Catheter Directed Thrombolysis for Not Immediately Threatening Acute Limb Ischaemia: Systematic Review and Meta-Analysis.

OBJECTIVE: This systematic review and meta-analysis reports the outcomes of catheter directed thrombolysis (CDT) in patients with not immediately threatening (Rutherford I) acute lower limb ischaemia (ALI). DATA SOURCES: PubMed, Embase, and the Cochrane Library. REVIEW METHODS: A systematic search of PubMed, Embase, and the Cochrane Library was performed to identify observational studies and trials published between 1990 and 2022 reporting on the results of CDT in patients with Rutherford I ALI. A meta-analysis was performed using a random effects model with 95% confidence intervals (CIs). The outcomes of interests were treatment duration, angiographic success, bleeding complications, amputation and mortality rates, primary and secondary patency, and functional outcome expressed as pain free walking distance. RESULTS: Thirty-nine studies were included, comprising 1 861 patients who received CDT for not immediately threatening ALI. Funnel plots showed an indication of publication bias, and heterogeneity was substantial. Data from 5 to 13 studies were included in the meta-analysis. The pooled treatment duration was 2 days (95% CI 1 - 2), with an angiographic success rate of 80% (95% CI 73 - 86) and a 30 day freedom of amputation rate of 98% (95% CI 92 - 100). The major bleeding rate was 5% (95% CI 2 - 14), with a 30 day mortality rate of 3% (95% CI 1 - 5). The amputation free survival rate was 71% (95% CI 62 - 80) at the one year and 63% (95% CI 51 - 73) at the three year follow up. Long term patency rates were retrieved from four studies: 48% at one year (95% CI 27 - 70). No data could be retrieved on patient walking distance. CONCLUSION: Although CDT in the treatment of not immediately threatening ALI showed high angiographic success, the long term outcomes were relatively poor, with low patency and a substantial risk of major amputation. Further research is required to interpret the outcome of CDT in the context of potential confounders such as age and comorbidities.

Oncological Outcomes After Anastomotic Leakage After Surgery for Colon or Rectal Cancer: Increased Risk of Local Recurrence.

OBJECTIVE: The aim of this study was to evaluate oncological outcome for patients with and without anastomotic leakage after colon or rectal cancer surgery. SUMMARY OF BACKGROUND DATA: The role of anastomotic leakage in oncological outcome after colorectal cancer surgery is still topic of debate and impact on follow-up and consideration for further treatment remains unclear. METHODS: Patients included in the international, multicenter, non-inferior, open label, randomized, controlled trials COLOR and COLOR II, comparing laparoscopic surgery for curable colon (COLOR) and rectal (COLOR II) cancer with open surgery, were analyzed. Patients operated by abdominoperineal excision were excluded. Both univariate and multivariate analyses were performed to investigate the impact of leakage on overall survival, disease-free survival, local and distant recurrences, adjusted for possible confounders. Primary endpoints in the COLOR and COLOR II trial were disease-free survival and local recurrence at 3-year follow-up, respectively, and secondary endpoints included anastomotic leakage rate. RESULTS: For colon cancer, anastomotic leakage was not associated with increased percentage of local recurrence or decreased disease-free-survival. For rectal cancer, an increase of local recurrences (13.3% vs 4.6%; hazard ratio 2.96; 95% confidence interval 1.38-6.34; P = 0.005) and a decrease of disease-free survival (53.6% vs 70.9%; hazard ratio 1.67; 95% confidence interval 1.16-2.41; P = 0.006) at 5-year follow-up were found in patients with anastomotic leakage. CONCLUSION: Short-term morbidity, mortality, and long-term oncological outcomes are negatively influenced by the occurrence of anastomotic leakage after rectal cancer surgery. For colon cancer, no significant effect was observed; however, due to low power, no conclusions on the influence of anastomotic leakage on outcomes after colon surgery could be reached. Clinical awareness of increased risk of local recurrence after anastomotic leakage throughout the follow-up is mandatory.Trial Registration: Registered with ClinicalTrials.gov, number NCT00387842 and NCT00297791.

Mitochondrial genomic data are effective at placing mycoheterotrophic lineages in plant phylogeny.

