Genomic variation landscape of the human gut microbiome.

Whereas large-scale efforts have rapidly advanced the understanding and practical impact of human genomic variation, the practical impact of variation is largely unexplored in the human microbiome. We therefore developed a framework for metagenomic variation analysis and applied it to 252 faecal metagenomes of 207 individuals from Europe and North America. Using 7.4 billion reads aligned to 101 reference species, we detected 10.3 million single nucleotide polymorphisms (SNPs), 107,991 short insertions/deletions, and 1,051 structural variants. The average ratio of non-synonymous to synonymous polymorphism rates of 0.11 was more variable between gut microbial species than across human hosts. Subjects sampled at varying time intervals exhibited individuality and temporal stability of SNP variation patterns, despite considerable composition changes of their gut microbiota. This indicates that individual-specific strains are not easily replaced and that an individual might have a unique metagenomic genotype, which may be exploitable for personalized diet or drug intake.

Role for urea in nitrification by polar marine Archaea.

Despite the high abundance of Archaea in the global ocean, their metabolism and biogeochemical roles remain largely unresolved. We investigated the population dynamics and metabolic activity of Thaumarchaeota in polar environments, where these microorganisms are particularly abundant and exhibit seasonal growth. Thaumarchaeota were more abundant in deep Arctic and Antarctic waters and grew throughout the winter at surface and deeper Arctic halocline waters. However, in situ single-cell activity measurements revealed a low activity of this group in the uptake of both leucine and bicarbonate (<5% Thaumarchaeota cells active), which is inconsistent with known heterotrophic and autotrophic thaumarchaeal lifestyles. These results suggested the existence of alternative sources of carbon and energy. Our analysis of an environmental metagenome from the Arctic winter revealed that Thaumarchaeota had pathways for ammonia oxidation and, unexpectedly, an abundance of genes involved in urea transport and degradation. Quantitative PCR analysis confirmed that most polar Thaumarchaeota had the potential to oxidize ammonia, and a large fraction of them had urease genes, enabling the use of urea to fuel nitrification. Thaumarchaeota from Arctic deep waters had a higher abundance of urease genes than those near the surface suggesting genetic differences between closely related archaeal populations. In situ measurements of urea uptake and concentration in Arctic waters showed that small-sized prokaryotes incorporated the carbon from urea, and the availability of urea was often higher than that of ammonium. Therefore, the degradation of urea may be a relevant pathway for Thaumarchaeota and other microorganisms exposed to the low-energy conditions of dark polar waters.

Orthologous gene clusters and taxon signature genes for viruses of prokaryotes.

Viruses are the most abundant biological entities on earth and encompass a vast amount of genetic diversity. The recent rapid increase in the number of sequenced viral genomes has created unprecedented opportunities for gaining new insight into the structure and evolution of the virosphere. Here, we present an update of the phage orthologous groups (POGs), a collection of 4,542 clusters of orthologous genes from bacteriophages that now also includes viruses infecting archaea and encompasses more than 1,000 distinct virus genomes. Analysis of this expanded data set shows that the number of POGs keeps growing without saturation and that a substantial majority of the POGs remain specific to viruses, lacking homologues in prokaryotic cells, outside known proviruses. Thus, the great majority of virus genes apparently remains to be discovered. A complementary observation is that numerous viral genomes remain poorly, if at all, covered by POGs. The genome coverage by POGs is expected to increase as more genomes are sequenced. Taxon-specific, single-copy signature genes that are not observed in prokaryotic genomes outside detected proviruses were identified for two-thirds of the 57 taxa (those with genomes available from at least 3 distinct viruses), with half of these present in all members of the respective taxon. These signatures can be used to specifically identify the presence and quantify the abundance of viruses from particular taxa in metagenomic samples and thus gain new insights into the ecology and evolution of viruses in relation to their hosts.

Prediction and identification of sequences coding for orphan enzymes using genomic and metagenomic neighbours.

