downloaded directly from the interface. One example of the polymorphic STR distributions on chromosome 6 with perfect STR quality settings is shown in Figure To comprehensively display the identified polymorphic STRs and provide detailed information on selected genes, the system has a look-up table. In this table, users can easily find detailed descriptions of the selected gene and identified STRs. This web page includes Ensembl gene ID, gene name, pattern(s) of polymorphic STRs, transcript ID(s), and STR locations within the corresponding chromosome. In addition, the system also provides sequence files for two assembled families and reference sequences from Ensembl. Because of alternative splicing mechanisms in genomes, genetic regions of identified STR patterns might be affected and result in different conclusions for different transcripts. To observe all possible scenarios, the system presents all polymorphic STRs according to transcript ID. Users can click on any transcript ID and the identified results are immediately shown on the web page. To rapidly identify polymorphic STR patterns, users can click on a detected polymorphic pattern within the gene information table to display a corresponding message that is annotated with the locations framed in red. To display global sequence alignments of the identified STRs among the 7 individuals, clicking on the identified STR pattern or "Alignment Result" automatically displays the alignment results. Through these alignment procedures, users can verify and understand the polymorphic distribution of STRs among sample genomes. The multiple sequence alignments are generated in the system by ClustalW .http://isp.cs.ntou.edu.tw/. The proposed algorithms started with performing reference mapping or de novo assembly of the imported NGS sequences, and the coordinate calibration was defined by mapping onto the Ensembl reference human genome. An integrated STR template profile was initially created to overcome the insertions and deletions that occurred in the reference genome or other target genomes. All possible polymorphic STR patterns could be detected automatically and precisely according to the aligned coordinate system. In this paper, polymorphic STRs from several different gene sets were applied to demonstrate the proof-of-concept, including the gene set selected by CODIS, the disease-related gene set caused by STR variations, the cross-species homologous gene set, and a human unique gene set as our evaluation datasets. In addition, all STR polymorphisms that were found within the 1000 Genomes Trio Project (6 genomes) were comprehensively identified and downloadable from the designed website. We also performed statistical analyses on both polymorphic and conserved STRs in each chromosome (except the Y chromosome), and occurrence frequencies for polymorphic STR variations between cross-species homologous genes and human-unique genes were compared for investigating the relationships between functional features or identifiable features of STR biomarkers. Therefore, STR variation frequencies for human-unique genes were clearly higher than those for cross-species conserved homologous genes, despite both gene sets exhibiting similar STR distributions and densities. This result provides an important implication in that mutations of STR elements tend not to appear within highly conserved genes among different organisms during evolutionary processes, and these cross-species conserved STRs could be considered more functionally related STRs. In other words, the polymorphic STRs that appeared within human-unique genes could be regarded as good candidates for identifiable biometric features. Focusing on the selected 477 polymorphic STRs from human-unique genes, three different categories were logically analyzed and suggested according to the 7 human genomes . Interestingly, we found some STR variation characteristics from human-unique genes possessing distinguishable features that could support CODIS STR verification. Furthermore, from genome-wide analysis and selection, we found a set of 26 polymorphic STRs retrieved from human-unique genes that displayed relatively higher distinguishability compared to other identified STRs. In order to understand the distributions of polymorphic STRs within each chromosome (except the Y chromosome), we compared densities of polymorphic STRs within each chromosome, and the results show that chromosome 19 had the highest density of polymorphic STRs, while chromosome 3 had the lowest density. The developed system has shown that our proposed methods could detect any polymorphic STR markers efficiently, and the proposed method could take advantage of NGS high-throughput sequencing technology and detect polymorphic STRs without manually curated and compared works. In order to efficiently provide a clear view of query results for polymorphic STRs for each gene, we have pre-processed all genes within all chromosomes (except the Y chromosome). Users will be able to perform customized sequence comparisons online for identifying all polymorphic STRs within a specified gene. In addition, users can upload their own query sequences to compare STR variations with 7 human genomes. We believe that the developed system can facilitate research involving the detection of novel STR biomarkers and the discovery of regulatory STR elements.In this study, an automated workflow for discovering STR polymorphisms from individual NGS sequencing data was proposed and the developed system is freely available at de facto standard format for representing reference mapping results [To demonstrate that the proposed method is capable of detecting STR polymorphisms from NGS data, we have downloaded NGS genomic data from the 1000 Genomes Project as benchmark datasets. The 1000 Genomes Project is an ongoing international research project, the goal of which is to provide population-scale and high-coverage sequencing data world-wide. In 2010, the project completed its first phase, which included 3 pilot projects: the Low Coverage Project for providing low-coverage, whole-genome sequencing data from 179 people; the Exon Project for providing high-coverage sequencing data from 697 people, with sequencing regions limited to exonic regions of 906 randomly selected genes; the Trio Project for supporting whole-genome, high-coverage sequencing data from two families in different populations . In the  results . BecauseThe human genome sequences of GRCh37 from the Ensembl FTP site were also downloaded as references, and Ensembl gene annotations from BioMart interfaces were retrieved to verify genetic locations of STR motifs . In the To verify the proposed method using previously published polymorphic STR motifs, we applied the well-known STR markers from the combined DNA index system (CODIS). The CODIS is a criminal forensic DNA database constructed by the U.S. Federal Bureau of Investigation (FBI). There were 13 highly polymorphic STR markers listed in the CODIS . Each deAll STR markers collected in the CODIS system were linked with neither gene regulation networks nor genetic diseases. However, several STR variations have been verified as crucial factors in causing lethal diseases, and many of the identified STRs play important roles as regulatory elements that affect gene expression. Though there are no individual medical records available for the acquired Trio samples, these verified STRs were detectable and it could be used to determine whether polymorphisms of known disease-related STR motifs occur among different individuals in the Trio Project.Quantity and quality of homologous genes provide powerful evidence for analyzing evolutionary relationships between two queried species. Investigation of STR conservation across different species has facilitated the discovery of functional STR motifs. Hence, we simultaneously collected well-defined homologous genes belonging to human, cow, dog, zebrafish, stickleback, macaque, mouse, medaka, tetraodon, and fugu as one of our experimental datasets. Through sequence alignment analysis and annotations from Ensembl, a total of 225 genes exhibiting orthologous relationships were collected, and these genes were applied to the analysis of STR polymorphisms within 7 human genomes. In contrast with the homologous gene analysis, we also collected human-unique genes by comparing all possible homologous relationships between human and the closest chimpanzee genomes. A total of 492 human-unique genes were collected for performing polymorphism analysis in this study. Polymorphic characteristics of identified STRs from human-unique genes among 7 different human genomes were considered potential candidates for STR biomarkers. To ensure the uniqueness of the collected genes, we further verified five mammalian species including gorilla, chimpanzee, macaque, orangutan, and mouse.de novo assembly approaches. Either approach or a combination of