BRCA1 and BRCA2 genes account for 20–25 % of inherited breast cancers and about 10 % of all breast cancer cases. Detection of BRCA mutation carriers can lead to therapeutic interventions such as mastectomy, oophorectomy, hormonal prevention therapy, improved screening, and targeted therapies such as PARP-inhibition. We estimate that African Americans and Hispanics are 4–5 times less likely to receive BRCA screening, despite having similar mutation frequencies as non-Jewish Caucasians, who have higher breast cancer mortality. To begin addressing this health disparity, we initiated a nationwide trial of BRCA testing of Latin American women with breast cancer. Patients were recruited through community organizations, clinics, public events, and by mail and Internet. Subjects completed the consent process and questionnaire, and provided a saliva sample by mail or in person. DNA from 120 subjects was used to sequence the entirety of BRCA1 and BRCA2 coding regions and splice sites, and validate pathogenic mutations, with a total material cost of $85/subject. Subjects ranged in age from 23 to 81 years , 6 % had bilateral disease, 57 % were ER/PR+, 23 % HER2+, and 17 % had triple-negative disease.Germline mutations in the A total of seven different predicted deleterious mutations were identified, one newly described and the rest rare. In addition, four variants of unknown effect were found.Application of this strategy on a larger scale could lead to improved cancer care of minority and underserved populations.The online version of this article (doi:10.1186/s13742-015-0088-z) contains supplementary material, which is available to authorized users. BRCA1 and BRCA2 genes result in predisposition to breast and ovarian cancers [Mutations in the  cancers –5; there cancers . In Cauc cancers –10.BRCA1/2 carriers is a critical component of breast and ovarian cancer prevention as there are multiple screening, surgical, and chemoprevention strategies that can be employed. Magnetic resonance imaging (MRI) screening is effective in detecting early cancers in BRCA1/2 carriers; mastectomy and oophorectomy can reduce ovarian cancers; and estrogen inhibition can reduce both type of malignancy [The identification of lignancy . HoweverBRCA1 and BRCA2 mutation screening from 2006 to 2008, found that 13–16 % of African Americans, Native Americans and Hispanics possess disease-causing mutations and a high rate of variants of unknown significance [BRCA1 or BRCA2 gene mutation, and a small Puerto Rican study reported a 52 % mutation rate [BRCA1 and BRCA2, exist in Hispanic/Latin American communities, which accounted for 35–45 % of the mutation carriers [Although African American and Hispanic women have a lower incidence of breast cancer, they have a higher mortality. Triple-negative breast cancer is more common in African Americans and Hispanic women, accounting, in part, for this health disparity –16. A stificance . An indeion rate , 19. Comcarriers –20.The BRCA1 and BRCA2 proteins play a role in the repair of double-stranded breaks in DNA. A synthetic lethal strategy for cancer therapy has been developed using DNA damaging chemotherapy agents to cause single-stranded breaks, combined with poly-ADP ribose polymerase (PARP) inhibitors, to inhibit single-stranded DNA repair. This approach may be particularly effective in BRCA mutation carriers, as the tumor will be unable to repair the double-stranded breaks , 22. CliBRCA1 and BRCA2 have been detected with a variety of techniques including multiple mutation scanning methods and Sanger sequencing (reviewed in [BRCA1 and BRCA2 genes we sought to apply this approach to a cohort of Hispanic/Latin American breast cancer patients.Mutations in iewed in ). Next-giewed in –32. A cliewed in . With thBRCA1 and BRCA2 genes in Hispanic subjects with breast cancer. The sequence was identified through amplification, library preparation and semi-conductor sequencing on an Ion Torrent Personal Genome Machine (PGM) Sequencer (Thermo Fisher Scientific) to an average  coverage in runs with 92 samples/chip and 466X with 46 samples/chip. Excluding samples giving fewer than 20,000 total reads, 1 amplicon of BRCA1 was below 100x average reads ; and 4 amplicons of BRCA2 had <100× average, covering 261 bp of coding region and 5 splice sites. Therefore, the coverage of the coding regions is 100 % for BRCA1 and 98 % for BRCA2.The data involve variants in the coding and flanking intron sequences of the human The data consist of raw sequence reads mapped to the human genome, and the resulting BAM files. These files were used to predict sequence variants using the Torrent Suite Variant Caller (TSVC) and a modified Genome Analysis Tool Kit (GATK) variant caller, optimized for PGM data. For SNPs, the two variant callers (VCs) give virtually identical results, each calling one intronic SNP the other missed . GATK is known to be ineffective at calling indels on the Ion Torrent platform. Parameter files for TSVC are given, as well as the raw and annotated variant files. Variants were manually examined in the Integrated Genome Viewer (IGV) and selected screen shots are provided. Rare variants were annotated to be Deleterious, Probably Benign, or Benign through inspection of appropriate databases see . DeleterClinical data consists of information on the pathology of the tumor extracted from pathology reports, and results of a questionnaire administered by study personnel. Data are managed in a FileMaker relational database, and information on mutation carriers was double checked for accuracy. The composite information is displayed in Table BRCA1 and BRCA2 testing we used data from Hall et al. [To estimate the participation rate of minorities in l et al. , on 64,7We designed a clinical trial to address some of these issues, with recruitment of Latin American women through community organizations, clinics with large Hispanic populations, public events, and the Internet. Study materials were available in Spanish and English, and the patients were protected by a certificate of confidentiality . We recrThe subjects had an age range at diagnosis of 23–81 years (mean 50.6 years); 60 % had a household income below $25,000; and 81 % were educated to high school level or less . The other six alleles were unique and, except for 189del11 in BRCA1 and E1308X in BRCA2, all represent mutations uncommonly seen in these genes  was used to amplify all coding exons and splice sites 24,143 bp) with an average coverage of 313–466X and 100 % coverage (>100X average) of the database  and with bp with BRCA1 E577Q and BRCA2 F266L). To further interrogate the variants of unknown significance, the Align GVGD site for evolutionary conservation [BRCA1 T826K) is listed as neutral in LOVD and the other three  are ranked as C0 (not conserved) in GVGD. It has been shown that an alignment of only primate sequences provides a potentially more appropriate model for human genetic variants [Several rare missense alleles were found, but by using data in BIC and ClinVar, all but four could be excluded as: known; likely non-pathogenic variants; or those found in a sample with an existing mutation Table . Two of ervation  and LOVDervation  databasevariants . TherefoP = 0.029). Two of the seven patients with deleterious mutations had triple-negative disease (29 %); this is not significantly different from those patients without identified mutations. Only two of the patients with identified mutations had a family history of breast cancer, with one second-degree relative in each case. This frequency of a family history, 29 %, was not significantly different to that of the cohort as a whole.Women with deleterious mutations were younger  and homopolymers (Ion Hi-Q Sequencing Chemistry Technology Access Program Information). We sequenced the same library of 91 DNA samples using both the standard and the Hi-Q enzyme, and analyzed the results according to the manufacturer’s instructions. With the Hi-Q enzyme the number of false positives was lower especially for 1 bp deletion alleles Table , Fig. 3.BRCA1 and BRCA2 mutational and allelic diversity are all higher in African American and Hispanic populations. Although these populations could benefit significantly from genetic testing and screening, the combination of lower average income; lower insurance coverage; reduced knowledge of the benefits of testing; and mistrust of medical and government agencies, have led to a large disparity in participation [Rates of mortality, triple-negative disease, and cipation –44. Fromcipation . A similcipation .a fully de-linked sample not requiring extensive counseling regarding BRCA testing;collection of saliva that can be completed in the home, community clinics or public events;bilingual study materials; andfull protection of confidentiality.To begin to address these issues in Hispanic populations, we designed a study with a number of potential advantages:Our recruitment success was very modest from online publicity , and few minority patients participate in public fund-raising events, such as Avon fundraising walks. However, partnerships with community groups such as Nueva Vida  and the Latino Community Development Agency (Oklahoma City) were successful in recruiting a number of Hispanic women of diverse backgrounds. The remainder of the population samples were collected at the Texas Tech University Health Science Hospitals in Lubbock and El Paso, Texas. By sending and receiving saliva kits through the US mail we were able to keep the cost of the collection/shipping and DNA preparation materials to under $20 per subject. Targeted sequencing, performed in batch sizes of 90 samples, has a materials cost of approximately $50, making the total reagent cost under $100, including Sanger validation but excluding labor. This is an important factor if sample sizes in the order of thousands of subjects are to be eventually attained.BRCA2 that performed poorly in most samples, having <100X average coverage, comprising 261 bp of coding region and 4 splice sites, and 1 splice site of BRCA1 poorly covered. The initial run of 92 samples had three that underperformed and required repetition (3 %); however, these samples performed well on a second run. Our second run of 46 samples allowed the average coverage to increase from 313–361X to 466X. Running 46 samples on a single chip would raise the cost/sample by $5, and may be advisable for clinical testing.We did identify amplicons in BRCA1 gene contains multiple mononucleotide repeats, for which it is challenging to accurately detect 1 bp indels; these regions are the most problematic to detect for all current methods [BRCA1 and BRCA2 genes  that are below this threshold in our data. In a small sample set, we show here that the Hi-Q formulation and current TVC analysis settings result in a substantial reduction in 1 bp indel calls, especially in false positive mononucleotide regions. Manual analysis of BAM files in IGV allows most of these variants to be excluded from consideration, although the difference in background between the two enzymes visible in