Fully mycoheterotrophic plants can be difficult to place in plant phylogeny due to elevated substitution rates associated with photosynthesis loss. This potentially limits the effectiveness of downstream analyses of mycoheterotrophy that depend on accurate phylogenetic inference. Although mitochondrial genomic data sets are rarely used in plant phylogenetics, theory predicts that they should be resilient to long-branch artefacts, thanks to their generally slow evolution, coupled with limited rate elevation in heterotrophs. We examined the utility of mitochondrial genomes for resolving contentious higher-order placements of mycoheterotrophic lineages in two test cases: monocots (focusing on Dioscoreales) and Ericaceae. We find Thismiaceae to be distantly related to Burmanniaceae in the monocot order Dioscoreales, conflicting with current classification schemes based on few gene data sets. We confirm that the unusual Afrothismia is related to Taccaceae-Thismiaceae, with a corresponding independent loss of photosynthesis. In Ericaceae we recovered the first well supported relationships among its five major lineages: mycoheterotrophic Ericaceae are not monophyletic, as pyroloids are inferred to be sister to core Ericaceae, and monotropoids to arbutoids. Genes recovered from mitochondrial genomes collectively resolved previously ambiguous mycoheterotroph higher-order relationships. We propose that mitochondrial genomic data should be considered in standardised gene panels for inferring overall plant phylogeny.

Assessment of stream macroinvertebrate communities with eDNA is not congruent with tissue-based metabarcoding.

Freshwater biomonitoring programmes routinely sample aquatic macroinvertebrates. These samples are time-consuming to collect, as well as challenging and costly to identify reliably genus or species. Environmental DNA (eDNA) metabarcoding has emerged as a surrogate to traditional collection techniques and has been used in whole-community approaches across several taxa and ecosystems. However, the usefulness of eDNA-based detection of freshwater macroinvertebrates has not been extensively explored. Few studies have directly compared bulk sample and eDNA metabarcoding at a local scale to assess how effective each method is at characterizing aquatic macroinvertebrate communities. Here, we collected both eDNA and kicknet samples at the same sample transect locations across nine different streams in southern Ontario, Canada. We observed minimal overlap in community composition between these paired samples. Bulk tissue metabarcoding resulted in a greater proportion of sequences belonging to metazoan taxa (over 99%) than eDNA (12%) and had higher OTU richness for macroinvertebrate taxa. We suggest that degenerate primers are not effective for eDNA metabarcoding due to the high degree of nontarget amplification and subsequently low yield of target DNA. While both bulk sample and eDNA metabarcoding had the power to detect differences between stream communities, eDNA did not represent local communities. Bulk tissue metabarcoding thus provides a more accurate representation of local stream macroinvertebrate communities and is the preferred method if smaller-scale spatial resolution is an important factor in data analyses.

Quality of life after rectal cancer surgery: differences between laparoscopic and transanal total mesorectal excision.

BACKGROUND: Transanal total mesorectal excision (TaTME) is a safe alternative to laparoscopic TME for mid and low rectal cancer. TaTME allows improved visualization of the surgical planes and margins, and may potentially improve oncological outcomes. However, functional results after total mesorectal excision (TME) are variable and there are currently only a few published studies that include functional data related to the outcomes of TaTME. METHODS: Fifty-four consecutive patients were included in this study: one group included 27 patients who underwent laparoscopic low anterior and the other included 27 patients who underwent TaTME. All patients were asked to complete five questionnaires related to quality of life (QOL) and function [EQ-5D-3L, EORTC-QLQ C30, EORTC-QLQ C29, Low Anterior Resection Syndrome score (LARS), and International Prostate Symptom Score IPSS]. All TaTME patients were operated on at The Gelderse Vallei Hospital by a single surgeon and had a follow-up of at least 6.6 months. RESULTS: The EORTC-QLQ C30 and EQ-5D-3L questionnaires showed comparable outcomes in terms of QOL between the two groups. Almost all items evaluated by the EORTC-QLQ C29, including sexual outcomes, were similar between the two groups. One item concerning fecal incontinence, however, was scored worse for TaTME. There were no significant differences between the groups in terms of LARS symptoms or urinary function. CONCLUSIONS: Patients undergoing laparoscopic or transanal TME showed comparable functional and QOL outcomes. Although the TaTME technique is still evolving, this study indicates that this technique is a safe alternative to laparoscopic surgery in terms of functional outcomes for mid and low rectal cancers.

Transanal Endoscopic Microsurgery with or without Completion Total Mesorectal Excision for T2 and T3 Rectal Carcinoma.