the two methods could be applied, depending on the target species for referencing sequences. (2) After extracting the consensus sequences from assembled contigs, each individual sequence was bias corrected, and its corresponding upstream and downstream segments were extracted. (3) Traditional in silico STR detection was performed on both reference and target individual consensus sequences to generate individual STR profiles. (4) Each individual sequence was aligned to a selected reference sequence in order to recognize and calibrate all corresponding locations of STR candidates. (5) Once a unified STR template profile could be constructed according to all previous STR profiles generated from imported NGS datasets, all potential polymorphic STRs were identified by automatically comparing the defined STR template profile against each individual target STR profile. (6) At the final step, a checking procedure was performed for evaluating overlapped and/or mis-recognized cases during STR retrieval processes under various parameter settings. The system was designed to include these overlapped candidates according to defined genetic locations and adjust the settings of retrieving modules  in ordereference mapping approach can be applied to assemble the sequence reads. Short reads are aligned to known reference sequences, and differences between reference sequences and query reads are annotated. This approach is usually applied to sequencing model species and medically related studies. On the other hand, if no reliable reference genome is available for the target organism, a de novo assembly approach should be applied to the sequenced short reads. A de novo assembly algorithm reconstructs the original sequencing reads using read contents only, which usually requires more computational resources. Many tools are publicly available for both reference mapping and de novo assembly [The NGS datasets are usually composed of a large amount of short reads accompanied by information regarding sequence quality. The length of short reads were usually formed from tens to hundreds of base pairs depending on various NGS machines and protocols. Since the exact location of each read is unknown, assembly processes to reconstruct the correct gene sequences from these segmented short reads were essential steps prior to genomic analysis. There are two main types of reconstruction methods available under different circumstances. If the genome of the query organism has been sequenced and published previously, a assembly .de novo assembly tools. After extraction of a consensus sequence, a reference sequence was assigned as the central representation prior to subsequent mapping processes. For the assembled results obtained from reference mapping, the reference sequences could be automatically applied. However, the output results from de novo assembly, i.e., the reference sequences, were picked from the individual sequencing results. A standard quality indicator such as N50 or average contig lengths could be applied for the reference selection in general. In order to compare upstream and downstream regions of target genes, we additionally collected 7500 bp from either side of each gene.At the first stage of our proposed workflow, sequence reconstruction was completed in a manner that depended on the origin of a specific sequence. The intermediate output at this stage was consensus sequences in the standard FASTA format that were extracted from mapped results or obtained from the Although the gene sequences among the different individuals were highly similar to each other, coordinates of assembled sequences could not be directly applied across various sequencing datasets. This issue was mainly due to that random insertions or deletions caused by evolution, mutations, or reconstruction errors occurred during genomic sequence analysis. Hence, we employed NCBI BLAST+ programs to perform quick searches to further identify vague locations of the target samples . Our purin silico STRs [There are several different tools available for retrieving ico STRs . The ideico STRs . STR motFor the problem of varied gene lengths mentioned in the previous section, sequence locations might appear as deviations within an STR profile. This location bias may lead to failed results in template-building procedures; thus, all corresponding STR segments among different individuals should be identified through an appropriate approach. Each sequence was therefore calibrated in advance regarding their system coordinates comparing to the assigned reference sequence through a global pairwise alignment. In this study, we applied the EMBOSS stretcher program to perform global alignment between the reference sequence and each individual target sequence. The aligned results were then employed as the data resource for coordinate transformation . Each diSince an STR profile contained all retrieved STR motifs from an individual genome under an identical coordinate system, the STR polymorphism could then be observed by comparing with all the remaining STR profiles. To efficiently and effectively list all polymorphic STR candidates, a representative and comprehensive STR template was built by union operations from the reference profile and all other individual profiles. It should be noted that all STR patterns were compared under rotational tolerance because the basic STR pattern might be shifted as a result of point mutations or insertion/deletion polymorphisms. Once the template profile was constructed, polymorphic STRs could be identified easily by comparing all STR records within the accumulated template profile against each individual STR profile. Since all coordinates of STRs were aligned to the reference sequence, the known gene annotations from the reference gene could be applied to each individual STR motif for assigning appropriate genetic location information. After constructing a comprehensive and annotated STR profile for each individual, we only have to judge the existence and the repeat number of a specific STR pattern at a corresponding location, and therefore the system could respond to a query in real time and verify all polymorphic STRs.Due to mutations and gap noises that appear within a repeat DNA sequence, polymorphic STRs could be erroneously divided into several segments. This situation caused statistical errors during cross-sample comparison. In order to avoid such errors, the system provides a merging function for neighboring segmented patterns according to their patterns and overlapped conditions. The merging module could reunite disconnected STR segments into one motif according to previously defined coordinate information. Another potential problem is N/A nucleotides; these require adjusting one of the parameters in CGSSR to find shorter STR patterns that might not have been found in previous steps. Through this proposed mechanism, a comprehensive STR profile for each gene could be successfully constructed.http://isp.cs.ntou.edu.tw/.To design an integrated system for customized services, the system provides users the ability to upload their own gene sequences and discover all polymorphic STRs against the benchmark human genomes. Once a customized sequence is uploaded, the designed system automatically blasts the query gene sequence against these genomes to identify its corresponding gene. The query sequence is then scanned to detect all STR motifs, and their corresponding STR profiles will be created according to previously introduced modules. The online system is freely available at Additional file Additional file STR - Short Tandem RepeatpSTR - Polymorphic STRTLgene - Total length of geneTLssr - Total length of SSRTNgene - Total number of geneTNpssr - Total number of polymorphism SSRTNssr - Total number of SSRUTR - Untranslated RegionChr/Chrom - ChromosomeBLAST - Basic Local Alignment Search ToolMbp - Mega base pairsNCBI - National Center for Biotechnology InformationENBL - EnsemblCODIS - Combined DNA Index SystemNGS - Next Generation SequencingBAM - Binary Alignment MapSAM - Sequence Alignment MapThe authors declare that they have no competing interests.CMC, CPS and TWP conceived the algorithms. CMC, CPS, and YLL implemented the algorithms, performed the experiments. CMC and CPS wrote the manuscript. TWP, HTC, and CHH evaluated the systems, and proofread and revised the manuscript. All authors read and approved the final manuscript.Supplementary Document I: A total of 477 polymorphism STR patterns retrieved from 492 human-unique genes. Sorted by Avg. Dev. and Max. Dev.; Chrom is an abbreviation of chromosome; Enbl represents Ensembl; NA12878 represents CEU child, NA12891 for CEU father, and NA12892 for CEU mother; NA19238 represents YRI mother, NA19239 for YRI father, and NA19240 for YRI child. Region: 1=Intron, 2=Upstream, 3=Downstream, 4 = 5'UTR, 5=Coding, 6 = 3'UTR, 7=Undefined exon.Click here for fileSupplementary Document II: A total of 26 STRs selected from 477 polymorphic STRs based on specific conditions. All STRs were sorted by different levets of family (Type I), individual (Type II), and ethnic (Type III). Chrom is an abbreviation of chromosome; Enbl represents Ensembl; NA12878 represents the CEU child, NA12891 the CEU father, and NA12892 the CEU mother; NA19238 represents the YRI mother, NA19239 the YRI father, and NA19240 the YRI child. The symbol of "*" in the GID column represents genetic variation of single nucleotide polymorphism (SNP).Click here for file