Despite the current wealth of sequencing data, one-third of all biochemically characterized metabolic enzymes lack a corresponding gene or protein sequence, and as such can be considered orphan enzymes. They represent a major gap between our molecular and biochemical knowledge, and consequently are not amenable to modern systemic analyses. As 555 of these orphan enzymes have metabolic pathway neighbours, we developed a global framework that utilizes the pathway and (meta)genomic neighbour information to assign candidate sequences to orphan enzymes. For 131 orphan enzymes (37% of those for which (meta)genomic neighbours are available), we associate sequences to them using scoring parameters with an estimated accuracy of 70%, implying functional annotation of 16,345 gene sequences in numerous (meta)genomes. As a case in point, two of these candidate sequences were experimentally validated to encode the predicted activity. In addition, we augmented the currently available genome-scale metabolic models with these new sequence-function associations and were able to expand the models by on average 8%, with a considerable change in the flux connectivity patterns and improved essentiality prediction.

Randomized controlled trial of osteoconductive fixation screws for anterior cruciate ligament reconstruction: a comparison of the Calaxo and Milagro screws.

PURPOSE: To compare the outcome of 2 bioabsorbable screws for tibial interference fixation in anterior cruciate ligament reconstruction with reference to rate of absorption, osteoconductive properties, and clinical outcome. METHODS: Patients undergoing primary anterior cruciate ligament reconstruction with hamstring autograft in a single unit were invited to participate in this study. Patients were randomized to receive either the Calaxo screw (Smith & Nephew, Andover, MA) or Milagro screw (DePuy Mitek, Raynham, MA) for tibial fixation. Patients were reviewed with subjective and objective evaluation by use of the International Knee Documentation Committee form, Lysholm score, KT-1000 arthrometry (MEDmetric, San Diego, CA), and clinical examination. Magnetic resonance imaging was performed at 1 year and computed tomography scanning at 1 week and at 6, 12, and 24 months. RESULTS: Sixty patients agreed to participate in the study, with 32 patients randomized to the Calaxo screw and 28 to the Milagro screw for tibial fixation. There was no significant difference in subjective or objective clinical outcome between the 2 groups. At 24 months, 88% of Calaxo screws showed complete screw resorption compared with 0% of Milagro screws (P < .001). Tibial cysts were present in 88% of the Calaxo group and 7% of the Milagro group (P = .001). At 24 months, the mean volume of new bone formation for the Calaxo group was 21% of original screw volume. Ossification of the Milagro screw was unable to be accurately assessed as a result of incomplete screw resorption. CONCLUSIONS: Both screws showed similar favorable objective and subjective outcomes at 2 years. The Calaxo screw resorbed completely over a period of 6 months and was associated with a high incidence of intra-tunnel cyst formation. The Milagro screw increased in volume over a period of 6 months, followed by a gradual resorption, which was still ongoing at 2 years. Both screws were associated with tunnel widening, and neither showed evidence of significant tunnel ossification. We conclude that, despite satisfactory clinical outcomes, the addition of "osteoconductive" materials to bioabsorbable screws is not associated with bone formation at the screw site at 2 years. LEVEL OF EVIDENCE: Level I, randomized controlled trial.

Transcriptional analysis of a Dehalococcoides-containing microbial consortium reveals prophage activation.

Chlorinated solvents are among the most prevalent groundwater contaminants in the industrialized world. Biodegradation with Dehalococcoides-containing mixed cultures is an effective remediation technology. To elucidate transcribed genes in a Dehalococcoides-containing mixed culture, a shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination and during starvation (no chlorinated compounds) by a microbial enrichment culture called KB-1. In both treatment conditions, methanol was amended as an electron donor. Subsequently, spots were sequenced that contained the genes most differentially transcribed between the VC-degrading and methanol-only conditions, as well as spots with the highest intensities. Sequencing revealed that during VC degradation Dehalococcoides genes involved in transcription, translation, metabolic energy generation, and amino acid and lipid metabolism and transport were overrepresented in the transcripts compared to the average Dehalococcoides genome. KB-1 rdhA14 (vcrA) was the only reductive dehalogenase homologous (RDH) gene with higher transcript levels during VC degradation, while multiple RDH genes had higher transcript levels in the absence of VC. Numerous hypothetical genes from Dehalococcoides also had higher transcript levels in methanol-only treatments, indicating that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. In addition, microarray results prompted biological experiments confirming that electron acceptor limiting conditions activated a Dehalococcoides prophage. Transcripts from Spirochaetes, Chloroflexi, Geobacter, and methanogens demonstrate the importance of non-Dehalococcoides organisms to the culture, and sequencing of identified shotgun clones of interest provided information for follow-on targeted studies.

