Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil.

Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this "uncultivated majority" remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages.IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.

PhyloToL: A Taxon/Gene-Rich Phylogenomic Pipeline to Explore Genome Evolution of Diverse Eukaryotes.

Estimating multiple sequence alignments (MSAs) and inferring phylogenies are essential for many aspects of comparative biology. Yet, many bioinformatics tools for such analyses have focused on specific clades, with greatest attention paid to plants, animals, and fungi. The rapid increase in high-throughput sequencing (HTS) data from diverse lineages now provides opportunities to estimate evolutionary relationships and gene family evolution across the eukaryotic tree of life. At the same time, these types of data are known to be error-prone (e.g., substitutions, contamination). To address these opportunities and challenges, we have refined a phylogenomic pipeline, now named PhyloToL, to allow easy incorporation of data from HTS studies, to automate production of both MSAs and gene trees, and to identify and remove contaminants. PhyloToL is designed for phylogenomic analyses of diverse lineages across the tree of life (i.e., at scales of >100 My). We demonstrate the power of PhyloToL by assessing stop codon usage in Ciliophora, identifying contamination in a taxon- and gene-rich database and exploring the evolutionary history of chromosomes in the kinetoplastid parasite Trypanosoma brucei, the causative agent of African sleeping sickness. Benchmarking PhyloToL's homology assessment against that of OrthoMCL and a published paper on superfamilies of bacterial and eukaryotic organellar outer membrane pore-forming proteins demonstrates the power of our approach for determining gene family membership and inferring gene trees. PhyloToL is highly flexible and allows users to easily explore HTS data, test hypotheses about phylogeny and gene family evolution and combine outputs with third-party tools (e.g., PhyloChromoMap, iGTP).

The ties that bind: perceived social support, stress, and IBS in severely affected patients.

BACKGROUND: This study assessed the association between social support and the severity of irritable bowel syndrome (IBS) symptoms in a sample of severely affected IBS patients recruited to an NIH-funded clinical trial. In addition, we examined if the effects of social support on IBS pain are mediated through the effects on stress. METHODS: Subjects were 105 Rome II diagnosed IBS patients (F = 85%) who completed seven questionnaires which were collected as part of a pretreatment baseline assessment. KEY RESULTS: Partial correlations were conducted to clarify the relationships between social support and clinically relevant variables with baseline levels of psychopathology, holding constant number of comorbid medical diseases, age, gender, marital status, ethnicity, and education. Analyses indicated that social support was inversely related to IBS symptom severity. Social support was positively related with less severe pain. A similar pattern of data was found for perceived stress but not quality of life impairment. Regression analyses examined if the effects of social support on pain are mediated by stress. The effects of social support on bodily pain were mediated by stress such that the greater the social support the less stress and the less pain. This effect did not hold for symptom severity, quality of life, or psychological distress. CONCLUSIONS & INFERENCES: This study links the perceived adequacy of social support to the global severity of symptoms of IBS and its cardinal symptom (pain). It also suggests that the mechanism by which social support alleviates pain is through a reduction in stress levels.

Motility of the antroduodenum in healthy and gastroparetics characterized by wireless motility capsule.

BACKGROUND: The wireless motility capsule (WMC) measures intraluminal pH and pressure, and records transit time and contractile activity throughout the gastrointestinal tract. Our hypothesis is that WMC can differentiate antroduodenal pressure profiles between healthy people and patients with upper gut motility dysfunctions. This study aims to analyze differences in the phasic pressure profiles of the stomach and small intestine in healthy and gastroparetic subjects. METHODS: Data from 71 healthy and 42 gastroparetic subjects were analyzed. The number of contractions (Ct), area under the pressure curve and motility index (MI = Ln (Ct *sum amplitudes +1)) were analyzed for 60 min before gastric emptying of the capsule (GET), (gastric window) and after GET (small bowel window) and results between groups were compared with the Wilcoxon rank sum test. KEY RESULTS: Significant differences were observed between healthy and gastroparetic subjects for Ct and MI (P < 0.05). Median values of the motility parameters in gastric window were Ct = 72, MI = 11.83 for healthy and Ct = 47, MI = 11.12 for gastroparetics. In the small bowel, median values were Ct = 144.5, MI = 12.78 for healthy and Ct = 93, MI = 12.12 for gastroparetics. Diabetic subjects with gastroparesis showed significantly lower Ct and MI compared with healthy subjects in both gastric and small bowel windows while idiopathic gastroparetic subjects did not show significant differences. CONCLUSIONS & INFERENCES: The WMC is able to differentiate between healthy and gastroparetic subjects based on gastric and small bowel motility profiles.