Haemophilus influenzae is an important cause of human disease. Strains were selected for genome sequencing to represent the breadth of nontypeable strains within the species, as previously defined by the electrophoretic mobility of 16 metabolic enzymes.Nontypeable  Haemophilus influenzae (NTHi) colonizes the human nasopharynx and is an opportunistic pathogen causing significant human disease. NTHi causes both mucosal and invasive infections, including otitis media, exacerbations of chronic obstructive pulmonary disease, and bacteremia . Samples were indexed according to standard protocols so that they could be pooled together and sequenced simultaneously in a single run on the Illumina MiSeq using paired-end 150-bp  or paired-end 250-bp  chemistry. Prior to sequencing, all libraries were run individually on the Agilent high-sensitivity DNA chip to confirm library quality and average insert size. Samples were pooled in equimolar amounts, and 8 pM the pool was run on the sequencer. Per Illumina’s recommendation, a PhiX control was spiked into the library pool prior to loading for quality control purposes. For the 300-cycle runs, 5 to 10% PhiX was used, and for the 500-cycle run, 1% PhiX was used, as per the Illumina protocols. Thirty to forty million reads in total were collected for each run. Raw sequence data were aligned to a reference isolate using CLC Genomics Workbench (CLC bio) to identify single-nucleotide polymorphisms (SNPs), and regions with insertions or deletions (indels) or raw data files were assembled using CLC Genome sequences for the eight strains described here have been deposited in GenBank under the accession numbers listed in


Understanding the dynamics of stem cell differentiation processes at the molecular level is a central challenge in developmental biology and regenerative medicine. Although the dynamic behaviors of differentiation regulators have been partially characterized, the architecture regulating the underlying molecular systems remains unclear.System-level analysis of transcriptional data was performed to characterize the dynamics of molecular networks in neural differentiation of stem cells. Expression of a network module of genes tightly co-expressed in mouse embryonic stem (ES) cells fluctuated greatly among cell populations before differentiation, but became stable following neural differentiation. During the neural differentiation process, genes exhibiting both differential variance and differential correlation between undifferentiated and differentiating states were related to developmental functions such as body axis development, neuronal movement, and transcriptional regulation. Furthermore, these genes were genetically associated with neuronal differentiation, providing support for the idea they are not only differentiation markers but could also play important roles in neural differentiation. Comparisons with transcriptional data from human induced pluripotent stem (iPS) cells revealed that the system of genes dynamically regulated during neural differentiation is conserved between mouse and human.The results of this study provide a systematic analytical framework for identifying key genes involved in neural differentiation by detecting their dynamical behaviors, as well as a basis for understanding the dynamic molecular mechanisms underlying the processes of neural differentiation.The online version of this article (doi:10.1186/s12918-015-0231-6) contains supplementary material, which is available to authorized users. Cell differentiation is a complex process requiring precise dynamic regulation of cellular components. The spatio-temporal heterogeneity of ES cells and iPS cells makes it hard to determine the molecular mechanisms of cell differentiation and establish efficient differentiation protocols , 2. Fromp = 3.61e-50). This result indicates that genes specifically expressed in ES cells in the undifferentiated state were more variable than those expressed in differentiating cells, even though it is likely that multiple types of cells are present in the population during neural differentiation. On the other hand, the genes that were differentially expressed between day 0 and subsequent days were not enriched in the skyblue module  from mouse ES cells undergoing neural differentiation; each time point was measured in eight replicates E-TABM-1108) . Two ind108 . Twoule Fig.  as much differential variance and correlation) genes. We detected 671 such genes , but the DVC genes did significantly overlap with the set of genes  and Nanog, and began to express the primitive ectoderm marker Fgf5 on day 3  in neural and non-neural lineages , 32, 36.Inactivation of HES1 in ES cells promotes rapid and homogeneous differentiation into neural progenitors . CCNG2 wHowever, it remains challenging to identify dynamic changes in molecular networks. The approach used in this study might not detect all the early warning signals of upcoming transitions. For example, genes previously shown to exhibit oscillatory expression in stem cells, such as Ascl1 and Dll1, were not identified in this study. One possible reason for this is that we measured mean values of gene expression in cell populations, potentially resulting in underestimation of gene expression variance among individual cells. A single-cell analysis of gene-expression profiles with a large number of replicates would help us to observe the transcriptional distribution of each gene across individual cells. Characterizing the transcriptional distribution in this manner could provide more accurate estimation of a gene expression variance between cellular states.There are, of course, genetic and epigenetic differences between humans and mice. For example, the molecular machineries that maintain the stemness of ES and iPS cells are not completely identical , 41. In Our system-level analysis of the mouse ES and human iPS cell transcriptomes demonstrates the existence of a transitional state during neural differentiation that exhibits fluctuations in gene expression and transcriptional regulation. These dynamic transcriptional changes, identified by unbiased systematic detection of early warning signals for upcoming neural differentiation, were genetically related to developmental functions. Our analysis provides a systems biology framework that could be used to gain insights into the mechanisms underlying dynamic developmental processes in stem cells.2 transformed and subjected to quantile normalization. Probes corresponding to genes that were expressed  and exhibited variance (SD > 0) across replicates in each time point or differentiation stage were used in the analysis. Differential co-expression and variation analyses were performed based on 30,035 and 12,364 probes from mouse ES cell and human iPS cell data, respectively. The gene expression signals were standardized to the Z-score  for each gene across replicates of each time point or differentiation stage.Transcriptional data of neural differentiation in mouse ES cells were obtained from the ArrayExpress database (E-TABM-1108) . TranscrThe differential co-expression and variation analysis was conducted using the Bioconductor package in the R language . To idenpe 3.1.2 . Correlahttp://www.ingenuity.com) software. Genes associated with differentiation-related phenotypes in knockout and transgenic mice were identified based on the Mouse Genome Informatics database [phyper function in R.Functional enrichment and upstream regulator analysis was performed using Ingenuity Pathways Analysis (IPA®, Qiagen, database . Enrichmp-value < 0.05, as in the definition of DVC genes. To compare the mouse ES cell and human iPS cell data, mouse orthologs of the DVC genes from the human iPS cell study were identified based on information in the HUGO Comparison of Orthology Predictions database [Welch’s t-test was applied to transcriptional data to identify genes differentially expressed between day 0 and subsequent days during neural differentiation process. F-test was carried out to evaluate differential variances of genes between day 0 and subsequent days. P-values were adjusted by the Benjamini–Hochberg method . Differedatabase . OverlapThe data sets supporting the results of this article are available in the ArrayExpress database under accession no E-TABM-1108 and the Gene Expression Omnibus repository under accession no GSE25542.