L-cell Arntl is required for rhythmic glucagon-like peptide-1 secretion and maintenance of intestinal homeostasis.

OBJECTIVE: Recent studies using whole-body clock-disrupted animals identified a disruption in the circadian rhythm of the intestinal L-cell incretin hormone, glucagon-like peptide-1 (GLP-1). Although GLP-1 plays an essential role in metabolism through enhancement of both glucose-stimulated insulin secretion and satiety, recent evidence has also demonstrated its importance in regulating intestinal and microbial homeostasis. Therefore, using in vivo and in vitro models, this study assessed the role of the core circadian clock gene Arntl in the regulation of time-dependent GLP-1 secretion and its impact on the intestinal environment. METHODS: Oral glucose tolerance tests were conducted at zeitgeber time 2 and 14 in control and inducible Gcg-Arntl knockout (KO) mice. Colonic intraepithelial lymphocytes were isolated, mucosal gene expression analysis was conducted, and 16S rRNA gene sequencing of colonic feces as well as analysis of microbial metabolites were performed. Time-dependent GLP-1 secretion and transcriptomic analysis were conducted in murine (m) GLUTag L-cells following siRNA-mediated knockdown of Arntl. RESULTS: Gcg-Arntl KO mice displayed disrupted rhythmic release of GLP-1 associated with reduced secretion at the established peak time point. Analysis of the intestinal environment in KO mice revealed a decreased proportion of CD4+ intraepithelial lymphocytes in association with increased proinflammatory cytokine gene expression and increased colonic weight. Moreover, increased Actinobacteria within the colonic microbiome was found following L-cell Arntl disruption, as well as reductions in the microbial products, short chain fatty acids, and bile acids. Finally, siRNA-mediated knockdown of Arntl in mGLUTag L-cells resulted in both impaired time-dependent GLP-1 secretion and the disruption of pathways related to key cellular processes. CONCLUSIONS: These data establish, for the first time, the essential role of Arntl in the intestinal L-cell in regulating time-dependent GLP-1 secretion. Furthermore, this study revealed the integral role of L-cell Arntl in mediating the intestinal environment, which ultimately may provide novel insight into the development of therapeutics for the treatment of intestinal and metabolic disorders.

Plant microbiome analysis after Metarhizium amendment reveals increases in abundance of plant growth-promoting organisms and maintenance of disease-suppressive soil.

The microbial community in the plant rhizosphere is vital to plant productivity and disease resistance. Alterations in the composition and diversity of species within this community could be detrimental if microbes suppressing the activity of pathogens are removed. Species of the insect-pathogenic fungus, Metarhizium, commonly employed as biological control agents against crop pests, have recently been identified as plant root colonizers and provide a variety of benefits (e.g. growth promotion, drought resistance, nitrogen acquisition). However, the impact of Metarhizium amendment on the rhizosphere microbiome has yet to be elucidated. Using Illumina sequencing, we examined the community profiles (bacteria and fungi) of common bean (Phaseolus vulgaris) rhizosphere (loose soil and plant root) after amendment with M. robertsii conidia, in the presence and absence of an insect host. Although alpha diversity was not significantly affected overall, there were numerous examples of plant growth-promoting organisms that significantly increased with Metarhizium amendment (Bradyrhizobium, Flavobacterium, Chaetomium, Trichoderma). Specifically, the abundance of Bradyrhizobium, a group of nitrogen-fixing bacteria, was confirmed to be increased using a qPCR assay with genus-specific primers. In addition, the ability of the microbiome to suppress the activity of a known bean root pathogen was assessed. The development of disease symptoms after application with Fusarium solani f. sp. phaseoli was visible in the hypocotyl and upper root of plants grown in sterilized soil but was suppressed during growth in microbiome soil and soil treated with M. robertsii. Successful amendment of agricultural soils with biocontrol agents such as Metarhizium necessitates a comprehensive understanding of the effects on the diversity of the rhizosphere microbiome. Such research is fundamentally important towards sustainable agricultural practices to improve overall plant health and productivity.

Circadian GLP-1 Secretion in Mice Is Dependent on the Intestinal Microbiome for Maintenance of Diurnal Metabolic Homeostasis.