IGV displays are not dramatic. The addition of overlapping amplicons in the most difficult regions could improve this result. Although we chose an amplicon-based method on Ion Torrent, the samples could also be run on multiple platforms or used in a capture-based method [https://getcolor.com/) now offers a clinical test of 19 breast cancer related genes for $249.The  methods , 40, 41.d method . Color GBRCA1 and BRCA2 in diverse cancers, as well as the active design of targeted therapies, further adds to the need to recruit minority subjects.While this initial phase of the project was designed to maximize participation and diversity at a modest cost, it has the disadvantage that subjects with mutations could not be retrospectively identified in order to benefit from the testing. A second phase, in which identifiers will be retained and patients counseled about testing, will now be initiated. We expect recruitment to increase as several individuals and groups declined to participate in the unlinked arm of the study. Population growth; advancement in age of the US Hispanic populations; and large family size make it imperative that innovative means be employed to increase participation in clinical and genetic studies. The increasingly recognized involvement of germline mutations in BRCA1 185delAG mutation. Of the six mutations we identified, only one, BRCA2 E1308X, has been reported in multiple studies. A much larger study, incorporating all the regional and ethnic diversities of Hispanic populations, will have to be carried out to fully understand mutational diversity, and to aid in the classification of VUS.To date there have been no large nationwide surveys of Latin American with breast cancer. In the report by Hall et al., only the identities of mutations reaching 4 % or greater were made available . The CliBRCA1 have been found in both Mexican and Puerto Rican breast cancer patients [Clearly, a comprehensive characterization of our samples will require copy number analysis to identify large insertion/deletion mutations. Recurrent large deletions in patients , 20. Rarpatients  or complBRCA1 and BRCA2 genes have numerous mononucleotide repeat regions, and these areas are rich in known mutations. Thus, a method to increase accuracy of sequencing in repeats would be welcome. We documented that the Hi-Q enzyme can achieve a significantly higher accuracy in sequencing through mononucleotide repeats. When combined with methods or specific assays for the most prevalent large deletions, a high percentage of germline mutations can be identified. A recent study with the same BRCA1/2 panel was tested in a diagnostics laboratory with high accuracy [While we and others have documented that the major next-generation sequencers do an excellent job in identifying single nucleotide variants, they can be deficient in the prediction of insertion and deletion variants, especially in mononucleotide repeats . The BRCaccuracy .Table 5PIn summary, we have succeeded in recruiting and anonymously testing diverse groups of Latin American women with breast cancer in the US, all at a materials cost of less than $100 for samples collection, shipment and sequencing, and confirmation. Using DNA sequencing, we found almost exclusively rare mutations, most of which have been observed in other studies. Expansion of this approach could be a component of a larger effort to improve the application of the benefits of genetic testing to Hispanic American women.BRCA1 and BRCA2 testing for women with breast and/or ovarian cancers, women with a family history and/or elevated risk, or even all women, are evident [BRCA1 and BRCA2 mutation carrier subjects occur at an earlier age, identification, education and implementation of risk reduction has a high cost-to-benefit ratio in favor of benefit. By reducing the cost of testing, simplifying sample collection, and working with organizations and clinics focusing on Hispanic communities, we address some of the barriers to utilizing this technology. Extending this approach to larger populations, employing counseling and analysis in approved clinical genetics laboratories, could contribute to reducing the higher mortality from these cancers in minority populations.The benefits of  evident . A rangeHispanic patients with breast cancer were recruited through community organizations, dedicated clinics, public events  and through online contacts. The study was approved by the National Cancer Institute (NCI) Institutional Review Board (IRB) as well as the IRBs of Texas Tech Health Science Center Hospitals at Lubbock and El Paso, and included a confidentiality agreement from the National Institutes for Health (NIH) . SubjectSaliva (~5 ml) was collected in Oragene collection devices , stored at room temperature, and shipped at ambient temperature through the US mail. A 0.5 ml aliquot was extracted according to manufacturer’s instructions and quantified by a NanoDrop (ND-1000) spectrophotometer . Per the IRB protocol, DNA samples and selected clinical data were given new numbers unlinked to patient identifiers.BRCA1 and BRCA2 genes [Starting with 30 ng of genomic DNA, samples were processed according to the standard protocol for Ampliseq target amplification and library preparation using the targeted, multiplex Ion AmpliSeq BRCA1 and BRCA2 Community Panel. The panel contains 167 amplicons, covers 16.3 kb and provides 98–100 % coverage of the coding regions of the A2 genes . The libA2 genes  using deA2 genes .Predicted variants were manually reviewed in IGV  and manuData was compiled in a relational database (Filemaker) and statistical analysis performed in STATA .GigaScience GigaDB [Supporting data including BAM files of standard and Hi-Q enzyme libraries and ABI Trace files of validated mutations are available from the e GigaDB . The new


Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six 14C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. , recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated 14C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 14C analysis.Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as  Analyses of both extant and extinct fauna are essential for understanding the evolutionary ecology of discrete regions through time. The two most important pieces of information required are: 1) accurate species identification, and 2) an accurate chronological framework. For archaeological and palaeontological material the former of these is typically obtained through morphological analyses of the bones, whereas the latter can be achieved through various dating methods, but most accurately through the use of radiocarbon dating (when samples are sub ~50 k BP). Both of these sources of information are affected by environmental conditions, including climate (temperature and humidity changes), and other taphonomic considerations such as deposition environment, and matrix and pore water geochemistry. The tropics, which are most noted for high biodiversity, conversely have the poorest survival record for faunal remains due to the high temperature and humidity that adversely affects protein (i.e. collagen) survival, thereby reducing the probability of fossilisation . AdvanceThe construction of a temporal framework of biodiversity patterns in faunal assemblages allows insight into the effects of ecological changes on habitats, including human-induced impacts. However, one of the most appropriate means to achieve such a framework—radiocarbon dating—is significantly costly, both in terms of equipment and time . Radiocaet al. [et al. [The degree of organic preservation remains a crucial concern when radiocarbon dating is applied to any bone material , and theet al.  used micet al. , 9). How [et al.  suggeste14C dating, such as the Oxford Radiocarbon Accelerator Unit (ORAU), occasionally measure the percentage of nitrogen (%N) in bone as a screening mechanism prior to dating [Laboratories that conduct o dating . As collo dating . Similaro dating , 3 and so dating .14C dates whilst removing some, but not all, exogenous contaminants (including organic acids) [14C dating can therefore provide supporting evidence to ensure dates have been acquired from endogenous collagen rather than external contaminants, thereby producing reliable and publishable dates.Routine collagen extraction procedures at radiocarbon laboratories often include ultrafiltration steps that work to improve c acids) . These cc acids) , 13. VanThe collagen fingerprinting technique, often referred to as Zooarchaeology by Mass Spectrometry (ZooMS) , utiliseThe best conditions for molecular preservation of Late Quaternary vertebrate remains in the tropics lies almost exclusively within cave systems . Within 14C) dates have been obtained from the Cayman cave systems in an attempt to age the sub-fossils [14C dating was conducted on the remains of Cuban crocodile, Crocodylus rhombifer, and on a femur of Capromys sp., yielding dates of 860 ± 50 yr BP and 375 ± 60 yr BP respectively [14C dates from Cayman Brac have been generated from land snail shell samples from three distinct substrate layers in Patton’s Fissure: 80–100 cm below the surface of the sediments, 11,180 ± 105 yr BP; 120–140 cm below surface, 13,230 ± 135 yr BP; and 160 cm below surface, 13,850 ± 135 yr BP [The British Overseas Territory of Cayman Brac is a carbonate island with numerous caves and rock fissures that have naturally accumulated remains of island fauna over time. These caves act as natural repositories, providing potential data documenting island biodiversity through time. To date, only five radiocarbon . This consolidated ZooMS methodology requires no more, and often less, destruction than sampling for radiocarbon dating would require. We utilise this technique to qualitatively assess the degree of collagen preservation within sub-fossil mammal bone collected from Cayman Brac (Cayman Islands) and thus apply it as a screening procedure prior to 14C dating. There are two key advantages of ZooMS collagen fingerprinting in this respect: 1) ZooMS offers faunal identification, which is particularly useful in sites that host limited and/or highly fragmented material, or intentionally modified material such as bone tools, and 2) ZooMS screening helps circumvent the acquisition of dates from external contaminants rather than bone collagen by confirming the quality of the surviving collagen.ZooMS and The specimens collected in this study are permanently reposited in the departmental collection of the School of Earth, Atmospheric & Environmental Sciences  within the Cayman Brac (CB) sub-fossil collection and are accessible to all researchers. Specimens 3–25 were labelled MB3-25 on initial submission for radiocarbon dating, with later samples 430 and 433 dated under the labels MB29 and MB30 respectively. No permits were required for the described study, which complied with all relevant regulations working in conjunction with the Department of Environment (Cayman Islands). The Cayman Islands’ legal system is based on English common law, locally enacted statutes and