RNA interference mediated by short interfering RNAs and microRNAs (miRNA), is a widespread anti-viral defense strategy. Importantly, viruses also encode their own miRNAs. In recent times miRNAs were identified as key players in host-virus interaction. Furthermore, viruses were shown to exploit the host miRNA networks to suite their own need. The complex cross-talk between host and viral miRNAs and their cellular and viral targets forms the environment for viral pathogenesis. Apart from protein-coding mRNAs, non-coding RNAs may also be targeted by host or viral miRNAs in virus infected cells, and viruses can exploit the host miRNA mediated gene regulatory network via the competing endogenous RNA effect. A recent report showed that viral U-rich non-coding RNAs called HSUR, expressed in primate virus herpesvirus saimiri (HVS) infected T cells, were able to bind to three host miRNAs, causing significant alteration in cellular level for one of the miRNAs. We have predicted protein coding and non protein-coding targets for viral and human miRNAs in virus infected cells. We identified viral miRNA targets within host non-coding RNA loci from AGO interacting regions in three different virus infected cells. Gene ontology (GO) and pathway enrichment analysis of the genes comprising the ceRNA networks in the virus infected cells revealed enrichment of key cellular signaling pathways related to cell fate decisions and gene transcription, like Notch and Wnt signaling pathways, as well as pathways related to viral entry, replication and virulence. We identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes; e.g., APOBEC family genes, in some virus infected cells. All these information are compiled in HumanViCe ( Caenorhabditis elegans  are small non coding RNAs (21–24 nucleotides) that play a major role in post-transcriptional gene regulation. Following their discovery in We collected human and viral mature miRNA sequence data from miRBase database . We considered transcripts with seed complementarity as well as one base mismatch tolerance (in position 2–8 or 2–7) in the seed region with 3′ compensatory complementarities. To reduce runtime involved in computation of target sites on huge set of transcripts (~25000 lncRNA+circRNA), we first searched for sites containing perfect 6-mer complementarity with miRNA seed from either miRNA 5′ end (2–7th base) or 3′ end (13–18th base). In case a match was found, then a 25 base window around the seed-matched site  was used for further alignment by the modified Smith-Waterman algorithm.For calculating duplex energy of the predicted miRNA-target duplex we used the Vienna RNA package lnE = Free energy of the target lncRNA .For partial validation and providing a measure of accuracy in our prediction of miRNA targets on host lncRNAs, we verified our miRNA target finding program on the dataset of Nam et al NCBI GEO accession no. GSE52530  out of a total of 2363 negative sets (FP+TN), which gave an FDR of 0.04. The low FDR was due to the stringency of the algorithm that required perfect seed matches (either from miRNA 5′ end or 3′ end) and a filtering for favorable miRNA-target duplex. Though the stringent conditions resulted in much false negative detection in the current dataset, we kept these conditions to ensure a low false positive rate that was particularly required for the present work.We used host (human) miRNA expression profiling data from EBV, HCMV, HIV1, and KSHV infected cells, collected from NCBI GEO database. miRNA expression profile from small RNA deep sequencing of EBV B95.8-infected LCLs was collected from GEO series accession no. GSE41437, high-throughput profiling of smRNAs, Ago1, and Ago2-associated miRNAs from HCMV-infected fibroblast cells was collected from GSE33584, miRNA expression profiles in the peripheral blood mononuclear cells (PBMCs) after HIV1 infection was collected from GSE44332, Ago HITS-CLIP in KSHV-infected PEL cell lines BCBL-1 and BC-3 was collected from GSE41357.p-value for each potential ceRNA pair by hypergeometric test considering the number of shared miRNAs between a ceRNA pair against the number of miRNAs targeting individual components of the ceRNA pair. The p-value was measured as:We implemented a measure to assess the likelihood of a ceRNA pair to regulate each other via shared miRNAs. This approach was similar to what has been used in the study of Sumazin et al.  and in Swhere,TM = Total number of miRNAs in the human genomepm = Number of miRNAs interacting with the first ceRNAnm = Total number of miRNAs interacting with the second ceRNAcm = Number of miRNAs shared between the ceRNA pair.The miRNA target finding algorithm was implemented in JAVA and the web interface of the database HumanViCe was developed using PHP-mySql. The collected dataset of viral miRNA-host mRNA interactions and host miRNA-host miRNA interactions along with our generated dataset of viral miRNA-host lncRNA and circRNA and host miRNA-host lncRNA interactions were stored in a mySQL database. The dataset of host miRNA expression profiles in virus infected cells were also stored in the database . For these four viruses, only the targets of host miRNAs expressed in the virus infected cells were displayed. Browsing of the database HumanViCe for viral miRNA targets or ceRNAs of a target transcript by users is enabled by mySql queries from PHP. Figure Presently HumanViCe contains targets for a total of 144 viral miRNAs encoded by 10 viruses known to infect human cells  for ceRNA networks corresponding to each virus, we found many common pathways to be enriched in all these networks. The most common enriched pathways include Axon Guidance , signaling by NGF , Hippo signaling pathway , MAPK signaling pathway , signaling by NOTCH , Proteoglycans in cancer , Wnt signaling pathway . The key cellular signaling pathways related to cell fate decisions and gene transcription, like Notch signaling and Wnt signaling pathways are frequently utilized or manipulated by viruses to suite their own need . These results suggest that host lncRNAs are likely to be targeted by viral miRNAs.We identified viral miRNA targets on host lncRNA loci from AGO PAR-CLIP dataset in EBV, HCMV, and KSHV infected human cells. Datasets of PAR-CLIP performed in EBV B95-8-infected LCLs was collected from NCBI GEO database under GEO accession id GSE41437. We identified 34 targets of 16 different EBV miRNAs on 29 different human lncRNA loci. Similarly, we found 1 HCMV miRNA target on 1 human lncRNA loci from AGO PAR-CLIP dataset from HCMV infected primary human fibroblast cells (GSE33584). From the PAR-CLIP dataset on two latently KSHV infected PEL cell lines, BCBL-1 and BC-3 (GSE41357), we identified 69 targets of 16 different KSHV miRNAs on the genomic loci of 55 different human lncRNA loci . The ceRNA network consisting of only host immune response related targets of host and viral miRNAs is shown in Figure Kaposi's sarcoma associated herpesvirus (KSHV) or Herrpesvirus 8 (HHV-8) is an oncovirus that causes Kaposi's sarcoma and PEL in human. We identified viral and host miRNA targets from PAR-CLIP datasets in KSHV infected PEL cells that may act as competing endogenous RNA (ceRNA). Computational analysis identified 762 protein-coding and 144 non-coding targets of 1717 distinct human miRNAs and 19 distinct KSHV miRNAs. We developed the whole predicted ceRNA network consisting of host mRNAs, lncRNAs and circRNAs targeted by human and viral miRNAs in KSHV infected cells Figure . We did http://gyanxet-beta.com/humanvice). This suggested an immensely complex regulatory circuit around host immune response associated genes in HIV1 infected PBMCs. As the presence of ceRNAs was known to have a diluting effect on the miRNA mediated regulation of a gene, the extensive predicted ceRNA network around the immune response related genes targeted by miRNAs in HIV1 infected PBMCs should have significant effects on fine tuning of their expressions in these cells. APOBEC family genes have been shown to inhibit HIV1 replication as part of the innate immune system. We looked into the miRNA target network around APOBEC family genes. Many of the miRNAs expressed in HIV1 infected PBMCs were predicted to target APOBEC family genes including APOBEC1, APOBEC2, APOBEC3G, and APOBEC4 . From a dataset of miRNA expression profiling in HIV1 infected PBMC GSE44332), out of 339 host miRNAs expressed in HIV1 infected PBMCs, we found 87 miRNAs potentially targeting 1370 human immune response associated gene transcripts out of total 1535 immune response associated gene transcripts listed in InnateDB  to act as ceRNA to the chosen transcript. The list of ceRNAs is sorted by the number of shared miRNAs. The miRNA targets for a particular lncRNA, mRNA, or circRNA can also be browsed in our database by choosing a particular lncRNA/mRNA/circRNA from the ceRNA list. The usage of HumanViCe is described in Figure Users can browse for viral miRNA targets on host protein-coding and non-coding genes by choosing a particular virus. The resulting page lists miRNAs encoded by the chosen virus along with its mRNA, lncRNA, and circRNA targets in host cells. Users can view details of the targets by choosing a particular viral miRNA. There is also provision for checking the host cellular miRNA targets on a particular viral miRNA target transcript. The users can check the