Improvement of the reverse transcription loop mediated isothermal amplification (RTLAMP) method for the detection of Peach latent mosaic viroid (PLMVd)

Peach latent mosaic viroid (PLMVd) is the most known peach viroid. Among the diagnostic techniques used for its detection, the most recent described being the reverse transcription loop-mediated isothermal amplification method (RT-LAMP). Several modifications were done on the basic protocol proposed by Boubourakas et al. (2009), additional experiments were preformed in order to further evaluate the method. Namely, the reaction time was further reduced and traces of leaf tissue, taken by a sterile toothpick, instead of tRNA were used as the intial material. Moreover, the AMV reverse transcriptase proved to be more effective than Thermoscript, while restriction enzyme analysis was performed on the RT-LAMP products in order to confirm that products had the respective sequences of the selected target. Finally, the extremely high efficiency and sensitivity of RT-LAMP proved to be sufficient for the detection of PLMVd in hosts other than peach. Keywords: PLMVd, RT-LAMP, peach, reverse transcriptase

Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA.

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Is prolonged infusion of piperacillin/tazobactam and meropenem in critically ill patients associated with improved pharmacokinetic/pharmacodynamic and patient outcomes? An observation from the Defining Antibiotic Levels in Intensive care unit patients (DALI) cohort

Objectives:We utilized the database of the Defining Antibiotic Levels in Intensive care unit patients (DALI) study to statistically compare the pharmacokinetic/pharmacodynamic and clinical outcomes between prolonged-infusion and intermittent-bolus dosing of piperacillin/tazobactam and meropenem in critically ill patients using inclusion criteria similar to those used in previous prospective studies.Methods: This was a post hoc analysis of a prospective, multicentre pharmacokinetic point-prevalence study (DALI), which recruited a large cohort of critically ill patients from 68 ICUs across 10 countries.Results: Of the 211 patients receiving piperacillin/tazobactam and meropenem in the DALI study, 182 met inclusion criteria. Overall, 89.0% (162/182) of patients achieved the most conservative target of 50% fT(> MIC) (time over which unbound or free drug concentration remains above the MIC). Decreasing creatinine clearance and the use of prolonged infusion significantly increased the PTA for most pharmacokinetic/pharmacodynamic targets. In the subgroup of patients who had respiratory infection, patients receiving beta-lactams via prolonged infusion demonstrated significantly better 30 day survival when compared with intermittent-bolus patients [86.2% (25/29) versus 56.7% (17/30); P=0.012]. Additionally, in patients with a SOFA score of >= 9, administration by prolonged infusion compared with intermittent-bolus dosing demonstrated significantly better clinical cure [73.3% (11/15) versus 35.0% (7/20); P=0.035] and survival rates [73.3% (11/15) versus 25.0% (5/20); P=0.025].Conclusions: Analysis of this large dataset has provided additional data on the niche benefits of administration of piperacillin/tazobactam and meropenem by prolonged infusion in critically ill patients, particularly for patients with respiratory infections

A Validated Age-Related Normative Model for Male Total Testosterone Shows Increasing Variance but No Decline after Age 40 Years

The diagnosis of hypogonadism in human males includes identification of low serum testosterone levels, and hence there is an underlying assumption that normal ranges of testosterone for the healthy population are known for all ages. However, to our knowledge, no such reference model exists in the literature, and hence the availability of an applicable biochemical reference range would be helpful for the clinical assessment of hypogonadal men. In this study, using model selection and validation analysis of data identified and extracted from thirteen studies, we derive and validate a normative model of total testosterone across the lifespan in healthy men. We show that total testosterone peaks [mean (2.5-97.5 percentile)] at 15.4 (7.2-31.1) nmol/L at an average age of 19 years, and falls in the average case [mean (2.5-97.5 percentile)] to 13.0 (6.6-25.3) nmol/L by age 40 years, but we find no evidence for a further fall in mean total testosterone with increasing age through to old age. However we do show that there is an increased variation in total testosterone levels with advancing age after age 40 years. This model provides the age related reference ranges needed to support research and clinical decision making in males who have symptoms that may be due to hypogonadism.Publisher PDFPeer reviewe

