The microbiome of diabetic foot ulcers: a comparison of swab and tissue biopsy wound sampling techniques using 16S rRNA gene sequencing.

BACKGROUND: Health-care professionals need to collect wound samples to identify potential pathogens that contribute to wound infection. Obtaining appropriate samples from diabetic foot ulcers (DFUs) where there is a suspicion of infection is of high importance. Paired swabs and tissue biopsies were collected from DFUs and both sampling techniques were compared using 16S rRNA gene sequencing. RESULTS: Mean bacterial abundance determined using quantitative polymerase chain reaction (qPCR) was significantly lower in tissue biopsies (p = 0.03). The mean number of reads across all samples was significantly higher in wound swabs [Formula: see text] = 32,014) compared to tissue ([Formula: see text] = 15,256, p = 0.001). Tissue biopsies exhibited greater overall diversity of bacteria relative to swabs (Shannon's H diversity p = 0.009). However, based on a presence/absence analysis of all paired samples, the frequency of occurrence of bacteria from genera of known and potential pathogens was generally higher in wound swabs than tissue biopsies. Multivariate analysis identified significantly different bacterial communities in swabs compared to tissue (p = 0.001). There was minimal correlation between paired wound swabs and tissue biopsies in the number and types of microorganisms. RELATE analysis revealed low concordance between paired DFU swab and tissue biopsy samples (Rho = 0.043, p = 0.34). CONCLUSIONS: Using 16S rRNA gene sequencing this study identifies the potential for using less invasive swabs to recover high relative abundances of known and potential pathogen genera from DFUs when compared to the gold standard collection method of tissue biopsy.

Genome and secretome analysis of jute endophyte Grammothele lineata strain SDL-CO-2015-1: Insights into its lignocellulolytic structure and secondary metabolite profile.

Grammothele lineata strain SDL-CO-2015-1, jute (Corchorus olitorius) endophyte has been reported to produce anti-cancer drug paclitaxel in culture condition. Here we investigated the genome using different bioinformatic tools to find its association with the production of commercially important compounds including taxol. Carbohydrate-active enzymes, proteases, and secretory proteins were annotated revealing a complex endophytic relationship with its plant host. The presences of a diverse range of CAZymes including numerous lignocellulolytic enzymes support its potentiality in biomass degradation. Genome annotation led to the identification of 28 clusters for secondary metabolite biosynthesis. Several biosynthesis gene clusters were identified for terpene biosynthesis from antiSMASH analysis but none could be specifically pinned to taxol synthesis. This study will direct us to understand the genomic organization of endophytic basidiomycetes with a potential for producing numerous commercially important enzymes and secondary metabolites taking G. lineata as a model.

Residents' readiness for out-of-hours service: a Dutch national survey.

BACKGROUND: Residents play a crucial role in out-of-hours service. Their perceived readiness for out-of-hours service, however, remains underexposed. This national exploratory study assesses whether or not Dutch residents feel sufficiently prepared to provide out-of-hours service at the time of their first shift, and aims to identify factors influencing perceived readiness. METHODS: An online questionnaire focussing on residents' working conditions was accessible from 21 September to 10 November 2015. Questions targeting perceived readiness for out-of-hours service were presented to all responding medical residents actively involved in out-of-hours service. Residents who felt sufficiently prepared were compared with residents who did not, exploring both individual characteristics and environmental factors. RESULTS: A total of 960 residents (mean age 32.5 years ±; 3.5, 72.4% female) from over 30 different medical specialties were included. Thirty-six percent of responding residents felt insufficiently prepared to provide out-of-hours service at the time of their first shift. Current junior status (p = 0.020), prolonged clinical experience prior to the first shift (p < 0.001), targeted training (p < 0.001), assessment of relevant skills and competencies (p < 0.001), and formal consequences following negative assessment (p = 0.001) were positively associated with perceived readiness. CONCLUSION: One-third of responding residents felt insufficiently prepared for their first out-of-hours shift. Our results emphasise the need for sufficient time to gain clinical experience as a new graduate, and underline the positive contribution of targeted training and assessment of skills and competencies relevant to out-of-hours service.

Draft Genome Sequence of Grammothele lineata SDL-CO-2015-1, a Jute Endophyte with a Potential for Paclitaxel Biosynthesis.