potential ceRNAs for a chosen transcript. The resulting page shows the potential ceRNA candidates that share one or more common cellular or viral miRNA(s), sorted by their probability  play important roles on host-viral interaction. The complex cross-talk between host and viral miRNAs and their cellular and viral targets form the environment for viral pathogenesis. The current study is aimed at unraveling the cross-talk-network of the viral and host miRNA targets in virus infected cells in human.In recent years, it has been observed that other than protein-coding transcripts, cellular miRNAs can also target other non-coding RNA like lncRNA and circRNA. Cellular transcripts  sharing target sites for one or more common miRNAs compete with each other for the limited pool of cellular miRNAs and thus affect the competing RNA's level, a phenomenon known as ceRNA effect. This ceRNA effect is known to play significant roles in important biological processes including many disease pathogenesis as crucial new determinants of gene expression regulation. As viral miRNAs have already been reported to interact with host cellular factors, and viral miRNAs may have potential interaction with cellular non coding RNAs as well. Furthermore, it is highly likely that viral miRNAs may exploit the host gene regulatory network via the ceRNA effect. A virus exploiting the cellular miRNA mediated gene regulatory network via ceRNA effect has been already reported. In a previous study Cazalla and his group reported that viral U-rich non-coding RNAs called HSUR expressed in primate virus HVS infected T cells are able to bind to three host miRNAs resulting in a striking alteration of the cellular levels of one of these miRNAs, miRNA-27, which in turn may impair the regulation of the cellular targets of that miRNA in HVS infected cells. In our study, we identified viral miRNA targets on host lncRNA loci from AGO PAR-CLIP dataset in EBV, HCMV, and KSHV infected human cells. circRNAs are reported to contain extensive miRNA binding sites for effective miRNA sponge activity. From our study we identified some circRNAs containing large number of binding sites for some viral miRNAs; e.g., human circRNA transcript of ANKRD11 (circRNA id: hsa_circ_002048) was found to bear 36 sites for EBV encoded miRNA ebv-miR-BART20-5p. Our computational analysis identified many protein-coding and non-coding transcripts targeted by common viral or host miRNAs that points to a possible ceRNA effect in the virus infected cells. When we looked into the predicted ceRNA networks for all the 10 viruses used in current study, we found genes enriched for cellular signaling pathways commonly exploited by viruses. The most common enriched pathways for all the viruses include axon guidance, NOTCH signaling pathway, MAPK signaling pathway, Wnt signaling pathway. These pathways are related to cell differentiation state, gene transcription and intracellular signaling and prone to be manipulated by viruses after infection. Also the enrichment of pathways related to viral entry, replication and virulence was observed. The genes in the ceRNA networks of EBV, HCMV, HIV, HSV1, HSV2, and KSHV were found to be enriched for endocytosis. The genes in the ceRNA networks of EBV, HIV, HSV1, HSV2, and KSHV were found to be enriched for membrane trafficking. ceRNA networks of EBV, HCMV, HSV2, and MCV were found to be enriched for focal adhesion. The genes in the ceRNA networks of HCMV, HIV, HSV1, and HSV2 were enriched for platelet activation, signaling and aggregation.We especially looked into the possible ceRNA networks around human immune response associated genes in KSHV and HIV infected cells. Our analysis suggested that transcripts potentially targeted by miRNAs in both KSHV infected human PEL cells and HIV infected PBMCs were enriched for having a ceRNA effect on the host immune response associated genes. Furthermore, we identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes in these virus infected cells. Importantly, we identified seed matched targets sites of 87 miRNAs expressed in HIV infected PBMCs on 1370 host immune response associated genes out of the total 1535 genes listed in InnateDB. Furthermore, extensive predicted ceRNA network was identified around these host immune response associated genes, which suggested of a complex regulatory circuit around host immune response associated genes in HIV infected PBMCs. Working toward understanding the regulatory effect of the ceRNA network in HIV infected cells will be our future direction.We developed HumanViCe, a repository of the putative viral and host (human) miRNA interaction with cellular protein-coding RNA, lncRNA, and circRNAs in virus infected cells in human. Potential ceRNAs in different virus infected cells can be browsed in HumanViCe, where we have also calculated a ceRNA score (measured using the number of shared miRNAs between a pair) for each potential ceRNA pair to show the likelihood for them to act as ceRNAs. Put together, HumanViCe can be a very useful tool for researchers working on host-virus interactions to understand the dynamics of host and viral miRNA mediated regulations in virus infected cells.http://gyanxet-beta.com/humanviceHumanViCe is available freely from The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The deleterious metabolic effects of high fructose loads appear to be a consequence of altered transcriptional regulatory networks impacting intracellular macronutrient metabolism and altering signaling and inflammatory processes. Uric acid generated by fructose metabolism may also contribute to or exacerbate these effects. Here we review data from human and animal intervention and stable isotope tracer studies relevant to the role of dietary sugars on NAFLD development and progression, in the context of typical sugar consumption patterns and dietary recommendations worldwide. We conclude that the use of hypercaloric, supra-physiological doses in intervention trials has been a major confounding factor and whether or not dietary sugars, including fructose, at typically consumed population levels, effect hepatic lipogenesis and NAFLD pathogenesis in humans independently of excess energy remains unresolved.Dietary sugar consumption, in particular sugar-sweetened beverages and the monosaccharide fructose, has been linked to the incidence and severity of non-alcoholic fatty liver disease (NAFLD). Intervention studies in both animals and humans have shown large doses of fructose to be particularly lipogenic. While fructose does stimulate  Therefore the use of [13C] sugars does not measure % DNL but rather gives an assessment of any changes/differences in the DNL pathway. In order to assess the contribution of dietary sugars to DNL, sugars labelled with et al. glucose or fructose as 0.75 g/kg body weight to healthy participants. The sugars were traced into TG-fatty acids and TG-glycerol, and although significantly more TG-fatty acids were derived from fructose than glucose, in the short term only a very small proportion of TG-fatty acids were derived from fructose (<0.5%), compared with 38% TG-glycerol formed de novo. Four hours after ingestion, newly synthesized fatty acids from fructose made up less than one percent of circulating VLDL-TG. For those individuals who synthesized detectable amounts of fatty acids, palmitate was the major end point of fatty acid synthesis  during a 12 h period after an evening meal acetate to measure DNL, it was shown that DNL was higher when subjects ingested a meal containing 50% glucose and 50% fructose (16%) compared with 8% when the sugar was only glucose [n = 6 subjects). Stanhope et al. [et al. [vs. a control diet without the drinks, fasting DNL was significantly higher (9% vs. 2%) [In this section we report studies that have measured %DNL in VLDL-TG as a proxy for liver TG. In an acute setting, healthy individuals consumed 85 g sugar added to a meal. Using Cacetate e et al.  measured vs. 2%) .The percentage term for DNL may be adjusted for the fatty acid pool size, and when combined with flux data, can give a quantitative result such as mg palmitate in VLDL-TG that has arisen from DNL per day , but thiDespite a large body of work the health and metabolic effects of fructose and other dietary sugars are unresolved. Multiple mechanisms have been described for how large doses of fructose are lipogenic and have adverse metabolic effects. The data taken together suggest that although fructose does stimulate DNL to some extent, quantitatively the lipogenic effect of fructose is not mediated exclusively by its provision of excess substrates for DNL. The deleterious metabolic effects of supra-physiological fructose loads are a consequence of altered transcriptional regulatory networks impacting intracellular macronutrient metabolism, including fatty acid oxidation and esterification, along with altering signaling and inflammatory processes. Increased production of uric acid may also contribute to or exacerbate these effects. Pragmatically, in the context of epidemic levels of obesity, reducing dietary sugar consumption is a prudent public health message. However, whether or not dietary sugars, including fructose, at typically consumed population levels, effects hepatic lipogenesis and NAFLD pathogenesis in humans independently of excess energy remains an unresolved question.