Orders-in-Council. Local principal statutes (laws) are passed by the Legislative Assembly and assented to by the Governor. The cave deposits studied and collected from the Cayman Islands are currently not protected by any local laws. Locality and horizon data are described below.The Cayman Islands comprise three small, low-lying subtropical islands located in the north-west of the Caribbean Sea, all of which are considered to be geographically part of the Greater Antilles and of the West Indies . Little Cayman and Cayman Brac lie within 7 km of one another, and are located about 130 km north-west of Grand Cayman.14C dating. These two samples were selected due to the quality of their collagen fingerprints, larger size and deposition location deep within Green Cave , Pebble Cave , Shelby’s Bolt Hole , Bedding Plane Cave I  and Bedding Plane Cave II . A total of twenty bones were radiocarbon dated in this study . Sample ™) water between each reagent. Crude collagen was gelatinised by heating at 75°C in 1 mM hydrochloric acid (HCl) solution (pH 3.0) for 20 h, filtered using a 100 μL polyethylene Eezi-filter™ and the acid-insoluble pellets discarded. Supernatant was ultrafiltered  and centrifuged (2500–3000 rpm) until 0.5–1 mL of the fraction remained. Resulting purified gelatin was freeze-dried and combusted in a CHN analyser, with separation of peptides >30 kDa. Results were applied to calibration plots using Oxcal computer programme (v4.2) of C. Bronk Ramsey, using the ‘IntCal13’ dataset as described by Ramsey et al. [Samples were dated using Accelerator Mass Spectrometry (AMS) at the Oxford Radiocarbon Accelerator Unit (ORAU), using a standard protocol –30. Geoly et al. .et al. [Materials and chemicals were purchased as in Buckley et al. : HCl, acet al. [et al. [14C dating was undertaken, to avoid issues caused by variability in preservation within each sample. Approximately 5 mg of matrix-free bone powder was acquired. Bone powder was subsequently demineralised in 1 mL 0.6 M HCl over 3 h at room temperature. Bone samples from the skeletal remains of known reference species, Capromys pilorides , were demineralised, as whole fragments or as drilled samples (required for samples of large size), in 1 mL 0.6 M HCl for 18 h at 4°C. Samples were then centrifuged for 5 min at 14,000 x g, and supernatant filtered using 10 kDa MWCO ultrafilters  and spun at 14,000 x g for 30 min. The ultrafilter supernatant for each sample was discarded and the retained collagen washed twice with 0.5 mL 50 mM ABC and centrifuged at 14,000 x g for each wash. The acid-soluble collagen was then resuspended using 100 μl 50 mM ABC and digested with 0.4 μg trypsin  for 18 hr at 37°C in a separate Eppendorf tube.Collagen peptide fingerprints were obtained following methods modified from Buckley et al.  and van  [et al.  utilisinet al. [et al. [2O 1:1 v/v), and air-dried. Mass spectra from the collagen digest fractions were obtained using a Bruker Ultraflex II MALDI mass spectrometer (MS) operating at up to 5000 laser shots per plate spot, over a m/z range of 700–3700, and calibrated against an adjacent MS standard spot containing five calibrant peptides of 0.9 to 3.7 kiloDalton range . Mass peptide fingerprints were analysed using mMass software (v5.5.0) and peaks were filtered with a signal-to-noise (S/N) threshold of 5.0. Spectra were deemed successful when the number of labelled peaks above m/z 1500 was >10 in the 10% fractionation, following the S/N filter, to alleviate false positive results caused by matrix peaks or weak collagen signals.Sample peptide mixtures were then purified  using C18 solid phase extraction pipette tips , through methodology presented by Buckley et al.  and van  [et al. , to alloCapromys sp., as revealed by ZooMS analysis  of the specimens tested ; a reaso0 of the analysis .14C dates also gave excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Moreover, in a second round of analysis, a batch of 81 bone fragments from various localities within Green Cave were subjected to ZooMS to pre-screen for 14C dating  indicated a positive result. ZooMS and 14C dating both rely on the presence of preserved collagen in a sample to achieve a positive result, thus there is 100% matching between samples which either passed both (✓) or failed both (X) analyses. Additionally, all the bone samples analysed here show PMFs that match that of the reference sample of Capromys pilorides, [as shown by the common peaks between ancient and modern samples—see Capromys sp., as yet undescribed from the Cayman Islands. It can be noted that peak height and area in modern samples is much greater than in ancient specimens, which is a common artefact of collagen degradation over time. Certain parameters are routinely tested at ORAU, including %yield, collagen C:N and %C, in order to assess a sample as worthy of dating  facilitate excellent preservation that is unusual for the tropics. 14C dates of up to 1588 ± 26 yr BP were obtained, which are comparable to other sites of exceptional preservation in similar equatorial climes, such as Puerto Rico [Surprisingly little work has been conducted on bone collagen preservation in the tropics, although it is well known that collagen survival is subject to a number of factors that work synergistically to facilitate degradation; namely time, temperature, bacterial presence, geochemistry and hydrology , 33. Thrrto Rico , and othrto Rico . On the 14C dating. The method has so far exhibited a 100% success rate with regards to successfully categorising samples as suitable for dating or not, a figure significantly larger than the success rates achieved via %N or C:N investigations [14C laboratories may be many months but can be saved with ZooMS analysis prior to dating, reducing the pressure of sample pre-screening on the laboratory. As both 14C and ZooMS techniques rely on collagen preservation for accurate results, a successful result in one technique is likely to produce a successful result in the other. PMF quality (peak number and height) is largely reflected by the quantity of preserved collagen, and a sample of <1% collagen yield that would be rejected for dating at ORAU for example, could still yield a collagen fingerprint, albeit very poor. The application of a signal-to-noise (S/N) filter and a threshold of 10+ peaks required above m/z 1500 during data analysis screens out samples that produce poor PMFs and would be more likely to fail radiocarbon dating.Radiocarbon dating can be a particularly costly pursuit in regions whereby collagen survival is limited and poorly understood. ZooMS provides an alternative rapid screening technique that can be utilised to identify collagen integrity see  and can igations . ZooMS c14C results. Within this first round, four samples (numbers 10 to 15) were tested from Green Cave, Chamber 5, all of which failed dating and ZooMS. In the second round of dating, samples 430 and 433 were selected from a suite of 81 bone fragments all from Green Cave, Chamber 5. Samples 430 and 433 both passed ZooMS analyses and went on to be successfully dated at ORAU , we demonstrate with samples 430 and 433 that ZooMS can be successfully utilised as a pre-screening technique to highlight samples likely to yield 14C dates.Fossil samples from Green Cave show a trend for increasing specimen age with increasing distance from the cave entrance see . In the  at ORAU . Therefo14C dating. It is also significant that %C and collagen C:N data for the eight samples are in keeping with thresholds that demonstrate dates are reliably yielded from endogenous collagen [It is noteworthy that samples collected from Chamber 4 and Chamber 5 of Green Cave are older than those from chambers nearer the cave entrance. This trend is not exclusive, as the two Chamber 5 samples, 430 and 433, are moderately younger (by approximately 400 years) than the successfully dated Chamber 4 specimen, sample 7. This is not a surprising finding due to the high levels of bioturbation within the cave systems of Cayman Brac. A fissure is also present at the furthest end of Chamber 5 and these younger dates may be when the fissure opened, allowing animals a new access to the cave. Older samples are likely to be more taphonomically altered, perhaps being deposited during a period when mineral encapsulation was not equally occurring, increasing the value of ZooMS in highlighting sample suitability for dating when faced with a large assemblage of bone fragments of unknown preservation status. Fresh bone naturally yields a collagen content (%yield) that is approximately 22% the total weight of the bone [collagen  dated stIt is noteworthy that all previous investigations on the faunal assemblages of Cayman Brac have relied exclusively on the presence of key morphological characteristics to undergo species identification, placing obvious constraints on the identification of remains that have become fragmented and/or heavily encased in matrix so that they have no morphologically identifiable regions visible. ZooMS is therefore uniquely suited to identifying taxa based upon fragmented samples such as those present in the cave systems of Cayman Brac.Through the application of ZooMS, we are able to rapidly determine faunal identity and collagen integrity within bone remains, the latter for the acquirement of a temporal framework. With such insight, we have the opportunity to investigate palaeobiodiversity through time on the Cayman Islands in order to understand and offer protection to the subterranean cave ecosystem on Cayman Brac. The data derived from this and future studies will be of paramount importance in better understanding anthropogenically-driven terrestrial extinctions and last occurrence dates. It is our hope that the results from this study will demonstrate the importance of the Cayman Island cave deposits, but more importantly inform decision makers who might generate legislation to protect these fragile cave systems within this globally important biodiversity hotspot.Zooarchaeology offers a unique perspective to understanding how biodiversity has changed in response to key parameters, such as geological time and prevailing geochemistry, along with climatic or anthropogenic fluctuations through time. Type I collagen is one of the hardiest proteins, and from it we can deduce species identification , age at deposition, and a suite of other informative applications. Such insight permits identification of taxa that have become introduced, extinct or extirpated within a temporal framework. This understanding can provide supporting evidence for reintroductions or culls, towards the regeneration of natural biotas. With such knowledge, we can also improve our understanding of anthropogenic impacts on Cayman Brac and to other comparable biomes, and be better positioned to identify and protect biodiversity at these key hotspots. More significantly, the screening technique has the potential to be of worldwide significance for building chronological frameworks in the late Pleistocene.S1 Appendix(DOCX)Click here for additional data file.


