Despite the disabling nature of eating disorders (EDs), many individuals with ED symptoms do not receive appropriate mental health care. Internet-based interventions have potential to reduce the unmet needs by providing easily accessible health care services.This study aimed to investigate the effectiveness of an Internet-based intervention for individuals with ED symptoms, called “Featback.” In addition, the added value of different intensities of therapist support was investigated.Participants (N=354) were aged 16 years or older with self-reported ED symptoms, including symptoms of anorexia nervosa, bulimia nervosa, and binge eating disorder. Participants were recruited via the website of Featback and the website of a Dutch pro-recovery–focused e-community for young women with ED problems. Participants were randomized to: (1) Featback, consisting of psychoeducation and a fully automated self-monitoring and feedback system, (2) Featback supplemented with low-intensity (weekly) digital therapist support, (3) Featback supplemented with high-intensity (3 times a week) digital therapist support, and (4) a waiting list control condition. Internet-administered self-report questionnaires were completed at baseline, post-intervention , and at 3- and 6-month follow-up. The primary outcome measure was ED psychopathology. Secondary outcome measures were symptoms of depression and anxiety, perseverative thinking, and ED-related quality of life. Statistical analyses were conducted according to an intent-to-treat approach using linear mixed models.The 3 Featback conditions were superior to a waiting list in reducing bulimic psychopathology =−0.31 to −0.01), symptoms of depression and anxiety , and perseverative thinking . No added value of therapist support was found in terms of symptom reduction although participants who received therapist support were significantly more satisfied with the intervention than those who did not receive supplemental therapist support. No significant differences between the Featback conditions supplemented with low- and high-intensity therapist support were found regarding the effectiveness and satisfaction with the intervention.The fully automated Internet-based self-monitoring and feedback intervention Featback was effective in reducing ED and comorbid psychopathology. Supplemental therapist support enhanced satisfaction with the intervention but did not increase its effectiveness. Automated interventions such as Featback can provide widely disseminable and easily accessible care. Such interventions could be incorporated within a stepped-care approach in the treatment of EDs and help to bridge the gap between mental disorders and mental health care services.Netherlands Trial Registry: NTR3646; http://www.trialregister.nl/trialreg/admin/ rctview.asp?TC=3646 (Archived by WebCite at http://www.webcitation.org/6fgHTGKHE) Eating disorders (EDs) are serious psychiatric disorders characterized by high rates of comorbidity, chronicity, mortality, and relapse -5. UnforNumerous Internet-based interventions for the prevention and treatment of ED have shown promising results -10. The Internet-based interventions can include many different components and can be provided with or without therapist support. In the field of depression and anxiety, it has been found that Internet-based interventions with therapist support were more effective than those without or those with only minimal therapeutic contact ,15. DireIn addition to the intensity of therapist support, another important factor is the way in which such support is provided. Tate et al  investigThis study evaluated self-help intervention “Featback” for individuals with ED symptoms. Featback comprises psychoeducation and a fully automated self-monitoring and feedback system. Self-monitoring is an important clinical technique that is often used in cognitive behavioral therapy , where iThe first aim of this study was to investigate the effectiveness of Featback in reducing ED psychopathology and comorbid symptoms. The second aim was to investigate the added value of therapist support and different intensities of therapist support. A randomized controlled trial was conducted comparing 4 conditions: (1) Internet-based intervention Featback, consisting of psychoeducation and a fully automated monitoring and feedback system, (2) Featback supplemented with low-intensity (weekly) therapist support, (3) Featback supplemented with high-intensity (3 times a week) therapist support, and (4) a waiting list control (WLC).This study was a 4-arm randomized control trial. Ethical approval was obtained from the Leiden University Medical Center Ethics Committee. This committee granted exemption for parental consent for individuals aged between 16 and 18 years of age. Detailed information on the study methods, including the design, intervention conditions, measures, and ethical precautions and crisis management, can be found in the published study protocol .Participants were recruited via the website of Featback  and the After Web-based completion of informed consent and the screening questionnaire including questions regarding the eligibility criteria, participants were invited to complete the baseline questionnaire. Thereafter, participants were randomly assigned to 1 of the 4 study conditions with a block size of 40 and an equal allocation ratio (1:1:1:1). An independent researcher who had no involvement in any other aspect of this study conducted the randomization allocation by means of computer-generated random numbers created in SPSS. She concealed the allocation sequence in a password-protected computer file from the main researchers until interventions were assigned, preventing researchers from having any prior knowledge of the upcoming condition assignments. Importantly, therapists were alternately assigned to low- versus high-intensity therapist support.All participants had access to the Featback website where comprehensive and general information on ED could be found , for example, the types of EDs and symptoms, risk factors, causes, and comorbid problems. This information served primarily to educate participants about EDs and stimulate recognition and acknowledgement. The psychoeducation was purely self-guided, meaning that participants were free in choosing when and what to read. The monitoring and feedback system comprised a weekly invitation by email to complete a monitoring questionnaire. This questionnaire consisted of 8 4-point Likert items assessing cognitive and behavioral correlates of the following 4 dimensions: (1) body dissatisfaction, (2) excessive concerns with body weight and shape, (3) unbalanced nutrition and dieting, and (4) binge eating and compensatory behaviors. After completion, an algorithm determines the patterns of change of each of these 4 dimensions: still in the functional or healthy range, still in the dysfunctional or unhealthy range, improvement from the dysfunctional to the functional range, or deterioration from the functional to the dysfunctional range. The 4 different patterns of change with respect to the 4 dimensions of ED symptoms result in 4×4×4×4= 256 possible scenarios regarding a participant’s status. For each possible scenario, 10 to 15 different feedback messages were preformulated in a database. After determining the status of a participant, the algorithm randomly selected 1 tailored feedback message out of this database and sent this to the participant accordingly. Hence, when a participant’s status does not change over time, one would not receive the same message over and over again. All the feedback messages contained social support by expressing interest in and concerns about the participants’ well-being. Positive reinforcement techniques such as encouragement were used to stimulate and maintain healthy behaviors and attitudes. Furthermore, the messages included tips and advice on how to counteract negative developments in reported ED-related symptoms. The following is an example of a feedback message, which could be sent to someone with dysfunctional overconcerns with body weight and shape, unbalanced nutrition and dieting , as well as deteriorations in body dissatisfaction and symptoms of binge eating and compensatory behaviors:We are concerned with the changes in your body image and eating behaviors, however, we know that you have the ability to make healthy changes. Your body image and eating habits are closely linked. This week, try to eat regular, well-balanced meals and snacks, which might help to prevent the binge eating and/or compensatory behaviors and help you to feel better. If you continue to have negative thoughts about your body, it may be helpful for you to talk to someone about it, maybe a family member? Or a friend? Take care!The fully automated self-monitoring and feedback system was developed in Germany, and for more detailed information on this system, see the study by Bauer et al . A reminParticipants received Featback as described previously supplemented with low-intensity (weekly) therapist support by means of email, chat and/or audio teleconference . Participants could schedule support sessions in a Web-based agenda where available time slots of the therapist were presented. For each support session, participants could choose their preferred medium of support. Therapists were instructed to send an email to participants in case they did not schedule any support session(s) or in case they did not show up at scheduled support session(s) and to repeat this process twice per nonresponse. Chat and teleconference sessions had a maximum duration of 20 minutes, whereas an email session contained 1 email reply from the therapist to the participant. The therapist support was independent of the monitoring and feedback system. The chat methodology was based on a 5-phase model: (1) a warm welcome, (2) clarifying the question, (3) determining the goal of the conversation, (4) concrete elaboration of the goal of the conversation, and (5) closing the circle . The emaParticipants received Featback, supplemented with high-intensity (3 times a week) therapist support by means of email, chat, and/or teleconference as described previously.Participants were placed on a waiting list for 5 months, after which they were offered Featback with low-intensity therapist support.In all 4 intervention conditions, participants were free to undergo any other type of intervention or treatment .The therapists were 7 females who were either Master of Science students in clinical psychology or individuals with a master’s degree in clinical psychology. All therapists underwent training in the delivery and methodology of Internet-based support. Furthermore, they received extensive information on EDs and practiced with case material and expert patients  before the start of the trial. Monthly face-to-face supervision sessions were organized by the main researcher (JA), a psychologist (MN), and an experienced psychotherapist (EvF) as a matter of routine professional and ethical care, as well as to reinforce adherence to the protocol. In addition, 2 individual supervision sessions were provided to all therapists during their first