Additionally, unprotected siRNAs are highly unstable once introduced into the circulation, with half-life of less than ten minutes, as they are quickly degraded by endogenous nucleases.RNA interference (RNAi) is a process that involves the delivery of small single stranded RNA molecules into mammalian cells, resulting in the sequence-specific cleavage of complementary mRNA and the silencing of specific gene expression. These small RNA molecules called small interfering RNAs (siRNAs) are the focus of intense research due to their potential therapeutic uses in various disease processes ranging from cancer to autoimmune and inflammatory conditions. However, critical barriers to the delivery of siRNAs Effective delivery of siRNAs to the desired cells/tissues requires specialized delivery strategies as naked siRNAs penetrate cell membrane poorly due to their negative charge. Different carrier systems have been developed to enhance siRNA delivery, including cationic lipid- and polymer-based nanoparticle formulations. However, potential toxicities have hindered the clinical translation of these platforms. Several cell-penetrating peptides (CPP) have also been used to enhance intracellular uptake of siRNAs. Excessive endosomal entrapment, however, remains the primary barrier to many CPP-mediated delivery systems.Melittin is a well-studied peptide with cell lytic potential through its lipid membrane binding and pore forming property. Our studies of the peptide structure-function relationships and peptide-lipid interactions led to modifications that yielded an amphipathic cationic peptide called “p5” that serves as a “peptide linker” capable of rapidly functionalizing lipid membranes with cargo such as targeting ligands, drugs, and imaging contrast agents but is now substantially less lytic  compared to native melittin -3. Furthin vivo delivery of siRNAs for the treatment of RA and potentially many other inflammatory conditions that require chronic and repeated injections.Rheumatoid arthritis (RA) is a chronic inflammatory condition affecting approximately 1% of the population worldwide. Despite recent advances in therapeutics, 30-40% of RA patients fail to respond to these new medications or are forced to cease treatment due to adverse effects. In our recent work , we exam


The hierarchical levels of genome architecture exert transcriptional control by tuning the accessibility and proximity of genes and regulatory elements. Here, we review current insights into the trans-acting factors that enable the genome to flexibly adopt different functionally relevant conformations. In its untangled, unfolded and completely linearized state, the human genome has a length of ~2 m. To fit it into a nucleus with a diameter of ~10 μm, DNA is wrapped around histone octamers, creating strings of nucleosomes that can be further organized into higher-order levels of compaction –5, whichTo exert stimulatory or repressive effects on transcription, accessible regulatory DNA elements must be in close spatial proximity to susceptible genes. Enhancers promote transcription by providing a binding platform for transcription factors  that canChromosomes are structured such that domains with a similar chromatin signature cluster spatially, a phenomenon first appreciated by traditional microscopy studies. Centromeres and flanking pericentromeric repeat regions of different chromosomes aggregate and form microscopically visible chromocenters in interphase nuclei . SimilarStudies using a derivative of chromosome conformation capture (3C) known as ‘Hi-C’ have revealed that long-range genomic contacts segregate TADs into an active (A) and inactive (B) compartment . Based oTo evaluate these issues, it is important to keep in mind how chromosome folding changes during cell division. Spatial genome organization is generally studied in non-synchronous cells, of which interphase cells make up the biggest proportion. In interphase nuclei, chromosomes are decondensed and organized hierarchically into the transcriptionally relevant structures described above. To prepare for cell division, chromosomes untangle and condense, while transcription ceases almost entirely. Mitotic chromosomes no longer show preferential higher-order contacts or compartmentalized TAD-based organization , and it As can be expected from stochastically reshuffled chromatin, inter-TAD and inter-chromosomal contacts showed high levels of variation between cells in a single-cell Hi-C experiment . MoreoveA chromatinized DNA fiber is assumed to behave essentially as a polymer, with a certain flexibility that allows random collisions between regions of the chromatin fiber. The likelihood for two sites to autonomously find each other in nuclear space decreases when their linear distance increases , 61. TheThe loops formed between TAD boundaries seem to exemplify the longest-range contacts that are stably and reproducibly formed between specific pairs of sequences. Although the mechanisms that underlie the looping of TAD boundaries are largely unknown, numerous reports have identified transcriptional repressor CTCF and the cohesin complex at the sites that anchor these loops , 18, 62.Cohesin is a protein complex that forms a large ring-like structure to hold the sister chromatids together after DNA replication. In recent years, cohesin has also been found to bind to chromatin in post-mitotic cells –74. CoheHox gene cluster allowed active chromatin marks to expand into a normally repressed domain [Depletion of CTCF through knockdown resulted in an increased inter-TAD contact frequency, whereas intra-TAD contact numbers were reduced . By contd domain . In summShh gene and its posterior limb-bud enhancer, which are located 1 Mb away from each other at opposite ends of a TAD, exist in such a pre-formed looped configuration that is stable throughout development and that is maintained even after deletion of the enhancer [Shh expression to ectopic sites elsewhere in the developing limb bud [Currently, two types of enhancer-promoter loops are distinguished: pre-established loops and loops formed de novo, or permissive and instructive conformations, respectively . Pre-forenhancer . Mutatiolimb bud , demonstlimb bud , 87. Thelimb bud . Bookmarlimb bud  in unrellimb bud .A recent study provided a first systematic insight into the genome-wide pervasiveness of each loop type across a series of cultured cell lines. While the majority of loops appeared conserved among most of the assessed cell lines and between species, hundreds of tissue-specific enhancer-promoter loops were also uncovered, which nearly always corresponded with strongly increased transcriptional output of the gene involved . FormatiFactors such as CTCF and cohesin seem to be major contributors to the formation and architecture of TADs, whereas other factors appear to be involved in the segregation of TADs into nuclear subcompartments containing similar types of chromatin domains. The rules that govern the relative positioning of TADs in the interphase nucleus must be considered in the context of the genome adopting an energetically favorable conformation upon unfolding after exit from mitosis. Because TADs are parts of much larger chromosomes, the engagement of stable inter-TAD contacts by one TAD imposes constraints on the sampling space of its neighboring TADs. Some regions, in particular the (peri-)centromeric parts of the chromosomes and the ribosomal DNA (rDNA) gene clusters, appear relatively dominant in determining their preferred genomic neighborhood because they find each other in nearly every cell. Others, which might include the olfactory receptor gene clusters and the PcG-bound regions, also retain some autonomy in choosing partners to contact in the nuclear space. Because of this, most of the remaining TADs (and genes) must passively adapt to the resulting configurations . In a prSwitches between A and B compartments occur for at least a third of the genome during early development, often in a