Lactococcus garvieae strain PAQ102015-99, a recently isolated strain from an outbreak of lactococcosis at a commercial trout farm in the northwestern United States. The draft genome comprises 14 contigs totaling 2,068,357 bp with an N50 of 496,618 bp and average G+C content of 38%.We announce the draft genome assembly of  Lactococcus garvieae, the causative agent of lactococcosis, is a highly virulent Gram-positive pathogen of rainbow trout (Oncorhynchus mykiss) and is recognized as one of the most significant disease threats to the European trout farming industry , which was isolated from the head kidney of a rainbow trout showing clinical signs of lactococcosis. Whole-genome shotgun sequencing was conducted using an Illumina MiSeq on a NexteraXT prepared library. Quality filtering, trimming, and de novo assembly were performed using CLC Genomics Workbench v8.5.1. Functional annotation was conducted with Prokka (L. garvieae genomes as the initial reference database for annotation.Here, we report the draft genome sequence of a representative United States N50 value of 496,618 and average G+C content of 38.0%. A total of 1,947 predicted CDSs, 47 tRNAs, and 5 rRNAs were identified after annotation. Six hundred fourteen CDS features were identified as hypothetical proteins while 31 were identified as phage related. Important features related to pathogenicity and virulence included genes for colicins and related iron acquisition and metabolism, antibiotic resistance, cell adherence and invasion, and hyaluronic acid metabolism. Availability of this United States strain genome sequence will allow comparison to the genomes of European and Asia strains and thus provide a better understanding of the evolutionary relationship among these strains and insight into the emergence of this pathogen in the United States.The draft genome consists of 14 contigs totaling 2,068,357 bp with an LXWL00000000.The raw sequencing and contig data are available from GenBank/ENA/DDBJ under BioProject accession number PRJNA320828. The annotated assembly is directly available under accession number


The study was performed on a large collection of 384 wheat genotypes grown under water stress , mild water stress , and full irrigation (FI). The average GY of two growing seasons was 2.4, 4.8, and 8.9 Mg ha−1 under WS, MWS, and FI, respectively. Chlorophyll content at anthesis was positively correlated with GY (except under FI in 2011) and the agronomical components kernels per spike (KS) and thousand kernel weight (TKW). The WSC content at anthesis (WSCCa) was negatively correlated with spikes per square meter (SM2), but positively correlated with KS and TKW under WS and FI conditions. As a consequence, the relationships between WSCCa with GY were low or not significant. Therefore, selecting for high stem WSC would not necessary lead to genotypes of GY potential. The relationship between Δ13C and GY was positive under FI and MWS but negative under severe WS (in 2011), indicating higher water use under yield potential and MWS conditions.Different physiological traits have been proposed as key traits associated with yield potential as well as performance under water stress. The aim of this paper is to examine the genotypic variability of leaf chlorophyll, stem water-soluble carbohydrate content and carbon isotope discrimination (Δ Since the Green Revolution the yields of wheat and other cereals have increased considerably in many regions of the world, including Chile  in kernels . However, there is evidence that phenotyping using physiological traits, as a complement to agronomic traits, may help in identifying selectable features that accelerate breeding for yield potential and performance under drought  cultivars and advanced lines of spring bread wheat there is high genotypic variability for agronomic and physiological traits. In addition, the yield performance of genotypes under drought conditions is associated with stem WSC accumulation, delayed leaf senescence, and carbon discrimination in grains.In this study we investigated the genotypic variability of flag leaf chlorophyll content (measured with a portable leaf meter), stem WSC accumulation at anthesis and the ΔTriticum aestivum L.), including 153 lines from the wheat breeding program of the Instituto de Investigaciones Agropecuarias (INIA) in Chile, 53 from the International Wheat and Maize Improvement Centre (CIMMYT) that were previously selected for adaptiveness to Chilean environments (these lines share common ancestors with the INIA-Chile breeding program), and 178 lines from INIA in Uruguay (Table S1). The objective with this set of lines was to create a germplasm base to breed for drier areas in Chile and subsequently other countries within the projects involved.A collection of 384 cultivars and advanced semidwarf lines of spring bread wheat  under the water stress (WS) typical of the rainfed at this site, and Santa Rosa  under full irrigation (FI) and moderate water stress (MWS) conditions achieved through support irrigation. Trials were assayed during two consecutive (2011 and 2012) crop seasons, except for the MWS trial, which was only set up during 2011. Cauquenes corresponds to the Mediterranean drought-prone area of Chile; the average annual temperature is 14.7°C, the minimum average is 4.7°C (July) and the maximum is 27°C (January). The evapotranspiration is 1200 mm , 90 kg ha−1 of potassium chloride (60% K2O), 200 kg ha−1 of sul-po-mag , 10 kg ha−1 of boronatrocalcite (11% B), and 3 kg ha−1 of zinc sulfate (35% Zn). Fertilizers were incorporated with a cultivator before sowing. During tillering an extra 153 kg ha−1 of N was applied. Weeds were controlled with the application of Flufenacet + Flurtamone + Diflufenican (96 g a.i.) as pre-emergence controls and a further application of MCPA (525 g a.i.) + Metsulfuron-metil (5 g a.i.) as post-emergents. Cultivars were disease resistance and no fungicide was used.The experimental design was an α-lattice with 20 incomplete blocks per replicate, each block containing 20 genotypes. In each replicate two cultivars (Don Alberto and Carpintero) were included eight times. Two replicates per genotypes were used, except at Cauquenes and Santa Rosa SI in 2011 where a single replicate was established. Plots consisted of five rows of 2 m in length and 0.2 m distance between rows. The sowing rate was 20 g mFurrow irrigation was used in Santa Rosa: one irrigation at the end of tillering  were determined for a 1 m length of an inside row, and the number of kernels per spike (KS) and 1000 kernel weight (TKW) were determined in 25 spikes taken at random. Grain yield was assessed by harvesting the whole plot.Days from emergence to heading (DH) were determined in Santa Rosa, through periodic (twice a week) observations, when approximately half of the spikes in the plot had already extruded. At maturity and for each plot the plant height (PH) of the different trials, up to the extreme of the spike (excluding awns), was measured, the number of spikes per m−1), and WSC content per unit of stem (mg CHO stem−1) and per unit grown area (g CHO m−2), respectively. In addition, the apparent WSC remobilization was calculated as the differences from anthesis to maturity in WSC content on a stem and land area basis.Chlorophyll content (SPAD index) was determined at anthesis and then during grain filling about 2 weeks after anthesis  in five flag leaves per plot using a SPAD 502  portable leaf chlorophyll meter. WSC concentration in stems (harvested at ground level and excluding leaf laminas and sheaths) was determined at anthesis and maturity, on five main stems per plot, using the anthrone reactive method  isotope ratio was measured in mature kernels using an elemental analyser  coupled with an isotope ratio mass spectrometer, at the Laboratory of Applied Physical Chemistry at Ghent University (Belgium). The 13C/12C ratios were expressed in δ notation sample/(13C/12C)standard −1 . Further, the carbon isotope discrimination (Δ13C) of kernels was calculated as: Δ13C (%¸) = (δ13Ca − δ13Cp)/[1+ (δ13Cp)/1000], where a and p refer to air and the plant, respectively  and irrigation conditions , respectively, and D and I are the mean yield of all genotypes growing under drought and irrigated conditions, respectively.The yield tolerance index (YTI), which combines the relative performance of a genotype under drought with its potential yield under irrigated conditions  an plant height >120 cm. ANOVAs for physiological and yield-related traits were performed for the whole set of genotypes using PROC MIXED of the SAS Institute Inc. Genotypes and environment (Cauquenes WS and Santa Rosa FI) were considered fixed effects, whereas blocks and incomplete blocks within each replication (in an α-lattice design) were considered random effects. Data from Santa Rosa MWS where not considered in the ANOVAs because there was no replication and only one year (2011) of observations. Correlation analysis was performed between agronomic and physiological traits, and also stepwise regressions between grain yield and related agronomical and physiological traits. Principal component analysis (PCA) was carried out for the 378 genotypes using the mean values for physiological and agronomical traits evaluated under severe water stress in Cauquenes and full irrigation in Santa Rosa, in two growing seasons, using IBM SPSS Statistics 19.P < 0.001) in both growing seasons, whereas for GY, PH, and KM2 was only in one growing season (Table P < 0.001) GxE interaction in both growing seasons, but stem weight and WSC concentration and content, and Δ13C of kernels was only in 2012  interaction was highly significant P < 0.00 in both −1 but some genotypes produced up to 12 Mg ha−1  reduced in Cauquenes, by 79 and 68% in 2011 and 2012, respectively, compared to Santa Rosa under FI  was 8–10 Mg ha1 Figure . Under MI Figure . Also, pI Figure .The reduction in SM2, KS and TKW under WS compared with FI was in general more pronounced in the first growing season; on average (of the two growing seasons) these traits were reduced by 25, 41, 21, and 18%, respectively, whereas KM2 was reduced by 53% . The yield tolerance index (YTI) of the 378 genotypes based on GY under WS and FI presented a wide range of values in both years, from 0.05 (very susceptible) to 0.65 (very tolerant genotypes). The frequency distribution of YTI had a left-skewed deviation in 2011 (mean YTI = 0.21) compared to 2012 (mean YTI = 0.32).The relationships for GY under FI and WS showed no significant correlation in both years (P < 0.001) in the SPAD index at anthesis and during grain filling was observed under WS in 2012.Days to heading, determined under FI, differed by about 20 days between the earliest and latest genotypes  at anthesis was highly correlated to the WSC concentration  and the stem biomass .The WSC concentration and content per stem at anthesis and maturity presented large genotypic variabilities in all the environments Table . The ste13C also exhibited genotypic variability under WS, MWS, and FI  with plant height in all the environments.GY was positively correlated with SM2 and KM2, but negatively correlated with TKW, in both water regimes and growing seasons Figure . GY was Days to heading (determined at FI) was not correlated with GY, but it was positively correlated with SM2 and negatively correlated with TKW, except under FI in 2012 Table . The SPA13C and GY was slightly negative under WS in 2011, but positive and highly significant in 2012, and also positive under MWS and FI in 2011 and 2012 .The relationship between Δ12 Table . Indeed,Y Figure . The cor13C presented the strongest association