month. Thereafter, therapists’ adherence to the protocol was regularly checked at random, by checking whether the chats and emails included the 5- and 3-phase model, respectively.All data were collected by means of Internet-administered self-report questionnaires at baseline, post-intervention (8 weeks after baseline), and at 3- and 6-month follow-up. Waiting list participants were offered Featback with low-intensity therapist support after the 3-month follow-up and were not assessed at 6-month follow-up.The primary outcome measure was ED psychopathology as measured by the SEED  and the Secondary outcome measures included ED-related quality of life as assessed by the ED-Related Quality of Life Questionnaire (ED-QOL), a validated 25-item questionnaire assessing the influence of eating behaviors and body weight in the psychological, physical and cognitive, financial and work- or school-related domain . The ED-Given that participants were free to undergo any other type of intervention, psychological health care service utilization  was assessed with the Trimbos/iMTA Questionnaire for Costs Associated with Psychiatric Illness: TiC-P) . User saAll data were analyzed in SPSS version 22 using 2-tailed tests and α=.05. A target sample size of 344 participants was calculated by the software program Power Analysis and Sample Size version 8.0 (2008) to yield 80% power to detect an expected between-group (pooled Featback conditions vs WLC) difference at post-intervention with an effect size of 0.3, α=.05, and an expected dropout rate of 30%  were calculated by dividing the unstandardized coefficients of interaction effects (time × condition) by the pooled within-group standard deviation (SD) of the outcome measure at baseline . The resχ2(3)=4.35, P=.23) and 6-month follow-up (χ2(2)=2.87, P=.24), although at 3-month follow-up, the WLC participants dropped out of the study less often than participants who received Featback without or with low-intensity therapist support (χ2(3)=15.69, P=.001). No differences in non-usage attrition were found among the 3 Featback conditions (χ2(2)=5.24, P=.07).Participants were recruited between November 7, 2012 and June 17, 2013. Follow-up was completed at March 3, 2014. P=.84). One hundred participants (40.2%) did not receive any psychological health care appointments during this period, whereas 149 participants (59.8%) did have such appointments (range 1-40).Baseline characteristics of participants are summarized in χ2(15)=19.33, P=.20).Participants in this study demonstrated severe levels of ED psychopathology: their EDE-Q scores were comparable to the overall norm for treatment-seeking patients with an ED in our clinical program . The meaP=.258). Participants in the 2 Featback conditions with therapist support received a total of 1407 support sessions, with email being the most popular medium , followed by chat  and teleconference . These proportions of email =−1.63, P=.11), chat =1.42, P=.16), and teleconference =0.53, P=.59) were similar for the 2 study conditions. The mean number of received therapist support sessions differed significantly between Featback with low- and high-intensity therapist support (t (175)=8.24, P<.001): participants in the former condition received on average 4.7  sessions, whereas participants in the latter condition received on average 11.2  sessions. Thus, we successfully created 2 different intervention conditions regarding the intensity of therapist support.Participants in the 3 Featback conditions completed a mean number of 5.6  of 8 weekly monitoring questionnaires, with no significant difference between the conditions =1.36, The outcome data for each of the 4 conditions over time can be found in In statistical models 2 and 3, we compared the intervention conditions and thus investigated the added value of therapist support, and higher versus lower intensities of therapist support, respectively. As shown in P values ≤.01). When comparing Featback without therapist support with the pooled Featback conditions with therapist support , the results demonstrated no significant differences between the conditions over time  and from post-intervention to 6-month follow-up  than participants who received Featback with low-intensity therapist support. This finding should be interpreted with caution because participants who received Featback without therapist support scored in between and thereby not significantly different from the 2 Featback conditions with therapist support  with respect to bulimic psychopathology, global ED psychopathology, ED-related quality of life, symptoms of depression and anxiety, and levels of perseverative thinking . Participants who received Featback without therapist support were significantly less satisfied  than participants who received Featback with low-  or high-intensity therapist support , whereas no differences between the latter 2 were found. Overall, participants were very satisfied with the therapist support , with no significant differences between the low- and high-intensity therapist support conditions = −0.34, P=.74). In addition, no significant differences in satisfaction with the different therapists were found =0.36, P=.902).Regarding participants’ experiences, significant differences in participants’ level of satisfaction with Featback were found =38.41, A total of 158 participants provided negative feedback, and 160 participants provided positive feedback to the open-ended questions regarding their satisfaction with the intervention. Participants’ most reported critical comments included statements about the limitations of the automated feedback , for example, it being too general or impersonal, as well as the lack of more personal or individual therapist support. Most of the positive comments  included complementary remarks regarding the individual therapist support, such as participants having received good advice and support, having enjoyed the empathy, warmth, and attention of the therapists, as well as the feeling that someone was looking after them. Approximately one third  of all positive comments included positive feedback on this system, for example, experiencing the system as a good checkup supporting moments of reflection. No adverse effects from Featback were reported.To our knowledge, this is the first randomized controlled trial to investigate an Internet-based fully automated self-monitoring and feedback intervention (Featback) and the added value of 2 different intensities of therapist support for individuals with ED psychopathology. The results demonstrated Featback to be superior to a WLC in reducing bulimic psychopathology , perseverative thinking, and symptoms of depression and anxiety. Thus, self-monitoring of ED-related attitudes and behaviors and receiving feedback by means of an automatic system can be effective in reducing psychopathology. No effects were found regarding anorectic psychopathology; hence, Featback may be more suitable for individuals with bulimic psychopathology. Interestingly, when comparing Featback with and without therapist support, no added value was found for therapist support in terms of the effectiveness of the intervention, although participants who received Featback with therapist support were significantly more satisfied.other areas in the field of psychiatry. Indeed, a recent study [Our findings add to the growing body of literature indicating the potential of ehealth interventions for individuals with (ED) psychopathology ,12,13,44nt study  demonstrThe finding that Featback was equally effective with and without therapist support is in line with that of several previous studies -48, howeIncreasing the frequency of therapist support did not significantly affect outcome, which is in line with the results of a study that experimentally investigated different intensities of therapist support in an Internet-based treatment for panic disorder . More frInterestingly, our results show a discrepancy between the added value of therapist support in terms of effectiveness  and satisfaction with the intervention . The fact that therapist support did increase the satisfaction of participants significantly might well be due to the empathy, warmth, and attention of the therapists. Individuals with ED are often ashamed about their ED and can feel isolated and unsupported, as well as misunderstood by their personal environment . AlthougAdding therapist support did not enhance study adherence because no differences between study dropout rates were found between the 3 Featback conditions. However, our results showed that at 3-month follow-up, participants in the waiting list condition dropped out less often than participants who received Featback without or with low-intensity therapist support. Presumably, participants in the waiting list condition were more motivated to complete the study questionnaires given their knowledge that they would receive Featback with low-intensity therapist support after completing this follow-up questionnaire.It is noteworthy that Featback produced significant reductions in psychopathology over and above usual care. Participants’ treatment status (yes or no) or number of received psychological health care appointments during the intervention period did not significantly differ between the study conditions and could furthermore not account for the superiority of Featback in comparison to WLC when entered as a predictor in the model. This suggests minimal self-help interventions such as Featback to be of interest for a broad population of individuals with ED symptoms. The small effect sizes match our expectations, given the type of intervention  and the fact that most participants received psychological health care during the intervention period. Interventions such as Featback could be incorporated within regular treatment settings , where it would enable accurate monitoring of patients’ well-being in treatment settings and in their everyday lives ,50. AlsoThis study has several strengths and limitations. Strengths include the large sample size, randomized controlled design, intent-to-treat analyses, and the use of multiple imputation methods as these have shown improved performance over alternative approaches such as complete case analysis or single imputation methods . LimitatIn conclusion, an Internet-based fully automated monitoring and feedback intervention was effective in reducing psychopathology and is an interesting means of providing care for individuals with ED symptoms. Supplemental therapist support enhanced satisfaction with the intervention but did not increase its effectiveness. An interesting next step is to economically evaluate Featback with and without therapist support to determine its cost-effectiveness in comparison to a waiting list. Also, examining potential predictors, moderators, and mediators of intervention response will help to inform the field regarding for whom and how Featback work(s). A final topic for future investigation is a focus on opening the black box of therapeutic support in Internet-based interventions: what do therapists actually do when providing Web-based support and can their behavior be linked to the effectiveness of such interventions?