AIM: Transanal endoscopic microsurgery (TEM) is used for the resection of large rectal adenomas and well or moderately differentiated T1 carcinomas. Due to difficulty in preoperative staging, final pathology may reveal a carcinoma not suitable for TEM. Although completion total mesorectal excision is considered standard of care in T2 or more invasive carcinomas, this completion surgery is not always performed. The purpose of this article is to evaluate the outcome of patients after TEM-only, when completion surgery would be indicated. METHODS: In this retrospective multicenter, observational cohort study, outcome after TEM-only (n = 41) and completion surgery (n = 40) following TEM for a pT2-3 rectal adenocarcinoma was compared. RESULTS: Median follow-up was 29 months for the TEM-only group and 31 months for the completion surgery group. Local recurrence rate was 35 and 11% for the TEM-only and completion surgery groups respectively. Distant metastasis occurred in 16% of the patients in both groups. The 3-year overall survival was 63% in the TEM-only group and 91% in the completion surgery group respectively. Three-year disease-specific survival was 91 versus 93% respectively. CONCLUSIONS: Although local recurrence after TEM-only for pT2-3 rectal cancer is worse compared to the recurrence that occurs after completion surgery, disease-specific survival is comparable between both groups. The lower unadjusted overall survival in the TEM-only group indicates that TEM-only may be a valid alternative in older and frail patients, especially when high morbidity of completion surgery is taken into consideration. Nevertheless, completion surgery should always be advised when curation is intended.

A Sequel to Sanger: amplicon sequencing that scales.

BACKGROUND: Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. RESULTS: By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion). CONCLUSIONS: SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.

On the brink: the highly reduced plastomes of nonphotosynthetic Ericaceae.

Ericaceae (the heather family) is a large and diverse group of plants that forms elaborate symbiotic relationships with mycorrhizal fungi, and includes several nonphotosynthetic lineages. Using an extensive sample of fully mycoheterotrophic (MH) species, we explored inter- and intraspecific variation as well as selective constraints acting on the plastomes of these unusual plants. The plastomes of seven MH genera were analysed in a phylogenetic context with two geographically disparate individuals sequenced for Allotropa, Monotropa, and Pityopus. The plastomes of nonphotosynthetic Ericaceae are highly reduced in size (c. 33-41 kbp) and content, having lost all photosynthesis-related genes, and are reduced to encoding housekeeping genes as well as a protease subunit (clpP)-like and acetyl-CoA carboxylase subunit D (accD)-like open reading frames. Despite an increase in the rate of their nucleotide substitutions, the remaining protein-coding genes are typically under purifying selection in full MHs. We also identified ribosomal proteins under relaxed or neutral selection. These plastomes also exhibit striking structural rearrangements. Intraspecific variation within MH Ericaceae ranges from a few differences (Allotropa) to extensive population divergences (Monotropa, Hypopitys), which indicates that cryptic speciation may be occurring in several lineages. The pattern of gene loss within fully MH Ericaceae plastomes suggests an advanced state of degradation.

Different HLA-B27 subtypes present the same immunodominant Epstein-Barr virus peptide.

An immunological basis has been postulated for the strong association between at least five subtypes of the HLA-B27 allele (B27.01, .02, .04, .05, and .06) and ankylosing spondylitis, namely that cytotoxic T lymphocyte (CTL) responses are induced against an "arthritogenic" peptide that these different subtypes can all present. This requires a degree of overlap between the peptide binding repertoires of different B27 molecules. The present work, using CTL responses to Epstein-Barr virus (EBV) as a model system in which to identify B27-restricted epitopes, provides the first direct evidence that different disease-related alleles can present the same immunodominant peptide. We first noted that EBV-specific CTL clones, whether from B27.05-, B27.02-, or B27.04-positive donors, were largely subtype-specific in their restriction, recognizing only EBV-transformed B cell lines of the relevant B27 subtype. However, when tested against targets expressing individual EBV proteins from recombinant vaccinia virus vectors, all B27.05-restricted, all B27.02-restricted, and a proportion of B27.04-restricted clones were reactive to the same viral nuclear antigen, Epstein-Barr nuclear antigen (EBNA)3C. In subsequent peptide sensitization assays, all the EBNA3C-specific clones tested and also the EBNA3C-specific component within polyclonal CTL preparations from B27.05-, B27.02-, or B27.04-positive donors recognized the same immunodominant viral peptide RRIYDLIEL (EBNA3C residues 258-266). This sequence accords well with the proposed B27.05 peptide motif and clearly must be accommodated within the different peptide binding grooves of B27.05, B27.02, and B27.04 molecules. Clonal analysis revealed a second component of the B27.04-restricted response that was not shared with other subtypes. This was directed against an EBV latent membrane protein LMP2 epitope whose sequence RRRWRRLTV satisfies some but not all requirements of the B27.05 peptide motif. We conclude that there is indeed a degree of functional overlap between different B27 subtypes in their selection and presentation of CTL epitopes.

Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies.

Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins.

Validation of COI metabarcoding primers for terrestrial arthropods.

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40-60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

Metabarcoding a diverse arthropod mock community.

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.

Finding the pond through the weeds: eDNA reveals underestimated diversity of pondweeds.

PREMISE OF THE STUDY: The detection of environmental DNA (eDNA) using high-throughput sequencing has rapidly emerged as a method to detect organisms from environmental samples. However, eDNA studies of aquatic biomes have focused on surveillance of animal species with less emphasis on plants. Pondweeds are important bioindicators of freshwater ecosystems, although their diversity is underestimated due to difficulties in morphological identification and monitoring. METHODS: A protocol was developed to detect pondweeds in water samples using atpB-rbcL and ITS2 markers. The water samples were collected from the Grand River within the rare Charitable Research Reserve, Ontario (RARE). Short fragments were amplified using primers targeting pondweeds, sequenced on an Ion Torrent Personal Genome Machine, and assigned to the taxonomy using a local DNA reference library and GenBank. RESULTS: We detected two species earlier documented at the experimental site during ecological surveys (Potamogeton crispus and Stuckenia pectinata) and three species new to the RARE checklist (P. foliosus, S. filiformis, and Zannichellia palustris). DISCUSSION: Our targeted approach to track the species composition of pondweeds in freshwater ecosystems revealed underestimation of their diversity. This result suggests that eDNA is an effective tool for monitoring plant diversity in aquatic habitats.

[Application of flexible transanal port for excision of rectal tumours]. / Toepassing flexibele toegangspoort voor excisie rectumtumoren.

In 2010 the first report of the application of the flexible transanal port ('operation platform') for the excision of rectal tumours was published. Due to the enhanced vision it provides, adenomas and small malignant rectal tumours can be radically resected with significantly fewer recurrences than with endoscopic mucosal resection or transanal excisions done without this platform. The application of this platform is cheaper and more intuitive than transanal endoscopic microsurgery, while the quality of the local resection, the risk of postoperative complications and the functional and oncological outcomes all appear to be comparable. This is the reason that this flexible platform is now in use in most Dutch hospitals. The flexible port has led to an increase in rectum-sparing treatment for low-risk T1 rectal carcinoma. Nowadays, this platform is also used for the transanal approach during radical rectal surgery for high-risk rectal carcinomas and for rectal operations in patients with benign conditions in the pelvis minor such as severe endometriosis or Crohn's disease.

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada.

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

PREMISE OF THE STUDY: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. METHODS: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. RESULTS: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). DISCUSSION: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.

Epstein-Barr virus-specific cytotoxic T lymphocyte responses in the blood and tumor site of Hodgkin's disease patients: implications for a T-cell-based therapy.

Approximately 40% of Hodgkin's disease (HD) cases carry EBV in the malignant Hodgkin-Reed Sternberg (H-RS) cells, with expression of viral latent membrane proteins (LMPs) 1 and 2. These viral proteins are targets for CTLs in healthy EBV carriers, and their expression in EBV-associated HD raises the possibility of targeting them for a CTL-based immunotherapy. Here we characterize the CTL response to EBV latent antigens in both the blood and tumor-infiltrating lymphocytes of HD patients using two approaches: (a) in vitro reactivation of CTLs by stimulation with the autologous EBV-transformed lymphoblastoid cell line; and (b) an enzyme-linked immunospot assay to quantify frequencies of CTLs specific for known LMP1/2 epitopes. We detected EBV-specific CTLs in blood and biopsy samples from both EBV-negative and EBV-positive HD patients. However, as in healthy EBV carriers, LMP-specific CTL precursors occurred only at low frequency in the blood of HD patients, and with the exception of one EBV-negative HD case, were undetectable in the tumor. These data give rise to two considerations: (a) they may explain why EBV-positive tumor cells persist in the presence of an existing EBV-specific immune response; and (b) they provide a rationale for selectively boosting/eliciting LMP-specific CTL responses as a therapy for EBV-positive HD.

Correction: Authentication of Herbal Supplements Using Next-Generation Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0156426.].

Authentication of Herbal Supplements Using Next-Generation Sequencing.

BACKGROUND: DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. METHODS: We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. RESULTS: All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. CONCLUSION: Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions.