The extent of damage in New York City following Hurricane Sandy in October 2012 was unprecedented. Bellevue Hospital Center (BHC), a tertiary public hospital, was evacuated and temporarily closed as a result of hurricane-related damages. BHC’s large primary care office-based buprenorphine clinic was relocated to an affiliate public hospital for three weeks. The extent of environmental damage and ensuing service disruption effects on rates of illicit drug, tobacco, and alcohol misuse, buprenorphine medication supply disruptions, or direct resource losses among office-based buprenorphine patients is to date unknown.A quantitative and qualitative semi-structured survey was administered to patients in BHC’s primary care buprenorphine program starting one month after the hurricane. Survey domains included: housing and employment disruptions; social and economic support; treatment outcomes (buprenorphine adherence and ability to get care), and tobacco, alcohol, and drug use. Open-ended questions probed general patient experiences related to the storm, coping strategies, and associated disruptions.There were 132 patients enrolled in the clinic at the time of the storm; of those, 91 patients were recruited to the survey, and 89 completed (98% of those invited). Illicit opioid misuse was rare, with 7 respondents reporting increased heroin or illicit prescription opioid use following Sandy. Roughly half of respondents reported disruption of their buprenorphine-naloxone medication supply post-event, and self-lowering of daily doses to prolong supply was common. Additional buprenorphine was obtained through unscheduled telephone or written refills from relocated Bellevue providers, informally from friends and family, and, more rarely, from drug dealers.The findings highlight the relative adaptability of public sector office-based buprenorphine treatment during and after a significant natural disaster. Only minimal increases in self-reported substance use were reported despite many disruptions to regular buprenorphine supplies and previous daily doses. Informal supplies of substitute buprenorphine from family and friends was common. Remote telephone refill support and a temporary back-up location that provided written prescription refills and medication dispensing for uninsured patients enabled some patients to maintain an adequate medication supply. Such adaptive strategies to ensure medication maintenance continuity pre/post natural disasters likely minimize poor treatment outcomes. Hurricane Sandy struck the Northeastern United States on Monday, October 29, 2012. It was the largest and second costliest hurricane in United States’ history that resulted in 117 deaths and approximately $50 to 65 billion in damages [The disaster led to the suspension of office-based operations of BHC’s primary care buprenorphine practice, along with a variety of citywide hospital, outpatient clinic, and pharmacy closures. Since 2006, BHC has operated a large primary care office-based buprenorphine practice serving predominantly Medicaid and uninsured patients ,4. Care On the second day after the Hurricane , while the inpatient units of the hospital were being evacuated, Bellevue ambulatory care staff downloaded patient schedule information for targeted groups of ‘high-risk’ patients. These patients included those on blood thinners (e.g. Coumadin) and those on chronic opioid maintenance (e.g. the buprenorphine clinic). Using this list of patient phone numbers, the buprenorphine program director attempted to make contact with patients in order of appointments scheduled, starting with those who had already had appointments cancelled that week. Since entrance to the hospital was barred for non-staff, arrangements were made to mail prescriptions for those who had Medicaid or other insurance; for others, there were initially a few ad-hoc patient-doctor meetings immediately outside the hospital entrance to provide prescriptions and directions to other public hospitals. Within the first week after the hurricane, arrangements were made to house a Bellevue-staffed outpatient walk-in clinic for Bellevue patients at an alternate public hospital location; BHC buprenorphine patients who needed to be seen were scheduled for appointments at this location on days/times that a buprenorphine-certified physician was present. Outside of these phone calls from the clinic director, some patients contacted the buprenorphine clinic physicians via phone numbers ‘saved’ from previous between-visit communications or via online searches for email addresses. BHC’s outpatient facilities, including the buprenorphine program, were reopened three weeks after the hurricane . Inpatient hospital services did not reopen until mid-February 2013.As providers and health services researchers, we were concerned with direct and indirect effects of the hurricane itself (i.e. flooding) and transit, employment, and health system disruptions on our patients and their opioid treatment outcomes. Previous research indicates that the relationship between disaster exposure and substance use patterns is complex. Psychiatric co-morbidities, cultural factors, and disaster-related exposures have been associated with diverse post-event substance use patterns. For instance, while post-traumatic stress disorder and resource loss have been associated with increased substance misuse post-disaster, cultural and social factors were suggested as potential deterrents to alcohol consumption following the 1995 Hanshin earthquake in Japan ,11.th attacks had higher rates of relapse post-event compared to the period preceding the attacks [Studies on post-disaster substance use among patients enrolled in addiction treatment programs have been limited. Participants in New York City completing alcohol detoxification prior to the September 11 attacks . Among o attacks . While r attacks , substanThe intention of this exploratory study was to determine self-reported illicit opioid use (other than illicitly-obtained buprenorphine); self-reported tobacco, alcohol, and drug misuse; coping strategies following buprenorphine supply disruption, and resource loss among opioid-dependent patients enrolled in BHC’s office-based buprenorphine clinic immediately following Hurricane Sandy.th, 2013. The New York University School of Medicine Institutional Review Board approved the study protocol.The population of interest consisted of opioid-dependent patients enrolled in the BHC primary care office-based buprenorphine program at the time of the event (N = 132). Eligible participants were adult opioid dependent patients with an active buprenorphine prescription from a BHC primary care provider on October 30, 2012. Consecutive sampling occurred between November 27th, 2012, and February 4A 52-item administered survey instrument was developed and refined by the research team following a review of the disaster, addiction, and post-traumatic stress literature. The survey assessed self-reported pre/post estimates of drug use, treatment adherence, health behaviors, and environmental effects of the hurricane using discreet and open-ended items. Baseline demographic characteristics  anchored items querying changes in housing, employment, income and social and government support post-event. Alcohol and drug  use items were adapted from the Addiction Severity Index . ParticiPatients were approached in the clinic waiting area or contacted by phone by the research team to participate in the study. They were informed that all responses would be kept confidential and that participation would have no impact on their regular medical care. Surveys were administered by the research team by phone or in person in a private clinic room; two of the interviewers were also buprenorphine clinic physicians, who attempted to minimize coercion and response bias by explaining that participation in the study and survey responses would not be shared with other buprenorphine providers or affect their clinical care in any manner. Interviews lasted approximately 15 minutes and were conducted in a private clinic space or by phone. Patients not surveyed during a clinic session were contacted by telephone. Five phone attempts were made to contact each patient as necessary. Interviews were recorded in writing and de-identified survey responses were entered into an electronic database. Analysis was descriptive . Open-ended responses were coded using thematic analysis for emergent themes.Of the 132 eligible patients enrolled in the clinic at the time of Sandy, a total of 91 patients were invited to participate in the study. No patients refused to participate; however, 2 of 91 participants refused to complete the entire survey. Most participants were surveyed within 2 months of the hurricane (71%), while the remaining 29% were surveyed 3–4 months post-event. The 41 patients not interviewed appeared to be a combination of: a) not in clinic and not reachable by phone (drop outs), or, b) active in clinic post-event but missed by the interviewers and not reachable by phone . The study sample was predominately male (82%), White non-Hispanic (42%). All were active buprenorphine maintenance patients with a mean time in treatment of 28 months and an average buprenorphine dose of 20 mg/day (Table Few self-reported increased or new-onset heroin (7%) or prescription drug use (1%) following the hurricane; only four participants reported illicit opioid misuse in the three months preceding the hurricane (4%). Two of the six heroin users and the one prescription opioid user reported illicit opioid misuse after running out of buprenorphine. Two participants reported using heroin regularly with no change pre/post; one participant reported decreased heroin use post-event. When asked about cost, purity, and access to heroin, two participants reported increased police presence surrounding street-level drug dealing, increased cost, and decreased purity following Sandy.Shortages in buprenorphine medication supplies were reported by 38 (43%) participants; however written or telephone refills were eventually obtained from either the clinic providers or an unaffiliated buprenorphine provider (71%), friends 24%), and/or drug dealers (8%) , which were well-known barriers to routine refills prior to the event. Respondents unable to reach clinic buprenorphine providers were unable to obtain guest doses of buprenorphine from emergency rooms (3%) or pharmacies (3%). Some respondents (12%) reported significant crowding and long wait-times at the alternate temporary clinic location, which was providing care in a facility not usually used for clinical care.Contact with BHC buprenorphine providers on an unscheduled basis or surrounding a previously scheduled appointment appeared to help patients cope with hurricane-associated difficulties. Nearly half of the respondents were called by a BHC primary care clinic buprenorphine provider or BHC employee following Sandy (49%). Most respondents had attempted calling BHC following the hurricane (64%), however only 19 (33%) participants reported having their needs addressed through the general information/operator number.Respondents were also asked about suggestions to improve clinical services in the event of a disaster or unexpected disruption in services. The most common suggestion was to improve physician-patient communication (36%). Specific suggestions included: providing disaster hotlines, access to buprenorphine providers’ mobile phone numbers and emails, and a frequently updated website with disaster-related information. Other suggestions included improving access to buprenorphine refills (12%) and providing additional supplies of buprenorphine prior to the hurricane (2%).Changes in self-reported tobacco, alcohol, and other drug use during the three months prior to Hurricane Sandy compared to the one-month period following Sandy are displayed in Figure Hurricane-related adverse material consequences were reported by 38 (43%) participants. These problems included financial difficulties (27%), prolonged power outages (24%), and loss of housing (9%). Of the 38 respondents reporting hurricane-related adverse effects, assistance was provided by family (34%), friends (26%), relief organizations including FEMA and Red Cross (11%), and peers from 12-step groups (3%). Six (16%) participants reported no receipt of any disaster relief. Neighbors and religious organizations were specifically not reported as sources of help by any participants. Respondents that received assistance were asked to elaborate on the type of help they received, and most commonly reported receiving temporary housing (9%), financial support (8%), food (5%), medical assistance (1%), household supplies (1%), and “other” (3%). Of the participants reporting receiving financial support, help was provided by FEMA (29%), personal savings (29%), illicit income (21%), family and friends (14%), employers (7%). Two participants refused to disclose their sources of financial support. Only two of the 48 respondents reported being paid by employers for lost wages (4%). Most participants resided in their primary residence during the night of the hurricane (78%). Four respondents were not in New York City at the time of the hurricane. Of the eight respondents requiring temporary housing due to storm damage or flooding, one participant reported having to leave one ‘three-quarter’ house for another. There was open drug use at the alternate housing location, to which he attributed his post-event heroin use.This cross-sectional survey among NYC public sector primary care office-based buprenorphine patients following Hurricane Sandy indicated low rates of new-onset or increased illicit opioid use, and high rates of buprenorphine treatment adherence despite the temporary disruption of regular follow-up visits due to hospital closure. New onset or increased heroin and prescription opioid misuse was rarely reported, despite many patients experiencing buprenorphine medication supply disruptions and lowered daily buprenorphine doses. These findings suggest office-based buprenorphine, in which patients are typically seen for office visits and medication refills weekly-to-monthly while maintaining a medication supply at home, is relatively adaptable to service disruptions following a natural disaster.th attacks [The minimal increase in new onset or increased illicit opioid use among surveyed participants is in line with Weiss’s findings that only one in nine respondents enrolled in a methadone treatment program reported heroin use after the September 11 attacks . A home Our findings revealed that close to half of patients experienced shortages in buprenorphine supply following the hurricane. Most patients reported being able to relatively quickly obtain refills from their usual BHC buprenorphine providers, most often by phone followed by mailed written prescriptions to area pharmacies. Temporarily relocating to an affiliate public hospital provided a physical back-up to phone-based refills and provided medication dispensing to uninsured patients. The communication link between patients and providers was an important factor in facilitating call-in refills to local pharmacies , obtaining prior authorizations, and arranging eventual follow-up visits. As shown in Table Downward adjustments to daily buprenorphine doses and obtaining additional supplies from friends or family was common following Sandy. A quarter of the participants in this study reporting supply disruption were able to obtain extra doses of buprenorphine from friends or family also receiving buprenorphine treatment. An additional third of surveyed patients reported reducing (“stretching”) their daily doses of buprenorphine. While some were forced to do so as a result of reduced supplies of buprenorphine, others proactively rationed their daily use in case of unexpected barriers to accessing refills. Simply defining such behaviors as “diversion” or “treatment non-adherence” fails to acknowledge patients’ earnest attempts to avoid illicit opioid misuse. A home supply of buprenorphine may offer a more robust source of support for patients, as well as their peers, that encounter disasters and disruptions. Policies permitting additional doses of buprenorphine prior to an anticipated disaster event or service disruption may prevent adverse events such as relapse.When asked how the clinic could improve services in the event of a future disaster, the most common response was to improve communication between patients and buprenorphine providers. This feedback was echoed throughout the survey. Patients that had a buprenorphine provider’s cell phone number highlighted how helpful this was throughout the post-disaster period. This was important for the treatment team to acknowledge, as it highlighted the critical role direct contact with primary care based buprenorphine providers had in the immediate disaster mitigation and more extended disaster recovery phases, as opposed to general hospital contact numbers. When asked about coping strategies in the post-disaster period, the starting point for patients requiring refills was not a local emergency room or disaster relief medical station, but their primary care buprenorphine provider.Prior disaster studies emphasized the importance of improving the continuity of care for those burdened with chronic medical and psychiatric conditions who are at risk of decompensating due to disruptions in transportation and reduced access to healthcare services -21. The Limitations to this analysis of self-reported survey data include response and recall biases. In general, our clinic has approximately a 13% baseline rate of inaccurate opioid misuse self-report compared to urine toxicology results (i.e. 13% of visits involve divergent self-report and urine toxicology results) and avoidance of opioid misuse and buprenorphine diversion are overall goals of treatment [Further investigation is needed into developing primary care based disaster preparedness and mitigation efforts for patients enrolled in a buprenorphine primary care program, particularly strategies that will strengthen physician-patient communication following service disruption. Additional studies are also needed to determine outcomes among opioid dependent patients enrolled in MTP, private practice buprenorphine treatment, and medication-free treatment. Our findings highlight the relative adaptability of a prescribed home supply of buprenorphine for opioid dependent patients encountering a disaster and temporary clinic closure. We found only minimal increases in self-reported substance use. Offering patients with a back-up location to provide refills, medical, and psychiatric treatment ensured minimal disruption to continuity of care. Ensuring a working phone hotline and immediate post-disaster telephone communication with patients may assist in providing an information clearinghouse and referral system for those requiring the most immediate assistance.Drs. Rotrosen and Lee are lead investigators for a multi-site NIDA-funded study that is receiving free study drug (Suboxone film) from Reckitt-Benckiser.BT, EG, JR, JDL made substantial contributions to conception and design. EG, BT, RB, ARW have made substantial contributions acquisition of data, BT, JDL, and EG contributed to the analysis and interpretation of data; BT, ARW, RB, JR, EG, JDL were involved in drafting the manuscript. All authors read and approved the final manuscript.


Firmicutes, Bacteroidetes and Actinobacteria representing around The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration.Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to  The human gastrointestinal tract (GIT) hosts one of the most complex microbial communities, which has come under intensive research in recent years by applying culture-dependent and -independent methods Firmicutes and Bacteroidetes phyla HGM is notoriously dominated by The study of natural microbial communities, particularly those associated to humans, is a hard task with major culture-related difficulties Firmicutes- and Bacteroidetes-related phylogroups which represent about Firmicutes or Bacteroidetes. The rare biosphere is actually a very discussed topic addressing questions about their maintenance and contribution to the whole community Fecal samples are generally studied using direct approaches like 16S rDNA clone libraries or metagenomics Based on membrane integrity, FCM-CS has been applied to quantify viable, injured and dead bacteria from fecal samples In the present work, we marked active microbial cells by means of a technique based on the presence of RNA using pyronin-Y, a fluorescent stain for total RNA. Thus, we have applied FCM-CS to sort active microbial fraction from fecal samples of four healthy volunteers. All fractions related to active and total bacteria have been characterized by 16S rDNA gene amplification and next generation sequencing (454 pyrosequencing), exploring the HGM diversity. The taxonomical distributions of ORB and URB were studied, with results indicating that in all samples active fraction differed remarkably from that obtained from raw DNA fecal samples. Several phylogroups appeared or disappeared on applying cell sorting. One of the most striking results concerns the high number of URB-related groups appearing only in the active fractions. Finally, the results suggest that the active population differs significantly from what is found by looking at the total GIT microbial population which is, up-to-date, the most common approach to gut microbiota descriptions.We performed a permutational multivariate analysis of variance using distance matrix (see Method section) in order to test the null hypothesis that variance between grouping factors was randomly distributed. The analysis confirmed that samples are significantly different between them as well as are active versus non-active fractions see . MoreoveBacteroidetes are those most represented in the total fraction, contrary to what is observed for Firmicutes, Actinobacteria and Proteobacteria. A more detailed view of the distribution of families is shown in Firmicutes  while up to 73 were identified from the three active fractions (HC plus LC plus PA). These results indicate that most of the families are visible only when cell sorting based on RNA content is employed. Using the inflection point identification method  at Ruminococcaceae, Clostridiaceae and Lachnospiraceae were the most commonly retrieved families from almost all samples  Clostridium-related reads have been found but were not common to all samples. Lachnospiraceae (from ORB) were significantly the most abundant in active fractions. For instance, reads related to