Grammothele lineata strain SDL-CO-2015-1, a basidiomycete fungus, was identified as an endophyte from a jute species, Corchorus olitorius var. 2015, and found to produce paclitaxel, a diterpenic polyoxygenated pseudoalkaloid with antitumor activity. Here, we report the draft genome sequence (42.8 Mb with 9,395 genes) of this strain.

Comparative genomics of Eucalyptus and Corymbia reveals low rates of genome structural rearrangement.

BACKGROUND: Previous studies suggest genome structure is largely conserved between Eucalyptus species. However, it is unknown if this conservation extends to more divergent eucalypt taxa. We performed comparative genomics between the eucalypt genera Eucalyptus and Corymbia. Our results will facilitate transfer of genomic information between these important taxa and provide further insights into the rate of structural change in tree genomes. RESULTS: We constructed three high density linkage maps for two Corymbia species (Corymbia citriodora subsp. variegata and Corymbia torelliana) which were used to compare genome structure between both species and Eucalyptus grandis. Genome structure was highly conserved between the Corymbia species. However, the comparison of Corymbia and E. grandis suggests large (from 1-13 MB) intra-chromosomal rearrangements have occurred on seven of the 11 chromosomes. Most rearrangements were supported through comparisons of the three independent Corymbia maps to the E. grandis genome sequence, and to other independently constructed Eucalyptus linkage maps. CONCLUSIONS: These are the first large scale chromosomal rearrangements discovered between eucalypts. Nonetheless, in the general context of plants, the genomic structure of the two genera was remarkably conserved; adding to a growing body of evidence that conservation of genome structure is common amongst woody angiosperms.

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming.

Induced pluripotent stem cells (iPSCs) are considered patient-specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c-Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical-grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non-integrating viral and non-viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.

Imaging findings of a cutaneous B-cell lymphoma.

We report a case of a slowly growing primary cutaneous B-cell lymphoma of the right upper arm in an 81-old-year-old female. Although primary cutaneous lymphoma is the second most common group of extranodal non-Hodgkin lymphomas, reports on the potential value of imaging in the diagnosis are very scarce in the radiological literature. Although the dermatologist usually relies on clinical and histological findings for a correct diagnosis, MR imaging may be useful in local staging of the tumor, whereas 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) is the imaging technique of choice for initial distant staging and follow-up after treatment.

Fast splice site detection using information content and feature reduction.

BACKGROUND: Accurate identification of splice sites in DNA sequences plays a key role in the prediction of gene structure in eukaryotes. Already many computational methods have been proposed for the detection of splice sites and some of them showed high prediction accuracy. However, most of these methods are limited in terms of their long computation time when applied to whole genome sequence data. RESULTS: In this paper we propose a hybrid algorithm which combines several effective and informative input features with the state of the art support vector machine (SVM). To obtain the input features we employ information content method based on Shannon's information theory, Shapiro's score scheme, and Markovian probabilities. We also use a feature elimination scheme to reduce the less informative features from the input data. CONCLUSION: In this study we propose a new feature based splice site detection method that shows improved acceptor and donor splice site detection in DNA sequences when the performance is compared with various state of the art and well known methods.

Splice site identification using probabilistic parameters and SVM classification.

BACKGROUND: Recent advances and automation in DNA sequencing technology has created a vast amount of DNA sequence data. This increasing growth of sequence data demands better and efficient analysis methods. Identifying genes in this newly accumulated data is an important issue in bioinformatics, and it requires the prediction of the complete gene structure. Accurate identification of splice sites in DNA sequences plays one of the central roles of gene structural prediction in eukaryotes. Effective detection of splice sites requires the knowledge of characteristics, dependencies, and relationship of nucleotides in the splice site surrounding region. A higher-order Markov model is generally regarded as a useful technique for modeling higher-order dependencies. However, their implementation requires estimating a large number of parameters, which is computationally expensive. RESULTS: The proposed method for splice site detection consists of two stages: a first order Markov model (MM1) is used in the first stage and a support vector machine (SVM) with polynomial kernel is used in the second stage. The MM1 serves as a pre-processing step for the SVM and takes DNA sequences as its input. It models the compositional features and dependencies of nucleotides in terms of probabilistic parameters around splice site regions. The probabilistic parameters are then fed into the SVM, which combines them nonlinearly to predict splice sites. When the proposed MM1-SVM model is compared with other existing standard splice site detection methods, it shows a superior performance in all the cases. CONCLUSION: We proposed an effective pre-processing scheme for the SVM and applied it for the identification of splice sites. This is a simple yet effective splice site detection method, which shows a better classification accuracy and computational speed than some other more complex methods.