lineage-restricted fashion . The obsIf not transcription, what is it that keeps active TADs together? Principles similar to those underlying local chromatin loop formation might well drive spatial juxtapositioning of TADs and set up tissue-invariant as well as tissue-specific higher-order topologies. Promoters marked by trimethylation on lysine 4 of histone 3 (H3K4me3) co-localize not only within TADs but also in the larger nuclear space in a largely tissue-invariant manner , 99, 100Loops, domains, and compartments define the shape of the genome, and all topological levels contribute to the functioning of the genome. Domain organization seems conserved and exhibits an invariance that is remarkable given the observation that TADs are not detected during mitosis . DespiteAt each level of structural organization, chromatin-associated factors shape the genome. CTCF and cohesin play important roles in chromatin looping—they anchor loops that create chromosomal domains (TADs) and loops that recruit enhancers to their target genes. CTCF might exert its action through self-dimerization or through recruiting cohesin. The observation that loops preferably form between convergent CTCF binding sites Fig.  might ha


Giant condylomata are not usually seen nowadays in developed nations, but such cases are still seen in the under-resourced countries. Condylomata acuminata are commonly transmitted through sexual intercourse. Generally diagnosed based on their appearance. Giant condyloma acuminata also named Buschke- Löwenstein tumour (BLT) is a slow growing cauliflower-like tumor, locally aggressive and destructive, with possible malignant transformation. Common clinical treatment of anogenital warts is conservative, however, in extreme cases conservative therapy is insufficient and surgical excision is required. A case of common presentation of giant condylomata in a 50 years old, divorced, multiparous woman is presented and the literature is reviewed. She presented with 15 years history of slowly progressive vulval lesion and associated itching, contact bleeding, malodorous vaginal discharge and difficulty in walking. She had previously been treated with podophyllin and cryosurgery without success. The growth measured 30×10 cm in each side and was successfully excised with no evidence of malignancy concomitant and reconstruction also done.  The immune system effectively repels the majority of HPV infections and is associated with marked localized cell mediated immune responses. However, approximately 10% of individuals develop a persistent infection, with risk of developing benign proliferative lesions, high-grade precursors and eventually invasive carcinomas.9 HPVs are classified into high- or low-risk types depending on oncogenic potential. Low-risk types 6 and 11 are isolated in approximately 90% of genital wart cases.3 The most common clinical treatment is conservative, with local chemical or physical destruction and immunological therapy.10 In more extreme cases conservative therapy is insufficient and surgical excision is required.Anogenital warts  are a common sexually transmitted disease among females and males.11,12 A complex histological pattern may exist with areas of benign condyloma intermixed with foci of atypical epithelial cells or well differentiated squamous cell carcinoma. GCA is mainly localized to the genital region, however, in rare cases the tumor is localized to distinct histological zones of the anorectal region. Due to infiltration of the underlying tissue, fistulae and abscesses may be observed. GCA is resistant to chemotherapy or radiotherapy and usually requires local radical resection for curative treatment. We report a case of 50-year-old lady with GCA of vulva who was successfully treated by simple vulvectomy and reconstruction of the defect by ‘‘two-advanment flap technique’’.Giant condyloma acuminata  is an extremely rare clinical form of genital warts, characterized by aggressive down growth into underlying dermal structures.A 50-year-old female presented with cauliflower-like growth over the uvula region. The growth had been diagnosed previously as condyloma acuminatum which was resistant to conservative therapy . The patOn examination, there was a huge friable, foul smelling growth on her vulva involving both labia majora, extending anteriorly to mons pubis and posteriorly to perianal region . There wA simple vulvectomy, achieving wide margins, was performed. Small lesions in perianal area and labia minora were also excised. Reconstruction of vulva was performed using ‘two flap advancement technique’. In this technique, two random pattern flaps in the dimension of 1:1 or 1:2 were raised adjacent to the defect and advanced medially to resurface the defect. This resulted in defect being transferred laterally, but this defect was smaller than the original defect and could be closed primarily  and 3.Final histopathology report confirmed the diagnosis of condyloma acuminatum with mild degree of dysplasia . Functio10 In rare cases, anogenital warts develop into extremely large tumor masses leading to deterioration of patient quality of life. An identified underlying histopathology of specific cases of giant condyloma is superficial planocellular carcinoma. Anogenital warts are the most common outcome of HPV genital infection. Therapeutics against this sexually transmitted disease are currently associated with low efficacy, due to a 30-70% recurrence rate identified six months following therapy administration.Patients with high susceptibility to local development and fast progression  and the highest rate of recurrence often exhibit various types of immunodeficiency. In addition, immunodeficiency leads to difficulties in evaluation of optimal therapeutic management. However, patients with no marked immunodeficiency and treatment-resistant genital warts have been identified. Furthermore, condyloma lesions occasionally form large exophytic masses, interfering with intercourse, normal urination, defecation or vaginal delivery.13 The method selected for reconstruction is crucial, particularly in neglected cases similar to the present case study. Local tissue availability and the patient’s condition and attitude towards the development of the disease are major factors for reconstruction with local fascio-cutaneous flaps. Five years following surgery, the present patient is disease-free with no recurrence.Commonly, GCA develops as cauliflower-like masses and the tumors exhibit histological features of pseudo-epitheliomatous proliferation and local invasion. In the absence of metastases, they are termed Buschke-Löwenstein tumors. Due to the aggressive local development of these masses, they belong to the verrucous carcinoma group, although a malignant histological alteration in the form of micro-invasive carcinoma or well-differentiated epidermoid keratinizing carcinoma has been reported. Due to the high frequency of local recurrence, radical surgical excision is the current treatment of choice as topical preparations and chemotherapy are generally considered ineffective.1,3 Other modalities include combined radiotherapy and chemotherapy, topical agents  and intralesional injections of interferons.4-7Many treatment strategies have been documented in the literature for management of GCA, but mainly in the form of case reports. Complete surgical excision with histologically clear margins, with or without adjuvants, is the mainstay of the treatment for GCA, including that of vulva.1,8-10 Flaps have been shown to give better results for reconstruction of vulva.9,8 To conclude, surgical treatment using wide local excision of the lesion with clear margins and reconstruction of the resultant skin defects by two transpose flap technique gives excellent cosmetic and functional results.The skin defects created after excision of GCA in the peri-anal region can be managed with mesh skin grafting, flaps and even healing by secondary intention.The authors declare no conflict of interest.