Coprococcus members  were retrieved prevalently in all active fractions from all samples; Coprococcus are gram-positive, anaerobic cocci active carbohydrates fermenters and producers of butyric and acetic acids with formic or propionic and/or lactic acids Lachnospiraceae were Hespellia porcina and H. stercorisuis, both able to grow on glucose, maltose, mannose, sucrose, fructose and xylose as energy sources, although the former is also able to metabolize arabinose and inositol while the latter can also grow on lactose, cellobiose, trehalose, amygdalin, and sorbitol Lachnospiraceae were Roseburia faecis, R. hominis, R. inulinovorans R. intestinalis, which are able to degrade starch with butyrate and lactate as some of the major products, and which were also described as the most active in metabolizing linoleic acid Ruminococcaceae is the other important Clostridiales family in which the most common reads retrieved from active fractions were related to Faecalibacterium prausnitzii or Hydrogenoanaerobacterium saccharovorans. The former is a recognized producer of short-chain fatty acids (SCFA) with anti-inflammatory properties and was found to be reduced in Crohn disease patients Ruminococcaceae were Ruminococcus bromii, R. flavefaciens and R. gauvreauii, which are known as normal commensal components of HGM, some of which play a protective role Blautia sp. were commonly found in all samples. Other underrepresented Clostridiales families were related to Clostridiales family XI which were found in higher abundance when associated to pathological status Clostridiales family XII whose genera are characterized by very heterogeneous phenotypes; Clostridiales family XIII, Clostridiales family XVIII, Gracilibacteraceae, Peptostreptococcaceae, etc.Bacillales and Lactobacillales, from Bacilli we did not find any family common to all fractions. Finally, Selenomonadales, from Negativicute, were URB retrieved from all samples.Although we identified several families belonging to the orders Bacteroidaceae were those most frequently recruited from total factions (FS and R). More specifically in Sample 1 and Sample 3 this family was represented about 9 and 5 times more  in total than in active fractions. Bacteroides dorei and B. uniformis were the only two families found in all samples. Despite the high recruitment of this phylogroup in the human gut, probably its real activity is low or related to other GIT districts. It is worth mentioning that several studies indicate that Bacteroidetes are more abundant when a low-calorie diet is maintained Bacteroides are known to be short-chain fatty acid producers with a proven role against gut inflammation Porphyromonadaceae, Prevotellaceae and Rikenellaceae from URB.In samples 1, 2 and 3, -Proteobacteria were found in almost all fractions as URB. Almost all families of -Proteobacteria were more abundant in active fractions, with the exception of Rhodospirillaceae, Methylocystaceae and Ricketsiaceae. Alcaligenaceae, from -Proteobacteria, were recruited in almost all samples but they were only significantly more abundant in active fractions in Sample 1. Meanwhile, -Proteobacteria were identified mostly in the active fractions. Moraxellaceae from -Proteobacteria was the sole family recruited in all samples, although with diverse species distribution and, also in this case, from active fractions. Pseudomonadaceae and Enterobacteriaceae were found in all samples except Sample 4. Finally, for Proteobacteria phylum, a common profile could not be identified at a deeper taxonomic level than family, but again, despite the very low frequency of identification, these bacteria were always present as active players of HGM.Proteobacteria are known to have very low representation in the HGM Actinobacteria counting Coriobacteriaceae, no family was identified as common to all samples. Members of Actinomycetaceae, Coriobacteriaceae, Corynebacteriaceae and Propionibacteriaceae were significantly more abundant in the active fractions than in the total one. Other reads belonging to Dermabacteraceae, Geodermatophilaceae, Gordoniaceae, Intrasporangiaceae, Micrococcaceae and Nocardiaceae were always found mainly in active fractions. Actinobacteria are reported to be underrepresented in gut phylogenetic descriptions, although there is evidence of their active role as demonstrated by FISH-related studies Sample 2 was the richest in The RNA-based cell sorting approach enables the HGM total and active population to be clearly differentiated. The pyronin-Y-based sorting methodology provides more detailed information of the complex HGM community, highlighting the presence of active underrepresented bacteria hidden by the over represented ones. At family level the active fractions tend to cluster together independently of the sample. Finally, altogether the data clearly show that the functional microbiome should not be deduced uniquely from DNA-based experiments using raw fecal samples.Fecal samples were obtained from four healthy human volunteers  between 25 and 35 years old, resident in Valencia (Spain). All volunteers follow a Mediterranean diet. The volunteers involved in this study provided their written informed consent. The study was approved by the Ethics and Research Committee of Centre for Public Health Research (CSISP) of Valencia, Spain. None of the volunteers had intestinal organic disorders or recent treatment with antibiotics. Samples were collected in sterile 30 ml screw-cap containers , containing 8 ml RNAlater (Ambion #AM7020) in order to preserve RNA. The samples were delivered to the laboratory within 24 h and stored at tube see . Upper PA subsample of 100 One microliter of pyronin-Y  was added to the sample from previous step (1 ml of volume) for total RNA staining and incubated for 20 min at Microbial cell sorting was carried out using the MoFloTM XDP cell sorter. The cytometer emission filter was the 580/30. The trigger was in FL2. The light sources were the Argon 488 nm (blue) laser (200 mW power) and the 635 nm (red) diode laser (25 mW power). The lasers were aligned using Flow-CheckOne microliter of each sample was diluted in 10 ml of 0.2 in-Y see . All parDNA extraction from all fractions was carried out using the CTAB method ad-hoc in our laboratory based on the last common ancestor (LCA) algorithm (available at https://github.com/emepyc/Blast2lca).Each fraction was initially dereplicated using CDHIT software Descriptive and statistical analyses were carried out with the R statistic package The analysis of variance  was carrA mathematical approach was employed to define if a given family belonged to the URB or ORB fraction respectively. To do so, for each sample/fraction, the list of family abundance values was sorted and the value corresponding to the last inflection point was used as cut-off. The reads belonging to active  and total fractions (R and FS) were respectively summarized and median values were used to perform the Welch Two Sample t-test which is an adaptation of the Student's t-test intended for use with two samples having possibly unequal variances.Flow cytometry data were analyzed with R package flowCore and flowViz from Bioconductor Sequences were deposited in NCBI Sequence Read Archive (SRA) database (SRP005393).Figure S1Clustering analysis. The figure shows the heatmap built on abundance values of families (in percentage) for each sample/fraction. Legend describes percentage ranges; blue gradient goes exponentially from 0.0001 to 1 representing URB distributions, brown gradient representing ORB. Dendrogram on top of the chart clusters the fractions. Abbreviations are defined as follow. Active fractions: PA, pyronin-Y activated; LC, low Cy5; HC, high Cy5. Total fractions: FS, Fecal Suspension; R, Ring fraction (see tion see  section.(PDF)Click here for additional data file.Figure S2Inflection points. Sample 1. Top panels show the second derivative used to calculate inflection points for each fraction. Bottom panels show ordered family distributions in percentages. Red lines describe the smoothed curve calculated for ordered family distribution data points. Dashed vertical lines mark the identified inflection points for each fraction (continue…).(PDF)Click here for additional data file.Figure S3Inflection points. Sample 2. (Follow): Top panels show the second derivative used to calculate inflection points for each fraction. Bottom panels show ordered family distributions in percentages. Red lines describe the smoothed curve calculated for ordered family distribution data points. Dashed vertical lines mark the identified inflection points for each fraction (continue…).(PDF)Click here for additional data file.Figure S4Inflection points. Sample 3. (Follow): Top panels show the second derivative used to calculate inflection points for each fraction. Bottom panels show ordered family distributions in percentages. Red lines describe the smoothed curve calculated for ordered family distribution data points. Dashed vertical lines mark the identified inflection points for each fraction (continue…).(PDF)Click here for additional data file.Figure S5Inflection points. Sample 4. (Follow): Top panels show the second derivative used to calculate inflection points for each fraction. Bottom panels show ordered family distributions in percentages. Red lines describe the smoothed curve calculated for ordered family distribution data points. Dashed vertical lines mark the identified inflection points for each fraction.(PDF)Click here for additional data file.Figure S6Microbial cell preparation from fecal samples. Microscopy photograph on the left (panel a) shows DAPI stained microbial cells obtained from R fraction recovered from (PDF)Click here for additional data file.Figure S7Cytometry dotplot. Fluorescence dotplot of pyronine-Y-activated cells. The X-axis describes the intensity of fluorescence emitted by each cell (arbitrary units), measured on the FL8 photomultiplier. The Y-axis describes the intensity of the fluorescence emitted by each cell passing over the FL2 discriminator (bacteria stained with pyronin-Y). The PA region was used to collect all pyronin-Y activated cells; LC region collected cells with low or null Cy5 fluorescence emission; HC region collected mainly cells hybridized with group-specific probes with high Cy5 fluorescence emission. Flow cytometry data were analyzed with R package flowCore and flowViz by Bioconductor (PDF)Click here for additional data file.Figure S8Protocol schema. Arrows define the work flow. Black arrows ideally represents all cells and particles contained in the samples. Red arrows represents the fraction of the microbiota hybridized to CY5 probes. Green arrows represents the fraction of cells labeled with pyronin-Y. Double colored arrows indicate cells stained simultaneously with pyronin-Y and CY5 fluorescent probes. Gray arrows represents the unstained fraction . In bold are represented the fractions obtained for downstream sequencing.(PDF)Click here for additional data file.Table S1Diversity indexes. Main diversity indexes calculated at family taxonomy rank for each sample/fraction.(PDF)Click here for additional data file.Table S2Probes used in this work .(PDF)Click here for additional data file.Table S3Multiplex Identifiers (MIDs) list and universal 16S rRNA primers used in this work .(PDF)Click here for additional data file.