The assessment of regional gut transit times in healthy controls and patients with gastroparesis using wireless motility technology.

BACKGROUND: Wireless pH and pressure motility capsule (wireless motility capsule) technology provides a method to assess regional gastrointestinal transit times. AIMS: To analyse data from a multi-centre study of gastroparetic patients and healthy controls and to compare regional transit times measured by wireless motility capsule in healthy controls and gastroparetics (GP). METHODS: A total of 66 healthy controls and 34 patients with GP (15 diabetic and 19 idiopathic) swallowed wireless motility capsule together with standardized meal (255 kcal). Gastric emptying time (GET), small bowel transit time (SBTT), colon transit time (CTT) and whole gut transit time (WGTT) were calculated using the wireless motility capsule. RESULTS: Gastric emptying time, CTT and WGTT but not SBTT were significantly longer in GP than in controls. Eighteen percent of gastroparetic patients had delayed WGTT. Both diabetic and idiopathic aetiologies of gastroparetics had significantly slower WGTT (P < 0.0001) in addition to significantly slower GET than healthy controls. Diabetic gastroparetics additionally had significantly slower CTT than healthy controls (P = 0.0054). CONCLUSIONS: In addition to assessing gastric emptying, regional transit times can be measured using wireless motility capsule. The prolongation of CTT in gastroparetic patients indicates that dysmotility beyond the stomach in GP is present, and it could be contributing to symptom presentation.

Comparison of gastric emptying of a nondigestible capsule to a radio-labelled meal in healthy and gastroparetic subjects.

BACKGROUND: Gastric emptying scintigraphy (GES) using a radio-labelled meal is used to measure gastric emptying. A nondigestible capsule, SmartPill, records luminal pH, temperature, and pressure during gastrointestinal transit providing a measure of gastric emptying time (GET). AIMS: To compare gastric emptying time and GES by assessing their correlation, and to compare GET and GES for discriminating healthy subjects from gastroparetics. METHODS: Eighty-seven healthy subjects and 61 gastroparetics enrolled with simultaneous SmartPill and GES. Fasted subjects were ingested capsule and [(99m)Tc]-SC radio-labelled meal. Images were obtained every 30 min for 6 h. Gastric emptying time and percentage of meal remaining at 2/4 h were determined for each subject. The sensitivity/specificity and receiver operating characteristic analysis of each measure were determined for each subject. RESULTS: Correlation between GET and GES-4 h was 0.73 and GES-2 h was 0.63. The diagnostic accuracy from the receiver operating characteristic curve between gastroparetics and healthy subjects was GET = 0.83, GES-4 h = 0.82 and GES-2 h = 0.79. The 300-min cut-off time for GET gives sensitivity of 0.65 and specificity of 0.87 for diagnosis of gastroparesis. The corresponding sensitivity/specificity for 2 and 4 h standard GES measures were 0.34/0.93 and 0.44/0.93, respectively. CONCLUSION: SmartPill GET correlates with GES and discriminates between healthy and gastroparetic subjects offering a nonradioactive, standardized, ambulatory alternative to scintigraphy.

Non-Mendelian inheritance of paralogs of 2 cytoskeletal genes in the ciliate Chilodonella uncinata.

Recognition of the role of non-Mendelian inheritance is on the rise, particularly as epigenetic phenomena are shown to shape the transformation of genomes into phenotypes. Ciliates provide a model system in which to explore the role of epigenetics because ciliates have both a germ line (micronuclear) and somatic (macronuclear) genome within every cell. In the ciliate Chilodonella uncinata, the macronucleus is extensively fragmented such that many genes end up on their own chromosomes. Hence, it is possible to track the fate of unlinked genes within macronuclei of C. uncinata. Here we demonstrate that the pattern of inheritance in isolates of C. uncinata is complex and involves both Mendelian transmission between micronuclei and macronuclei and epigenetic phenomena. The macronuclei from 2 isolates of C. uncinata and their progeny share identical rDNA loci and 2 identical beta-tubulin paralogs, yet have different actin paralogs and some beta-tubulin paralogs that are not shared. We propose a model in which all the divergent paralogs are present in the ciliate micronuclei. Under this model, different paralogs are retained in developing macronuclei following conjugation. We further speculate that an epigenetic mechanism, such as RNA interference, is involved in selective retention of specific paralogs within lines. This system allows the exploration of epigenetic phenomena that shape somatic genomes and provides parallels to studies of the development of somatic nuclei within animals.

Repeat surfactant therapy for postsurfactant slump.