Effects of inositol hexaphosphate on growth and differentiation in K-562 erythroleukemia cell line.

Inositol hexaphosphate (InsP6) has recently been shown to inhibit experimental cancers in vivo. Since the lower phosphorylated forms of InsP6 are important in cell growth in a wide variety of mammalian cells, we tested the efficacy of InsP6 in growth reduction of K-562 human erythroleukemia cells in vitro. We report that InsP6 decreases the K-562 cell population by 19-36% (P less than 0.001) concomitant to an increased differentiation as evidenced by ultrastructural morphology and increased hemoglobin synthesis. Pilot experiments to study the mechanism of action of InsP6 show that following treatment with InsP6, the concentration of intracellular [Ca2+] ([Ca2+]i) is increased by 57% (P less than 0.02). Likewise, a 41% increase (P less than 0.05) in InsP3 and a 26% decrease (P less than 0.02) in InsP2 were noted 1 h following treatment with InsP6. Contrary to the dogma that cell division is associated with increased [Ca2+]i, our data show that reduced cell growth and enhanced differentiation is associated with increased [Ca2+]i and increased InsP3 in the presence of InsP6.

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.

Stability of fecapentaene-12 and its carcinogenicity in F-344 rats.

Carcinogenicity studies of the fecal mutagen fecapentaene-12 (FP-12) have been hampered because of its apparent instability. We report here that: (i) contrary to the popular belief, FP-12 is quite stable, particularly at micromolar to nanomolar concentration; and (ii) its characteristic spectrophotometric absorbance spectrum is a function of the solvent or vehicle. Using synchronous fluorescence spectrophotometry (SFS), we have determined that at delta lambda 36.5 nm FP-12 gives a characteristic single emission peak between 413 and 423 nm, allowing us to identify FP-12 in DNA when reacted in vitro. We also report an increased incidence (statistically not significant) of fibrosarcomas and mammary carcinomas in male F-344 rats following intrarectal instillation of FP-12. In the in vitro human colon explant model, direct addition of FP-12 results in alteration in mucin histochemical changes typical of precancer and cancer. Our results support the contention that FP-12 is a naturally occurring carcinogen and may be responsible for human cancer(s).

Inositol-phosphate-induced enhancement of natural killer cell activity correlates with tumor suppression.

In recent studies, we have demonstrated that inositol hexaphosphate (InsP6) inhibits experimental colon carcinogenesis. Since natural killer (NK) cells are involved in tumor cell destruction, we investigated the effect of InsP6 on murine NK cell activity. We show that; (i) 1,2-dimethylhydrazine (DMH), a colon carcinogen, depresses NK activity; (ii) in vivo treatment of mice with InsP6 enhances baseline NK activity and reverses DMH-induced depressed NK activity with an inverse correlation (r = -0.9811) with tumor incidence, (iii) short-term in vitro treatment of spleen cells and NK-enriched fraction with InsP6 also enhances NK cytotoxicity in a dose-dependent manner, (iv) inositol potentiates the action of InsP6. Our data suggest yet another important role of inositol phosphates in the regulation of cellular activity.

Phorbol ester-induced loss of membrane sialic acid: implications for tumor cytolysis by natural killer cells.

Freeze-fracture analysis has shown that treatment of cells with phorbol myristate acetate (PMA) results in a loss of intramembranous particles (IMP) associated with the external leaflet of their plasma membranes. It has also been demonstrated that phorbol esters markedly enhance the sensitivity of tumor targets to natural killer (NK) cells, although the mechanism underlying this phenomenon has remained unexplained. Since the ability of NK cells to recognize neoplasms appears to be inversely related to the concentration of sialic acid on the target cell surface, it seemed possible that phorbols affect membrane glycoproteins which have terminal carbohydrates bearing sialic acid residues. To investigate whether phorbol treatment could be responsible for the loss of sialic acid, four tumor cell lines were examined before and after exposure to PMA. A reduction in surface sialic acid was established by four different methods: 1) standard thiobarbituric acid analysis of cell hydrolysates, 2) metabolic labelling of cells with [3H]-mannosamine followed by treatment with neuraminidase, 3) chromatography of membrane extracts, and 4) freeze-fracture analysis of lectin-labelled intact cells. These observations suggest a mechanism whereby phorbols may facilitate NK-cell-mediated cytolysis. In addition, an entirely novel effect of these tumor-producing agents may have been uncovered.