Tylonycteris pachypus) is a nonmigratory Asian bat species that roosts in small groups within the internodes of bamboo stems and the habitats are fragmented. Our previous parentage analyses revealed considerable extra-group mating in this species. To assess the spatial limits and sex-biased nature of gene flow in the same population, we used 20 microsatellite loci and mtDNA sequencing of the ND2 gene to quantify genetic structure among 54 groups of adult flat-headed bats, at nine localities in South China. AMOVA and FST estimates revealed significant genetic differentiation among localities. Alternatively, the pairwise FST values among roosting groups appeared to be related to the incidence of associated extra-group breeding, suggesting the impact of mating events on fine-scale genetic structure. Global spatial autocorrelation analyses showed positive genetic correlation for up to 3 km, indicating the role of fragmented habitat and the specialized social organization as a barrier in the movement of individuals among bamboo forests. The male-biased dispersal pattern resulted in weaker spatial genetic structure between localities among males than among females, and fine-scale analyses supported that relatedness levels within internodes were higher among females than among males. Finally, only females were more related to their same sex roost mates than to individuals from neighbouring roosts, suggestive of natal philopatry in females.Population genetic structure has important consequences in evolutionary processes and conservation genetics in animals. Fine-scale population genetic structure depends on the pattern of landscape, the permanent movement of individuals, and the dispersal of their genes during temporary mating events. The lesser flat-headed bat ( Numerous studies of wild animal populations have revealed the importance of behavioral and ecological traits in shaping population genetic structure Bats are a highly speciose (more than 1100 species) and ecologically diverse group in which contrasting behaviours show good correspondence to different patterns of dispersal and population genetic structure Tylonycteris pachypus (Chiroptera: Vespertilionidae) is one of the smallest mammals in the world Gigantochloa scortecluni) and normally forms small groups, where females typically produce twins P>0.05), although males appeared to travel over longer distance  The lesser flat-headed bat, T. pachypus revealed a similar pattern of individual movements T. pachypus in South China, in which offspring belonging to full sibships were often found to be sired by males not present in the group at the time of sampling. More generally, approximately half (43.5%) of sets of twins were of mixed paternity while the other half shared the same fathers Mark-recapture data from adult T. pachypus resembles the commonly seen mammalian system of female natal philopatry combined with male-biased natal dispersal, then (i) mtDNA haplotypes distribution would show a different pattern between the sexes T. pachypus.To further characterize this system of breeding and social structure, and assess its consequences for wider gene flow, we sequenced adults from the same study populations at mtDNA ND2 gene, and combined our results with existing multi-locus microsatellite genotype data. We hypothesized that if This study was carried out in strict accordance with the guidelines of Regulations for the Administration of Laboratory Animals . All bats were released immediately the wing membrane tissue biopsies (diameter: 3 mm) were taken. We obtained approval for this study from the Guangdong Entomological Institute Administrative Panel on Laboratory Animal Care. Permission from the landowners was also obtained.Tylonycteris pachypus were sampled from Chongzuo district, Guangxi Province in South China . Details of amplification and sequencing were also provided in Hua et al. FIS) and among (FST) possible subpopulations were evaluated in T. pachypus. FIS, allelic richness corrected for unequal sample size, the observed and expected heterozygosity were calculated with microsatellite data in FSTAT v2.9.3.2 P values were adjusted with Bonferroni correction for multiple comparisons FST among localities were estimated and the significance was tested using 1000 permutations in GENETIX with both microsatellite and mtDNA data . Overall population differentiation was tested by exact G-test FST between roosting groups with extra-group paternity  and those with no extra-group paternity with Mann-Whitney test. In this analysis, only roosting groups comprising five or more individuals were analyzed and our published data on paternal sibships results was used F-statistics designed to quantify the partitioning of genetic diversity within (FST/(1−FST) and Log10 (geographic distance) between localities was tested with Mantel test in IBDWS v3.23 Analyses of molecular variance (AMOVA) from both microsatellite and mtDNA data were used to estimate the genetic variation among localities, among roosting groups within each locality and within localities as implemented in ARLEQUIN v3.01 FIS, FST, and HS, each based on pooled samples from all localities FST due to similar allele frequencies, yet a higher FIS due to a deficit of heterozygosity resulting from the combined sampling of residents and immigrants (Wahlund effect). We also calculated the probability of corrected mean population assignment (mAIc) and its variance (vAIc) vAIc, the ratio), 10000 randomizations were used. All analyses were performed in FSTAT program.Several specific tests of male-biased dispersal were also carried out with microsatellite data. We compared sex-specific estimates of T. pachypus, we estimated pairwise relatedness (R) within roosting groups with microsatellite data To offer new insights into sex-biased dispersal in r provides a measure of the genetic similarity between pairs of individuals whose geographic separation falls within the specified distance class. A correlogram is produced that shows the autocorrelation coefficient r as a function of distance class. To assess the genetic similarity and spatial distance, the observed r values are compared to expected values (rp) based on 1000 permutations. When r is significantly larger than rp, positive spatial genetic structure is accepted for specified geographic distance class.Spatial autocorrelation analysis to measure the genetic structure across the entire sampling site with microsatellite data was carried out in GENALEX v6 r values In this study, global spatial autocorrelation was measured for all 54 roosting groups of bats within the nine localities. Pairwise linear geographic and squared genetic distance matrices were calculated in GENALEX. Linear Euclidean distances were measured from latitude/longitude coordinates of samples. Genetic distances for codominant data were calculated under the assumption of statistical independence across loci according to the method proposed by Peakall et al. Tylonycteris pachypus, we analyzed genetic data from nine localities comprising 69 males and 227 females. We detected a total of 279 alleles, ranging from three to 21 alleles per locus at the 20 microsatellite loci scored. Tests for Hardy-Weinberg equilibrium within localities across all loci indicated six out of 180 tests were significant for heterozygote deficit after Bonferroni correction (lower than expected by chance when α = 0.05). In total, 146 out of 1710 loci pairs deviated from linkage equilibrium (P<0.05), but only six were detected after Bonferroni correction (P<2.9×10−5). High genetic variation within localities was detected in terms of both observed heterozygosity (HO: 0.668 to 0.770) and expected heterozygosity  and pairwise FST ranged from 0.009  to 0.064 . When the population differentiations were restricted between pairs of populations within the same region, all pairwise FST were less than 0.03, suggesting higher gene flow among localities within regions. All pair of localities indicated significant divergence .Analyses of microsatellite data revealed significant genetic differentiation across all localities . The variance among regions contributed to 2.25% variation . AMOVA with ND2 sequences demonstrated similar results  in allele frequencies was within localities . 2.33% w results .FST/(1−FST) and Log10 (geographic distance) showed strong signal of isolation by distance pattern when all localities were included . Given the large geographic isolation and discontinuity between Longzhou region  and Ningmin region .Mantel test with pairwise T. pachypus, FST values among roosting groups comprising five or more adults within localities were also evaluated. We found the FST values with extra-group paternity were significantly lower than FST with no extra-group paternity . Furthermore, the pairwise relatedness values within internodes were greater than zero for both females and males . In spite of this, we cannot detect the difference of relatedness levels between males within internodes and among internodes . Distributions of relatedness values for different sexes demonstrated females were more related to each other than to males within roosting groups , with the highest relatedness was in TL locality  within internodes was significantly greater than the null expectation of zero  and mAIc values . The tests of FIS, HS and vAIc were not statistically significant .Five one-tailed tests for male-biased dispersal were run separately. Females showed significantly greater r  of T. pachypus makes its flying performance a lot more limited compared to many other bat species T. pachypus is relatively limited and the dispersal distance is considered short Usually, in the roosting internodes of T. pachypus has revealed that the number of migration events between localities was very low  Mark-recapture data collected in natal localities of FST is lower than other animals when assessed with nuclear genetic markers FST value of T. pachypus was 0.024 among all localities, suggesting high gene flow, but still dispersal barriers among localities. On the other hand, all pairwise FST were significant. The results indicated that although the shortest geographic distance was less than 1 km among localities, there still existed strong genetic substructure in most cases. Significant genetic differentiation on a fine scale support our expectation that fragmented habitat combined with the specialized social organization of T. pachypus results in the limited movement of individuals among localities in this species.Usually, in flying, migratory or ocean-dwelling species , the population differentiation estimated by Myotis septentrionalis, Rhinolophus monoceros and Nyctalus noctulaMyotis bechsteiniiSaccopteryx bilineata, also a nonmigratory bat, showed female-biased dispersal T. pachypus, strong male-biased dispersal was detected from FST and mAIc statistics. According to Goubet et al. FIS, HS and vAIc values between sexes.In most mammals and birds, dispersal is biased towards one sex . Male-biased dispersal has been found in some migratory bat species, such as T. pachypus is also expected to limit the gene flow among localities (bamboo forests). Most bamboo forests were small and separated from each other by agricultural land or urban development, etc. T. pachypus. In addition, because of the economy value, many bamboo forests were felled. All of these suggest that T. pachypus is highly reliable on their habitats (bamboo forests), the populations are patchy, and in some regions, likely to be suffering from habitat loss In the common frog populations, Johansson et al. found more pronounced genetic differentiation in the fragmented than in the continuous habitat of this species. On the other hand, the mean values of the fitness related traits and the amount of microsatellite variation was positive related. The results suggest the potential importance of habitat fragmentation in limiting gene flow and impairing future adaptive potential of natural populations In bats, fine-scale genetic structure was unexpected, given many bats are highly vagile and/or migratory, but the multivariate spatial autocorrelation methods such as the multilocus, multi-allele method have proved highly sensitive for detecting unexpected fine-scale genetic structure r values among intra-locality females were a little weaker than that for females, but not significantly different . However, a significant difference between females and males was detected. The extent of genetic structure in females (4.9 km) was greater than that in males (864 m). The more biased dispersal in males reduced the genetic correlation with spatial distance, even within localities. Our results further confirmed the influence of male bias in dispersal behaviours on the local pattern of genetic structure in T. pachypus.Spatial correlation analysis of females revealed similar results to that of all individuals combined. The Plecotus auritus, Myotis bechsteinii, M. nattereri, have been reported to mate at swarming sites during autumn Many nonmigratory bats species, such as T. pachypus does not follow the swarming site mating pattern, their mating events may occur mostly in bamboo internodes T. pachypus have demonstrated that related females preferred to mate with the specific individual males FST among roosting groups, our results revealed that the genetic differentiation among groups was lower significantly when extra-group mating occurred Click here for additional data file.


Rates of SNPs (single nucleotide polymorphisms) and cross-species genomic sequence conservation reflect intra- and inter-species variation, respectively. Here, I report SNP rates and genomic sequence conservation adjacent to mRNA processing regions and show that, as expected, more SNPs occur in less conserved regions and that functional regions have fewer SNPs.  Single nucleotide polymorphisms (SNPs) are intra-species sequence variation. Conversely, cross-species genomic conservation reflects longer term inter-species evolution. SNPs mark genomic locations where intra-species variability is permissible; fewer SNPs should occur in regions encoding sequence-dependent functions. Similarly, insofar that many functional molecular processes are sequence dependent and behave similarly across species, higher genome sequence conservation should occur in functional regions. Therefore, SNP occurrence rates and genomic conservation should be anti-correlated but similarly delineate functional regions. Indeed, there is an emerging field to actively integrate intra- and inter-genomic variation, with applications ranging from disease to phenotype to functional molecular biology to evolution to crop breeding to conservation Each step in mRNA-processing relies on specific sequences. After transcription, the spliceosome binds RNA to splice exons into a mature transcript; sequence at the transcript is recognized and polyadenylated. The ribosome scans the mature transcript, starting protein translation at a start codon and ending translation at a stop codon. One pathway for mRNA degradation involves miRNA recognition of sequences in the mRNA 3′ untranslated region (3′ UTR).The human and mouse genomes were assembled in 2001 Here, I integrate these genomic coordinates to determine SNP rates and conservation scores surrounding mRNA processing elements. I expected that, for example, SNPs would occur more frequently in codon position three, the degenerate position, and that conservation would be higher at this position. The results indeed confirm expectations and clearly show that SNP rates and conservation are anti-correlated, that both mark functional elements, and demonstrate the single-nucleotide, sequence-level genetic constraints imposed by protein-coding, by splicing, and by transcript processing.Using mouse and human data available in the UCSC genome databases, I calculate SNP rates and conservation scores at single nucleotide resolution across six mRNA processing regions, for both species . The SNPWithin the region adjacent mouse protein coding start sites, the SNP rate is lowest and the conservation highest at the start codon. A phase three periodicity in SNP rate and conservation levels occurs after the start codon, with more frequent SNPs and less conservation at codon position three. Within each codon, the majority of positions two, and not positions one, show the lowest SNP rate. After peaking at the start codon to near 0.7, conservation stays high in the coding region at 0.6. The SNP rate is on average lower in positions one and two after the start codon, while the SNP rate in position three is similar before and after the start codon.The set of mouse RefSeq transcripts contain 50,779, 40,890, and 22,078 phase zero, one, and two introns, respectively, adjacent protein coding exons greater than 60 nt long. The corresponding human genomic regions show results similar to the mouse results . As for Near mouse stop codons, the SNP rate is lower and conservation higher before the stop codon. The SNP rate and conservation score display a phase three periodicity before the stop codon. Position three shows a greater SNP frequency and lower conservation; position two shows the lowest SNP rate. The region with both the highest SNP rate and least conservation occurs from 5 to 20 nt after the stop codon, which is found in both human and mouse, suggesting that this region is under less evolutionary control.TargetScan The UCSC genome databases list transcript 3′ ends for mouse transcripts and predicted polyadenylation sites for human transcripts Using the predicted human polyA site coordinates, I extracted the sequence from the UCSC genome databases and searched for the polyA signal motifs AAUAAA and AUUAAA. The motif occurs between 30 and 17 nt before the polyA site  inset inHere, I used mouse and human genomic resources to examine SNP rates and interspecies genomic sequence conservation in RNA-relevant regions. The results confirm expectations that the SNP rate and conservation score show anti-correlation: fewer SNPs occur in conserved regions. This is in agreement with our understanding that both processes mark regions under evolutionary conservation: SNPs reflect allowed intra-species genome variation and cross-species conservation demarcates long-term evolutionary stability.Mus musculus domesticus) or a variation between sub-species (e.g. between M. m. domesticus and M. m. musculus). The latter represent variation somewhat between a true intra-species polymorphism and cross-species variation.Additional biases may result from the genome of the laboratory mouse genome. Inbred laboratory mouse strains have a genome that is mosaic of two sub-species Furthermore, changes in mutation rate variation could introduce biases in these observations e.g., . The datPredicted miRNA binding sites The profiles of conservation scores and composite SNP rates delineate established functional elements. Conservation can be used to identify individual elements whereas SNP rates can identify elements when consolidated across the genome. The sharp peak in conservation and the SNP rate trough occurring 20 nt before the polyadenylation site identifies a region highly enriched for the polyA signal AA[U/A]AAA. Motif enrichment, conservation, and the low SNP rate all occur over a narrow range from 30 to 17 nt preceding the polyadenylation site. Variations near 5′ and 3′ splice sites may impact pre-mRNA splicing and can cause disease Low genomic variability exists in protein coding regions: SNP rates are lower and conservation higher in all protein coding regions examined. Near the protein start site, conservation is sharply higher precisely at the start site and maintains a high level into the protein coding region. Similar profiles, but in reverse, occur at the protein stop site. However, while the stop codon shows a low SNP rate, conservation at the stop site codon itself is not higher than at previous positions, unlike the spike at the start codon. This may reflect either the stop codon degeneracy or that a mutated stop codon will likely be followed by a second stop codon. Furthermore, compared to the conservation in the internal protein coding exons e.g., , the conAdditionally, a 15 nt trough in conservation and corresponding high in the SNP rate occur from 5 to 20 nt after the stop site in both mouse and human results. That it occurs in both SNPs and conservation suggests that it is real and, taken at face value, shows that more changes have occurred in the region between the stop codon and 20 nt into the 3′ UTR. Previous work has shown that miRNA binding sites at positioned within the 3′UTR occur at least 15 nt from the stop codon Inter-species (conservation), intra-species (SNPs), and intra-genome (webLogos) data all demonstrate the low variation at splice sites. The SNP rate increases almost 3-fold from within the coding exons to the rate at 100-nt into introns. However, the low variability occurs not only in protein-coding regions but also into the adjacent intron, accentuating the need to study variation beyond non-synonymous changes e.g., . Indeed,Finally, all protein coding regions display a phase three periodicity with increased variability at position three. The periodicity is not observed outside of the protein coding regions, such as adjacent polyadenylation sites. That the SNP rates are highest at codon position three is undoubtedly a result of the degeneracy for amino acid coding at the third position (19 of 22 amino acids are degenerate at codon position 3). Moreover, codon degeneracy is slightly higher at codon position one relative to position two: I correspondingly find a lower SNP rate at the second position. Thus, more SNPs occur in degenerate positions for amino acid encoding, and are thus less likely to have a functional impact Together, these findings reinforce our understanding of evolution and functional elements in the genome. These include that SNPs occur more frequently in less conserved regions; that both genomic conservation and aggregated SNPs rates can be used to identify functional elements; and that codon position three in protein coding regions is more degenerate.Genomic coordinates were downloaded from UCSC databases For the human predicted polyadenylation sites, I selected only those polyadenylation sites falling within 100 nucleotides of the 3′ terminus of a RefSeq transcript. This excluded alternative polyadenylation sites occurring in the interior of 3′ UTRs and also allowed a strand assignment to each polyadenylation site, based on the RefSeq transcript orientation.I used RefSeq “NM” transcript alignments which include exon boundaries, transcription start and stop coordinates, coding start and stop coordinates, and intron phase. I required at least 60 nt of coding sequence after and before start and stop codons, respectively. For the 5′ and 3′ splice sites, I included only internal exons longer than 60 nt surrounded by introns greater than 300 nt long.I examined several mRNA processing regions : protein