with GY, except under the severe WS in 2011  explained >50% of the observed variability, under WS and FI conditions Figure . KS was 1 Figure . The SPA13C) traits indicated that under water stress conditions, the contribution of the agronomical trails was greater than the physiological ones, but under full irrigation conditions WSCCa and Δ13C contributed similarly to the agronomical traits to GY  and physiological . However, some genotypes were able to produce more than 4 Mg ha−1 under such WS conditions and showed high values of YTI (>0.50). Actually, YTI was highly correlated  with GY under WS in Cauquenes. Under the full irrigation conditions of Santa Rosa some genotypes achieved extremely high yields (12 Mg ha−1), for a Mediterranean environment. Large genotypic variability in GY and its agronomical components has also been found in 127 recombinant inbred lines (Dharwar Dry × Sitta) of wheat growing under severe water stress in Obregon, Mexico , but the correlation coefficients for each environment were not as high as has been reported by several authors  and TKW in WS  and FI  conditions. The PCA indicated that KS was better associated with GY in both WS and FI conditions , followed by KS (21%), and as a consequence the number of kernels per m1%, follos Figure . Other ss Figure  and alsos Figure .Triticum turgidum ssp. durum) stay-green mutants growing under glasshouse conditions remained green for longer and had higher rates of leaf photosynthesis and seed weight  negative correlation  between SM2 and stem weight at anthesis. Thus, genotypes with lower number of stems have probably more light transmission through the canopy and therefore higher rates of photosynthesis per stem, leading to higher stem weight and WSC content (more reserves), and greater numbers of grains per spike and kernel size. A significant and positive correlation between accumulated WSC at anthesis and kernel weight has been also observed in recombinant inbred lines (RILs) from the Seri/Babax population  in modern cultivars compared with old varieties has been found in bread  and GY have been frequently reported for cereals under Mediterranean conditions  or enrichment (Δ18O) and grain yield have been reported in bread wheat under fully irrigated conditions  may be discarded, since heading date was not correlated with Δ13C (P > 0.05) in none of the environments. Actually, the positive correlations between Δ13C and GY was also found when the relationship were studied within subset of 212 genotypes with similar heading duration (80–85 days); r = 0.50 for WS and 0.42 for WI in 2012.The relationship between Δ13C and GY was positive under MWS or FI but was negative under WS Figure . Other s2 and GY under terminal water stress and non-stress conditions. Additionally, genotypes with delay in leaf senescence (a higher SPAD index) would lead to higher KS and TKW, particularly under water stress, and to a lesser extent at full irrigation. In the case of yield potential conditions, this is probably the consequence of greater amounts of fixed carbon available for grain filling, whereas under water stress stay-green it is an indicator of resilience to stress conditions. In addition, genotypes with higher carbon discrimination values are associated with higher GY under MWS and full irrigation, indicating that more water is used by the crop. In addition, selection for a higher WSC at anthesis may bring negative consequences in terms of yield potential and adaptation to MWS conditions. This study clearly illustrates the importance of defining the target environment for wheat breeding before determining the set of phenotyping traits for selection.The identification of genotypic variability for agronomical and physiological traits under water stress conditions and full irrigation is of great interest for breeders because selected genotypes with favorable traits can be used as parents in future crosses. Among these, genotypes with higher numbers of fertile tillers would lead to higher numbers of kernels per mAD and IM designed the experiments, selected the germplasm and participated on field evaluations. AY and GT were in charge of carbohydrate determinations. DC was in charge of the management of the experiments and evaluation of agronomic traits. LS and JA contributed to analysis of the data. AD was in charge of the writing up but all the authors contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Compared to recently-studied novel morphological measures, conventional small nerve fiber functional tests have not been systematically studied for identification of diabetic sensorimotor polyneuropathy (DSP). We aimed to determine and compare the diagnostic performance of cooling detection thresholds (CDT) in a cross-sectional type 1 diabetes cohort.FLARE). Area under the curve (AUC) and optimal thresholds were determined by receiver operating characteristic (ROC) curves in the type 1 diabetes cohort.136 subjects with type 1 diabetes and 52 healthy volunteers underwent clinical and electrophysiological examination for DSP classification concomitantly with the Toronto Clinical Neuropathy Score (TCNS) and three small fiber function tests: CDT, heart rate variability (HRV), and laser doppler imaging of axon-mediated neurogenic flare responses to cutaneous heating  and AUCHRV , but exceeded AUCLDIFLARE . The threshold of <25.1°C was equivalent to the lower bound of the healthy volunteer 95% distribution  and performed with 83% sensitivity and 82% specificity.Type 1 diabetes subjects were 42±17 years of age with mean HbA1c 7.9±1.7%. Fifty-nine (45%) met the case definition for DSP. CDT values were lowest in cases with DSP (18.3±8.4°C) compared to controls without DSP (28.4±3.5°C) and to healthy volunteers . AUCAkin to novel small fiber morphological measures, CDT is a functional test that identifies DSP with very good diagnostic performance. These findings support further research that revisits the role of CDT in early DSP detection. Diabetic sensorimotor polyneuropathy (DSP) represents the most common complication of diabetes and predisposes to neuropathic pain, sensory and autonomic dysfunction and ultimately to sequelae such as limb amputation.Tests for abnormality in small fiber function are appealing for use as early DSP screening tests because the prevailing hypothesis on the pathophysiology of neuropathy is that the initial damage to small, unmyelinated or thinly myelinated Aδ and C-type nerve fibers precedes large fiber damage.FLARE) have been considered.The most commonly used small nerve fiber function test in specialized neuropathy assessment is quantitative sensory testing using cooling detection threshold (CDT) measurement which evaluates the perception of cold stimulus.The concurrent validity of CDT for the identification of DSP has not been previously evaluated in a type 1 diabetes cohort. Nested within the baseline examinations in the Toronto Neuropathy Cohort,We examined 136 subjects with type 1 diabetes and 52 healthy volunteers from an ongoing longitudinal cohort (JDRF grant No. 17-2008-715) evaluating the concurrent and predictive validity of small nerve fiber measures. The protocol and consent procedures were conducted in accordance with the World Medical Association's Helsinki Declaration and were approved by the Multidisciplinary Research Ethics Board of the Toronto General Hospital Research Institute. All participants provided written informed consent. Type 1 diabetes subjects were accrued using the Toronto Clinical Neuropathy Score (TCNS), a validated grading assessment that uses elements of history and physical examination to estimate severity of neuropathy.Comprehensive medical and neurological examinations were conducted on each subject and involved assessment of neuropathy-related symptoms and signs , lifestyle factors, comorbidities (including smoking), biochemical tests , and tests of small and large nerve fiber function. Type 1 diabetes subjects were included if they were ≥ 18 years of age, had a diagnosis of diabetes as defined by the 2008 (and 2013) Canadian Diabetes Association Guidelines and the ability to understand and cooperate with study procedures.CDT, a quantitative sensory threshold test, was assessed using the Medoc device . A stimulator was applied to the dorsum of the foot at a temperature of 32°C and gradually decreased to the first level detected by the patient as cooler than the preceding level. Subjects depressed a button when they perceived the cooling sensation and the sensory threshold was recorded. The test was performed five times bilaterally on the great toe. The five trials from each foot were averaged to establish mean CDT thresholds and compared to age-matched normative data; a catch trial involving a null stimulus was inserted between the five trials at random to ensure patient understanding of the procedure.HRV was measured using the Dantec Keypoint Workstation  according to a defined protocol described elsewhere.FLARE technique was measured using the MoorLDI2 .FLARE area was calculated in centimeters squared.Axon reflex-mediated neurogenic vasodilation in response to cutaneous heating by the LDIType 1 diabetes subjects were stratified into three subgroups: clinical DSP cases (the reference standard), preclinical DSP cases , and controls without DSP. Clinical DSP cases were defined according to published clinical and electrophysiological criteria.FLARE, HRV, and the TCNS. In order to test the ability of CDT and the other measures to detect either late- or early-stage disease, ROC curves were generated using two methods: one used clinical DSP as the gold-standard for disease identification, while the other used preclinical DSP. A statistical comparison of each tests' area under the curve (AUC) was performed using the method of Pencina.Statistical analysis was performed using SAS version 9.3 for Windows  and R statistical software (version 2.10.1). Baseline clinical characteristics are reported for each variable as either mean ± standard deviation, frequency and percent, or median and interquartile range, as indicated in To address concerns over the effects of age and body mass index (BMI) on thermal thresholds,FLARE generally showed incrementally lower values across the categories from left to right in Characteristics of the 52 healthy volunteers and 136 type 1 diabetes subjects are presented in FLARE . Pertinent negative associations were observed for two potential confounder variables: the Spearman rank-correlation for age was −0.24 (p = 0.09) and for BMI was −0.08 (p = 0.61). Among type 1 diabetes subjects, more impaired (lower) CDT was associated with older age , longer diabetes duration , higher BMI , higher systolic blood pressure , and higher TCNS . Among the diabetes subjects, as observed in healthy volunteers, lower CDT was also associated with more abnormal large fiber measures including sural nerve amplitude potential  and conduction velocity , and peroneal nerve amplitude potential , conduction velocity , and f-wave latency . Furthermore, lower CDT levels were associated with more abnormal small fiber measures including lower LDIFLARE  and lower HRV .Results of a linear correlation analysis demonstrate that in healthy volunteers higher CDT was associated with lower TCNS , more normal large fiber measures including sural nerve amplitude potential  and conduction velocity , peroneal nerve amplitude potential , conduction velocity , and f-wave latency , and the small fiber measure LDICDT was the highest at 0.863. Compared to AUCCDT, AUCTCNS was 0.858 (p = 0.24) and AUCHRV was 0.788 (p = 0.05). However, AUCLDIFLARE was significantly lower than CDT . CDT had the best