Pleurocapsa-like cyanobacteria at microscale. Multivariate statistical analyses showed a strong positive correlation of Pleurocapsales and Chroococcales with aragonite formation at macroscale, and suggest a potential causal link. Despite the previous identification of intracellularly calcifying cyanobacteria in Alchichica microbialites, most carbonate precipitation seems extracellular in this system.Cyanobacteria are thought to play a key role in carbonate formation due to their metabolic activity, but other organisms carrying out oxygenic photosynthesis (photosynthetic eukaryotes) or other metabolisms , may also contribute to carbonate formation. To obtain more quantitative information than that provided by more classical PCR-dependent methods, we studied the microbial diversity of microbialites from the Alchichica crater lake (Mexico) by mining for 16S/18S rRNA genes in metagenomes obtained by direct sequencing of environmental DNA. We studied samples collected at the Western (AL-W) and Northern (AL-N) shores of the lake and, at the latter site, along a depth gradient . The associated microbial communities were mainly composed of bacteria, most of which seemed heterotrophic, whereas archaea were negligible. Eukaryotes composed a relatively minor fraction dominated by photosynthetic lineages, diatoms in AL-W, influenced by Si-rich seepage waters, and green algae in AL-N samples. Members of the Gammaproteobacteria and Alphaproteobacteria classes of Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacterial taxa, followed by Planctomycetes, Deltaproteobacteria (Proteobacteria), Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi. Community composition varied among sites and with depth. Although cyanobacteria were the most important bacterial group contributing to the carbonate precipitation potential, photosynthetic eukaryotes, anoxygenic photosynthesizers and sulfate reducers were also very abundant. Cyanobacteria affiliated to Pleurocapsales largely increased with depth. Scanning electron microscopy (SEM) observations showed considerable areas of aragonite-encrusted  Calcifying microbial mat communities may result in the formation of microbialites . These cin situ precipitation of minerals, mostly carbonates, due to microbial activity. Whereas the accretion of particles, essentially by filamentous cyanobacteria (2+ or Mg2+) and the presence of nucleation centers , the discovery of anoxygenic photosynthesis in Chloroflexi, also present in calcifying mats and stromatolites, revealed the importance of other photosynthesizers in these systems . Today, However, even if recent molecular analyses based on amplicon sequencing provide qualitative information about the diversity of archaea, bacteria and eukaryotes, the relative inter-domain proportions remain essentially unknown because primer pairs used to amplify 18S and 16S rRNA genes are generally domain-specific. Moreover, in addition to common biases that affect all kind of molecular analyses, such as differential cell lysis, DNA purification yield or gene copy number, classical 16S/18S rDNA amplification and cloning strategies are affected by both PCR-  and clon5(CO3)4(OH)2.4(H2O)] and aragonite (CaCO3),  crater lake located at high altitude , which harbors conspicuous microbialites consisting essentially of hydromagnesite  and aragonite (CaCO3). In addition, we were able to identify hydrotalcite [(Mg 0.667 Al0.333)(OH)2 (CO3)0.167 (H2O)0.5] and illite (K4Al16Si8O48) as minor phases in some samples . Moreover, based on scanning electron microscopy (SEM) analyses  and the present chemical analyses, we considered an additional silicate phase with a talc-like composition: Mg3Si4O10(OH)2. Based on this mineralogical assemblage and bulk chemical analyses of the samples, we could calculate the proportion of these three different phases. SEM observations of ultrathin sections coated with carbon were done in angle selected backscattered electron mode using a Zeiss Ultra 55 FEG-SEM operating at 15 kV at a working distance of 7.5 mm.Concentrations of major elements, total organic carbon, and total sulfur were determined at the TM DNA Isolation Kit  according to manufacturer’s instructions. To facilitate the lysis of cells within the carbonate matrix and limit DNA adsorption to carbonate, which seriously diminish DNA yield , to remove residual EPS. Because of the limiting amount of genomic DNA for AL-N samples, one paired-end Illumina library was prepared for AL-N-1 to 15. Three different libraries were prepared for the AL-W sample, which can be considered as some kind of technical triplicates. Two of them (AL-W-1a and AL-W-1b) were prepared from DNA fragments that had undergone a purification step to select for DNA fragments in the 150–250 bp size-range. AL-W-1a and AL-W-1b differed in the starting DNA amount used to construct the paired-end Illumina libraries . The AL-W-1c paired-end Illumina library was constructed, as the four AL-N libraries, using a classical protocol with DNA fragments ranging from 100 to 500 bp in length. DNA from the seven metagenomic libraries was sequenced in two independent runs (AL-N and AL-W samples) using Illumina HiSeq2000 v3 (2x100 bp paired-end reads) by Beckman Coulter Genomics . The total number of paired end reads ranged from 78 to 131 millions, i.e., 7.8–13.1 Gbp per library and orientation .DNA extraction took place immediately upon return. Microbialite grains were ground using a sterile agate mortar and the material that was not immediately used for DNA purification stored at –20°C. DNA purification from individual microbialite powder aliquots of 0.2 g was carried out using the Power BiofilmNA yield , we prevNA yield . Briefly1, release 108. Although it has been shown that 16S rDNA amplicons as short as 100 bp are sufficient for accurate affiliation to high ranking taxonomic groups . Sequences from a cluster were discarded if the coverage of their representative sequence with its best hit was lower than 95%. Similarly, singletons having a coverage lower than 95% and a sequence identity lower than 98% with their best hit were discarded. In general, because of the short length of the sequences retained (130–190 bp), taxonomic affiliation was considered reliable only above the Order level. The clustering process was only used to affiliate sequences to taxa; for quantitative analysis of relative abundances, we subsequently considered the number of sequences included in the clusters. Chloroplast sequences were considered independently. Statistics regarding the total number of reads and paired-end sequences per sample, the number of predicted 16S/18S rRNA genes and the average sequence lengths retained are presented in Table 2. The sequences analyzed in this work have been deposited in GenBank under the BioProject number PRJNA286185.For each library, forward and reverse reads of a same DNA fragment (paired-end sequences) were assembled using the software FLASH  with defc groups , we applroups  was applied to test whether microbial diversity was significantly correlated to the environmental variables. Mantel tests were done using the ‘Vegan’ package. Canonical Correspondence Analyses (CCA) including cyanobacterial order frequencies and mineral components were calculated with the ‘Ade4’ package.Statistical analyses were carried out using the R software3 . Non-met3  between 3  implemenFigure 1). The AL-N series corresponded to four microbialite fragments collected along a depth gradient from 1 to 15 m depth. We chose this site to be able to compare our high-throughput and more quantitative metagenome-based diversity results with those of a previous study based on PCR amplification, cloning, and Sanger sequencing analysis conducted in the same site and at similar depths , the microbial biofilm that covered its surface was less mineralized and had a more glutinous aspect . This resulted in a much higher DNA yield for AL-W, which allowed us to construct three independent genomic libraries for Illumina sequencing that we subsequently treated as technical replicates. Two of them, AL-W-1a and AL-W-1b, were constructed from size-fractionated DNA fragments comprised between 150 and 250 bp; AL-W-1c corresponded to a genomic library without DNA size fractionation.We searched for 16S/18S rRNA gene sequences in seven metagenomic datasets produced in parallel from a total of five microbialite samples collected at two sites in the Northern and Western lake shores (Table 2). In the case of AL-W-1c and the AL-N series, between 22 and 42% of these reads could be merged in paired end sequences applying strict overlapping criteria , which produced sequences of 150 bp on average. Unsurprisingly, libraries of size-selected DNA fragments (AL-W-1a and AL-W-1b) yielded much higher merging rates for paired end reads (∼95%). The merged paired-end datasets thus obtained for the different samples were mined for 16/18S rRNA genes belonging to the three domains of life. The proportion of predicted rDNAs was comparable among samples, ranging from 0.02 to 0.06%, which represented, despite the use of stringent quality filters, as many as 13,533–35,038 16S/18S rRNA gene sequences (Table 2).Sequencing resulted in 78–131 million paired-end (forward and reverse) reads for each sample (Table 2). Comparing the phylogenetic affiliation and relative abundance of clustered sequences and singletons should reveal if patterns of phylogenetic diversity differed significantly between high and low abundant organisms. However, the distribution of relative proportions of singletons and of more abundant sequences among major bacterial and eukaryotic taxa were remarkably similar . We then chose the longest sequence as representative of the whole cluster and BLAST it against public databases to get a more accurate taxonomic affiliation of the cluster. However, for subsequent quantitative analysis, only the number of individual sequences was taken into account. Individual sequences that did not match any other sequence in each of our metagenomic datasets were analyzed independently. In the following, we refer to these unique sequences as ‘singletons’ by analogy with unique amplified sequences in amplicon studies. However, contrary to singletons in amplicon studies, a portion of which corresponds to sequencing artifacts or PCR chimeras, metagenomic singletons are unlikely to be artifacts and likely reflect lower abundances of the corresponding taxa. Metagenomic singletons accounted for ca. 5–15% of the predicted 16S/18S rRNA genes in our datasets . These values are comparable to those found in metagenomic datasets from Cuatro Ciénegas  and will not be commented further. Both Thaumarchaeota and Euryarchaeota sequences were detected in all samples, except AL-N-10 and AL-N-15 where only Thaumarchaeota were present (data not shown). This corroborated the presence of the two archaeal lineages previously detected . The eukaryotic diversity covered a large variety of high-rank taxa in all the samples, but was numerically dominated by photosynthetic taxa that contributed between 75 and 95% of the microbial eukaryotic sequences. However, whereas AL-W metagenomes were dominated by diatom sequences , samples from the AL-N vertical profile were all dominated by green algal sequences . This radical difference between the two sites does not appear related to the mineral composition of the substrate (Table 1) but may be related to the local hydrochemistry, which seems locally influenced by seepage activity and perhaps also by occasional spring discharge at the steepest part of the crater. Indeed, the chemical analysis of seepage water collected at this site in 2007 revealed much less saline  but far more Si-rich water. Si was on average 40 times more concentrated (694 μmol/l) than the rest of the water lake . In addition to diatoms and green algae, likely photosynthetic euglenids  were present in the two sites, having a noticeable percentage (6–8%) in the AL-W site. The heterotrophic component of the microbial eukaryotic community was more varied and encompassed protist grazers  and fungi. Some apicomplexan sequences were identified, which might correspond to parasites . The analysis of singletons revealed the presence of some groups not detected in high abundance; for instance, haptophytes were identified in AL-N-1, AL-N-10, and AL-N-15 . Although in varying proportions, Cyanobacteria, Gammaproteobacteria and Alphaproteobacteria, and Bacteroidetes were generally the most abundant groups, collectively accounting for up to 70 or 80% of all bacterial sequences, depending on the metagenome. Gammaproteobacteria were nevertheless much less abundant in AL-W samples  as compared to the AL-N samples, where they accounted for 20% up to more than 50%. Bacteroidetes, on the contrary, were more abundant in the AL-W samples, accounting for 20% of sequences. Cyanobacteria were less abundant in surface microbialites (around 10%), but increased their proportions to 15–30% in deeper samples. Other relatively abundant groups (5–10% sequences) comprised the Planctomycetes, Deltaproteobacteria, Chloroflexi, Verrucomicrobia and, in the AL-N samples, also the Gram positive Actinobacteria and, in the deeper sample AL-N-15, Firmicutes. Many other phyla and candidate divisions were detected in minor amounts. The phylogenetic distribution of singletons was consistent with that of more abundant sequences . AL-W samples were largely dominated by filamentous cyanobacteria of the order Oscillatoriales, followed by Nostocales. However, in the AL-N samples, although AL-N-1 was also dominated by Oscillatoriales, their proportion clearly diminished with depth. On the contrary, members of the Pleurocapsales were much more abundant in the 15 m deep sample, shifting from 20% to almost 80% of the cyanobacterial 16S rRNA gene sequences identified. Therefore, our more quantitative metagenome-based analyses clearly confirm the trend previously observed  and Gammaproteobacteria    . Similarrosinus)  are sulfFigure 4). This implies that the remaining rRNA gene sequences  corresponded to non-photosynthetic organisms, most of which are likely heterotrophs carrying out aerobic or anaerobic respiration or fermentation. Their metabolic activity would tend to counterbalance the carbonate-promoting action of photosynthesis and sulfate reduction , photosynthetic eukaryotes accounted for higher proportions among the organisms with carbonate precipitation potential, reaching up values of 20 to 50% in some samples . Although anoxygenic photosynthesizers varied, they reached similar relatively high (∼15%) proportions in AL-W and all AL-N samples but AL-N-15, where they were less abundant. Finally, sulfate reduction, although relatively minor in all the microbialite fragments analyzed, was relatively abundant in AL-N-1, ca. 20%. The higher proportion of 16S rRNA genes affiliated to anoxygenic phototrophs and sulfate reducers in this sample might in principle suggest that the sampled microbialite fragment  contained larger portion of anoxic zones than the other microbialite fragments. However, there might be alternative or additional explanations not related to global anoxia, since typically anaerobic sulfate reducers have been conspicuously observed to be abundant and very active in oxic areas of microbial mats . This suggests that the potential diatom input to the microbialite silicon content  is not different from that of the rest of microbialites in the lake or, alternatively, that Si in the microbialites has not a diatom origin. On the contrary, the AL-N samples were strongly mineralized with many cyanobacteria and green algae intimately linked with the mineral substrate. This suggests that at least part of this diversity may be directly involved in biomineralization.Nonetheless, oxygenic photosynthesis seems prevalent in modern Alchichica microbialites. Overall two different kinds of patterns were observed, with eukaryotic photosynthesis dominating AL-W and cyanobacterial photosynthesis dominating AL-N samples. Around 50% of the 18S rRNA genes belonging to taxa with carbonate-induction potential corresponded to photosynthetic eukaryotes, essentially diatoms, in AL-W. In this sample, the number of chloroplast 16S rRNAs as compared with the nuclear 18S rRNA genes of photosynthetic eukaryotes was higher, suggesting more chloroplasts per cell than in the AL-N samples . First, ordination analyses of bacterial plus eukaryotic community composition at the level of major taxa  using NMDS clearly separated the different samples according to their location and depth . As expected, the three AL-W replicate samples clustered together, and were segregated from the AL-N samples along the NMDS1 axis. AL-N samples were discriminated along the second axis (NMDS2) according to depth. Similar results were observed when eukaryotes and prokaryotes were analyzed separately (data not shown).We looked for potential correlations between the community composition and the local physico-chemical parameters measured, essentially depth and the chemical composition of the microbialite fragments that we analyzed in parallel . Remarkably, Mantel tests revealed a significant correlation between depth and overall microbial community composition . Even if the number of points is limited at this stage, these results indicate that at least depth is an important determinant of community structure. However, it is likely that microbial community structure depends also on other local abiotic parameters. For instance, the influence of seepage water on local hydrochemistry  and the chemical composition of the microbialites , we looked for potential correlations between the chemical and mineral composition of microbialites and the cyanobacterial taxa identified. CCA revealed relationships between some cyanobacterial orders and particular chemical parameters. Pleurocapsales and Chroococcales strongly and positively correlated with the calcium content of microbialites  and, therefore, with the only mineral phase carrying Ca in these microbialites, i.e., aragonite . On the opposite trend, the Nostocales correlated with magnesium and its associated mineral in modern Alchichica microbialites, hydromagnesite . Prochlorales and Oscillatoriales did not show any particular correlation with the chemical/mineralogical composition. The latter is not surprising. Prochlorales are typically small planktonic cyanobacteria, particularly well studied in oceans , suggesting a contamination from the surrounding lake plankton. Oscillatoriales, on the contrary, were the dominant cyanobacteria in surface samples from the AL-W and AL-N sites although their global presence is limited since, in those samples, cyanobacterial genes accounted for less than 10% of the total bacterial 16S rRNA genes . Oscillatoriales are typical filamentous cyanobacteria that live on the surface of the microbialites and often depend on high light intensities , indicating a very strong correlation and arguing in favor of causality. Previous studies at microscale have shown a specific connection between members of the Pleurocapsales and aragonite using a variety of microscopy and spectroscopy techniques . Our CCA analysis based on metagenome-inferred diversity and the mineral composition of microbialites show that same trend at macroscale, reinforcing the idea that cyanobacteria belonging to the Pleurocapsales are indeed responsible for the biomineralization of aragonite in Alchichica microbialites. Interestingly, Chroococcales showed a similar positive correlation with aragonite. Since members of the Chroococcales are coccoid cyanobacteria endowed with thick mucilaginous sheaths, properties shared with the Pleurocapsales, and are often associated with microbialites and microbial mats in extreme environments , which induce aragonite formation extracellularly before being filled by aragonite post-mortem , it seembialites . Howeverbialites  and despbialites .Pleurocapsa-like cells  will allow testing whether our hypothesis of a causal link holds true. In addition, we also hypothesize that Chroococcales, less abundant than Pleurocapsales but following similar trends, may be involved in aragonite biomineralization. By contrast, despite the previous detection of intracellularly calcifying cyanobacteria in Alchichica microbialites, their proportion in our metagenome-based diversity data is negligible, suggesting that extracellular precipitation is the major source of biomineralization in these structures.In this manuscript, we make a series of observations about the diversity of different taxa associated to living microbialites based on metagenome sequence data and try to infer potential players in carbonate precipitation. Function cannot be directly inferred from metagenomic sequence data without making a series of assumptions that are reasonable (based on known diagnostic metabolisms for specific lineages) but that may be affected by many different biases that are common to all molecular- and metagenomic-based studies . This cautionary note implies that our metagenome-based diversity data allow postulating functional hypotheses that need to be validated by complementary approaches. In our case, by mining metagenomes generated by direct high-throughput Illumina sequencing of microbialites from the Alchichica lake collected at two distinct sites and at different depths in search of 16S and 18S rRNA gene sequences, we have been able to establish a more quantitative and deeper estimation of the microbial diversity associated to these structures. Our results confirm and extend previous observations based on classical gene amplification, cloning, and sequencing , which rke cells , (ii) obThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine the vision-related quality of life (VR-QOL) after surgery for macula-off rhegmatogenous retinal detachment (RRD) in relation to visual acuity, contrast acuity, and color vision.In a prospective observational study, we included 55 patients with a macula-off RRD. Best corrected visual acuity (BCVA), color vision (saturated and desaturated color confusion indices (CCI)) and contrast acuity were measured at 12 months postoperatively in both the RRD eye and the fellow control eye, and the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25) was filled out.Operated and fellow control eyes differed significantly in mean LogMAR BCVA (P<0.0001), median Log contrast acuity (P<0.0001), saturated CCI (P = 0.009), and desaturated CCI (P = 0.016). Significant correlations were observed between the NEI VFQ-25 overall composite score and postoperative LogMAR BCVA , contrast acuity , saturated CCI , and desaturated CCI .A lower VR-QOL was highly correlated to a worse postoperative BCVA and contrast acuity and to a lesser extent to color vision disturbances. Rhegmatogenous retinal detachment (RRD), which refers to a detachment of the neurosensory retina from the underlying retinal pigment epithelium due to a defect in the retina Not only