Emerging evidence showed that long non-coding RNAs (lncRNAs) play important roles in a wide range of biological processes and dysregulated lncRNAs are involved in many complex human diseases, including cancer. Although a few lncRNAs’ functions in cancer have been characterized, the detailed regulatory mechanisms of majority of lncRNAs in cancer initiation and progression remain largely unknown. In this review, we summarized recent progress on the mechanisms and functions of lncRNAs in cancer, especially focusing on the oncogenic and tumor suppressive roles of the newly identified lncRNAs, and the pathways these novel molecules might be involved in. Their potentials as biomarkers for diagnosis and prognosis in cancer are also discussed in this paper. Genome-wide transcriptome studies have revealed that there exist a large number of non-coding RNAs (ncRNAs), including short and long non-coding RNAs ,2. ShortRecent studies estimated that the number of lncRNAs in humans is approximately 15,000 and found that most lncRNAs displayed tissue-specific expression patterns . Based oBecause lncRNAs, like miRNAs, function as key regulators in gene regulation, it is not surprising that dysregulated lncRNAs are involved in many complicated human diseases including cancer. Mounting evidence showed that many lncRNAs, similar to miRNAs, have altered expression in various types of human cancer and dysregulated lncRNAs function as tumor suppressors or oncogenes ,19. UndoIt has been known that ncRNAs function as a new class of regulators involved in a complicated molecular network, controlling gene expression and cellular activity. Among them, well-documented miRNAs were known to regulate gene expression at either the transcriptional or the post-transcriptional level usually by base-pairing to the 3′-untranslated region (3′ UTR) of target mRNAs with the miRNA 5′-proximal “seed” region (positions 2-8) in animals . Howeverin cis (on neighboring genes of the same chromosome) or in trans (on distantly located genes of the same or different chromosome) [in trans. It has been shown the lncRNA HOTAIR mediates the epigenetic repression by increasing PRC2 and LSD1 recruitment to the genomic positions of target genes [in trans[Evidence showed that lncRNAs can bind and ‘guide’ chromatin modifying complexes to specific genomic sites to induce epigenetic changes and regulate gene expression omosome) . Severalomosome) ; AIR andomosome) ,25; HOTTomosome) . LncRNAset genes . In addiet genes -29. Inte[in trans.Another regulation mechanism of lncRNAs is to serve as scaffolds. Studies demonstrated that lncRNA KCNQ1OT1 could bind both G9a and PRC2, and lncRNA HOTAIR could interact with PRC2 and LSD1, revealing these lncRNAs can serve as scaffolds for chromatin-modifying complexes besides their guide role ,26. SimiLncRNAs regulate gene expression at transcriptional level via multiple mechanisms. It was reported that by competing for binding sites, the lncRNA GAS5 (growth arrest specific 5) interacts directly with the DNA binding domain of glucocorticoid receptors (GR), preventing them from binding to their DNA response elements, thus functions in transcription inhibition as a molecular decoy . In addiThe well-known example of mRNA splicing mediated by lncRNAs is that lncRNA MALAT1 interacts with the serine/arginine-rich splicing regulatory (SR) proteins involved in alternative splicing, suggesting that the lncRNA MALAT1 may serve a function in the regulation of alternate splicing . AnotherTranscriptional profiling has revealed highly aberrant lncRNA expression in human cancers . Though Seeking novel molecular biomarkers of malignancy is always important and helpful for clinical diagnosis and management. Like proteins, mRNAs and miRNAs, lncRNAs show their potentials as novel independent biomarkers for early diagnosis and prognosis prediction in cancer.It has been found that lncRNA HOTAIR is highly upregulated in breast cancer, hepatocellular carcinoma (HCC), colorectal cancer and gastrointestinal stromal tumors (GIST). Furthermore high HOTAIR gene expression is correlated with metastasis in these four types of cancers, poor survival rate in breast cancer, increasing risk of recurrence after hepatectomy in HCC and high-risk grade in GIST, indicating HOTAIR may be a useful biomarker of poor prognosis and tumor metastasis in these cancers ,44-47. IGenome-wide association studies (GWAS) have revealed a large number of genetic variants related to diseases, including cancer. Recently, genetic variations in lncRNAs and their associations with cancer susceptibility have been reported, suggesting single nucleotide polymorphisms (SNPs) may also serve as biomarkers for diagnosis and prognosis. Studies showed that SNP rs2151280 in lncRNA ANRIL was significantly associated with higher number of plexiform neurofibromas (PNFs) in neurofibromatosis type 1 (NF1) patients, which suggests SNP rs2151280 in lncRNA ANRIL is a potential biomarker for PNF susceptibility . InteresLike protein-coding genes and miRNAs, lncRNAs can function as oncogenes or tumor suppressors during cancer progression. A further investigation of the roles and mechanisms of lncRNAs in cancer will provide novel lncRNA-based strategies for the treatment of human cancers. Some well-studied lncRNAs, such as HOTAIR ,48, MALAin vitro and in vivo[in vitro and in vivo, indicating lncRNA CUDR functions as an oncogene and may serve as a novel therapeutic target for bladder cancer [The newly identified lncRNA BANCR was found overexpressed in melanoma and required for full migratory capacity of melanoma cells by upregulating CXCL11, an important gene involved in cell migration, revealing the potential functional role of lncRNA BANCR . Anotherd in vivo. In addir cancer . Comparer cancer , was recr cancer . Very inr cancer . Furtherr cancer .Differentially expressed lncRNAs between cancer and normal tissues indicate these lncRNAs may exert a key role in cancer development. Understanding the pathways lncRNAs involved in helps to provide further insights into the pathogenesis of cancer. In HCC, PKA-mediated phosphorylation of CREB leads to formation of the CREB-p300-Brg I complex, loading of the CREB-p300-Brg I complex onto the lncRNA HULC promoter, and consequent activation of HULC expression . In addiRecent studies revealed lncRNAs, like miRNAs, function as important regulators in gene regulatory networks and exert crucial roles in cancer progression. LncRNAs have become new and important players and demonstrated potential applications in clinical diagnosis and treatment. Although a few lncRNAs’ functions have been reported, there are still many challenges that remain to be solved. In the future, it would be necessary to further investigate the functional motifs and secondary or tertiary structure of lncRNAs, to fully elucidate the diverse gene regulatory mechanisms of lncRNAs, and to develop new and effective methods to predict target genes of lncRNAs using bioinformatics, etc. These endeavors will provide new insights into the complicated gene regulatory network involving lncRNAs, and ultimately provide novel strategies for cancer diagnosis and therapy.The authors declare that they have no competing interests.All authors have contributed to write and revise the manuscripts. All authors have read and approved the final manuscript.


Haloferax and Haloarcula. Ease of cultivation and the existence of well-developed genetic and biochemical tools for several diverse haloarchaeal species make haloarchaea a model group for the study of archaeal biology. The unique physiological properties of these organisms also make them good candidates for novel enzyme discovery for biotechnological applications. Seven genomes were sequenced to ∼20×coverage and assembled to an average of 50 contigs (range 5 scaffolds - 168 contigs). Comparisons of protein-coding gene compliments revealed large-scale differences in COG functional group enrichment between these genera. Analysis of genes encoding machinery for DNA metabolism reveals genera-specific expansions of the general transcription factor TATA binding protein as well as a history of extensive duplication and horizontal transfer of the proliferating cell nuclear antigen. Insights gained from this study emphasize the importance of haloarchaea for investigation of archaeal biology.We report the sequencing of seven genomes from two haloarchaeal genera,  In recent years, the Archaea have been shown to play major roles in global element cycling Halobacteriacea  in the phylum Euryarchaeota has enormous potential to serve as a model group for the study of archaeal biology. As aerophilic mesophiles, many haloarchaea are easily cultivated in the laboratory, making them one of the most widely studied archaeal groups and leading to the development of a variety of biochemical, genetic and genomic tools for several diverse haloarchaeal species. Although united by their ability to thrive at high salinities, haloarchaea possess a wide range of physiologies  The family In addition to serving as a model group for study of the Archaea in general, the Haloarchaea possess unique properties making them important objects of study in their own right. For example, understanding the genetic basis for these organisms’ ability to thrive in hypersaline environments  will inform efforts to develop salt-tolerant crop plants for growth in currently non-arable land. The Haloarchaea are also promising sources of salt and ionic liquid tolerant enzymes for various industrial processes, including biofuels manufacturing http://www.ncbi.nlm.nih.gov/genomes]. This discrepancy in the number of available bacterial and archaeal genomes is particularly striking when one considers that archaeal and bacterial genomes are similar in size and coding density, both lacking the extensive DNA repeats which complicate sequencing and assembly of eukaryotic genomes. Of the 116 completely sequenced archaeal genomes, fourteen are from the Haloarchaea, spanning thirteen genera, making this one of the most deeply sequenced archaeal clades. These haloarchaeal genomes average 3.3 Mbp, ranging from 2.67–5.44 Mbp. Distinguishing features of haloarchaeal genomes include multiple replicons, high GC content (averaging >60%), and highly acidic proteomes (pI ∼5), thought to be an adaptation to life at low water activity As of September 2011, the National Center for Biotechnology Information (NCBI) lists 1,628 completed bacterial, 37 eukaryotic and 116 archaeal genomes [Haloferax and Haloarcula), each with a previously sequenced member  and five Haloferax  - were sequenced on a single GS FLX Titanium run, with the previously sequenced Haloferax volcanii included as a control. After removal of low-quality nucleotides, mean read-length for each genome was between 410 and 439 base pairs . SequencHaloferax volcanii sequencing control and the published genome for this species, for an average of one differently called base per 305 Kbp. In addition, the newly assembled Hfx. volcanii genome was missing 123 regions present in the published genome, with a median length of 1126 bp and a total length of 119,245 bp of missing sequence. The sequencing control also had 140 short sequences not present in the published genome with a median length of 3 bp and a total of 1,319 bp additional sequence. While it is tempting to use this data to calculate error rates for the sequencing technology used in this study, we can not exclude the possibility that a portion of the differences between these two assemblies may be due to natural variation within the Haloferax volcanii DS2 population, errors in the previously published genome, and/or evolution of this organism between sequencing efforts.There were thirteen single base call discrepancies between the Har. sinaiiensis and Hfx. mediterranei, resulting in assembly of Hfx. mediterranei into five scaffolds. As the previously sequenced member of this genus, Hfx. volcanii, possesses four chromosomes and a plasmid Hfx. mediterranei into five scaffolds may indicate complete assembly for this species. To test whether our scaffolds represented complete replicons, we designed primers to the ends of each scaffold and looked for formation of short PCR products, indicating circularization of the replicon near our scaffold ends  while the smallest plasmids in Har. marismortui are 33 Kbp, and it is therefore likely that more scaffolding is required.The assembly of caffolds . While iHaloferax genomes were on average 11.6% smaller and encoded 13% fewer protein-coding genes than Haloarcula genomes, for an average of 578 fewer genes. The IMG annotation system predicted functions for an average of 66% of protein coding genes in each genome based on matches to a combination of