Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

Логістичні центри онлайн-торгівлі: пошук оптимального розташування

Закриття урядами країн міжнародних кордонів у відповідь на спалах пандемії COVID-19 спричинило значні збитки у міжнародному товароперевезенні та активізувало необхідність трансформації логістичних операцій з доставки товарів у системах електронної торгівлі. Метою статті є пошук оптимального місця розташування логістичних центрів онлайн-торгівлі. Для досягнення поставленої мети використано модифіковану модель Штейнера-Вебера, що дозволяє визначити оптимальне місце розташування центрального логістичного складу компанії та мінімізувати витрати при розподілі продукції від центрального складу до філій, розташованих в окремих країнах. Об’єктом дослідження обрано компанії у сфері онлайнторгівлі. Встановлено, що 86% досліджуваних компаній планують використовувати складські приміщення протягом найближчих трьох-п’яти років та зосереджуватимуть свою діяльність на розбудові логістичної системи. Емпірично обґрунтовано ефективність менеджменту компаній Amazon у США та Європі, а також та Alibaba в Китаї з оптимального розташування їх логістичних центрів. Основним факторами ефективного перетворення товарообороту у логістичних центрах є оптимізація виробничих затрат, своєчасність доставки товарів, розвиток високоавтоматизованих центрів обробки замовлень (зокрема, сервіс Amazon Prime дозволяє споживачам отримати замовлений товар у той же день). Дослідження емпірично підтверджує та теоретично доводить необхідність і специфіку створення логістичних центрів онлайн-торгівлі в окремих містах країн Європейського Союзу. Результати проведеного дослідження можуть бути корисними для власників, менеджерів компанії, які надають послуги з вантажоперевезення автомобільним, залізничним, морським та авіатранспортом, де важливими базовими факторами ефективності функціонування є відповідність вимогам до обсягів товарів, мінімізація транспортних витрат, своєчасне врахування зовнішніх обмежувальних факторів тощо.Since the COVID-19 pandemic hit last year, countries locked their borders. Thus, international shipping deteriorates drastically. Simultaneously, social distancing increased the need for immediate online consumption and fast home delivery. In the non-digital world, products still need to be shipped to their destination using trucks, trains, airplanes, and ships. Simultaneously, requirements for volumes of goods, transport costs, external limiting factors, etc., must be precisely defined. The article aims to find the optimal location selection solution based on the created mathematical model of the Modified Steiner-Weber Problem with restrictive conditions. The model allows for the central warehouse's optimal location and minimizes distribution costs from the central warehouse to subwarehouses/branches located in individual EU countries. The mathematical model has been applied to a case study of a selected e-commerce dealing, which has established branches in capital cities but does not have an established central warehouse. Systematization of literature sources and approaches to solving the problem of e-commerce distribution center location showed that 86% of the studied companies plan to use on-demand warehousing in the next three to five years. Therefore, the need for warehousing would be preserved. The authors noted that they do not necessarily need to have it in-house. Consequently, fulfillment centers and warehouses would likely continue to be a significant component in the future logistics system. This research would like to stress how important the management of the effective optimization of e-commerce distribution center location is and how to achieve it. The success of Amazon in the US, Europe, and Alibaba in China has genuinely redefined consumer expectations. With the emergence of services like Amazon Prime, consumers now expect same-day delivery. The solution enabling this evolution has been a mix of manufacturing where the production costs are optimal, just-in-time shipping, highly automated fulfillment centers, and mobile connectivity growth. The proposed model results showed that the best location for a central location and storage center concerning the e-commerce environment, including minimum annual transport costs, is near Bristol in the United Kingdom. Eighty-six percent of the companies in the study plan to use ondemand warehousing in the next three to five years, and the solution enabling this evolution has been a combination of manufacturing where the production costs are optimal, just-in-time shipping, highly automated fulfillment centers, and, to a growing extent, mobile connectivity

Surface characterization of plasma treated polymers for applications as biocompatible carriers

The objective of this work was to determine surface properties of polymer surfaces after plasma treatment with the aim of further cytocompatibility tests. Examined polymers were poly(ethyleneterephthalate) (PET), high-density polyethylene (HDPE), poly(tetrafluoro-ethylene) (PTFE) and poly(L-lactic acid) (PLLA). Goniometry has shown that the plasma treatment was immediately followed by a sharp decrease of contact angle of the surface. In the course of ageing the contact angle increased due to the reorientation of polar groups into the surface layer of polymer. Ablation of polymer surfaces was observed during the degradation. Decrease of weight of polymer samples was measured by gravimetry. Surface morphology and roughness was studied by atomic force microscopy (AFM). The PLLA samples exhibited saturation of wettability (aged surface) after approximately 100 hours, while the PET and PTFE achieved constant values of contact angle after 336 hours. Irradiation by plasma leads to polymer ablation, the highest mass loss being observed for PLLA. The changes in the surface roughness and morphology were observed, a lamellar structure being induced on PTFE. Selected polymer samples were seeded with VSMC (vascular smooth muscle cells) and the adhesion and proliferation of cells was studied. It was proved that certain combination of input treatment parameters led to improvement of polymer cytocompatibility. The plasma exposure was confirmed to significantly improve the PTFE biocompatibility

Immune response to E7 protein of human papillomavirus type 16 anchored on the cell surface

To target the E7 protein of human papilloma virus 16 to the cell surface, a fusion gene was constructed. It encodes the signal peptide, part of the immunoglobulin (IgG)-like domain, the transmembrane anchor of vaccinia virus (VV) hemagglutinin (HA), and the complete E7-coding sequence. The fusion gene was expressed under the HA late promoter by a recombinant VV, designated VV-E7-HA. The E7-HA protein was displayed on the surface of cells infected with the recombinant virus and was more stable than unmodified E7. The biological properties of the VV-E7-HA virus were compared with those of a VV-E7 virus that expressed the unmodified E7 and with a VV expressing the Sig-E7-LAMP fusion protein. While the first two of these recombinants were based on VV strain Praha, the third was derived from the WR strain of VV. Infection of mice with the VV-E7-HA virus induced the formation of E7-specific antibodies with the predominance of the IgG2a isotype, whereas the other two viruses did not induce the formation of E7-specific antibodies. Unlike the other two viruses, VV-E7-HA did not induce a response of cytotoxic T lymphocytes or Th1 cells and did not protect mice against the growth of E7-expressing tumors. Thus, VV-E7-HA induced a differently polarized immune response to the E7 protein than the other two viruses.status: publishe

A spiroketal-enol ether derivative from Tanacetum vulgare selectively inhibits HSV-1 and HSV-2 glycoprotein accumulation in Vero cells

The inhibitory effects of Tanacetum vulgare rhizome extracts on HSV-1 and HSV-2 in vitro replication were assessed. Unlike extracts obtained from the aerial parts, adsorption inhibition and virucidal activities seemed not to be relevant for the observed antiviral action of tansy rhizome extracts. Instead, the most significant effects were the inhibition of virus penetration and a novel mechanism consisting of the specific arrest of viral gene expression and consequently the decrease of viral protein accumulation within infected cells. Through a bioactivity-guided fractionation protocol we isolated and identified the spiroketal-enol ether derivative (E)-2-(2,4-hexadiynyliden)-1,6-dioxaspiro[4.5]dec-3-ene as the active compound responsible for this inhibitory effect

Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.Stemcel biology/Regenerative medicine (incl. bloodtransfusion