best corrected visual acuity (BCVA) is compromised in these cases, other aspects of central visual function are also compromised after macula-off RRD Previous studies identified a strong relation between postoperative visual function and post-operative vision-related quality of life (VR-QOL) as measured by the National Eye Institute Visual Functioning Questionnaire-25 (NEI VFQ-25) in patients operated on for various vitreoretinal disorders, including RRD We could not find previous studies addressing post-operative quality of life specifically in macula-off RRD patients in relation to BCVA, contrast acuity and color vision. Therefore, the purpose of the present study is to determine the postoperative VR-QOL after macula-off RRD one year after successful reattachment of the retina, and to assess which aspects of postoperative visual function  are most closely related herewith. In addition, we evaluated whether pre-operative, intra-operative, and postoperative factors are associated with a difference in postoperative VR-QOL.We conducted a prospective observational study in patients with a first presentation of macula-off RRD who had an attached retina at 12 months after the first surgical procedure. Reattachment was obtained by one or more surgical procedures. The research protocol was approved by the University Medical Center Groningen (UMCG) review board ethics committee, and was carried out in accordance with the tenets of the declaration of Helsinki. The study was registered with the Dutch Trial Register (NTR839). All patients were operated on at the ophthalmology department of the UMCG. The study was carried out over a three-year period .Adult patients visiting the ophthalmology department of the UMCG with a first presentation of unilateral macula-off RRD of 24 hours to 6 weeks duration were invited to participate in this study. Included in the study were patients of 18 years and older who had given their written informed consent. Patients had to be able to pinpoint their drop in VA to a specific 24-hour period in case of a 24-hour to 1 week macular detachment, and to a period of less than one week in case of a macular detachment of one to six weeks. The cut-off point of ≤1 week or >1 week is conform the available literature We acquired the following patients' characteristics: age, gender, affected eye, ophthalmic history and family history for RRD. In addition, we scored the number of retinal quadrants detached at presentation, and the presence, and grade of PVR Duration and height of macular detachment were determined using the following scoring system. Macula-off RRDs of less than one week duration were scored per day, and of more than one week duration they were scored as 11 days (1–2 weeks duration), 18 days (2–3 weeks duration), 25 days (3–4 weeks duration), 32 days (4–5 weeks duration), and 39 days (5–6 weeks duration), respectively.To measure the height of the detachment at the position of the central macula by ultrasonography the relative positions of the central macula and the optic nerve head were determined before performing a ultrasonography. For this purpose, digital fundus photographs of both eyes were made using the TRC-50 IX fundus camera (Topcon 9B ltd. UK). On both fundus photographs, the distance between the optic nerve head and fovea was measured using the software package IMAGEnet2000 2.53. The measured distance in the affected eye was used to determine the central position of the macula and at this position the height of the macular detachment was measured by ultrasonography. In those cases , in which the measurement of the distance between the macula and optic nerve head could not be performed on the photograph of the affected eye, the measurement of this distance in the fellow eye was used Based on clinical presentation, patients were either operated on by an external procedure (i.e. encircling band and/or buckle) or by 20 Gauge PPV (with or without an encircling band). In PPV cases, either a short acting tamponade (i.e. sulfur hexafluoride gas (SF6)) or a long acting tamponade was used (i.e. octafluoropropane (C3F8) or silicone oil). Collected data refer to the first surgical procedure in all cases.At 12 months postoperatively, we measured BCVA using the ETDRS chart At 12 months postoperatively, patients were requested to self-administer the validated Dutch version Because of an increased risk of cataract development after PPV, which may influence postoperative measurements, we scored the level of cataract using the lens opacities classification system III (LOCS III) in both eyes at pre-determined post-operative intervals Data were analysed using SPSS software package, version 16.0 . A one-tailed paired Student's t-test  or Wilcoxon signed rank test was used to explore statistical differences in visual function parameters between operated and fellow control eyes depending on the distribution of the variable. Spearman's correlation coefficients were calculated to explore significant correlations between the different postoperative visual function parameters. The relationships between age, preoperative factors, postoperative visual function tests  and the NEI VFQ-25 scores were examined using Spearman's correlation coefficients. To determine differences in NEI VFQ-25 OCS and subscores in subgroups we used a Mann-Whitney U test in case of two groups or a Kruskal-Wallis test in case of more than two groups. In the latter case post-hoc analyses were performed for pairwise comparisons between subgroups. All tests were considered statistically significant at a p-value of less than 0.05, except for the Kruskal-Wallis post-hoc analysis, for which a significance threshold of 0.05 divided by the number of groups was used.A total of 56 patients gave their written informed consent and were included. In 46 patients retinal re-attachment was obtained after one surgical procedure, whereas ten patients had one or more re-detachments. One patient died during the study period and was therefore excluded from analysis. In all remaining 55 patients, the retina was still surgically attached twelve months after the initial surgical procedure. Missing data further consisted of: visual function tests at 12 months (n = 1), saturated (n = 2) and desaturated (n = 3) CCI, because of color blindness (n = 2) and unknown reasons (n = 1).Postoperative BCVA, Log contrast acuity, saturated and desaturated CCI in operated eyes were significantly worse than in fellow control eyes . We obseIn summary, even though only small differences existed between vision related quality of life OCS and subscale scores, lower scores on vision related quality of life may be related to more extensive surgery, long-term intraocular tamponades and re-detachment. Patients with a more extensive retinal detachment had lower scores on the subscales ocular pain (i.e. experienced more ocular pain) and vision specific role difficulties. Primary surgical success was associated with higher scores on the subscale near activities and OCS. A higher number of re-detachment surgeries was associated with lower scores on ocular pain (i.e. more pain) and vision specific mental health. Patients operated on by PPV had lower scores on vision specific role difficulties, and patients in whom a PPV was combined with an encircling band scored higher on vision specific mental health. Patients receiving a shorter acting gas tamponade (SF6) instead of longer acting gas tamponade (C3F8) or silicone oil were observed to have higher scores on general vision, ocular pain, near activities, vision specific mental health, driving and on the OCS. Preoperatively pseudophakic patients (RRD eye or fellow eye) had higher scores on the vision specific mental health subscale than phakic patients.In our study on patients with macula-off RRD, NEI VFQ-25 OCS and subscale scores were relatively high with a mean OCS of 88.5. The best possible score on each question was 100 and the second best 75 or 80. This means that most patients scored between the best and second best score. Although the scores were somewhat lower than those in a normal working population and in a population with posterior vitreous detachment (PVD) For example, Okamoto et al. We observed correlations between NEI VFQ-25 OCS, and subscale scores and postoperative BCVA, contrast acuity and post-operative CCI. All tested postoperative visual function parameters were highly correlated with each other. This suggests that BCVA, contrast acuity, and color vision represent interdependent aspects of macular function. However, our observations suggest that - of all tested variables - postoperative BCVA and contrast acuity in the RRD-eye are the most important determinants of postoperative VR-QOL.In contrast to postoperative visual functioning, other patient and surgery related aspects showed very few correlations with NEI VFQ-25 outcomes. Correlations observed related to more extensive surgery, long-term intraocular tamponades and re-detachment. We identified that preoperatively pseudophakic patients (RRD eye or fellow eye) had higher scores on the vision specific mental health subscale than phakic patients. The mental health subscale consists of questions about troubling thoughts about the future and the eyesight. Perhaps, patients with a history of cataract extraction do not have to worry about undergoing a cataract extraction anymore.We observed a significant correlation between post-operative BCVA and NEI VFQ-25 OCS and the subscale scores general vision, ocular pain, near activities, distance activities, mental health, role difficulties and driving. In contrast, Okamoto et al. did not find such a relationship Generally, VA is considered as a major determinant of VR-QOL. This has been suggested in studies on macular hole, epiretinal membrane and diabetic retinopathy In line with the study by Okamoto et al., we observed a significant correlation between contrast acuity and NEI VFQ-25 OCS Although we observed significant correlations between reduced CCI and VR-QOL parameters, we did not find any papers on a possible relation between both aspects. The observed correlations were small, and we therefore assume that unilateral, mild color vision defects have less impact on patients' well-being and visual functioning than unilateral defects in VA and contrast acuity. Also, it could be that the fellow eye compensates better for the defect in color vision in the affected eye than it does for the other two aspects of visual functioning.Previously, we observed that both the duration and the height of macular detachment have a profound effect on the postoperative recovery of visual function, particularly BCVA and CCI Our study highlights important aspects of the postoperative VR-QOL in macula-off RRD patients. Some limitations include a possible selection bias towards highly motivated patients, because they would have been more likely to have participated in this study. In addition, the studied population is modest. Our sample size is considered adequate for overall analyses At 12 months postoperatively, BCVA, contrast acuity, and CCI's in macula-off RRD eyes were still significantly worse for the operated eyes compared to their fellow control eyes. A lower VR-QOL (OCS and subscores) had the highest correlation with a worse postoperative BCVA and contrast acuity (in the RRD-eye). Although less pronounced, postoperative color vision disturbances (saturated and desaturated CCI) were significantly correlated with the NEI VFQ-25 OCS.


The search for new therapies providing cardiorenal protection in chronic kidney disease (CKD) has led to treatments that combine conventional renin-angiotensin-aldosterone-system inhibitors with other drugs that exhibit potential in disease management.In rats made uremic by renal ablation, we examined the effects of addition of the endothelin-A receptor antagonist atrasentan to a previously examined combination of enalapril (angiotensin converting enzyme inhibitor) and paricalcitol (vitamin D receptor activator) on cardiac and renal parameters. The effects of the individual and combined drugs were examined after a 3-month treatment.A decrease in systolic blood pressure, serum creatinine and proteinuria, and improvement of renal histology in uremic rats were attributed to enalapril and/or paricalcitol treatment; atrasentan alone had no effect. In heart tissue, individual treatment with the drugs blunted the increase in cardiomyocyte size, and combined treatment additively decreased cardiomyocyte size to normal levels. Perivascular fbrosis was blunted in uremic control rats with atrasentan or enalapril treatment.We found distinct cardiac and renal effects of atrasentan. Combination treatment with atrasentan, enalapril and paricalcitol provided positive effects on cardiac remodeling in uremic rats, whereas combination treatment did not offer further protective effects on blood pressure, proteinuria or renal histology. Cardiovascular disease (CVD), a frequent complication of renal disease, is the major cause of mortality and morbidity in end-stage renal failure . The ren3) and its analogs have a therapeutic potential in attenuating experimentally-induced kidney diseases [Calcitriol  and endothelin receptor B (ETB). ETA receptors have vasoconstrictor and growth-promoting functions, whereas ETB receptors mediate vasodilation and inhibition of growth and inflammation via release of nitric oxide. Atrasentan is a highly selective endothelial receptor agonist (ETAR) with ∼1800-fold greater affinity for the ETA receptor than the ETB receptor. A recent clinical trial demonstrated that atrasentan exhibited additive effects in decreasing the urine albumin-to-creatinine ratio in patients with type 2 diabetic nephropathy who were receiving ACEIs or ARBs [Endothelial cells modulate vascular tone by releasing the vasodilation factor nitric oxide and the potent vasoconstrictor protein endothelin-1, the proinflammatory, mitogenic, and fibrotic effects of which contribute to the progression of renal failure . Endothe or ARBs .The aim of this study is to investigate the effect of atrasentan, enalapril and paricalcitol, individually and in combination, on cardiac and renal histology and function in the 5/6 nephrectomized uremic rat model.The Animal Studies Committee at Washington University School of Medicine approved all experimental protocols in accordance with federal regulations. Uremia was induced in a group of female Sprague-Dawley rats  by 5/6 nephrectomy. Nephrectomized rats were divided into 5 groups and preventive treatment started 1 day after surgery as follows: uremic control (UC); uremic + enalapril ; uremic + atrasentan ; uremic + paricalcitol ; and uremic + enalapril + paricalcitol + atrasentan (UEAP). Normal rats served as control (NC). Doses of enalapril and paricalcitol were chosen based on our previous studies , 19. TheSerum P and creatinine were measured by an autoanalyzer . Total serum calcium was measured by atomic absorption spectrophotometry . Ionized Ca was measured using a Nova 8 electrolyte analyzer .Formalin-fixed, paraffin sections of kidney tissue were stained with Hematoxylin/Eosin and Masson's Trichrome to assess glomerulosclerosis, interstitial inflammation, interstitial fibrosis, tubular atrophy, tubular dilation and calcifications as previously described . Each hiImmunohistochemical staining for CD-68 (Cluster of Differentiation 68), a marker of monocyte/macrophage cells was performed as previously described , with thT technique was used to calculate the ratio of target gene to GAPDH, with respect to normal control rats. Results were reported as percent of normal control values, which represented 100%.Kidney samples were analyzed by real-time (RT) quantitative PCR. Total RNA was isolated using RNAzol Bee . Reverse transcription of the RNA was carried out using oligo-dT primer and SMART MMLV reverse transcriptase . RT-PCR was performed using Fast SYBR Green Master Mix  in an Applied Biosystems 7900HT Fast Real-Time PCR System. QuantiTect Primer Assays obtained from Qiagen  were obtained for quantifying rat transforming growth factor-beta 1 , the chemokine monocyte chemoattractant protein ,), nuclear factor kappa-b subunit 1  and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The ΔΔCCross-sectional cuts of formalin-fixed, paraffin-embedded left ventricle were stained with Masson's Trichrome and analyzed using Image-Pro Plus software, Version 7.0 . Perivascular fibrosis was assessed by calculating the percentage of Trichrome-stained collagen deposits surrounding the vessel to the total perivascular area using the software's color cube function. Approximately 10 vessels were assayed for each rat (n=6 rats/group). The area of individually circumscribed cardiomyocytes was determined using the ImagePro software. The average cardiomyocyte size (pixels) from ∼100 cells/rat was expressed as unit area.t-test was used to compare the normal control groups with individual uremic groups or when only two groups were compared. The Kaplan-Meier method was used to construct survival curves, and a log-rank (Mantel-Cox) test was used to analyze differences between the groups . P ≤ 0.05 was considered significant.All data are expressed as mean SEM. One-way analysis of variance (ANOVA) with the Tukey's post-test, unless stated otherwise, was used for comparison between uremic groups. Student's nonpaired ++ with paricalcitol treatment (5.46 ± 0.07 for UP and 5.29 ± 0.06 mg/dl for UEAP) compared to normals (4.84 ± 0.04 mg/dl) and uremic controls (4.90 ± 0.04 mg/dl). Serum phosphorus and CaxP product were significantly elevated in all uremic rats, except for those treated with enalapril alone.Results from serum chemistries are shown in A time-course for urinary protein levels of spot-urine samples collected pre-surgery and at 1, 2, and 3 months of the study is shown in Systolic blood pressure was measured throughout the study and is shown in Kaplan-Meier survival curves are shown in Macrophage infiltration in the kidney was assessed by immunohistochemistry for the macrophage/monocyte marker CD-68. Representative images of the staining and quantitation are shown in Expression of NFkB-1 mRNA in the uremic control kidney was significantly increased 2.5-fold compared to the normal kidney (p≤0.05) as shown in Perivascular fibrosis in the left ventricle was assessed by deposition of collagen around the vasculature. AR to a treatment regimen consisting of an ACE inhibitor and VDRA would further improve cardiac and renal functions in the uremic rat model.Despite recent advances in CKD treatment, progression to end-stage renal disease and the development of cardiovascular events are still major problems for many CKD patients. The search for new therapies that will prevent, or even reverse, the progression of kidney disease has led to treatments that combine conventional RAAS inhibitors with other drugs that have shown promise for cardiorenal protection in CKD patients. Recent work from our group has demonstrated that combination therapy of paricalcitol and enalapril improved renal function and histology, suppressing the progression of renal failure via mediation/inhibition of the TGFβ-1 signaling pathway , 18, 19.ARs  have been investigated for their protective effects on cardiac and renal systems. Here, we found that atrasentan had distinct effects in the heart and kidney. Regarding cardiac remodeling, individual treatment with atrasentan, enalapril or paricalcitol blunted the increase in cardiomyocyte size, and combined treatment significantly decreased cardiomyocyte size to levels comparable to controls. The finding that cardiomyocyte size, but not LV weight, was blunted by atrasentan and paricalcitol may be attributed to changes in LV mass resulting from not only cardiomyocyte size, but also an increase in non-cardiomyocytes  or extracellular matrix. We previously showed that proliferation of cardiac interstitial cells dramatically increased in uremic animals [Since chronic activation of the endothelin system in CKD contributes to the rate of progression of the disease, targeted blocking of this system may aid in the management of kidney disease. Highly specific ET animals . In the  animals . However animals  recentlyThe inflammatory process resulting in renal fibrosis is complex, involving a multitude of cytokines/chemokines, growth factors, adhesion molecules and signaling pathways . Renal iARs, however, have produced variable results. In one study using streptozotocin-induced diabetic mice, atrasentan reduced diabetic urine flow, proteinuria and plasma creatinine levels, and positively affected myocardial dysfunction [Atrasentan had a very modest (though non-significant) decrease in mortality and no effect on proteinuria, blood pressure, or kidney histopathology. The lack of an effect of atrasentan on proteinuria is in contrast to recent clinical trials. In patients with type-2 diabetic nephropathy receiving ACEIs and/or ARBs, treatment with atrasentan produced an additive effect in decreasing the urine albumin-to-creatinine ratio . In anotfunction . In expefunction , 24 was function . Combinifunction .More consistent with our findings were the results of a study in rats with type-2 diabetes, in which cardiorenal effects were examined after treatment with sitaxsentan, ramipril, or combination treatment . RamipriIn the present study, atrasentan improved cardiac fibrosis, yet had no effect on renal fibrosis. We speculate that this difference may be due to the nature of damage inflicted upon the respective tissues. The method used for inducing uremia is an immediate, harsh insult to the renal tissue, whereas the effect on cardiac tissue is a consequence of the subsequent kidney damage. An immediate vs. delayed damage to renal and cardiac tissue, respectively, as well as possible differences in the pathways that lead to fibrosis in each tissue may have resulted in differential effects of atrasentan. Further studies are necessary to clarify this issue. Our study has several limitations. First, since it is a preventative study, it is difficult to translate our findings into a setting with patients who arrive for treatment after damage has already taken place. Second, a dose response of the effect of atrasentan on study parameters should have been performed before deciding on a final dose of the drug for the study. Third, a control group of enalapril and paricalcitol combination treatment should have been included.ARs. We found that protective effects on blood pressure, proteinuria and renal histology were due to enalapril and/or paricalcitol treatment, in agreement with previous reports. However, treatment with atrasentan alone or in combination with enalapril and paricalcitol provided positive effects on cardiac remodeling in uremic rats. An in-depth analysis of the effect of combination treatment of atrasentan, enalapril and paricalcitol on additional cardiovascular parameters is highly warranted.Clearly, there are distinct cardiac and renal effects of atrasentan and other ET