functional annotation databases  and an IMG native collection of functional roles (IMG terms). Of the predicted protein coding genes in the newly sequenced genomes, 67% matched an entry in the COG database, 51% had one or more significant matches to Pfam domains, and 87% matched an InterPro family or superfamily. The fraction of genes with functional predictions in these genomes is considerably lower than for well-studied bacterial species such as Escherichia coli K12 (87%), Bacillus subtilis BSn5 (74%), and Pseudomonas aeruginosa 2192 (82%) but comparable to previously sequenced haloarchaeal genomes (average 59%).The newly-sequenced genomes were similar in size to previously sequenced haloarchaea, ranging from 3.37 to 4.45 Mbp with GC content between 60.3% and 66.3% . HaloferHaloferax and Haloarcula species revealed that several functional categories were differentially enriched between the two genera, as determined by the non-parametric Wilcoxon rank-sum test  or CheY-like response regulators . Previous studies have found that, in bacteria, the proportion of an organism’s genes dedicated to certain functions grows disproportionately with increasing genome size Haloarcula genomes, in comparison with the smaller genomes of Haloferax species, could be explained in terms of a general phenomenon of enrichment for signal transduction in large genomes  had an annotated function. While ambiguities associated with automated annotation make predicting the precise function of many of these genes uncertain, several broad functional categories were nevertheless highly represented. These included many genes predicted to be involved in transport (32), including of heavy metals (7) as well as a number of predicted transcriptional regulators (14) and signal transducers (8)   (Table Scers (8) . Deeper As has been discussed previously Haloferax denitrificans-sulfurifontis-volcanii clade (HVO_B0083 in Hfx. volcanii). As previous work has concluded the presence of a functioning semi-phosphorylated ED pathway in Haloarcula vallismortis and Haloferax mediterranei based upon evidence for activity of key enzymes in this pathway Hfx. sulfurifontis. The six haloarchaeal BLAST hits recovered had relatively high E-values (∼0.001) and low query coverage, with the hit from Har. marismortui (rrnAC0850) matching the query with only 64% coverage. The putative gluconolactonase from Haloarcula marismortui belongs to the beta-propeller clan , the same clan to which the Hfx. gluconolactonases belong. However, this clan contains a large number of families and domains  with a wide variety of functions and it is unclear whether rrnAC0850 and the predicted Hfx. gluconolactonases share a function.A semi-phosphorylated alternative to the Entner-Doudoroff pathway, which bypasses the first six steps in glycolysis, has been previously described for the haloarchaea Haloferax volcanii (HVO_B0083) with many additional high-quality matches to bacterial gluconolactonases . Although these bacterial matches had low E-values and high query coverage (mean 92%), the percent identity was low (mean 31%). These results suggest that this gene may have been introduced into the Hfx. denitrificans-sulfurifontis-volcanii clade via a horizontal transfer event from the Bacteria. The low degree of identity between Haloferax and bacterial gluconolactonase may reflect rapid evolution in response to selective pressure for an acidic pI, which is required for protein function in the extremely saline cytoplasm of haloarchaea. Due to the apparent absence of gluconolactonase in other haloarchaea, we propose that haloarchaeal species previously described as using the semi-phosphorylated ED pathway may use an alternative entry of metabolites into this pathway, possibly including a novel mechanism for gluconate production. Further genetic and biochemical work will be required to decipher this unique pathway for sugar metabolism in the haloarchaea as well as the function of the bacterial gluconolactonase-like protein in the Haloferax denitrificans-sulfurifontis-volcanii clade.To investigate whether these results could stem from a high level of sequence divergence of this enzyme, a BLASTp search against NCBI’s non-redundant protein database was conducted with the same query. The only archaeal match was to Several recent studies have highlighted the unique metabolic capabilities of the haloarchaea and have uncovered enzymes with potential utility in several industrial processes, including biofuels manufacturing. Here we expand upon previous studies by surveying twenty-one haloarchaea for genes of biotechnological importance as well as genes involved in several metabolic processes unique to the haloarchaea.Halorhabdus utahensis (Hu-CBH1) Haloarcula species, however, notably lacking these enzymes , a potential renewable and biodegradable substitute for petroleum-derived plastics jeotgali , demonstHar. marismortui in 2011 Haloarcula marismortui, genes involved in the methylaspartate cycle are co-localized on the genome (rrnAC0683-rrnAC0690), with genes rrnAC0684-rrnAC0690 forming an operon, and succinyl-CoA:mesaconate CoA-transferase (rrnAC0683) being upstream of and anti-parallel to the operon Haloarcula species, along with Halomicrobium mukohataei, Natrialba magadii, Haloterrigena turkmenica, and Halopiger xanaduensis. The gene for succinyl-CoA:mesaconate CoA-transferase was missing in Halobacterium species, suggesting that the methylaspartate cycle may not be functional in these organisms. Other haloarchaea were found to possess the key enzymes required for carrying out acetyl-CoA assimilation via the glyoxylate cycle, including Natronomonas pharaonis, Haloquadratum walsbyi, Halogeometricum borinquense, and all Haloferax species   and Har. marismortui (YP_136594). The lack of annotation for this gene in Har. californiae may be due to the high number of contigs in this assembly. We have also identified, for the first time, an opsin belonging to a member of the Haloferax genus, which has previously been found to lack these proteins Haloferax mucosum, but not in the other Haloferax species included in this study.We performed a genomic screen to identify opsin homologs in the twenty-one currently sequenced haloarchaeal genomes. One or more opsins were found in fourteen of the twenty-one genomes, with most species lacking at least one of the four canonical opsin classes. In fact, only nAC0559) . The absPyrobaculum aerophilum (a crenarchaeote) and Thermococcus kodakaraensis (a euryarchaeote) has raised the possibility for specialization of redundant copies of this multi-purpose protein Proliferating cell nuclear antigen (PCNA), also referred to as DNA sliding clamp in archaea, plays an essential role in many aspects of DNA metabolism, serving as a processivity clamp for the replicative DNA polymerase and also acting as a scaffold for recruitment of proteins with diverse roles in DNA metabolism Har. californiae. The phylogenetic distribution of these multiple PCNA homologs precludes their origin from a single duplication or horizontal gene transfer event and suggests a more complex history , it is possible to begin disentangling the evolutionary history of the multiple PCNA homologs. The origin of the second Natrialba magadii PCNA (green) has been traced to Natrialba phage PhiCh1 (NP_665977) indicating that it has arisen from HGT. The origins of the additional PCNA homologs of Har. californiae (orange), Halogeometricum borinquense , and Halalkalicoccus jeotgali  are less clear. These sequences may have arisen through HGT from within the haloarchaea or from a closely related euryarchaeal clade. Additionally, the branching position of the second Halogeometricum borinquense PCNA homolog could be consistent with origin in a duplication and divergence event  is not conserved among haloarchaea, with about half of haloarchaeal PCNAs having a Lys Thr mutation at this position. We also note, however, that the majority of haloarchaeal PCNA sequences share a different set of three basic amino acid residues, also located in the central pore in analogous positions to three of the eukaryotic residues known to be involved in DNA binding . We hypothesize that these residues may interact with DNA in a manner similar to that observed in eukaryotes. Subsequent genetic and structural analysis will be required in order to confirm the importance of these residues to DNA binding.Previous work has shown The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is a recently discovered method of viral immunity present in a variety of bacterial and archaeal genomes. Although previous studies have reported CRISPR arrays in approximately 40% of tested bacterial and 90% of archaeal genomes Haloferax and all but one Haloarcula species  had no predicted CRISPRs or Cas genes, supporting previous work Haloterrigena turkmenica, Haloarcula vallismortis, Halogeometricum borinquense, and Natrialba magadii - were found to be lacking any of the known CRISPR-associate genes, even though they were predicted to possess one or more CRISPR loci. As the function of many of the Cas genes and their contributions to the CRISPR/Cas phage-defense system are still unknown Haloquadratum walsbyi DSM 16790, which was not identified in our analysis Haloquadratum walsbyi C23 was found to possess three CRISPR loci, including one homologous to the vestigial CRISPR locus in strain DSM 16790 Haloquadratum walsbyi DSM 16790, which was isolated via two years of serial enrichment, as compared with strain C23 which was isolated over a three-week period via the extinction dilution method The twenty-one sequenced haloarchaeal genomes available at the time of this study were searched for CRISPRs and CRISPR-associated (Cas) genes. Twelve of the twenty-one species were found to possess both CRISPRs and one or more Cas genes, including all  species . HoweverHalogeometricum borinquense and Natrialba magadii) but excluding Haloterrigena turkmenica and Har. vallismortis. This suggests that Halogeometricum borinquense and Natrialba magadii may have gained CRISPR loci in isolation from Cas genes through HGT with other haloarchaea, or lost Cas genes due to disuse of the CRISPR/Cas system as discussed above. In contrast, it appears that Haloterrigena turkmenica and Har. vallismortis may have gained their CRISPR loci through more distant HGT events.The direct repeats (DRs) of the CRISPR array have been previously shown to be highly conserved across CRISPR loci both within and between closely related species of haloarchaea Alignment of the conserved haloarchaeal DRs shows many highly conserved positions and a few positions (mainly in the center of the repeat) with no apparent conservation. This conservation pattern suggests a conserved stem-loop in the secondary structure, which was confirmed with the program RNAFold Halobacterium sp. NRC-1, which with six TBP and seven TFB homologs is the most GTF-rich archaeal species known, has shown promoter-specific binding of individual GTFs leading to differential regulation of transcription in different simulated environments Archaeal transcription initiation is dependent upon two general transcription