The treatment of the rare enteric nervous system (ENS) manifestations of paraneoplastic syndromes, which are most frequently associated with small cell lung cancer (SCLC), is poorly understood and described. Patients with neuroendocrine-derived tumors can develop B-cell reactivity towards the tumor with cross-reactivity for neurons located in the submucosal and myenteric ganglia of the ENS. The ensuing autoimmune neuritis causes aperistalsis and severe gastrointestinal (GI) dysfunction. Immune-directed therapy is not the standard of care but may be paramount for patient recovery. Our patient, a 63-year-old man with recent symptoms of esophageal dysmotility and newly diagnosed SCLC was hospitalized with nausea, emesis, and constipation. After an extensive work-up that included laparoscopy and celiotomy with bowel resection, we diagnosed what we refer to as Autoimmune Paraneoplastic Chronic Intestinal Pseudoobstruction (AP-CIPO). Unlike the few clinically similar reports, SCLC and AP-CIPO were diagnosed in our patient within weeks of each other, which presented the dilemma of treating the two processes simultaneously. In this report, we review the relevant literature and describe our patient's course. We believe standard chemotherapy is not effective treatment for AP-CIPO. Based on evidence discussed herein, we suggest initiating autoimmune-directed therapy before or simultaneous with cancer-directed therapy. Impaired intestinal motility, chronic intestinal pseudoobstruction (CIPO), paraneoplastic intestinal pseudoobstruction, and similar names are found in the few case reports that refer to a rare clinical condition associated with certain cancers that result in severe GI dysmotility. This disease is most commonly associated with small cell lung cancer (SCLC) . EvidencA 63-year-old male with COPD and a long smoking history presented with new symptoms of left sided facial pain and increasing shortness of breath. Biopsy of a left supraclavicular lymph node showed metastatic SCLC (immunohistochemistry positive for thyroid transcription factor-1 (TTF-1), synaptophysin, and chromogranin). His PET-CT showed a large fludeoxyglucose-avid lesion in the left upper lobe and FDG-avid left supraclavicular lymph nodes; outpatient chemotherapy was arranged. He was readmitted to the hospital two weeks later with a two-day history of diffuse abdominal pain, nausea, and bilious emesis. A computed tomography (CT) scan showed distended loops of small bowel without a clear transition point. No hernias were noted and he had no prior abdominal surgery. He was treated conservatively for partial small bowel obstruction versus ileus with a nasogastric tube (NGT). After continued high NGT output and lack of bowel movement for a week, he was taken to the operating room for a diagnostic laparoscopy. The large intestine appeared grossly normal; 20–30 cm of ileum proximal to the ileocecal (IC) valve was peristalsing but remained full of digestive material. Proximal to this, there was mildly dilated small bowel that was aperistaltic. At this time, our differential was ileus versus obstruction at the IC valve. The procedure was terminated without further intervention because there was no clear indication for bowel resection or bypass. Erythromycin was added to his regimen of senna and bisacodyl suppository for threedays without effect. A GI series showed no progression of contrast through the intestines for one week after the initial operation. The patient was then taken back to the operating room for an ileocecectomy with resection of 5–10 cm of bowel proximally and distally with primary anastamosis. All of the inspissated fecal material was either included in the specimen or milked out of the remaining bowel. Initial tissue microscopy showed no pathology.Postoperatively, the patient was started on TPN. He remained stable but had no signs of bowel function recovery despite treatment with methylnaltrexone, erythromycin, metoclopramide, senna, enemas, and bisacodyl suppositories. The patient developed paroxysmal atrial flutter and atrial fibrillation and his blood pressure and heart rate were markedly labile. Initially, intravenous metoprolol and later oral scheduled metoprolol controlled his heart rate. After his atypical course, we requested reexamination of the proximal portion of resected bowel . This showed lymphoplasmacytic infiltrate consistent with myenteric ganglioneuritis Figures  and 2. TOur patient never regained bowel function with chemotherapy over the following month. Abdominal X-ray showed little to no progression of contrast through his intestines . He acutParaneoplastic syndromes can affect any organ system in the body. Neurological manifestations are the least common and are almost always associated with SCLC . GI maniThe diagnosis of AP-CIPO is typically delayed and patients often undergo extensive evaluations and multiple noncurative bowel resections –9 as theThe treatment of AP-CIPO is poorly defined. We found four case reports in the literature describing different treatment regimens. One manuscript reported success with octreotide treatment . AnotherIn our patient, the decision was made to start chemotherapy without further delay and without directed immunosuppressive therapy. Our patient had no GI improvement with octreotide or through effects of cisplatin and etoposide directly on the immune system or indirectly through tumor suppression. It is conceivable that the added stress of being malnourished without GI function contributed to our patient's death and initiating immune-targeted therapy could have improved our patient's outcome.Bowel resection is another key issue in patients with AP-CIPO. All of the reported cases have described bowel resection during their diagnosis/treatment course. As with our patient, bowel resection was a response to failed conservative management of presumed partial small bowel obstruction. Bowel resection likely has no initial role in the initial treatment of AP-CIPO. However, after autoimmune-directed medical therapy, it is possible that some sections of bowel may not recover in every patient, and, guided by nerve function testing or direct visualization, bowel resection may be beneficial.Autoimmune Paraneoplastic Chronic Intestinal Pseudoobstruction (AP-CIPO) is a very rare disease seen in patients with nerve cell-derived tumors, most commonly SCLC. Although recognizing the disease is difficult due to its rarity, diagnosis can be made based on clinical findings (symptoms of bowel obstruction and autonomic nervous system dysfunction), intestinal pathology, and autoantibody testing. Based on disease mechanisms, our experience, and the combined experience gained from the few case reports available, we would promote treating AP-CIPO with autoimmune-directed therapy using rituximab.


The Page kidney is a rare phenomenon that refers to hypertension resulting from any external compression of a kidney by a hematoma, tumor, lymphocele, or urinoma. The activation of the renin-angiotensin-aldosterone system is believed to be the main mechanism responsible for development of resistant hypertension in Page kidney.We reported a patient with chronic pancreatitis who presented with hypotension due to splenic artery aneurysmal bleed; following the resuscitation, accelerated hypertension secondary to Page kidney caused by perinephric hematoma presented. Early diagnosis by contrast-enhanced computed tomography of the abdomen and renal angiogram was followed by therapeutic angioembolization. However, ultrasound guided aspiration was not done because of denial by the patient for further treatment. Follow-up showed normalization of blood pressure and resolution of hematoma on subsequent abdomen ultrasound evaluation.Splenic artery aneurysm is a very uncommon cause of Page kidney and to our knowledge, it was the first case of its kind ever reported in the literature. The Page kidney is a rare condition that occurs due to any external compression of kidney by a hematoma, lymphocele, tumor, or urinoma leading to hypertension (HTN). This high-renin state HTN occurs due to activation of the renin-angiotensin-aldosterone system (RAAS) induced by renal hypoperfusion and ischemia . Most co+, 2.6-3.4 mmol/L) while serum magnesium was normal . Ultrasonography of the whole abdomen showed left perinephric hematoma with normal-sized left kidney (11 cm); contralateral kidney was also normal. Subsequently, patient underwent contrast-enhanced computed tomography (CECT) of the abdomen with computed tomography angiography which showed large perinephric hematoma measuring 16.8 × 14.7 cm, secondary to splenic artery aneurysmal bleeding , with normal findings on renal function tests . Serum electrolyte assessment showed persistent hypokalemia  or aldosterone receptor blocker (ARB) with the aim of normalizing high blood pressure. The inability of a single agent to control the blood pressure necessitates the combination of multiple drugs such as beta blockers and calcium channel blockers . EarlierTo summarize, Page kidney is a rare cause of HTN secondary to RAAS activation. Although splenic artery aneurysm due to chronic pancreatitis is a very uncommon cause of Page kidney, early evaluation by CECT of abdomen and renal angiogram are diagnostic and less invasive procedures such as angioembolization and percutaneous drainage promise favorable outcome.