The incretin glucagon-like peptide 1 (GLP-1) is secreted by the intestinal L cell upon nutrient ingestion. GLP-1 also exhibits a circadian rhythm, with highest release at the onset of the feeding period. Similarly, microbial composition and function exhibit circadian rhythmicity with fasting-feeding. The circadian pattern of GLP-1 release was found to be dependent on the oral route of glucose administration and was necessary for the rhythmic release of insulin and diurnal glycemic control in normal male and female mice. In mice fed a Western (high-fat/high-sucrose) diet for 16 weeks, GLP-1 secretion was markedly increased but arrhythmic over the 24-h day, whereas levels of the other incretin, glucose-dependent insulinotropic polypeptide, were not as profoundly affected. Furthermore, the changes in GLP-1 secretion were shown to be essential for the maintenance of normoglycemia in this obesogenic environment. Analysis of the primary L-cell transcriptome, as well as of the intestinal microbiome, also demonstrated time-of-day- and diet-dependent changes paralleling GLP-1 secretion. Finally, studies in antibiotic-induced microbial depleted and in germ-free mice with and without fecal microbial transfer, provided evidence for a role of the microbiome in diurnal GLP-1 release. In combination, these findings establish a key role for microbiome-dependent circadian GLP-1 secretion in the maintenance of 24-h metabolic homeostasis.

Fifteen-Year Survival of Endoscopic Anterior Cruciate Ligament Reconstruction in Patients Aged 18 Years and Younger.

BACKGROUND: The current body of literature surrounding anterior cruciate ligament (ACL) survival and the variables contributing to further ACL injuries after primary ACL reconstruction in children and adolescents is limited, with no long-term evidence examining the incidence and contributing factors of further ACL injuries in this younger patient population. PURPOSE: To determine the long-term survival of the ACL graft and the contralateral ACL (CACL) after primary reconstruction in patients aged ≤18 years and to identify the factors that increase the odds of subsequent ACL injuries. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Patients having undergone primary ACL reconstruction at age ≤18 years between 1993 and 1998 who were included in a prospective database by a single surgeon were considered for this study. Single-incision endoscopic ACL reconstruction was performed with either an autologous bone-patellar tendon-bone graft or a hamstring tendon graft. At a minimum of 15 years after ACL reconstruction, patients completed a subjective survey involving the International Knee Documentation Committee (IKDC) questionnaire in addition to questions regarding current symptoms, further ACL injuries, family history of ACL injury, and current level of activity. RESULTS: A total of 288 adolescents (age range, 13-18 years) met the inclusion criteria, of whom 242 (84%) were reviewed at a mean of 16 years and 6 months after ACL reconstruction. Of these patients, 75 (31%) sustained a further ACL injury: 27 (11.2%) suffered an ACL graft rupture, 33 suffered a CACL injury (13.6%), and 15 sustained both an ACL graft rupture and a CACL injury (6.2%) over 15 years. Survival of the ACL graft was 95%, 92%, 88%, 85%, and 83% at 1, 2, 5, 10, and 15 years, respectively, and survival of the CACL was 99%, 98%, 90%, 83%, and 81%, respectively. Survival of the ACL graft was less favorable in those with a family history of ACL injury than in those without a family history (69% vs 90%, respectively; hazard ratio [HR], 3.6; P = .001). Survival of the CACL was less favorable in male patients than in female patients (75% vs 88%, respectively; HR, 2.1; P = .03) and in those who returned to competitive team ball sports than in those who did not (78% vs 89%, respectively; HR, 2.3; P = .05). CONCLUSION: After ACL reconstruction in patients aged ≤18 years, a further ACL injury occurred in 1 in 3 patients over 15 years. The 15-year survival rate of the ACL graft was 83%, and the 15-year survival rate of the CACL was 81%. The ACL graft and CACL were most vulnerable within the first 5 years after index surgery. A family history of ACL rupture significantly increased the risk for ACL graft ruptures, and a CACL injury was more common in male patients and those who returned to team ball sports. High IKDC scores and continued participation in sports were maintained over the long term after ACL reconstruction in the adolescent population.

Twenty-Year Outcome of a Longitudinal Prospective Evaluation of Isolated Endoscopic Anterior Cruciate Ligament Reconstruction With Patellar Tendon or Hamstring Autograft.

BACKGROUND: Long-term prospective studies of isolated endoscopic anterior cruciate ligament (ACL) reconstruction are limited and may include confounding factors. PURPOSE: This study aimed to compare the outcomes of isolated ACL reconstruction using the patellar tendon (PT) autograft and the hamstring (HT) autograft in 180 patients over 20 years. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 180 participants undergoing isolated ACL reconstruction between 1993 and 1994 were prospectively recruited. Evaluation was performed at 1, 2, 5, 7, 10, 15, and 20 years after surgery and included the International Knee Documentation Committee (IKDC) knee ligament evaluation with radiographic evaluation, KT-1000 arthrometer side-to-side difference, and subjective scores. RESULTS: Over 20 years, there were 16 patients (18%) and 9 patients (10%) with an ACL graft rupture in the HT and PT groups, respectively (P = .13). ACL graft rupture was associated with male sex (odds ratio [OR], 3.9; P = .007), nonideal tunnel position (OR, 3.6; P = .019), and age <18 years at the time of surgery (OR, 4.6; P = .003). The odds of a contralateral ACL rupture were increased in patients with the PT graft compared with patients with the HT graft (OR, 2.2; P = .02) and those aged <18 years at the time of surgery (OR, 3.4; P = .001). The mean IKDC scores at 20-year follow-up were 86 and 89 for the PT and HT groups, respectively (P = .18). At 20 years, 53% and 57% of the PT and HT groups participated in strenuous or very strenuous activities (P = .55), kneeling pain was present in 63% and 20% of the PT and HT groups (P = .018), and radiographic osteoarthritic change was found in 61% and 41% of the PT and HT groups (P = .008), respectively. CONCLUSION: Compared with patients who received the HT graft, patients who received the PT graft had significantly worse outcomes with regard to radiologically detectable osteoarthritis, kneeling pain, and contralateral ACL injury. At 20-year follow-up, both HT and PT autografts continued to provide good subjective outcomes and objective stability. However, further ACL injury is common, particularly in male individuals, younger patients, and those with tunnel malposition.

Twenty-year outcomes of a longitudinal prospective evaluation of isolated endoscopic anterior cruciate ligament reconstruction with patellar tendon autografts.

BACKGROUND: Long-term prospective follow-up studies of single-incision endoscopic anterior cruciate ligament (ACL) reconstruction are limited and may include confounding factors. PURPOSE: This longitudinal prospective study reports the outcomes of isolated ACL reconstruction using middle-third patellar tendon autografts in 90 patients over 20 years. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Between January 1993 and April 1994, a total of 90 patients met study inclusion criteria: evaluation at 1, 2, 3, 4, 5, 7, 10, 15, and 20 years after surgery. Exclusion criteria were associated ligamentous injuries requiring surgery, previous meniscectomy or meniscal injuries requiring more than one-third meniscectomy, chondral injuries, and an abnormal contralateral knee. RESULTS: At 20 years, 32 (36%) patients had sustained another ACL injury: 8 (9%) to the index limb and 27 (30%) to the contralateral limb (3 injuring both knees). The mean International Knee Documentation Committee (IKDC) score was 86. Of the patients, 50% participated in strenuous/very strenuous activities, and kneeling pain was present in 63%. Radiographic degenerative change was found in 61%; 20% had IKDC grade C, and 0% had grade D. The IKDC clinical examination revealed that 95% had a normal/nearly normal knee. Significant sex differences existed: when compared with male patients, female patients were less likely to reinjure the reconstructed ACL (18% vs 2%, respectively; P = .01), reported poorer IKDC subjective scores (90 vs 83, respectively; P = .03), had more activity-related pain (20% vs 57%, respectively; P = .02), and were less likely to participate in strenuous activities (66% vs 35%, respectively; P = .009). ACL graft survival was not related to age. Patients <18 years old had an increased odds ratio (3.2) for rupturing the contralateral ACL. A coronal graft angle <17° increased the risk of failure compared with an angle >17° (77% vs 96% survival, respectively) by a factor of 8.5. CONCLUSION: Injuries more commonly occurred in the contralateral ACL than in the reconstructed ACL graft, and the most significant predictor of a contralateral ACL injury was age <18 years. The most significant predictor of an ACL graft rupture was a coronal graft angle <17°. Female patients had lower rerupture rates, poorer subjective scores, and decreased participation in strenuous activities, putting the graft at a lower risk of failure. Kneeling pain remained persistent over 20 years. Radiographic osteoarthritis was evident in 61% of patients, but symptomatic osteoarthritic symptoms were rarely reported.

