Purification of phage display-modified bacteriophage T4 by affinity chromatography

<p>Abstract</p> <p>Background</p> <p>Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by <it>in vivo </it>phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system.</p> <p>Results</p> <p>Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 10²-10⁵times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml.</p> <p>Conclusions</p> <p>Affinity tags can be successfully incorporated into the T4 phage capsid by the <it>in vivo </it>phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.</p

thrombosis of the portal venous system in cirrhotic patients

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The surface of contact lenses and the safety and comfort of use

Soczewki kontaktowe są coraz popularniejszą metodą korekcji i coraz więcej specjalistów sięga po takie rozwiązanie dla swoich pacjentów. Mimo postępu technologicznego cały czas mamy jednak do czynienia z porzuceniami soczewek kontaktowych. Przyczyna, która jest wskazywana jako najważniejsza, to dyskomfort. Badania jednoznacznie pokazują, że komfort użytkowania soczewek jest związany z ich powierzchnią – powierzchnie zmodyfikowane, o dużym współczynniku lubrykacji dają dużo lepsze efekty. Co więcej, sama powierzchnia soczewek jest odpowiedzialna za gromadzenie się osadów, a to ma już bezpośredni wpływ na bezpieczeństwo.Contact lenses are becoming more popular method of correction and more specialists choose this solution for their patients. However, despite advances in technology we still have to deal with dropouts. The most popular reasons for its discomfort. Technology of lens surface is really important – lubrication and wettability cooperate with comfort

Weathering of siderite in the polar conditions abstract /

Tyt. z nagłówka.Art. jest abstr. referatu z konferencji.Bibliogr. s. 85.Rapid retreat and thinning of glaciers perturb balance of polar ecosystems. These processes influence global climate (Anderson et al. 2000, Płonka 2009). For better understanding of changes and their consequences for global climate, it is necessary to extend our knowledge about processes occurring contemporary in the forelands of retreating glaciers. Numerous researches were carried out on this subject (Anderson et al. 1997, Bukowska-Jania et al. 2003, Pulina et al. 2003). For example, upliftdriven climate change hypothesis assumes that decrease of temperature results from regression of glaciers and is connected with chemical denudation of silicate sediments, which acts as a sink of CO2 from the atmosphere (Płonka 2009). Despite of this, many processes and dependences in polar environment are poorly known. Retreating glaciers and high chemical denudation observed in Polar Regions play significant role in weathering of regolith. Fast retreat of many glaciers exposures great amounts of fresh and fine sediment. Weathering of rock-forming minerals is a source of ions in waters as well as secondary minerals and nutrients in initial soils. These issues are object of interest recently because intensified weathering in Polar Regions may affect global cycle of many elements, particularly Fe (Anderson et al. 1997, 2000, Brenasconi 2008). This study is a part of large research, concerning mechanisms, budget and transport of iron from the foreland of fast retreating arctic glacier. The objective of this research is to determine processes and products of siderite weathering in the polar condition. The area of this study is located on the SW part of the Spitsbergen, in the foreland of Werenskioldbreen. It is hypothesized that in Arctic these processes differ significantly from their equivalents in Alpine regions. Field experiment on Spitsbergen lasted eight years. Samples of the siderite were buried in the surface layer of bottom moraine (in the initial polar soils) 100 m, 250 m and 2000 m from glacier front. This represents a chronosequence of sediment uncovered from underneath the glacier ca. 5-, 10-, and 90-years ago. Samples were recovered after 1 year and after 8 years of burial. Optical microscopy in transmitted and reflected light, scanning electron microscopy SEM/EDS and powder X-ray diffractometry were applied to characterize both, control sample and experimental samples. Siderite with manganese substitutions sampled from the outcrops of metamorphic carbonate in the vicinity of Werenskiold glacier was used for the experiment. This resembles mineral fragments eroded and transported by the glacier. Characterization of control sample (siderite before the experiment) indicates that it already exhibits advanced stages of transformation into secondary phases. Secondary minerals include oxides and hydroxides of iron, such as: goethite, hematite and lepidocrocite. The same mineral phases were identified in experimental recovered after burial. Additionally, 8-year sample shows traces of dissolution. Weathering of siderite in polar initial soils in the foreland of retreating glacier leads to formation of secondary goethite, hematite and lepidocrocite. These phases appear to be the most stable products of siderite weathering in this environment. There is no difference between the samples buried in very young and in older, more evolved initial soil. Additionally, transformations of siderite are identical in initial polar soils and in the outcrops of siderite rock. Traces of dissolution noticed on the surface of older samples indicate that part of iron is permanently removed from the system by solutions. This process, probably mediated by microorganisms, requires further investigation.Dostępny również w formie drukowanej.KEYWORDS: global climate, ecosystem, CO2