In vitro study of the effect of quercetin on acute myeloid leukemia cells

Acute myeloid leukemia (AML) at older age is associated with several biologic and clinical characteristics. Hence, it may arise from an early level of hematopoietic stem cells and has a high frequency of blast cells with multidrug resistance glycoprotein MDR1 expression and particularly a high incidence of poor prognostic karyotypes. These factors, rather than age per se, underlie the poorer outcome as compared with younger cases. Therefore, new treatment strategies need to be developed. Today, flavonoids receive the greatest attention as potent anticancer agents. Quercetin (3, 3', 4', 5, 7- pentahydroxyflavone), a common component of our diet, is one of the most studied flavonoids due to its potent antiproliferative effect and ability to induce apoptosis in cancer cell lines. The induction of programmed cell death also known as apoptosis, is the common outcome of successful cytotoxic therapy for many different types of cancer, including AML. Multiple genetic alterations that result in the disruption of the physiological regulation of apoptosis are thought to account for the ability of leukemic cells to grow autonomously and for their clinical resistance to therapy. Recently, a new family of downstream inhibitors of caspases, the IAP (inhibitor of apoptosis protein) family, has emerged as a potential key player in the regulation of apoptosis in cancer. Survivin is a member of the IAP family and a bifunctional protein that suppresses apoptosis and regulates cell division. This protein has garnered great interest as a potential drug target because its expression is among the most tumor-specific of all human gene products. While fetal tissues contain abundant survivin mRNA and protein, most normal adult tissues do not. In contrast, the vast majority of tumors express survivin protein at high levels, suggesting that reactivation of SURVIVIN gene expression occurs commonly in cancers. The aim of this study is to investigate in vitro the effect of quercetin on viability, growth and apoptosis of AML cells from elderly patients, as well as to find out if and how the expression of antiapoptotic protein survivin is affected. For this reason we used long term bone marrow cultures (LTBMCs) from AML patients, where leukemic cells were grown without (control cultures) and with the addition of quercetin at doses of 50 μΜ and 100 μΜ in various time points (24, 48, 72 hours and 1 week). Cell viability was evaluated by double counting of trypan blue dye-excluding cells under a light microscope and cells counts were performed with a hemocytometer. Apoptosis was determined by the use of annexin V expression. Intracellular staining with anti-human survivin monoclonal antibody was used to detect survivin protein. Apoptosis and survivin expression were estimated using flow cytometry after gating on blast population, on the basis of their low SS and CD45 dim staining. We also used short term bone marrow cultures, after quercetin addition on LTBMCs for 24 hours, in order to evaluate the effect of quercetin on progenitor cells. Twenty elderly AML patients (14 with de novo AML and 6 transformed from MDS) were included in this study. The patients (12 men and 8 women), over 60 years of age (mean age 68.8±9.2 years), were at initial stage of AML, without any prior treatment. According to FAB criteria the classification was Μ0: 1, Μ1: 3, Μ2: 8, Μ4: 4, Μ5: 3, Μ6:1. Our results showed: • Quercetin decreased the viability of leukemic cells in a dose and time dependent manner. • Quercetin inhibited the growth of leukemic cells in a dose and time dependent manner. • Quercetin increased apoptosis of leukemic cells in a dose and time dependent manner. • All the AML patients expressed the survivin protein. • Quercetin enhanced the expression of the antiapoptotic protein survivin in leukemic cells in a dose dependent • Quercetin decreased the number of colonies, especially at the dose of 100 μΜ. In conclusion, it is obvious from this study that although the mechanism of action of quercetin is not clear, it is certain that this substance promotes apoptosis of leukemic cells. Given that many chemotherapeutic agents act via apoptosis induction, it is possible that compounds such as quercetin would have greater antileukemic efficacy if combined with conventional cytotoxic chemotherapy.