factors (GTFs) orthologous to eukaryotic TATA-binding protein (TBP) and transcription factor II B  Haloferax which is eclipsed in number of TBP paralogs only by Halobacterium, with most Hfx. species possessing four distinct TBP homologs. Phylogenetic analysis reveals that the dramatic expansion of the TBP family in these two genera are independent, with the Haloferax and Halobacterium TBPs being orthologous, not paralogous, with one another . Hence, although the presence of multiple TBPs in each genus indicates a sophisticated method for differential regulation of transcription initiation, we expect these mechanisms to be quite different from one another, involving different binding motifs and different sets of co-regulated genes in the two genera. In line with the predicted role for GTFs in niche adaptation predicted by Turkarslan et al Our analysis reveals that, although TBP and TFB expansions are widespread across the haloarchaea , several another . AdditioHaloarcula and Haloferax genera has further enriched our ability to conduct genomic analyses of the haloarchaea, for example, by enabling identification of genomic flux events at a finer time-scale than previously possible. In addition to providing a broad outline of the history of genomic evolution in this clade, these comparisons have identified several areas of specific interest for future investigation into the unique metabolic and regulatory capabilities of the haloarchaea.Comparisons of the twenty-one currently available haloarchaeal genomes have revealed dynamics of genome evolution at scales ranging from horizontal transfer and duplication of individual genes to major gene loss events and large-scale expansion of functional groups. Deep sequencing of the Strains were acquired as desiccated cells from the American Type Culture Collection (ATCC) in Manassas, Virginia and were rehydrated in recommended media according to ATCC protocols. Strains were grown to stationary phase at 37°C in liquid culture and genomic DNA harvested with the Wizard Genomic DNA purification (Promega).Haloarcula  and five from the genus Haloferax , and sequenced on a single GS FLX Titanium run following standard protocols (454 Life Sciences - http://454.com/). Hfx. volcanii was included as a sequencing control, as its genome had been completed previously Har. sinaiiensis and Hfx. mediterranei, 8 Kb pair-end libraries were constructed and the terminal 100 bp of each end was sequenced, according to standard protocols. The paired-end information and any trimming information are specified using annotation strings on the description line of the reads. Reads were assembled using the Genome Sequencer De Novo assembler (454 Life Sciences - http://www.my454.com/). Final assemblies are available at our website Fragment libraries were constructed for eight species of the family Halobacteriacea, three from the genus http://img.jgi.doe.gov/cgi-bin/geba/.Genomes were annotated with Rapid Annotation Using Subsystems Technology (RAST) Haloferax and Haloarcula genera using the whole genome alignment method progressiveMauve Haloferax volcaniiHaloarcula marismortuiHaloferax alignment were searched against all proteins in each SHT from the Haloarcula genomes using BLAST In order to determine phylogenetic distribution of haloarchaeal genes, a gene presence/absence matrix was constructed by the following process. Independent multi-genome alignments were made for the Hidden Markov Models (HMMs) were generated for each SHT using HMMER 3, resulting in 13,276 HMMs. The 1,303 completed archaeal and bacterial genomes available from NCBI as of March 15, 2011 were downloaded and a single genome from each genus selected at random, resulting in 396 genomes. Each SHT HMM was searched against these 396 genomes and the eight halophile genomes generated for this study using HMMER 3. Each gene was counted as belonging to the HMM if it had an E-value below 0.0001 and the hit covered greater than 80% of the length of both the gene and the HMM. If a gene hit more than one HMM it was counted only for the HMM with the best E-value. These hits were then used to generate a 13,276 x 405 presence/absence matrix. The genomes and HMMs were clustered using the ‘ctc’ library in R Three methods were used to determine the phylogenetic relationships among the newly sequenced haloarchaea. First, a tree was constructed using a set of twenty-eight highly conserved marker genes identified with Amphora Haloarcula and Haloferax clades were aligned using MUSCLE A second method was also used wherein the proteins within each of the 398 SHTs conserved across the seven newly-sequenced haloarchaeal genomes and the two previously sequenced representatives of the rpoB (in this case subunit rpoB’’) Haloarcula and Haloferax species were identical in these three trees, with the exception that polytomies were formed by the Har. californiae-sinaiiensis-marismortui and the Hfx. volcanii-sulfurifontis-denitrificans clades in the rpoB’’ tree. As only 36.8% of conserved genes supported the branching order shown for the Har. californiae-sinaiiensis-marismortui clade in the BUCKy and Amphora trees, it is not surprising that the rpoB’’ tree failed to resolve the branching order of these species. Presence/absence of particular genes of interest was determined by searching RAST-annotated GenBank files using in-house scripts, available at our website rpoB’’ tree.Thirdly, a tree was constructed based on the commonly used molecular marker Haloferax genomes (including a re-sequenced Hfx. volcanii DS2) was used to infer historical rates and patterns of segmental gain and loss using the GenoPlast software The multiple genome alignment of six Opsin homologs were gathered from the RAST-annotated GenBank files using in-house scripts available at our website Halalkalicoccus jeotgali possesses a predicted homolog with insignificant matches to both PCNA_N  and PCNA_C . The combined probability of finding both of these domains by chance is 0.000187, which is below the significance threshold. An alignment was constructed and phylogeny determined for PCNA homologs from the twenty-one sequenced haloarchaea, along with other euryarchaeota, crenarchaeota and eukaryotes using the Phylogeny.fr workflow Haloferax volcanii PCNA structure (PDB ID 3IFV) PCNA homologs were gathered from the RAST-annotated GenBank files using in-house scripts, available at our website CRISPRs were identified for the twenty-one haloarchaeal genomes included in this study using the PILER-CR CRISPR prediction program Protein sequences of the predicted general transcription factors (GTFs) TATA-binding protein (TBP) and transcription factor B (TFB) were extracted from the RAST-annotated GenBank files using in-house scripts, available at our website Halalkalicoccus jeotgali. Cells were grown to confluence, and washed twice with PBS. Several µL of cell suspension was smeared on a glass slide, air dried, heat fixed, stained with Nile Blue A for 10 min at 55 oC, washed with water and glacial acetic acid, stained with DAPI Gold Bond (R), covered with glass coverslip, dried overnight, and sealed with nail polish. Slides were visualized on a Leica DM600B compound microscope with a 100×objective. PHA and DNA were visualized using the Y3 and A4 filtercubes, respectively.Acid-hydrolyzed rice hull obtained from MicroMidas (Sacramento) was neutralized to pH ∼7 and inoculated with Figure S1Scaffold completeness in Haloferax mediterranei. Results of PCR-based test for circularization of scaffolds. Scaffold 2 (A), scaffold 4 (B), scaffold 5 (C), scaffold 3 (D), scaffold 1 (E), 2-log ladder (L). Approximate positions of PCR products from scaffolds B, C and D are marked along the Y axis. See (EPS)Click here for additional data file.Figure S2Halalkalicoccus jeotgali PHA stain. Polyhydroxyalkanoate (PHA) granules (red) and DNA (blue) in heat-fixed Halalkalicoccus jeotgali visualized with Nile Blue A and DAPI, respectively, under 100×objective.(EPS)Click here for additional data file.Figure S3Multi-genome alignment of Haloferax genomes. Whole genome alignment of Haloferax genomes with contigs re-ordered to reflect order of published Haloferax volcanii genome. Colored blocks are regions of predicted homology. Blocks lying below the center line in each panel are in reverse orientation with respect to Hfx. volcanii genome. Height of vertical bars within each colored block denote conservation within each homologous region.(PDF)Click here for additional data file.Figure S4Multi-genome alignment of Haloarcula genomes. Whole genome alignment of Haloarcula genomes with contigs re-ordered to reflect order of published Haloarcula marismortui genome. Colored blocks are regions of predicted homology. Blocks lying below the center line in each panel are in reverse orientation with respect to Hfx. marismortui genome. Height of vertical bars within each colored block denote conservation within each homologous region.(PDF)Click here for additional data file.Figure S5Presence/absence matrix of Syntenic Halophile Tribes (SHTs). Presence (red) and absence (black) of 13,276 hidden Markov models in 405 genomes including the eight genomes generated for this study as well as one genome randomly selected from each of the 396 genera in the NCBI genomes database. Matrix available as.cdt file through our website and as Dataset S1.(PNG)Click here for additional data file.Figure S6Amphora tree. Maximum likelihood tree based on twenty-eight highly conserved molecular marker genes identified by Amphora. Bootstrap support values over 0.50 shown for 500 bootstrap iterations.(EPS)Click here for additional data file.Figure S7BUCKy tree. Concordance tree constructed with Mr. Bayes from individual gene trees for all 398 SHTs conserved across Haloferax and Haloarcula species. Branch support values represent the percentage of individual gene trees for which each clade is observed.(PDF)Click here for additional data file.Table S1Primers for scaffold circularization experiment. Primer sequences for PCR-based test of scaffold completeness in Hfx. mediterranei.(XLSX)Click here for additional data file.Table S2COG enrichment. Percent CDSs assigned to COG groups in each species. Genera-wide means and P-values from Wilcoxon rank-sum test are shown.(XLSX)Click here for additional data file.Table S3Comparison of COG functional group enrichment with study of Konstantinidis et al. . P-values shown are from Wilcoxon rank-sum test.(XLSX)Click here for additional data file.Table S4List of genes differentially present in two Haloferax clades. Annotations associated with genes present in the Hfx. mucosum-Hfx. mediterranei clade but absent in the Hfx. volcanii-denitrificans-sulfurifontis clade according to the Syntenic Halophilic Tribes presence/absence matrix (Dataset S1).(XLSX)Click here for additional data file.Dataset S1Syntenic Halophilic Tribes matrix.(CDT)Click here for additional data file.Dataset S2Full alignment of Proliferating Cell Nuclear Antigen (PCNA) homologs. Untrimmed alignment of sixty-one PCNA homologs from fifty-seven archaeal and eukaryotic species constructed with MUSCLE.(TXT)Click here for additional data file.