operating characteristics of all measures. This corresponded to an optimal threshold value of 25.1°C, indicating that CDT values <25.1°C were associated with 83% sensitivity and 82% specificity for the identification of DSP cases. Positive and negative predictive values were 79% and 86%, respectively. Positive and negative likelihood ratios were 4.9 and 0.2, respectively. The optimal threshold value of 25.1°C for identification of DSP among diabetes subjects was identical to the lower bound of the 95% distribution of CDT values in the healthy volunteers.ROC curves for the identification of clinical DSP by CDT and other small nerve fiber function tests in the type 1 diabetes cohort are shown in CDT was highest at 0.800; AUCTCNS was 0.741 (p = 0.20 for comparison with AUCCDT), AUCHRV was 0.692 (p<0.0001), and AUCLDIFLARE 0.618 (p<0.0001). CDT had the best operating characteristics, corresponding to an optimal threshold value for preclinical DSP of <28.5°C, which had 76% sensitivity and 76% specificity for the identification of preclinical DSP cases. Positive and negative predictive values were 89% and 54%, respectively. Positive and negative likelihood ratios were 3.1 and 0.3, respectively.In the exploratory ROC analysis using preclinical DSP as the outcome measure, each of the small fiber function tests and TCNS showed similar AUC for identifying preclinical DSP, when compared to the results seen for clinical DSP. Specifically, AUCThe covariate-adjusted ROC curves for CDT in the identification of clinical DSP yielded high AUC: the age-adjusted ROC curve had AUC of 0.834, and the age- and BMI-adjusted ROC curve had AUC of 0.788. The multivariate logistic regression model using clinical DSP as the outcome (dependent) variable, and age & BMI the predictor (independent) variables yielded an AUC of 0.849, while the model including CDT as an additional predictor variable yielded an AUC of 0.898.FLARE), and even to measures of small fiber morphologyWe performed a cross-sectional analysis in a cohort of subjects with type 1 diabetes with a broad spectrum of nerve injury to determine the concurrent validity of CDT for identification of DSP and we described the distribution of values within a healthy volunteer cohort. In type 1 diabetes, an optimal CDT threshold value of <25.1°C was associated with very good operating characteristics  for the identification of clinical DSP, it was quantitatively associated with DSP severity, and it was identical to the lower bound of the 95% distribution in healthy volunteers. In context, the single measure of CDT had diagnostic performance comparable to an extensive clinical evaluation (the TCNS score), to quantitative measures of small fiber function (HRV and LDIThe hypothesis on the pathophysiology and progression of DSP in subjects with diabetes is that nerve fiber damage is progressive and affects small thinly myelinated Aδ and unmyelinated C fibers at the onset.FLARE, though we note that a definition of preclinical DSP has not been validated and our definition was therefore considered an exploratory analysis. Additionally, the association of CDT with nerve conduction study results suggests, as in previous studies In the current study we found CDT levels to be substantially lower in DSP cases compared to healthy volunteers, as well as compared to subjects with diabetes who did not have DSP. Furthermore, among those without DSP, those that had subclinical evidence of neuropathy as indicated by large fiber abnormality in at least one nerve conduction parameter had lower CDT values compared to those without any such large fiber abnormality. The ROC analysis suggested that CDT performed better at identifying established clinical DSP cases than it did identifying preclinical cases. This observation also applied to the other functional small fiber tests HRV and LDIDespite the very good concurrent validity of CDT for identification of the presence of clinical DSP, there is a major need for subclinical measures of DSP that can predict the future onset of neuropathy. This is a major priority for the design of future clinical trials that are needed to evaluate putative disease-modifying interventions in patients at high future risk of neuropathy, as well as a priority for identifying such high-risk patients in clinical practice in order to further modify their risk factors for neuropathy, such as further intensification of glycemic control.Although the current analysis was well-designed to evaluate the concurrent validity of CDT, it has limitations. First, it was cross-sectional and therefore conclusions about predictive validity can only be inferred from associations seen with preclinical DSP: validation will require longitudinal study. Second, this study was limited to subjects with type 1 diabetes and healthy volunteers. As such, generalizability of the diagnostic performance of CDT in subjects with type 2 diabetes, for whom small fiber damage may be more prevalent early in their natural history,We conclude that CDT is a traditional functional small fiber test that in a contemporary, systematic cross-sectional analysis of patients with type 1 diabetes identifies DSP with very good diagnostic performance, even akin to the cross-sectional performance of novel small fiber morphological measures such as in vivo corneal confocal microscopy.


Theileria is a genus of tick-borne protozoan that is globally widespread and infects nearly all ungulates in which they cause either latent infection or lethal disease. Wild animals are considered reservoir hosts of many species of Theileria and their diversity in wildlife species is increasingly becoming of interest. The molecular characterization and identification of Theileria infecting wildlife has been studied in a few species including buffalo, which are considered reservoir host for Theileria parva infecting cattle. In this study, we sequenced Theileria species infecting wildebeest (Connochaetes taurinus) and used molecular-genetic and phylogenetic analysis of the 18 Small Subunit of the Ribosomal RNA (18S rRNA) to identify their relationships with known species of Theileria. Our results revealed three new Theileria haplotypes infecting wildebeest. Phylogenetic analysis revealed that haplotype 1 and 2 clustered in the same clade as Theileria separata and with Theileria sp. isolated from other small to medium sized antelopes. Haplotype 3 clustered close to the Theileria ovis clade. This is the first molecular description and characterization of Theileria species infecting blue wildebeest in East Africa. This study demonstrates the potential for Theileria transmission between wildebeest and small domestic ungulates, such as sheep and goats. Theileria are protozoan parasites that infect a wide range of wild and domestic ungulates worldwide, causing diseases that are of great economic importance because of their impact on livestock health and production [Theileria species are transmitted among hosts by several species of Ixodid tick vectors [Theileria species and infected individuals are usually asymptomatic carriers [Theileria infections in many wildlife populations studied so far suggests that wildlife species are reservoir hosts for Theileria species infecting domestic ungulates [Theileria species) has stimulated research on the molecular characterization, identification and epidemiology of Theileria infecting wildlife. Most of these studies however, have focused on the role of buffalo as a reservoir for Theileria parva lawrenci, which causes lethal corridor disease in cattle. More recently, studies on wildlife Theileria has expanded to include a number of wildlife host species, such as grants’ gazelle (Nanger granti) [Kobus ellipsiprymnus) [Giraffa camelopardis) [Equus quagga) [Diceros bicornis) [Theileria gorgonis [Theileria species infecting wildebeest, which are particularly important as conduits of livestock diseases because of their annual long distance migrations for example in the Serengeti-Mara ecosystem in East Africa and in the Kalahari [et al. [Saiga tatarica tatarica) influenced the spread of helminths within sheep populations along their migratory route [oduction ,2,3. In  vectors . Many wicarriers ,6,7. Thengulates ,6,7,8,9. granti) , waterbuprymnus) , giraffeopardis)  zebras ( quagga) ,14, and icornis) . Early mKalahari ,17. LongKalahari . For exa [et al.  demonstrry route . Historiry route . The rolTheileria infections are often latent but can progress to fatal disease under conditions of either nutritional or translocation stress [Theileria infection have been documented in black rhinoceros and plains zebra as well as several species of antelopes, such as roan, sable, eland, greater kudu, gray duiker and tsessebe [Theileria have also been implicated as a cause for high calf mortality responsible for the decline in populations of roan and sable antelopes in South Africa [Although wildlife harbors a great diversity of parasites, usually in latent form, they still exert deleterious effects on their hosts and influence their host populations directly or indirectly ,22. Liken stress ,25,26,27tsessebe ,29,30. Ih Africa .Theileria and also in the revelation of new hosts for Theileria species that were thought to be host specific [Theileria is essential in designing molecular-genetic probes for the detection of species and strains of Theileria of economic importance to livestock production and wildlife conservation [The use of polymerase chain reaction (PCR) and sequencing in the diagnosis and detection of protozoans has led to the discovery of unparalleled species diversity in the genus specific . Knowledervation ,33.Theileria infecting migratory and resident wildebeest in the Serengeti-Mara ecosystem.Here, we used molecular genetic techniques, involving PCR and sequencing of the 18s rRNA gene to detect and characterize species of Theileria or Babesia since the primers we used could amplify both species. The amplicon size was found to range between 450–500 bp  was identified in one resident and one migratory wildebeest; Theileria sp. wildebeest haplotype 2  was identified in migratory wildebeest only while Theileria sp. wildebeest haplotype 3  was identified in five resident wildebeest and 19 migratory wildebeest. Polymorphism in the three haplotypes was elevated in the first 100 bp of the V4 region of 18s rRNA, a property shared by other species  were discarded from further analysis. Out of the 29 good sequences, 23 were from migratory wildebeest and six were from resident population of wildebeest. We analyzed the sequences and all the 29 good sequences were positive for  species .Theileria separata while Haplotype 3 clustered with Theileria ovis  and with sequences of known identity showed a clustering of Haplotype 1 and Haplotype 2 with ria ovis .The phylogenetic network analysis confirmed that our haplotype clustering was consistent with the topology of the phylogenetic tree .Theileria haplotypes detected in wildebeest and Theileria species they clustered with was variable. There was very limited divergence, especially between Theileria ovis and haplotype 3, but greater divergence and heterogeneity between Theileria separata with haplotypes 1 and 2 , common tsessebe  and sable antelope (Hippotragus niger) [In this study, we present the first genetic identification of  cluster . T. ovisnd goats ,36. Howes niger) .Theileria species are multi-host parasites, wildebeest, because of their large populations [Theileria prevalence, can therefore pose perennial risks of theileriosis to livestock. Several species of Theileria have recently been identified in wild mammalian species in Kenya, of which the majority have been novel