Η οξεία μυελογενής λευχαιμία (ΟΜΛ) στους ηλικιωμένους ασθενείς συνδέεται με ορισμένα βιολογικά και κλινικά χαρακτηριστικά. Προκαλείται από κλωνική διαταραχή των αρχέγονων αιμοποιητικών κυττάρων, συνοδεύεται από μεγάλο αριθμό βλαστικών κυττάρων που διηθούν το μυελό και το αίμα και εκφράζουν πολυφαρμακευτική ανθεκτικότητα, ενώ εμφανίζει ιδιαίτερα αυξημένο αριθμό καρυοτύπων πτωχής πρόγνωσης. Αυτά τα χαρακτηριστικά και όχι τόσο η ηλικία αυτή καθαυτήν, οδηγούν στο πτωχότερο θεραπευτικό αποτέλεσμα σε σχέση με τους νεότερους ασθενείς. Καθίσταται λοιπόν αναγκαίο να βρεθούν νέες θεραπευτικές στρατηγικές προσέγγισης των ασθενών αυτών για τη βελτίωση της πρόγνωσης και της αντιμετώπισής τους. Σήμερα τα φλαβονοειδή κερδίζουν μεγάλη αποδοχή ως πιθανοί αντικαρκινικοί παράγοντες. Η quercetin (3, 3', 4', 5, 7-πεντοϋδροξυφλαβόνη) ένα κοινό συστατικό της διατροφής μας είναι το πλέον μελετημένο φλαβονοειδές λόγω της ισχυρής αντιπολλαπλασιαστικής της δράσης σε διάφορες καρκινικές κυτταρικές σειρές και της ικανότητάς της να αυξάνει την απόπτωση σ' αυτές. Η έναρξη του προγραμματισμένου κυτταρικού θανάτου γνωστή και ως απόπτωση, είναι η κοινή έκβαση της επιτυχημένης κυτταροτοξικής θεραπείας διαφόρων τύπων καρκίνου συμπεριλαμβανομένης και της ΟΜΛ. Πολλαπλές γενετικές μεταβολές που έχουν ως αποτέλεσμα την απορρύθμιση της φυσιολογικής απόπτωσης, πιστεύεται ότι οφείλονται στην ικανότητα των λευχαιμικών κυττάρων να πολλαπλασιάζονται αυτόνομα και να αντιστέκονται στη θεραπεία. Πρόσφατα, μια νέα οικογένεια αναστολέων των κασπασών, η οικογένεια των IAPs (inhibitor of apoptosis proteins), εμφανίστηκε ως ένας σημαντικός παράγοντας για τη ρύθμιση της απόπτωσης στον καρκίνο. H survivin ανήκει στην οικογένεια των IAPs και είναι μια πρωτεΐνη με διττό ρόλο, που καταστέλλει την απόπτωση και ρυθμίζει την κυτταρική διαίρεση. Η πρωτεΐνη αυτή έχει κερδίσει μεγάλο ενδιαφέρον, θεωρούμενη θεραπευτικός στόχος λόγω του γεγονότος ότι η έκφρασή της είναι από τις πλέον ογκο-ειδικές πρωτεΐνες. Σημαντικό είναι ότι το mRNA και η πρωτεΐνη της survivin εκφράζονται έντονα από τους εμβρυϊκούς ιστούς ενώ δεν εκφράζονται από τους περισσότερους ενηλίκους ιστούς. Σε αντίθεση, η πλειονότητα των κακοηθειών εκφράζει την πρωτεΐνη της survivin σε υψηλά επίπεδα, γεγονός που υπαινίσσεται την ενεργοποίηση του γονιδίου SURVIVIN. Σκοπός της εργασίας αυτής είναι να μελετήσουμε in vitro την επίδραση της quercetin στη βιωσιμότητα, στην ανάπτυξη και στην απόπτωση των κυττάρων ηλικιωμένων ασθενών με ΟΜΛ, καθώς επίσης εάν και κατά πόσο επηρεάζεται η έκφραση της αντιαποπτωτικής πρωτεΐνης survivin από αυτήν. Για το σκοπό αυτό εφαρμόσαμε υγρές καλλιέργειες μυελού των οστών ασθενών με ΟΜΛ, στις οποίες τα λευχαιμικά κύτταρα αναπτύσσονταν χωρίς (καλλιέργειες μάρτυρες) και με την επίδραση της quercetin σε δόσεις των 50 μΜ και 100 μΜ, σε διαφορετικά χρονικά διαστήματα (24, 48, 72 ώρες και 1 εβδομάδα) και εκτιμήθηκε η βιωσιμότητα των κυττάρων. Η απόπτωση προσδιορίστηκε με τη χρήση της έκφρασης της αννεξίνης V. Για τον προσδιορισμό της ενδοκυττάριας πρωτεΐνης survivin χρησιμοποιήθηκε το μονοκλωνικό αντίσωμα anti-human survivin. Η απόπτωση και η έκφραση της survivin εκτιμήθηκαν με τη μέθοδο της κυτταρομετρίας ροής. Mετά την επίδραση της quercetin στις υγρές κυτταροκαλλιέργειες για 24 ώρες, εφαρμόσαμε ημίρρευστες κυτταροκαλλιέργειες, ώστε να ερευνηθεί η επίδραση της ουσίας στα δεσμευμένα προγονικά κύτταρα. Μελετήθηκαν 20 ηλικιωμένοι ασθενείς με ΟΜΛ, αμέσως μετά τη διάγνωση και πριν από τη χορήγηση οποιασδήποτε αγωγής, ηλικίας άνω των 60 ετών (μέση ηλικία 68,8 ± 9,2 έτη), από τους οποίους οι 14 είχαν de novο ΟΜΛ και οι 6 δευτεροπαθή μετά από ΜΔΣ. Τα αποτελέσματά μας έδειξαν ότι η quercetin προκαλούσε, με ένα δοσο- και χρονο-εξαρτώμενο τρόπο, ελάττωση της βιωσιμότητας, αναστολή του πολλαπλασιασμού και αύξηση της απόπτωσης των λευχαιμικών κυττάρων. Όλοι οι ασθενείς με ΟΜΛ εξέφραζαν την πρωτεΐνη της survivin. Η quercetin προκαλούσε αύξηση της έκφρασης της αντι-αποπτωτικής πρωτεΐνης survivin στα λευχαιμικά κύτταρα με ένα δοσοεξαρτώμενο τρόπο όπως επίσης και μείωση του αριθμού των αποικιών, ιδιαίτερα στη δόση των 100 μΜ. Συμπερασματικά, από τη μελέτη αυτή προκύπτει ότι αν και δεν έχει καθοριστεί επακριβώς ο μηχανισμός της δράσης της quercetin έχει διαπιστωθεί ότι η ουσία αυτή προάγει την απόπτωση των λευχαιμικών κυττάρων. Δεδομένου ότι αρκετοί χημειοθεραπευτικοί παράγοντες δρουν μέσω της έναρξης της απόπτωσης, είναι δυνατόν χημικές ουσίες όπως η quercetin να προκαλούν μεγαλύτερη αντιλευχαιμική δράση εάν συνδυαστούν με τη συμβατική κυτταροτοξική θεραπεία