Proceedings on the granting and payment of family benefits

Tematem pracy jest postępowanie w sprawie przyznawania i wypłacania świadczeń rodzinnych. Praca skupia się na przedstawieniu najważniejszych kwestii odnoszących się do świadczeń rodzinnych. Została w niej zawarta geneza świadczeń rodzinnych, podstawowe pojęcia odnoszące się do świadczeń rodzinnych, analiza rodzajów świadczeń rodzinnych, postępowanie w sprawie przyznawania i wypłacania świadczeń rodzinnych oraz wybrane zasady postępowania administracyjnego, które mają zasadniczy wpływ na to specyficzne postępowanie.The subject of the work is the procedure for granting and paying family benefits. The work focuses on presenting the most important issues related to family benefits. This work contains the genesis of family benefits, basic terms relating to family benefits, analysis of family benefits, proceedings on the granting and payment of family benefits and selected rules of administrative proceedings, which have a significant impact on this specific procedure

Allergy in contact lens practice

Choroby alergiczne stają się istotnym problemem medycznym i społecznym, gdyż dotykają coraz większej liczby pacjentów na całym świecie. Z tego powodu niezwykle istotne jest odpowiednie podejście do pacjentów kontaktologicznych z alergią i właściwe ich prowadzenie w codziennej praktyce okulistycznej. Alergia to problem wymagający rozpatrzenia zarówno w aspekcie tego, czy osoba obciążona wywiadem lub aktywnym stanem alergicznym może nosić soczewki kontaktowe, jak i w aspekcie stanów alergicznych związanych z użytkowaniem soczewek kontaktowych, jakie mogą pojawić się u osób zdrowych. Jednakże obecnie dzięki dostępności nowoczesnych materiałów, właściwej strategii doboru soczewek oraz odpowiedniemu prowadzeniu pacjenta ten rodzaj korekcji jest już dostępny również dla alergików.Allergic diseases are an important medical and social problem touching a growing number of patients around the world. For this reason, it is extremely important appropriate approach for contact lenses wearers with ocular allergy. For contact lens wearer allergy creates a comfort reduction, a wearing time reduction, dryness problems and lens awareness symptoms, that would lead at the end to contact lens drop-outs, patients who cease contact lens wear. However, nowadays, availability of modern materials, the right strategy and the appropriate fitting contact lenses causes of this type of correction is also available for patients with allergies. Studies have shown that daily disposable contact lenses can be a healthy option for contact lens wearers in general, and can be ideal for some people with eye allergies

Report from the BCLA conference (9–11 June 2017, Liverpool)

Konferencja BCLA jest jedną z  najważniejszych konferencji kontaktologicznych w Europie. W trakcie tegorocznej edycji, która odbyła się wiosną 2017 r. w Liverpoolu, poruszono kluczowe tematy dotyczące przyszłości kontaktologii. Jednym z dominujących zagadnień była ortokeratologia jako metoda terapeutycznej kontroli krótkowzroczności. Jedną z ciekawszych sesji poświęcono tematyce suchego oka. Na jednym z  wykładów omówiono wpływ soczewek kontaktowych na film łzowy. Ważnym elementem tej sesji było przedstawienie długo wyczekiwanego raportu Dry Eye Workshop (DEWS II), który zawiera zbiór rekomendacji dotyczących terapii zespołu suchego oka.The BCLA hosts the UK’s largest Clinical Conference and Exhibition dedicated to contact lenses and the anterior eye every 2 years. This year, the 40th Clinical Conference & Exhibition took place in Liverpool between 9th and 11th of June. The main topics covered the future of the contact lenses industry and orthokeratology – a method to prevent progression of myopia. One of the objectives of the conference was to discuss the TFOS DEWS II Report that changes the definition and management of the dry eye syndrome

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro

Table_1_Review of the epidemiology and variability of LRRK2 non-p.Gly2019Ser pathogenic mutations in Parkinson’s disease.docx

Parkinson’s disease (PD) is a heterogenous neurodegenerative disorder. Genetic factors play a significant role, especially in early onset and familial cases. Mutations are usually found in the LRRK2 gene, but their importance varies. Some mutations, such as p.Arg1441Cys or other alterations in the 1441 codon, show clear correlation with PD, whereas others are risk factors found also in healthy populations or have neglectable consequences. They also exhibit various prevalence among different populations. The aim of this paper is to sum up the current knowledge regarding the epidemiology and pathogenicity of LRRK2 mutations, other than the well-established p.Gly2019Ser. We performed a review of the literature using PubMed database. 103 publications met our inclusion criteria. p.Arg1441Cys, p.Arg1441Gly, p.Arg1441His, p.Arg1441Ser are the most common pathogenic mutations in European populations, especially Hispanic. p.Asn1437His is pathogenic and occurs mostly in the Scandinavians. p.Asn1437Ser and p.Asn1437Asp have been reported in German and Chinese cohorts respectively. p.Ile2020Thr is a rare pathogenic mutation described only in a Japanese cohort. p.Met1869Thr has only been reported in Caucasians. p.Tyr1699Cys, p.Ile1122Val have only been found in one family each. p.Glu1874Ter has been described in just one patient. We found no references concerning mutation p.Gln416Ter. We also report the first case of a Polish PD family whose members carried p.Asn1437His.</p