species or haplotypes [Theileria species and/or haplotypes.Wildlife is considered important reservoir of up to 77% of the livestock diseases . Since mulations ,38 and hplotypes ,11,14,15Theileria haplotypes across spatially distinct host populations in the Serengeti-Maasai Mara ecosystem. The population of resident wildebeest is estimated to be 31,000, whereas the migratory population that mostly resides on the Serengeti is estimated to be 1.2 Million [In the present study, two haplotypes (haplotype 1 and 3), were shared between the resident and migratory wildebeests. This suggests that migration of this massive population is potentially playing a central role in disease spread and in homogenizing the distribution of  Million ,38. Migr Million .Theileria species, we could not confidently confirm their species identity except for haplotype 3, which clustered with Theileria ovis. This haplotype had limited sequence divergence from T. ovis. The Theileria separata cluster which contained haplotypes 1 and 2 displayed greater sequence divergence within the cluster suggesting that there is either greater genetic heterogeneity in this species or that the cluster contains distinct species that are closely related to T. separata. We could not confirm the relationship between our haplotype 1 and 2 with previously confirmed species of Theileria (Theileria gorgonis) known to infect blue wildebeest due to the lack of sequence data for this species in GenBank [T. parva infecting buffalo and cattle, which have shown that these two hosts can maintain almost independent variants of T. parva even when opportunities for cross transmission in sympatry do occur [Although our haplotypes clustered closely with known  GenBank . Supportdo occur ,41.Samples used in this research were collected by Kenya Wildlife Service (KWS) for the surveillance of Malignant Catarrhal Fever in wildebeest as part of routine disease surveillance mandate of the Veterinary Services Department of the Kenya Wildlife service. Scientific and ethical approval was provided by the Scientific Research Committee of Kenya Wildlife Service, Reference number KWS/BRM/5001. The protocols and procedures for animal handling and care used during this study follow the Kenya Wildlife Veterinary guidelines and ethical practice 2006.Loxodonta africana), lion (Panthera leo), leopard (Panthera pardus), African buffalo (Syncerus caffer), black rhinoceros (Diceros bicornis), wildebeests (Connochaetes taurinus), and several antelope species [Maasai Mara National Reserve (MMNR) is located on the South-Western part of Kenya along the Kenya–Tanzania border between 1°13′ and 1°45′ South and 34°45′ and 35°25′ East. It occupies an area of approximately 1510 square kilometers and hosts a high diversity of wildlife including large mammals such as the African elephant  and Xylazine hydrochloride . The animals were reversed using a combination of Diprenophine and Atipamazole. Venous blood was drawn from the jugular vein and placed into 10 mL EDTA tubes, which was aliquoted into cryovials for storage in Liquid Nitrogen. A total of 32 blood samples were collected and preserved in ultra-cold freezer (−80 °C) at the Veterinary Services Laboratory of Kenya Wildlife Service (KWS), Nairobi. Twenty six samples were from migratory wildebeest whereas six were from resident wildebeest. Resident wildebeest and migratory wildebeest were identified because they occupy spatially distinct areas within the reserve.Wildebeests were immobilized by darting using a combination of Etorphine hydrochloride , and DNA was stored at −20 °C until subsequent analysis. We amplified the hypervariable V4 region of the 18S rRNA gene of the genera Theileria and Babesia from genomic DNA using a nested polymerase chain reaction. We used two sets of primers, one set for primary amplification and a second set for secondary amplification. Primary amplification was performed using primers; ILO-9029 (Forward), (5′-CGGTAATTCCAGCTCCAATAGCGT-3′) and ILO-9030 (Reverse) (5′-TTTCTCTCAAAGGTGCTGAAGGAGT-3′). The conditions for the primary amplification consisted of an initial denaturation step of 5 min at 95 °C, followed by 30 cycles of denaturation each at 95 °C for 30 s, annealing for 30 s at 53 °C, extension for 1 min at 72 °C and terminated by a final extension for 8.5 min at 72 °C. We carried out the secondary amplification by ILO-9029 (Forward), (5′-CGGTAATTCCAGCTCCAATAGCGT-3′) and ILO-7782 (Reverse) (5′-AACTGACGACCTCCAATCTCTAGTC-3′). The conditions for secondary amplification consisted of an initial denaturation step of 5 min at 95 °C, and 30 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 50 °C, extension for 1 min at 72 °C and terminated by a final extension for 8.5 min at 72 °C. We included extraction controls and PCR-product negative (water) in each PCR reaction as negative controls. We carried out both primary and secondary amplifications based on a final volume of 10 μL which consisted of 1.5 μL of genomic DNA, 0.1 μL of each primer, 5 μL of Thermo Scientific™ DreamTaq™ Green PCR Master Mix (2×) and 3.3 μL of water.All frozen blood samples were thawed prior to DNA Extraction. DNA was extracted from 200 μL of blood, using the Qiagen DNeasy blood and tissue extraction kit  following the manufacturers protocol. DNA yields were determined with a NanodropThe final PCR product (1 μL) was separated using gel electrophoresis on 1.5% agarose gel stained with GelRed™ Nucleic acid gel stain. The gel was visualized for positive amplification of the target region on a UV trans-illuminator, and photographed. One kb DNA Ladder was used to identify the approximate size of the molecule run on a gel. All PCR products that were positive upon visualization on agarose gel were purified and sequenced at Macrogen in Europe. The primers used to amplify the 18S rRNA during PCR was used to sequence PCR products in both the forward and reverse directions using Sanger method in an ABI 3730 Analyzer .−10. All our haplotypes matched closely to Theileria species. In order to classify our haplotypes into species or clusters of species, at least three representative sequences of each known Theileria species from GenBank were obtained for comparison. Altogether, we had three sequences from wildebeests and 50 sequences from GenBank (NCBI) subjected to phylogenetic analysis.The chromatograms were visualized and edited using ChromasLite v 2.1.1. Primers were trimmed out using the software for Molecular Evolutionary Genetics Analysis (MEGA) v.6 . The conPrior to phylogenetic analyses, multiple sequence alignment was performed using the program MUSCLE. MEGA v.6 was used to determine the model of sequence evolution as well as the rate heterogeneity of aligned sequences . The phyTheileria species infecting wildebeest and their phylogenetic relationships with genetically characterized species in wild and domestic animals. This study contributes to the knowledge on the diversity of Theileria infecting wildlife and the new genotypes revealed by this study will be useful in PCR diagnostics for understanding cross transmission between domestic and wildlife species. The sharing of Theileria haplotypes between resident and migratory species suggests that migration of the wildebeests plays a central role in the spread of pathogens within the transboundary ecosystem and most likely posing transmission risks beyond conspecifics but also across heterospecifics, especially livestock (goats and sheep).With the use of advanced molecular techniques we were able to identify Theileria and Babesia, which is likely to alter several aspects of previously held notions, such as their host range, diversity, phylo-geography and ultimately classification.Further studies need to be conducted to determine the role of wildebeest in the transmission of multiple pathogens and whether the species found in wildebeest are also in livestock. The advent of molecular tools and increasing interest in wildlife pathogens is revealing numerous previously unknown species and variants of


Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10−2 dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab  Polymerase chain reaction (PCR) is a powerful, rapid method for the diagnosis of microbial infections and genetic diseases, the detection of microorganisms in environmental and food samples, forensics, and the amplification of DNA sequences for phylogenetic and ecological studies . ApplicaIn light of the ubiquity of inhibitors and the need for correcting steps in processing, the vast majority of molecular studies finding PCR inhibitors have largely considered such substances as coincidental contaminants in the samples (see above reviews). Given that many of the inhibitors isolated to date have other known biological functions, interference of PCR by these substances is considered incidental. This has resulted in an overall lack of work aimed at understanding the potential role of such inhibitors in controlling the replication of DNA foreign to the organisms producing them. Yet, it is plausible that molecules blocking DNA replication may have other functions, for example, as cytostatic or cytotoxic agents arresting cell division of microbial pathogens.Petrolisthes rufescens (Anomura: Porcellanidae). This species is a filter feeder commonly found in the intertidal zones of the Red Sea, Persian Gulf, East Africa, Arabian Sea, and Indian Ocean  were collected at low tide from underneath rocks in the Ain Sukhna intertidal, Gulf of Suez, Egypt (29°57′N 32°32′E). Individuals were kept alive in plastic tubs with fresh seawater and transported to The American University in Cairo, where they were individually placed in plastic bags and frozen at −20 °C. Animals used were mature adults. Despite its broad geographic distribution and potentially dense populations . These were dissected to separate foregut, midgut, hindgut, muscle, and gills under a dissecting microscope, as necessary (see below). During dissection, animals were placed on a Petri dish kept cold above a layer of ice. DNA from each tissue was extracted using the DNEasy tissue extraction kit (Qiagen cat # 69504) and the DNEasy spin column protocol. Each tissue sample was placed in a 1.5 ml microcentrifuge tube and ground under 180 µl of Buffer ATL, before adding 40 µl of proteinase K and incubating at 56 °C for 1 h. Proteinase K was added at twice the specified concentration (unless otherwise indicated below) because we expected the digestive system environment to be high in proteins that could potentially inhibit PCR. Samples were periodically vortexed during digestion, and then finally for 15 min, before adding 200 µl of Buffer AL and 200 µl of ethanol, and vortexing again. DNA was purified by centrifuging serially in a DNeasy Mini spin column in three one-minute steps, including transfer to two purification buffers, as per manufacturer specifications. All transfer procedures were performed inside a sterile hood. Separate amplifications were periodically performed (without crab or fish tissues) to assess contamination of the buffers and sterile water used in the procedures.Extraction of DNA was performed on frozen crabs (NA yield  and N = Upeneus nigromarginatus) was extracted using the same protocols and served as positive control in various experiments as explained below. Henceforth, the use of the word “extract” will refer to aliquots resulting from DNA extraction procedures. A NanoDrop 3300 fluorospectrometer  was used to quantify DNA extracted using the Quant-iT PicoGreen dsDNA assay kit . As per manufacturer specifications, this instrument and technique can detect DNA concentrations down to 0.001 ng/µl. To assess the efficiency