Environmental impact of phycocyanin recovery from Spirulina platensis cyanobacterium

Multifunctional extracts from Spirulina platensis are suggested as food additives, due to their high content in functional ingredients and specifically phycocyanin. The recovery of phycocyanin from the microalgal biomass is performed by using ultrasounds and polar solvents such water, ethanol or buffer. The application of drying pretreatment in combination with the use of different solvents presents variation in the yields, affecting the actual recovery of the protein and hence the environmental impact of the production of 1 kg phycocyanin. Life cycle analysis on the recovery techniques for the isolation of the desired phycocyanin was performed in order to evaluate the selected extraction processes' sustainability. Drying exhibited increased environmental footprint due to the energy demand, while at the same time affecting not only the yielding but also the quality of the extracts. The use of aqueous solvents can lead to an environmental and efficient extraction, replacing organic solvent systems sufficiently. Industrial relevance Phycocyanin is a pigment-protein complex which is used into various food products to enhance their nutritional qualities acting as food colorant, antioxidant and emulsifier, which can sufficiently replace or reduce the use of synthetic additives. For the effective recovery of phycocyanin, the nutrient should be extracted from the microalgae biomass of Spirulina platensis. The steps to achieve that include the cultivation and harvesting of the microalgae, the drying of the biomass if necessary and the extraction process. However, these steps are resource and energy demanding processes which can affect the environmental footprint and the cost of the final product. Looking for more efficient practices combinations of materials (wet or dried biomass) and solvents (water, buffer and ethanol), which are currently used industrially, were examined in order to evaluate and suggest the most sustainable production line for phycocyanin. © 2017 Elsevier Lt

Essential oils of Phlomis species growing in Greece: Chemical composition and antimicrobial activity

The essential oils obtained from the aerial parts of Phlomis fruticosa, P. cretica and P. samia, growing in Greece, were analysed by GC and GC-MS. From the 72 identified constituents, representing 84.3%, 90.4% and 67.4% of the oils, respectively, α-pinene, limonene, β-caryophyllene, linalool, (E)-β-farnesene, germacrene D, (Z)-γ-bisabolene and cis-β-ocimene were the major components. Furthermore, all the oils exhibited an interesting antimicrobial profile after they were tested against two Gram-positive and four Gram-negative bacteria and three pathogenic fungi. Copyright © 2004 John Wiley & Sons, Ltd

Samioside, a new phenylethanoid glycoside with free-radical scavenging and antimicrobial activities from Phlomis samia

A new phenylethanoid glycoside, samioside, was isolated from the aerial parts of Phlomis samia and identified as 1-O-3,4-(dihydroxyphenyl)ethyl β-D-apiofuranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-4-O- caffeoyl-β-D-glucopyranoside (1). In addition, one known phenylethanoid glycoside and three known flavonoids were identified as acteoside (2), apigenin, chrysoeriol, and ermanin, respectively. The structure of 1 was elucidated on the basis of its spectroscopic data. Samioside (1) demonstrated scavenging properties toward the DPPH radical and antimicrobial activity against Gram-positive and -negative bacteria