OBJECTIVE: To evaluate repeat surfactant therapy for the treatment of respiratory failure associated with postsurfactant slump in extremely low birth weight infants (ELBW) by characterizing the population of premature infants who develop postsurfactant slump and measuring their response to a secondary course of surfactant therapy. STUDY DESIGN: A retrospective analysis of a cohort of all patients admitted over a 3-year period with birth weights <1000 g (ELBW infants). Information was collected by chart review and the patients were categorized into three distinct groups for analysis. Initial surfactant only, patients who received surfactant replacement therapy only for respiratory distress syndrome (RDS); repeat surfactant, patients who received both initial surfactant replacement for RDS and repeat surfactant therapy for postsurfactant slump (defined as respiratory failure after 6 days of age), and no surfactant, patients in whom no surfactant was ever administered. A respiratory severity score (RSS) was used to measure the severity of lung disease and response to surfactant therapy. RESULTS: Over 3 years, there were 165 ELBW infants who could develop postsurfactant slump and be eligible for repeat surfactant therapy. There were 39 infants who never received any surfactant therapy estimated gestational age (EGA) 27.7 +/- 1.7, birth weight 856 +/- 109 g) either at birth or after 6 days of life. There were 126 patients treated for RDS with initial surfactant replacement therapy (EGA 25.6 +/- 1.9 weeks, birth weight 713 +/- 179 g). Out of these RDS patients, 101 improved with an initial course of surfactant therapy (EGA 26 +/- 1.8, birth weight 751 +/- 143 g), but 25 (20% of the patients with RDS) developed postsurfactant slump and received a repeat course of surfactant therapy (EGA 24.7 +/- 1.2, birth weight 647 +/- 120 g). The repeat surfactant group (postsurfactant slump) was significantly more premature and had significantly lower birth weights compared to both the initial surfactant only group and the no surfactant ever group. Logistic regression analysis revealed that lack of antenatal steroids, earlier gestational age, and the receiving of 2 or more doses of surfactant to treat the initial RDS were significantly associated with receiving repeat surfactant therapy for postsurfactant slump. Of the 25 patients treated with a repeat course of surfactant therapy more than 70% of patients (n = 18) had an improvement in their lung disease with a 15% reduction in their RSS. This improvement was significant at all time points evaluated (12, 24, and 48 h). CONCLUSION: We found that a repeat course of surfactant therapy, after day of life 6, led to a significant improvement in hypoxemic respiratory failure in premature infants with postsurfactant slump. Infants who received repeat surfactant therapy were born at a significantly earlier gestational age, had significantly smaller birth weight and had significantly worse lung disease. They were significantly less likely to have received antenatal steroids and were significantly more likely to have received multiple doses of surfactant to treat their initial RDS. A repeat course of surfactant therapy for patients with postsurfactant slump appeared beneficial in the short-term. These initial findings would support performing randomized control trials of repeat surfactant therapy for postsurfactant slump.

Mutations in PITX2 may contribute to cases of omphalocele and VATER-like syndromes.

Omphalocele is a congenital anomaly with substantial morbidity. Rieger syndrome, an autosomal dominant disorder, is characterized by craniofacial abnormalities and abdominal wall defects. PITX2 mutations are etiologic in >40% of cases of Rieger syndrome. We demonstrate that the birth prevalence of omphalocele is significantly higher in Rieger syndrome than in the general population, with omphaloceles found in 0.03% in the Iowa newborn population and 4.3% of patients with Rieger syndrome. Our objective was to screen coding and conserved non-coding regions of PITX2 for mutations in 209 patients with omphalocele. We identified remarkable evolutionarily conserved regions by comparing the 3'UTR of Pitx2 in 13 vertebrate and 3 invertebrate species. No mutations changing the amino acid sequence were found within the omphalocele population. In one case of omphalocele with VATER-like additional anomalies, a three nucleotide deletion was found in the 3'UTR. This deletion was not seen in 1,186 controls. Also in the 3'UTR, we identified a single nucleotide polymorphism at a highly conserved residue. Our findings suggest additional studies of PITX2 conserved regions will be valuable. We also screened the omphalocele cases for mutations in exon 5 of the gene FLNA. Mutations in FLNA have been shown to cause a broad range of congenital malformations, including otopalatodigital syndrome type 2 in which a missense mutation occurring in exon 5 of FLNA results in omphalocele as part of the phenotype. We did not find any mutations in exon 5 of FLNA in 179 omphalocele cases studied.

Phylogenetic position of Sorogena stoianovitchae and relationships within the class Colpodea (Ciliophora) based on SSU rDNA sequences.

The ciliate Sorogena stoianovitchae, which can form a multicellular fruiting body, has been classified based upon its ultrastructure and morphology: the oral and somatic infraciliature of S. stoianovitchae most closely resemble those of members of the order Cyrtolophosidida in the class Colpodea. We characterized the small subunit ribosomal DNA (SSU rDNA) gene sequence from S. stoianovitchae and compared this sequence with those from representatives of all ciliate classes. These analyses placed S. stoianovitchae as either sister to members of the class Nassophorea or Colpodea. In an in-group analysis, including all SSU rDNA sequences from members of the classes Nassophorea and Colpodea and representatives of appropriate outgroups, S. stoianovitchae was always sister to Platyophrya vorax (class Colpodea, order Cyrtolophosidida). However, our analyses failed to support the monophyly of the class Colpodea. Instead, our data suggest that there are essentially three unresolved clades: (1) the class Nassophorea; (2) Bresslaua vorax, Colpoda inflata, Pseudoplatyophrya nana, and Bursaria truncatella (class Colpodea); and (3) P. vorax and S. stoianovitchae (class Colpodea).

Evolution of nuclear dualism in ciliates: a reanalysis in light of recent molecular data.

Ciliates are defined by the presence of dimorphic nuclei. Following conjugation, both the functional macronucleus (MAC) and the transcriptionally inactive germline micronucleus (MIC) develop from a zygotic nucleus. As the MAC develops, germline chromosomes are processed by excision of internal sequences, fragmentation and amplification of the remaining chromosomes. The extent of processing varies among lineages and, in all but one class of ciliates, the resulting MACs divide by an unusual process termed 'amitosis'. Research on these chromosomal rearrangements, largely from studies of only a handful of taxa from two of the nine classes of ciliates, has failed to find evidence of homologous processing among ciliate lineages. This observation, coupled with the structural diversity of MAC genomes among ciliates, led to the hypothesis of multiple origins of at least two MAC properties: (1) the ability to divide and (2) the mechanisms underlying chromosomal processing. Applying this logic to a more inclusive analysis of ciliate lineages, where an even greater diversity of MAC structure is observed, increases the potential number of origins of these MAC characteristics. Here, it is proposed that a single origin of a relatively plastic mechanism underlying MAC development better explains the observed diversity in MAC structure and processing among ciliates. Such a mechanism is suggested by the demonstration of epigenetic effects during MAC development in Paramecium and Tetrahymena.

Widespread distribution of extensive chromosomal fragmentation in ciliates.

Ciliates are a diverse group of eukaryotes characterized by their division of nuclear function into a "germ line" micronucleus and a "somatic" macronucleus. After conjugation, chromosomes in the transcriptionally active macronucleus develop by fragmentation, elimination, and amplification of germ line chromosomes. Extensive chromosomal processing that generates a macronucleus with gene-sized fragments has thus far been well documented in members of only one class of ciliates, the Spirotrichea. Here we establish the broad distribution of extensive fragmentation among members of the class Phyllopharyngea and the genera Metopus (order Armophorida) and Nyctotherus (order Clevelandellida). Moreover, analyses of small-subunit rDNA genealogies indicate that gene-sized chromosomes occur in members of the three separate clades: (1) the class Spirotrichea, (2) the class Phyllopharyngea, and (3) the two orders Clevelandellida and Armophorida. Together, these data indicate that the generation of gene-sized chromosomes is widespread and demonstrate multiple origins of extensive fragmentation within ciliates.

Survival after multimodality treatment for stage IIIC endometrial cancer.

OBJECTIVE: Our purpose was to review our results of multimodality treatment of lymph node metastasis in endometrial cancer (stage IIIC). STUDY DESIGN: All patients underwent surgical staging for endometrial cancer with complete pelvic and aortic lymphadenectomy. All macroscopic nodal metastases were resected. Patients with microscopic nodal metastasis received adjuvant teletherapy, whereas those with macroscopic nodal metastasis received chemotherapy (carboplatin AUC 5 and paclitaxel 135 mg/m2 every 3 weeks for 6 courses) followed by teletherapy. RESULTS: Twenty-one patients had stage IIIC disease, and one had stage IVB (inguinal nodal metastasis). Sixty-four percent of tumors were poorly differentiated. Fifty-five percent of patients had pelvic nodal metastasis only and 41% had macroscopic nodal metastasis. At a median follow-up of 3.8 years, 32% of patients had recurrence, all extrapelvic. Overall mean survival was 48 months and progression-free survival was 40 months. Overall survival for microscopic nodal metastasis was >60 months versus 35 months for macroscopic metastasis. Overall survival for pelvic nodal metastasis was 53 months versus 42 months for aorticinguinal metastasis. There were no complications from lymphadenectomy, a 22% chemotherapeutic toxicity, and a 14% radiation toxicity. CONCLUSION: Our surgical, chemotherapeutic, and radiation treatment protocol for stage IIIC endometrial cancer produced minimal toxicity and good survival.

Genetic structure of the blue ridge dusky salamander (Desmognathus orestes): inferences from allozymes, mitochondrial dna, and behavior.

The plethodontid salamander Desmognathus orestes, a member of the D. ochrophaeus species complex, is distributed in southwestern Virginia, eastern Tennessee, and western North Carolina. Previous allozyme analyses indicate that D. orestes consists of two distinct groups of populations (D. orestes 'B' and D. orestes 'C') with extensive intergradation and probable gene flow between these two groups. Spatially varying allele frequencies can reflect historical associations, current gene flow, or a combination of population-level processes. To differentiate among these processes, we use multiple markers to further characterize divergence among populations of D. orestes and assess the degree of intergradation between D. orestes 'B' and D. orestes 'C', specifically investigating variation in allozymes, mitochondrial DNA (mtDNA), and reproductive behavior among populations. On a broad scale, the mtDNA genealogies reconstruct haplotype clades that correspond to the species identified from previous allozyme analyses. However, at a finer geographic scale, the distributions of the allozyme and mtDNA markers for D. orestes 'B' and D. orestes 'C' are discordant. MtDNA haplotypes corresponding to D. orestes 'B' are more broadly distributed across western North Carolina than predicted by allozyme data, and the region of intergradation with D. orestes 'C' indicates asymmetric gene flow of these markers. Asymmetric mating may contribute to observed discordance in nuclear versus cytoplasmic markers. Results support describing D. orestes as a single species and emphasize the importance of using multiple markers to examine fine-scale patterns and elucidate evolutionary processes affecting gene flow when making species-level taxonomic decisions.

Principles of dialysis: special issues in women.

Nephrologists are frequently responsible for the primary care of their female patients. As such, they must be aware of medical issues that are unique to women. Although many of the medical considerations are similar to those in women without renal disease, there are a number of special considerations unique to end-stage renal disease (ESRD) patients. Women comprise a smaller proportion of the dialysis population and have better survival rates than men do. The improved survival is less marked than seen in the general population and may be function of differential susceptibility to disease processes, socio-cultural factors, or gender differences in acceptance or transplantation rates. A variety of factors are important in choosing dialysis modality including lifestyle issues and previous abdominal surgery. Women with ESRD are at high risk of both sexual and gonadal dysfunction, for which the latter may be treated with replacement hormones. Pregnancy is rare and requires an increase in the dose of dialysis and the care of a team of experienced physicians. Finally, awareness and implementation of routine health maintenance recommendations is essential in the care of female dialysis patients.

Prevention and Treatment of Hypertension Study (PATHS): effects of an alcohol treatment program on blood pressure.

OBJECTIVE: To determine whether blood pressure is reduced for at least 6 months with an intervention to lower alcohol intake in moderate to heavy drinkers with above optimal to slightly elevated diastolic blood pressure, and whether reduction of alcohol intake can be maintained for 2 years. DESIGN: A randomized controlled trial. METHODS: Six hundred forty-one outpatient veterans with an average intake of 3 or more alcoholic drinks per day in the 6 months before entry into the study and with diastolic blood pressure 80 to 99 mm Hg were randomly assigned to a cognitive-behavioral alcohol reduction intervention program or a control observation group for 15 to 24 months. The goal of the intervention was the lower of 2 or fewer drinks daily or a 50% reduction in intake. A subgroup with hypertension was defined as having a diastolic blood pressure of 90 to 99 mm Hg, or 80 to 99 mm Hg if recently taking medication for hypertension. RESULTS: Reduction in average weekly self-reported alcohol intake was significantly greater (P<.001) at every assessment from 3 to 24 months in the intervention group vs the control group: levels declined from 432 g/wk at baseline by 202 g/wk in the intervention group and from 445 g/wk by 78 g/wk in the control group in the first 6 months, with similar reductions after 24 months. The intervention group had a 1.2/0.7-mm Hg greater reduction in blood pressure than the control group (for each, P = .17 and P = .18) for the 6-month primary end point; for the hypertensive stratum the difference was 0.9/0.7 mm Hg (for each, P = .58 and P = .44). CONCLUSIONS: The 1.3 drinks per day average difference between changes in self-reported alcohol intake observed in this trial produced only small nonsignificant effects on blood pressure. The results from the Prevention and Treatment of Hypertension Study (PATHS) do not provide strong support for reducing alcohol consumption in nondependent moderate drinkers as a sole method for the prevention or treatment of hypertension.