In the United States, there are limited studies regarding use of prehospital ultrasound (US) by emergency medical service (EMS) providers. Field diagnosis of life-threatening conditions using US could be of great utility. This study assesses the ability of EMS providers and students to accurately interpret heart and lung US images.We tested certified emergency medical technicians (EMT-B) and paramedics (EMT-P) as well as EMT-B and EMT-P students enrolled in prehospital training programs within two California counties. Participants completed a pre-test of sonographic imaging of normal findings and three pathologic findings: pericardial effusion, pneumothorax, and cardiac standstill. A focused one-hour lecture on emergency US imaging followed. Post-tests were given to all EMS providers immediately following the lecture and to a subgroup one week later.We enrolled 57 prehospital providers . The mean pre-test score was 65.2%±12.7% with mean immediate post-test score of 91.1%±7.9% . Scores significantly improved for all three pathologic findings. Nineteen subjects took the one-week post-test. Their mean score remained significantly higher: pre-test 65.8%±10.7%; immediate post-test 90.5%±7.0% , one-week post-test 93.1%±8.3% .Using a small sample of EMS providers and students, this study shows the potential feasibility for educating prehospital providers to accurately identify images of pericardial effusion, pneumothorax, and cardiac standstill after a focused lecture. The use of bedside point-of-care ultrasound (US) in the emergency department (ED) has been increasing over the past two decades, and is now routinely used by emergency physicians as part of the diagnostic workup of sick patients and screening of trauma victims. It has decreased the time to life-saving interventions for many conditions. For example, use of the extended-focused assessment with sonography for trauma (E-FAST) exam by emergency physicians accurately identifies fluid in the abdomen requiring urgent blood transfusion or exploratory laparotomy, pericardial effusion requiring immediate evacuation, or pneumothorax requiring immediate decompression.6Emergency medical service (EMS) providers have the opportunity to diagnose, initiate treatment, and stabilize life-threatening conditions within the first critical minutes of a patient’s decompensation. US has been used by physicians, flight nurses, and EMTs, on both ground and air ambulance teams in several countries in EuropePrior studies have demonstrated that flight medics and ground EMS providers can obtain and interpret images for abdominal aortic aneurysm assessment, FAST exam screening, and cardiothoracic US images.16There remain significant gaps and limitations in existing studies regarding the ability of prehospital providers to acquire and interpret point-of-care US images. Here, we aimed to determine if EMS providers would be able to 1) accurately identify the presence or absence of pericardial effusion, pneumothorax, and cardiac standstill after a one-hour didactic course, and 2) retain the ability to interpret the images over time.We conducted a prospective, observational study of certified emergency medical technicians (EMT-B) and EMT-paramedics (EMT-P) as well as students enrolled in prehospital training programs within two counties in California. The institutional review board approved the study. Participants were recruited from four EMS training programs, and study sessions were held at each of these training programs with written consent obtained from participants.Inclusion criteria for the study were age greater than or equal to 18 years, enrollment in an EMS training course and/or current certification as an EMS provider, and ability to attend all sessions  during the study. Subjects were excluded if they were below the age of 18, were not involved within the county EMS system as either an actively enrolled student or certified EMS provider, did not consent to participation in the study, and/or were unable to attend the required sessions.Study sessions were held prior to or after scheduled classes for the local prehospital training programs; certified EMS providers were also invited to attend these study sessions. Study participants were first asked to complete an anonymous demographics questionnaire including gender, age, educational status, EMS affiliation, and prior US experience. This was followed by a multiple-choice question (MCQ) pre-test that included 16 full-motion and still US clips of normal and abnormal pathology. They then received a one-hour didactic lecture covering basic scanning technique, normal US anatomy, and image interpretation of both normal and pathologic heart and lung imaging videos. This included presence or absence of pericardial effusion, pneumothorax, and cardiac standstill. Immediately following the lecture, study participants were given a post-test consisting of the same video clips in different order with different questions from the pre-test. For one of the local prehospital training classes, the same post-test was administered one-week later. The test contained 16 image-questions, with six of the images having been shown in the lecture and 10 novel images. The images were originally obtained in the ED setting by emergency physicians with prior US training and knowledge. While the repeat post-test did contain the same questions from the immediate post-test, subjects were not given answers or feedback on their initial tests. Participants were asked to self-rate their confidence level with US interpretation and given one minute to answer each MCQ on both the pre- and post-tests. Scores were determined as percentage of questions answered correctly on the test. Subjects did not acquire any of the US images.Both the pre- and post-tests were validated using a population of emergency medicine physicians (both attendings and residents) knowledgeable on bedside point-of-care US, but who had no prior knowledge of the test images. The validation tests were administered to the physician group without receipt of the lecture intervention and prior to utilization of the tests for the study participants. All data were analyzed using two-tailed, paired t-tests in SPSS 11.0 .We enrolled 57 prehospital providers  consisting of 19 EMT-B students, 16 EMT-P students, 18 certified EMT-B providers, and 4 certified EMT-paramedics . There wThere was a significant improvement for all subjects between the pre- and post-tests with a mean pre-test score of 65.2%±12.7% and a mean immediate post-test score of 91.1%±7.9% . Scores significantly improved for all three individual pathologies as shown in Among the certified EMS providers (N=22), scores showed significant increases in mean score pre-test (63.9%±16.6%) to immediate post-test . Among these providers, scores for identification of pneumothorax and pericardial effusion showed significant increases after subjects received the focused lecture. There was no significant change for identification of cardiac standstill: pre-test score 90.9%±18.1% and post-test score 98.9%±5.3%  .The repeat post-test was administered one week later to 19 EMT-B students. Post-test scores remained significantly higher than pre-test scores . There wAmong all subjects, self-reported confidence with point-of-care US increased after the study intervention. During the pre-test, 53 participants (96%) reported no or low confidence with US interpretation. During the immediate-post test, only 8 participants (15%) reported no or low confidence whereas 46 subjects (85%) reported some or high confidence with US interpretation .Although use of bedside ultrasonography within United States EDs is now common, the use of this technology in the field by EMS providers is limited. Use of bedside ultrasonography in the prehospital setting has the potential to provide EMS providers with important diagnostic data and assist with difficult treatment and transport decisions in the field. Furthermore, as EMS systems within the United States evolve to focus on transport to specialty centers, such as trauma or cardiac care centers, prehospital US could play a role in guiding these decisions, just as they have in Europe.Our study findings concur with previous reports supporting the feasibility of formal US instruction and subsequent accurate US interpretation by EMS providers. Previous studies, however, were primarily conducted among international EMS subjects,Knowledge retention among the cohort of EMT-B students was high one week after the teaching session, and this effect was seen across all three pathologies tested. This complements prior work that showed retention of pneumothorax identification by prehospital providers in a cadaveric model up to nine months after initial teaching by prehospital providers.19Our results suggest that incorporation of a succinct and relevant one-hour didactic on prehospital point-of-care US into the EMS training curriculum could have a large and lasting impact on the providers’ skill set, even at a junior level, without much burden on already stretched curricula. In addition, the didactic material can be focused in scope, emphasizing those emergencies for which early identification might change treatment or triage decisions. Given a growing body of literature regarding potential impacts of prehospital identification of pneumothorax, pericardial effusion or cardiac standstill, we chose to focus our one-hour curriculum on these three modalities.Our findings validate European studies on pneumothorax evaluation, which demonstrated prehospital providers’ ability to correctly identify lung sliding.27Presence of cardiac standstill during non-traumatic arrest in the field could affect the need for transport to the ED given the growing body of evidence that patients with cardiac standstill have a nearly zero percent chance of survival to hospital discharge.15Our results also provide validation that EMS providers can identify the presence of pericardial effusion after receiving brief instruction. The significance of this modality in the prehospital setting is especially relevant among penetrating thoracic trauma patients, in whom early identification of an effusion or tamponade may expedite thoracotomy or pericardiocentesis upon ED arrival.Our study represents the evaluation of prehospital providers’ ability to interpret US images. The results do show that these providers can acquire and retain US interpretation skills and confidence. Limitations include the fact that this was an observational trial using a convenience sample of EMS volunteers, with limited sample sizes. It is possible that these volunteers may not represent the rank-and-file EMS student. Demographics and confidence data were acquired by self-report, which could be skewed by bias. However, this would not affect the objective statistical improvement in scores after our study intervention. Because participation was voluntary and did require attendance at both the didactic and post-testing sessions for inclusion in the retention cohort, it is possible that the retention cohort contained selection-bias from individuals more enthusiastic about US thus affecting results with a bias toward improvement. Additionally, this cohort had a smaller sample size due to our ability to have a follow-up session one week later with only one EMS class, thus potentially impacting statistical significant and generalizability of results. Similarly, our specific analysis of certified providers should be taken in the context that the majority of these providers were EMT-B trained with very few EMT-P trained individuals. While the analysis combined all certified providers into one cohort, the statistical significance of our findings could be skewed by the small sample of EMT-P individuals, and further, it would be difficult to analyze the results as to whether US may be more easily taught to either EMT-B or EMT-P certified individuals.Our study utilized test images that were obtained within the ED setting and although they were validated prior to use, the images may represent idealized versions of findings when compared with those that are obtained by EMS providers themselves or subject to other environmental factors while obtaining US in the prehospital setting. Though this may limit the strength of our findings, several prior studies have shown that EMS providers can obtain adequate images,Our study demonstrates the potential ability of EMS students and certified providers to acquire and retain knowledge of US interpretation with regard to specific pathologies. However, the impact on patient care and transport remains to be determined. Given studies noting the changes in management and transport to appropriate levels of care by European providers, the same may hold true for EMS US use and decision-making in the United States.20There remains a tangible cost to implementation of US in the EMS field, with the need to obtain and maintain equipment. Although our chosen modalities – pneumothorax, pericardial effusion, and cardiac standstill – could likely have potential benefits for triage, transport, and treatment of prehospital patients, the true impact and cost-effectiveness of such decisions has yet to be determined. Ongoing studies will need to assess prehospital providers’ acquisition of US, and potential delays in treatment or transport within EMS systems in the United States. There may also be differences in utility and clinical impact within urban versus rural communities. Future study could also focus on longitudinal tracking of individual EMS providers to evaluate the number of USs being completed, image acquisition ability, and also longitudinal knowledge retention and skills.This study showed potential promise for training prehospital EMS providers in accurate US interpretation through a one-hour didactic lecture focused on US technique and anatomy for the assessment of pericardial effusion, pneumothorax, and cardiac standstill. Using a small group of EMS students and providers, subjects’ performance on US image interpretation increased from 60% to 90% after training and was maintained at one week. Additionally, subjects reported increased confidence in their comfort level with US interpretation. Although limited, our findings lend potential support to existing data that demonstrate prehospital providers may be able to sufficiently gain and retain knowledge of point-of-care US interpretation for pericardial effusion, pneumothorax, and cardiac standstill.