of our DNA extraction protocols, preliminary quantification was performed on parts from 6 randomly selected crabs and on the fish DNA used as control. Two serial dilutions per sample were done, 10−1 and 10−2, and an equal volume of the dye was added to each dilution before measuring absorbance 530. We also used NanoDrop fluorospectrometry throughout the study to standardize the amount of DNA in our PCR reactions to 2 ng of extracted crab DNA and 2 ng of control fish DNA. Early experiments  were diluted serially by decreasing the amount of extracted DNA added to the PCR reaction in a total volume of 20 µl per reaction. In this initial exploration, NanoDrop readings showed extracted DNA amounts of 18.7, 4.1, and 0.93 ng in undiluted crab foreguts  gel imaging system. PCR conditions for each primer used are provided in P. rufescens foreguts (N = 3) using a fixed amount of foregut DNA extract (2 ng) and control fish DNA (2 ng) added to the reactions. Using the same primers for amplification described above (28s), PCR reactions were run along a control containing 2 ng of fish DNA in the reaction, but no extracted crab foregut (positive control) and a negative control lacking any DNA. Prior assays showed positive amplification at even higher amounts of DNA than 4 ng  of: (1) midguts, hindguts, and gills pooled per crab and (2) foreguts, midguts, hindguts and gills pooled per crab. Samples were adjusted so that a total of 2 ng of DNA (based on NanoDrop readings) were used in each PCR reaction: 1 ng of DNA from the foregut mixed with a total of 1 ng of DNA from midgut, hindgut and gills together. A negative control  was run simultaneously to assess potential microbial contamination of reagents during the experimental process.To assess whether the inhibitor was systemic in the crabs, amplification was further performed on DNA extracted from various parts of the crabs . For this experiment, we used universal bacterial primers and, therefore, the experiment also provided indirect information on the presence of microbes in the gut and other organs of  primers  Bac8f  and then added to the PCR reactions, which were then amplified using the 18s primers 18E-F (5′-CTGGTTGATCCTGCCAGT-3′) and 18sR3 (5′-TAATGATCCTTCCGCAGGTT-3′) (N = 3) aliquots, plus four serial dilutions of these same aliquots, along with two positive controls  and a negative control  for assessing contamination .The limits of activity of the inhibitor were assessed by progressively reducing the amount of foregut extract in PCR reactions containing 2 ng aliquots of fish DNA. For this, the amount of crab foregut extract was sequentially diluted in multiples of 10 from initial (2 ng) concentrations (10GGTT-3′) . Thus, fIf the inhibitor served a functional role in the foregut, and its activity was generalized for DNA replication, it was expected that regulation of some sort would keep the compound from affecting other parts of the digestive tract, where it could inhibit other potentially beneficial microbes or cells. To test for this, two experiments were performed by comparing the inhibition of PCR by foregut extracts versus that of foreguts mixed with midgut suspensions. Our hypothesis was that enzymatic degradation  of the inhibitor occurred in the midgut, the contiguous digestive chamber of the crab. Because our interest was to account for enzyme activity, we did not add proteinase K to the samples. Other protocols were kept the same. In the first experiment, undiluted extracts of foreguts (2 ng) were mixed with 2 ng control fish DNA. These were compared to samples from the same crabs in which foreguts and midguts were mixed in equal amounts before adding the fish DNA. All reactions were processed by using the 18s primers previously described. Foreguts and midguts were homogenized in 100 µl of Ultrapure, DNase and RNase free water (Invitrogen inc.). The foreguts were extracted as above.−1, 10−2, 10−3, 10−4) was added. The mixtures were incubated at 42 °C for two hours on a shaking thermoblock. This temperature was chosen to represent a potential extreme observed in areas of Egypt during summertime. Crabs often spend several hours above water when the tide recedes, where they are surrounded by tan to dark rocks and gravel (N = 3) had five PCR reactions . For this one and the previous assay samples were normalized using masses because the midguts were not extracted, but rather macerated in water and suspended in buffer, keeping us from normalizing using NanoDrop readings.The follow-up experiment aimed at establishing the minimum activity of the midgut suspensions that would arrest the activity of the inhibitor. This experiment followed the protocols above, but after the extraction, 50 µl of the foreguts were divided into five equal portions (10 µl each). To each portion an equal volume of either the midgut initial suspension from that same crab or one of its dilutions  were extracted with the same protocols previously described and tested. A dilution of 20−1 for each sample using TE buffer was made in a total volume of 400 µl. The absorbance of each sample at 320 nm was measured using a UV spectrophotometer. The measurement was repeated twice for each sample, using 196 µl each time and the two readings were subsequently averaged. The absorbances of the six groups were compared using one-way ANOVA. While this method does not quantify absolute melanin content in the absence of a melanin standard, or non-soluble melanin present in the tissues, it does provide reliable information on relative amount of melanin dissolved among samples  there was only a faint band, suggesting that the dilution of the midgut was approaching the limit of activity against the inhibitor. For these two experiments, both positive and negative controls showed the expected patterns . One-way ANOVA showed no significant difference between any of the six treatment groups in terms of potential melanin content  and microbial genes  suggests that the activity of the inhibitor is general and potentially effective against both eukaryotes and prokaryotes. Excess or critically low amounts of DNA in the samples did not explain inhibition of PCR (P. rufescens. That the inhibitor was found only in the foregut (or at least found in effective concentrations to arrest PCR) was confirmed by amplifying bacterial 16s rRNA genes from midgut and hindgut, along with gills. Clear amplification only happened when foreguts were absent from the mixture , or heating foregut samples at 99 °C for 1 h before amplifying by PCR, reduced the inhibitory activity . MedicalP. rufescens foregut, midgut, hindgut, gills, and muscle, and fish muscle , and several individuals (ca. 15) from the underside of a rock . Crabs were abundant among tan and dark rocks that were exposed at low tide and heated by full sunlight for hours. The surrounding coastal environment is the characteristic desert at this region of the Gulf of Suez (photos by E Cruz-Rivera).A single Click here for additional data file.10.7717/peerj.689/supp-3Figure S2Amplification of fish DNA using 28s primers in the presence or absence of foregut (FG) extracts. Three amounts of extracted foregut aliquots from three different crabs were added to the PCR reactions, but these were not standardized by mass using NanoDrop  and were done strictly by volume. The positive control contained only fish DNA (2 ng) for amplification and the negative control  contained no crab or fish DNA. Empty lanes in the gel are not labeled.Click here for additional data file.10.7717/peerj.689/supp-4Figure S3P. rufescens. DNA amounts ranged between 0.52 and 47.63 per PCR reaction in these experiments.Two separate gels showing amplification of bacterial DNA, from four different crabs, using universal 16s primers. All parts in all crabs, except the foreguts, yielded positive amplification products. These (among other) preliminary observations strongly suggested the presence of a PCR inhibitor restricted to the foregut of Click here for additional data file.10.7717/peerj.689/supp-5Supplemental Information 5DNA yield of extracted crab parts.Click here for additional data file.10.7717/peerj.689/supp-6Supplemental Information 6Absorbances for spectrophotometric melanin test.Click here for additional data file.


Hazards and potential complications associated with pedicle screw insertions have been reported. In contrast, complications due to implant removal are rarely described. An unreported case of acute vertebral body compression fracture following pedicle screw removal in a young man occurred during an episode of forceful coughing. Spinal implants need to be removed in cases of complications, pain or tissue irritation, and removal is mandatory when fixation involves L2 or the lower segments. Complications associated with spinal implant removal are rare but possible, and patients must be informed of this potential risk. In thoracolumbar fractures, the choice between conservative or surgical treatment and the final outcome depend on several factors: type of fracture, presence of neurological impairment, the patient’s general condition and comorbidities, and associated injuries , 2.Conservative treatment, especially bed rest, is not advisable in polytrauma patients, or in cases of claustrophobia, psychological disease, venous disease or previous deep venous thrombosis, obesity and bronchopulmonary diseases. In these situations percutaneous minimally invasive surgery can be an option . This teImplant removal after percutaneous stabilization should restore a “normal” spine, provided screws are placed without damaging the facet joints, but this beneficial aspect has yet to be demonstrated. We report a case of acute vertebral body compression fracture of L1 following removal of pedicle screws previously placed to treat L2 fracture by means of percutaneous fixation. To the best of our knowledge, this type of fracture in a young man has not previously been reported.A 29-year-old man was treated by percutaneous stabilization from L1 to L3 (pedicle screw diameter: 5.5 mm in L1 and 6.5 mm in L3) for a L2 burst fracture  scan and magnetic resonance imaging demonstrated an acute wedge fracture A1.2) of the superior plate on L1 . C-reactive protein was normal.The fracture was treated conservatively by a 3-point brace and analgesics for 3 months and healed without further progression of the kyphotic deformity Fig. c. The paIn the management of thoracolumbar fractures, percutaneous pedicle screw fixation is used to reduce the approach-related morbidity of the open technique: iatrogenic muscle denervation, increased intramuscular pressures, ischemia, pain and functional impairment .Hazards and potential complications associated with pedicle screw insertions have been reported .In contrast, complications due to implant removal are rarely described. Vanichkachorn et al.  reportedTo our knowledge, we describe the first acute vertebral compression fracture after percutaneous pedicle screw implant removal in a young man which occurred during an episode of forceful coughing.Only one case of herniated lumbar disc associated with whooping cough is reported, probably as a result of the pressure effect from the tremendous force produced in the thoracic and abdominal cavity during a coughing access . VertebrIn the treatment of thoracolumbar fractures, percutaneous pedicle screw stabilization seems to be a good option.Spinal implants need to be removed in cases of complications, pain or tissue irritations, and removal is mandatory when fixation involves L2 or the lower segments.Complications associated with spinal implant removal are rare but possible, and patients must be informed of this potential risk.