New insights into Dehalococcoides mccartyi metabolism from a reconstructed metabolic network-based systems-level analysis of D. mccartyi transcriptomes.

Organohalide respiration, mediated by Dehalococcoides mccartyi, is a useful bioremediation process that transforms ground water pollutants and known human carcinogens such as trichloroethene and vinyl chloride into benign ethenes. Successful application of this process depends on the fundamental understanding of the respiration and metabolism of D. mccartyi. Reductive dehalogenases, encoded by rdhA genes of these anaerobic bacteria, exclusively catalyze organohalide respiration and drive metabolism. To better elucidate D. mccartyi metabolism and physiology, we analyzed available transcriptomic data for a pure isolate (Dehalococcoides mccartyi strain 195) and a mixed microbial consortium (KB-1) using the previously developed pan-genome-scale reconstructed metabolic network of D. mccartyi. The transcriptomic data, together with available proteomic data helped confirm transcription and expression of the majority genes in D. mccartyi genomes. A composite genome of two highly similar D. mccartyi strains (KB-1 Dhc) from the KB-1 metagenome sequence was constructed, and operon prediction was conducted for this composite genome and other single genomes. This operon analysis, together with the quality threshold clustering analysis of transcriptomic data helped generate experimentally testable hypotheses regarding the function of a number of hypothetical proteins and the poorly understood mechanism of energy conservation in D. mccartyi. We also identified functionally enriched important clusters (13 for strain 195 and 11 for KB-1 Dhc) of co-expressed metabolic genes using information from the reconstructed metabolic network. This analysis highlighted some metabolic genes and processes, including lipid metabolism, energy metabolism, and transport that potentially play important roles in organohalide respiration. Overall, this study shows the importance of an organism's metabolic reconstruction in analyzing various "omics" data to obtain improved understanding of the metabolism and physiology of the organism.

Classification and quantification of bacteriophage taxa in human gut metagenomes.

Bacteriophages have key roles in microbial communities, to a large extent shaping the taxonomic and functional composition of the microbiome, but data on the connections between phage diversity and the composition of communities are scarce. Using taxon-specific marker genes, we identified and monitored 20 viral taxa in 252 human gut metagenomic samples, mostly at the level of genera. On average, five phage taxa were identified in each sample, with up to three of these being highly abundant. The abundances of most phage taxa vary by up to four orders of magnitude between the samples, and several taxa that are highly abundant in some samples are absent in others. Significant correlations exist between the abundances of some phage taxa and human host metadata: for example, 'Group 936 lactococcal phages' are more prevalent and abundant in Danish samples than in samples from Spain or the United States of America. Quantification of phages that exist as integrated prophages revealed that the abundance profiles of prophages are highly individual-specific and remain unique to an individual over a 1-year time period, and prediction of prophage lysis across the samples identified hundreds of prophages that are apparently active in the gut and vary across the samples, in terms of presence and lytic state. Finally, a prophage-host network of the human gut was established and includes numerous novel host-phage associations.

Endoscopic anterior cruciate ligament reconstruction in children using living donor hamstring tendon allografts.

BACKGROUND: The incidence of anterior cruciate ligament (ACL) injuries in children is increasing, but ACL reconstruction with traditional autograft sources is associated with high rates of further ACL injury when compared with adult populations. The outcome of ACL reconstruction using an alternative graft source, the living donor hamstring tendon (HT) allograft, has not been reported. PURPOSE: To determine the outcome of endoscopic transphyseal single-tunnel ACL reconstruction using living donor HT allografts. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Between 2007 and 2008, 32 children underwent endoscopic transphyseal single-tunnel ACL reconstruction using living donor HT allografts. The HT allograft was harvested from a parent. At a minimum 2 years' follow-up, full International Knee Documentation Committee (IKDC) knee ligament examination was performed on the children including instrumented testing using the KT-1000 arthrometer. Donors underwent subjective review at a minimum 2 years' follow-up. RESULTS: Thirty-one children (97%) were able to be contacted for review. Of these, 2 (6%) sustained an ACL graft rupture within 2 years after surgery. Twenty-nine children completed clinical and subjective review. The mean age at ACL reconstruction was 13 years (range, 8-16 years). The mean HT graft size was 7.2 mm (range, 6-8 mm). The mean IKDC subjective score was 97 (range, 84-100). Twenty-eight patients (97%) had a normal or nearly normal IKDC ligament grade. At 2 years after surgery, 27 patients (93%) reported regularly participating in very strenuous or strenuous activities. There were no cases of limb malalignment. Twenty eight (97%) of the donors reported that they would undergo the same procedure again under the same circumstances. CONCLUSION: Excellent clinical and subjective outcomes were achieved with high levels of return to desired activities. This technique allows a more predictable size of the HTs compared with an autograft from the child and maintains an intact neuromuscular hamstring structure in the child. Finally, the child's own HTs are reserved for future use. Anterior cruciate ligament reconstruction using living donor allografts should be considered a viable choice in children.

Individuality and temporal stability of the human gut microbiome.

INTRODUCTION: The breakthrough of next generation sequencing-technologies has enabled large-scale studies of natural microbial communities and the 16S rRNA genes have been widely used as a phylogenetic marker to study community structure. However, major limitations of this approach are that neither strain-level resolution nor genomic context of microorganisms can be provided. This information, however, is crucial to answer fundamental questions about the temporal stability and distinctiveness of natural microbial communities. MATERIAL AND METHODS: We developed a methodological framework for metagenomic single nucleotide polymorphism (SNP) variation analysis and applied it to publicly available data from 252 human fecal samples from 207 European and North American individuals. We further analyzed samples from 43 healthy subjects that were sampled at least twice over time intervals of up to one year and measured population similarities of dominant gut species. RESULTS: We detected 10.3 million SNPs in 101 species, which nearly amounts to the number identified in more than 1,000 humans. CONCLUSION: The most striking result was that host-specific strains appear to be retained over long time periods. This indicates that individual-specific strains are not easily exchanged with the environment and furthermore, that an individuals appear to have a unique metagenomic genotype. This, in turn, is linked to implications for human gut physiology, such as the stability of antibiotic resistance potential.

Clinical results and risk factors for reinjury 15 years after anterior cruciate ligament reconstruction: a prospective study of hamstring and patellar tendon grafts.

BACKGROUND: There is a lack of prospective studies comparing the long-term outcome of endoscopic anterior cruciate ligament (ACL) reconstruction with either a patellar tendon or hamstring tendon autograft. PURPOSE: This prospective longitudinal study compared the results of isolated endoscopic ACL reconstruction utilizing a 4-strand hamstring tendon (HT) or patellar tendon (PT) autograft over a 15-year period with respect to reinjury, clinical outcomes, and the development of osteoarthritis. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Ninety consecutive patients with isolated ACL rupture were reconstructed with a PT autograft, and 90 patients received an HT autograft, with an identical surgical technique. Patients were assessed at 2, 5, 7, 10, and 15 years. Assessment included the International Knee Documentation Committee (IKDC) knee ligament evaluation including radiographic evaluation, KT-1000 arthrometer testing, and Lysholm knee score. RESULTS: Patients who received the PT graft had significantly worse outcomes compared with those who received the HT graft at 15 years for the variables of radiologically detectable osteoarthritis (grade A: 46% in PT and 69% in HT; P = .04), motion loss (extension deficit <3°: 79% in PT and 94% in HT; P = .03), single-legged hop test (grade A: 65% in PT and 92% in HT; P = .001), participation in strenuous activity (very strenuous or strenuous: 62% of PT and 77% of HT; P = .04), and kneeling pain (moderate or greater pain: 42% of PT and 26% of HT; P = .04). There was no significant difference between the HT and PT groups in overall IKDC grade (grade A: 47% of PT and 57% of HT; P = .35). An ACL graft rupture occurred in 17% of the HT group and 8% of the PT group (P = .07). An ACL graft rupture was associated with nonideal tunnel position (odds ratio [OR], 5.0) and male sex (OR, 3.2). Contralateral ACL rupture occurred in significantly more PT patients (26%) than HT patients (12%) (P = .02) and was associated with age ≤18 years (OR, 4.1) and the PT graft (OR, 2.6). CONCLUSION: Anterior cruciate ligament reconstruction using ipsilateral autograft continues to show excellent results in terms of patient satisfaction, symptoms, function, activity level, and stability. The use of HT autograft does, however, show better outcomes than the PT autograft in all of these outcome measures. Additionally, at 15 years, the HT graft-reconstructed ACLs have shown a lower rate of radiological osteoarthritis.