Methanocorpusculum sp. and Methanosarcina sp., which co-occurred with Bacteroidetes environmental groups, such as Vadin HA17, in places where the organic matter was more degraded. The metabolic potential for organic matter decomposition by Ruminococcaceae was prevalent in samples with more pyrolyzable carbon. Rhodobium- and Hyphomicrobium-related genera within the Rhizobiales order that have the metabolic potential for dark hydrogen fermentation and methylotrophy, and unclassified Comamonadaceae were the dominant Proteobacteria. The unclassified environmental group Sh765B-TzT-29 was an important Delta-Proteobacteria group in this BCR that co-occurred with the dominant Rhizobiales operational taxonomic units. Organic matter degradation is one driver for shifting the microbial community composition and therefore possibly the performance of these bioreactors over time.Sulfidogenic biochemical reactors (BCRs) for metal removal that use complex organic carbon have been shown to be effective in laboratory studies, but their performance in the field is highly variable. Successful operation depends on the types of microorganisms supported by the organic matrix, and factors affecting the community composition are unknown. A molecular survey of a field-based BCR that had been removing zinc and arsenic for over 6 years revealed that the microbial community was dominated by methanogens related to  Using passive or semi-passive microbial reactors to remove metals from mine or metallurgical waste seepage is very attractive due to the vast repertoire of microbial metabolic responses to metals Gadd, . Both diThe type of nutrient source used is key and much previous work has taken place to compare different types and combinations of waste materials, such as manure, compost, wood, sawdust, silage, leaf litter, hay, and pulp and paper biosolids, for example, for their efficacy at supporting sulfate-reducing bacteria and removing metals  of each of the nine cores were produced for Archaea and bacteria using primers targeting the archaeal domain A4F (5′-TCCGGTTGATCCTGCCRG) and U1492R (5′-GGTTACCTTGTTACGACTT) and the bacterial domain B27F (5′-AGAGTTTGATCCTGGCTCAG) and U1492R (5′-GGTTACCTTAGTTACGACTT), respectively, using gradient PCR methods described in detail in the Supplemental Material. Cloned inserts were sequenced bi-directionally with M13F (5′-GTAAAACGACGGCCAG) and M13R (5′-CAGGAAACAGCTATGAC) primers at the Michael Smith Genome Sciences Centre . Sequences were assembled and checked for chimeras as described in the Supplemental Material.Different aliquots of the homogenized samples were used for the pyrotag libraries. The DNA was extracted using the Power Soil DNA isolation Kit  following the manufacturer’s instructions. DNA from adjacent 5-cm intervals was combined to yield 38 samples from all cores and all depths that were used for PCR amplification of the V6–V8 variable SSU rRNA region: 926f (5′ AAACTYAAAKGAATTGRCGG 3′) and 1392r (5′ ACGGGCGGTGTGTRC 3′) as described in detail in the Supplemental Material. Purified PCR products were sent to the McGill University Innovation Centre  for pyrosequencing using a Roche GS-FLX Titanium Series sequencer. Pyrotag reads were filtered using the following quality control criteria: minimum length 200 bp, no ambiguous base reads, no missing quality scores, mean quality score greater than 25, no more than six nucleotide length homopolymer runs, and no mismatches in reverse primer. Raw pyrotag sequences were deposited in the sequence read archive of the National Center for Biotechnology Information (NCBI) under project PRJNA239169  weighted UniFrac . Samples were preserved using 0.3% zinc acetate for sulfate analysis later in the laboratory using standard method 2 evolved during pyrolysis. Residual carbon (RC) refers to the remaining organic matter that is combusted. Additionally, mineral carbon (MINC) is quantified also in the Rock-Eval-6 process. More details and example Rock-Eval-6 spectra are given in the Supplementary Material.Influent and effluent metal and sulfate concentrations were measured every 2 weeks by the operators using inductively coupled mass spectroscopy (ICP-MS) in an industrial metallurgical laboratory. At the time of sampling, pore water pH, temperature, dissolved oxygen, and oxidation–reduction potential were measured using a 6600V2 sonde with appropriate probes . Pore water nitrite, nitrate, ammonia, total phosphorous, sulfide, and Fe3/month except for August 2008 when an exceptional amount of seepage  entered the system. Based on estimated hydraulic retention times (~4 weeks) through each of the stages in the whole treatment system, most of the zinc was removed in the first BCR  mg/L, 40 (8) mg/L, and 650 (100) mg/L, respectively  characteristics were determined so as to estimate the degree of degradability of the matrix at the time of sampling. Rock-Eval-6 is a pyrolysis method used to study the composition of organic material with different maturities; most often coal and other HCs, but also organic matter in soils and sediments , Bacteria (32), and Eukaryota (3) domains. The pyrotag reads revealed that 15 phyla dominated with Methanomicrobiales and Methanosarcinales closely related to Methanocorpusculum labreanum, M. parvum, Methanosarcina vacuolata, and M. barkeri. Close environmental relatives came from anaerobic digesters . Unclassified Methanobacteriaceae were highly prevalent although none of the clone library sequences were classified in this family.The Archaea clone SSU rRNA sequences formed five major clades . The majority of sequences were classified as Class II methanogens in the orders y Figure A with MeFirmicutes, Bacteroidetes, Proteobacteria, and Spirochetes according to both the clone library and pyrotags .Most bacteria were classified into the phyla s Figure . Apart fRuminococcaceae and Erysipelotrichaceae were highly represented in the Firmicutes phylum . Clone library sequences related to cellulose degrading and fermenting Clostridium species were well represented.Bacteria associated with organic matter digestion, such as Bacteroidetes phylum . At the time of writing, there were 44 environmental clones in the NCBI nucleotide database that were closely related to the predominant BCR Rhodobium-classified clone (>97% identity). Of these, 17 came from wetland soils, 11 from natural or agriculture soils, 12 from contaminated soils  and 4 from marine and cold-water sediments. OTUs classified in the environmental group Sh765B-TzT-29 were phylogenetically distinct from all the other Proteobacteria sequences . Putative sulfate-reducing bacteria were rare, with some pyrotag sequences classified as Desulfovibrio and Desulfobulbus genera . Clostridia-related OTUs classified as Desulfosporosinus and Desulfotomaculum were present also .Microbes classified in the Armatimonadetes-related reads were predominant . At the time of writing, there were no other environmental clones more than 94% homologous to the bioreactor Armatimonadetes-related clone. Fibrobacteres (Fibrobacter), Planctomycetes (AKAU3564 sediment group), and Synergistetes (Synergistaceae) were three other phyla well represented in the bioreactor.p-values <0.001), and to a lesser extent the S1 fraction  . UniFrac distances could not be used for correlation with the organic matter characteristics and Bray–Curtis dissimilarity indices based on OTU read counts were used instead . Statistically significant fits of microbial community composition with Rock-Eval-6 variables PC and, to a lesser extent, S1 were obtained for the July and October samples, but not with the April samples. The prevalent OTUs clustered similarly in the July and October samples.Proteobacteria-, Firmicutes- (Ruminococcaceae), and Actinobacteria-related OTUs co-occurred with each other . OTU read counts plotted versus PC/TOC fraction confirmed prevalence of the most abundant Methanocorpusculum- and VadinHA17-related OTUs with the samples containing more recalcitrant organic matter, and the greater prevalence of Proteobacteria and Ruminococcaceae where there was more PC . Prevalent C Figure .Archaea in their survey. Unpublished work from our laboratory indicates that in some field-based BCRs, sulfate-reducers are more abundant than methanogens , although some acetoclastic groups were also present . Rhodobium-related species have been found to be involved in dark carbon fermentation, which produces hydrogen  and their relatives present in the BCR might have been involved in methane metabolism . The availability of methane and nitrate may explain the prevalence of this group in the BCR.A pyrolysis-based technique proved to be useful for characterizing the degree of degradation of organic matter in the BCR. Other studies have used Rock-Eval to characterize soil organic matter. Deconvolution of the S2 peak revealed several overlapping peaks due to different classes of chemical compounds releasing at different temperatures  also found in other high metal environments . The first member of WCHB1-69 was a clone found in an aquifer contaminated with HCs and chlorinated-solvents that was undergoing intrinsic bioremediation (NCBI accession number AF050545; Dojka et al., Bacteroidetes-related OTUs were closely related to cultured species that produce acetate, H2, CO2, and possibly volatile sulfur compounds using elemental sulfur or nitrate as electron acceptors (Grabowski et al., The BCR contained other novel taxonomic groups that were predominant but whose role is unknown, such as those classified as Rhizobiales order. Predominant taxonomic groups revealed metabolic potential for methane production, metal-tolerance, and resistance, and many different types of fermentation including possibly dark fermentation to produce hydrogen. Sulfate-reduction potential according to known SRB taxonomic groups was less represented. Novel groups predominant within the BCR may contain organisms involved in sulfur cycling and metal resistance. The microbial community broadly delineated according to the degree of organic matter degradation with methanogens and putative syntrophs, dominant in more recalcitrant material and Proteobacteria, Actinobacteria, Armatimonadetes, and Planctomycetes prevalent in places with more PC.Overall, the microbial community in the BCR consisted of highly specialized groups closely related to other organisms associated with metal-rich environments as well as highly degraded HC reservoirs experiencing terminal carbon degradation by methanogens and possibly microbes within the Desulfobulbus, Desulfovibrio, Desulfosporosinus, and Desulfotomaculum. The bioreactor was dominated by methanogens, with a Methanocorpusculum-related OTU most prevalent. Correlation of methanogen-related OTUs with more degraded material suggests that as these types of bioreactors age, methanogens might become more prevalent. Many novel, uncharacterized taxonomic groups were found and suggest that the metabolic potential for organic matter degradation or even metal transformation is much more diverse than previously thought.This work has contributed to opening the “black box” of bioremediation of metal and sulfate-contaminated water. It was revealed for this particular system that sulfate-reducers, desired for metal sulfide precipitation, were rare and restricted to a few specific genera: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fbioe.2015.00027/abstractThe Supplementary Material for this article can be found online at This includes details of the molecular biology materials and methods, a table of the putative sulfate-reducing bacteria found in the BCR pyrotag library, a table of the highly correlated co-occurring OTUs, a simple cartoon of the BCR, details for the Rock-Eval-6 analysis, phylogenetic trees clone library SSU rRNA 97% homology cut-off OTUs, charts of the relative abundance of the most highly prevalent pyrotag OTUs, UniFrac principal component analysis of the microbial communities, Bray–Curtis dissimilarity detrended correspondence analysis of microbial communities faceted according to Phylum, Bray–Curtis dissimilarity detrended correspondence analysis of samples split according to season and a network of the most highly correlated OTUs.Click here for additional data file.