Outcome of anatomic transphyseal anterior cruciate ligament reconstruction in Tanner stage 1 and 2 patients with open physes.

BACKGROUND: Anterior cruciate ligament (ACL) injuries are being seen with increasing frequency in children. Treatment of the ACL-deficient knee in skeletally immature patients is controversial. PURPOSE: To determine the outcome of all-arthroscopic transphyseal anatomic single-bundle ACL reconstruction in Tanner stage 1 and 2 patients at a minimum of 2 years after surgery. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Between 2007 and 2008, 16 prepubescent patients underwent ACL reconstruction using soft tissue grafts. All patients were Tanner stage 1 and 2. Outcomes were assessed at a minimum of 2 years after surgery and included limb alignment, limb length, instrumented testing with the KT-1000 arthrometer, and International Knee Documentation Committee (IKDC) score. RESULTS: Mean age at the time of surgery was 12 years (range, 8-14 years). Graft choices included the following: living donor-related hamstring tendon allograft (n = 14), hamstring tendon autograft (n = 1), and fresh-frozen allograft (n = 1). Mean IKDC subjective score was 96 (range, 84-100). All patients had a stable knee postoperatively. Eleven patients had a negative Lachman test result, and 14 had a negative pivot-shift test result. The remainder had grade 1 Lachman and pivot-shift test findings, respectively. At 2 years after surgery, all patients had returned to strenuous activities, and normal or nearly normal overall IKDC score was documented in 94% of patients. There were no cases of limb malalignment or growth arrest. CONCLUSION: We present a case series of transphyseal anatomic single-bundle ACL reconstruction in Tanner stage 1 and 2 patients at a minimum of 2 years after surgery. Excellent clinical outcomes were obtained with high levels of return to desired activities. Importantly, no growth disturbances were seen in this series of patients.

Survival of the anterior cruciate ligament graft and the contralateral ACL at a minimum of 15 years.

BACKGROUND: The risks for primary anterior cruciate ligament (ACL) rupture have been established. What is less well known is the risk of graft rupture after reconstruction and also the risk of a primary ACL rupture in the contralateral knee. PURPOSE: To determine the long-term survival of the ACL graft and the contralateral ACL (CACL) after reconstruction and to identify factors that increase the odds of subsequent ACL injury. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: All patients having undergone primary ACL reconstruction in 1993 or 1994 by a single surgeon in a single unit were considered. Patients were contacted to complete a subjective interview by telephone or e-mail questionnaire at a minimum of 15 years after surgery. RESULTS: A total of 755 patients met the inclusion criteria, and ACL reconstruction was performed using a single-incision endoscopic technique with either autologous bone-patellar tendon-bone graft (BPTB; n = 314) or hamstring tendon graft (HT; n = 359) and metal interference screw fixation. Of these patients, 673 (89%) completed the questionnaire; 23% had sustained either a graft rupture or CACL rupture. Expected survival of the ACL graft was 95%, 93%, 91%, and 89% at a respective 2, 5, 10, and 15 years after reconstruction. Expected survival of the CACL was 97%, 93%, 90%, and 87%, respectively. Survival of the ACL graft was less favorable in men than in women (P = .007); ACL graft survival was not significantly different between the HT (88%) or BPTB (91%) groups (P = .149). Rupture of the CACL occurred twice as frequently as graft rupture in the BPTB group (graft survival, 84% vs 89%; P = .003). A positive family history of ACL rupture doubled the odds of both ACL graft and CACL rupture. The mean International Knee Documentation Committee subjective score at 15 years was 85. Return to preinjury sport levels was reported in 73% of patients, and 51% were still participating in strenuous or very strenuous activities at 15 years. CONCLUSION: Fifteen years after ACL reconstruction, expected survival of the ACL graft was 89% and expected survival of the CACL was 86%. Graft choice did not affect ACL graft rupture, but using BPTB increased the risk of CACL rupture compared with HT. Men had a less favorable survival rate of the ACL graft than did women, and a family history of ACL rupture increased the risk of both ACL graft and CACL rupture.

Assessment of metagenomic assembly using simulated next generation sequencing data.

Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition. For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities. Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available.