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Adenovirus Dodecahedron, a VLP, Can be Purified by Size Exclusion Chromatography Instead of Time-Consuming Sucrose Density Gradient Centrifugation.
Get PDFAdenoviral dodecahedron (Dd) is a virus-like particle composed of twelve pentameric penton base (Pb) proteins, responsible for adenovirus cell penetration. It is generated spontaneously in the baculovirus system upon expression of the Pb gene of adenovirus serotype 3. This particle shows remarkable cell penetration ability with 2,00,000-3,00,000 Dd internalized into one cell in culture, conceivably delivering several millions of foreign cargo molecules to the target cell. We have used it in the past for delivery of small drugs as well as a vaccination platform, in which Dd serves as a particulate vaccine delivery system. Since development of new biomedicals depends strongly on the cost of their expression and purification, we attempted, albeit unsuccessfully, to obtain Dd expression in bacteria. We therefore retained its expression in the baculovirus/insect cells system but introduced significant improvements in the protocols for Dd expression and purification, leading to considerable savings in time and improved yield
Novel molecular markers for the detection of methanogens and phylogenetic analyses of methanogenic communities.
Get PDFMethanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia
Updates on Metabolism in Lactic Acid Bacteria in Light of “Omic” Technologies
Get PDFLactic acid bacteria (LAB) comprise highly diverse bacterial genus and species characterized with a common feature, the ability to produce lactic acid from the fermentation of carbohydrates. Sugar metabolism of LAB together with other metabolic capacities such as proteolytic activity, production of aroma compounds and bioactive peptides, have been exploited throughout the world in the processing by fermentation of various food types, including milk, meat and plants. These bacteria are essential for the preservation of food and for maintaining and/or enhancing its organoleptic and nutritional quality.
Studies on the wide metabolic capacity of LAB considering the use of different carbon and nitrogen sources and exploring their complex regulatory circuits are the main areas of current research on these bacteria. Moreover, high-throughput technologies and the exponentially growing data bases of complete genome sequences of different LAB species, as well as all other large-scale omic-techniques combined with systems biology, bring about a constant need for updating current knowledge in LAB biology. Furthermore, it addresses strategies to exploit such knowledge into the rational design and use of LAB strains with increased industrial and consumer functionality
A Simple and Rapid Procedure for the Detection of Genes Encoding Shiga Toxins and Other Specific DNA Sequences
Get PDFA novel procedure for the detection of specific DNA sequences has been
developed. This procedure is based on the already known method employing PCR with
appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent
6-carboxylfluorescein (FAM) at the 5′ end and the fluorescence quencher BHQ-1 (black hole
quencher) at the 3′ end. However, instead of the detection of the fluorescence signal with the
use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence
polarization readers, as in all previously-reported procedures, we propose visual observation
of the fluorescence under UV light directly in the reaction tube. An example for the specific
detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga
toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost
effective. It may be suitable for use in research laboratories, as well as in diagnostic units of
medical institutions, even those equipped only with a thermocycler and a UV transilluminator,
particularly if rapid identification of a pathogen is required
Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain.
No full textPhosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development
Wpływ zwiększania suchej masy w surowcu na teksturę i mikrostrukturę twarogu kwasowego
Get PDFCelem badań była ocena wpływu zwiększenia zawartości suchej masy w surowcu na
teksturę i mikrostrukturę twarogu kwasowego. Surowcem do produkcji twarogu było mleko pasteryzowane
o zawartości 2% tłuszczu, zagęszczane metodą wyparną lub ultrafiltracji (UF) do ok. 25% suchej masy.
Twaróg kontrolny produkowano z mleka niezagęszczonego. Produkcję twarogu realizowano, stosując kulturę
doświadczalną IBBB3. W twarogach oznaczono zawartość suchej masy, białka ogółem, laktozy, tłuszczu
oraz popiołu. W badaniach tekstury dokonano pomiarów twardości i adhezyjności twarogów. Mikrostrukturę
odwodnionych liofilizacyjnie twarogów badano przy użyciu mikroskopu elektronowego skaningowego.
Między twarogami z mleka niezagęszczonego, zagęszczonego wyparnie oraz koncentratu UF występowały
istotne różnice w podstawowym składzie chemicznym. Twarogi z mleka niezagęszczonego oraz koncentratu
UF charakteryzowały się porównywalnym udziałem białka, tłuszczu oraz laktozy w składzie suchej masy,
natomiast twaróg z mleka zagęszczonego wyparnie wykazywał znacznie mniejszy udział białka i tłuszczu
oraz ponad 3-krotnie większy laktozy w suchej masie w porównaniu do twarogu z mleka niezagęszczonego
i koncentratu UF. Twarogi uzyskane z surowca o zwiększonej zawartości suchej masy zawierały ok. 1,7-2,0-
krotnie więcej popiołu w porównaniu do produktu z mleka niezagęszczonego. Twarogi z mleka zagęszczonego
wyparnie i koncentratu UF wykazywały istotnie mniejszą twardość i adhezyjność w porównaniu do
twarogów z mleka niezagęszczonego. Twardość i adhezyjność twarogów zwiększała się ze wzrostem zawartości
białka w ich składzie. Twarogi z mleka niezagęszczonego wykazywały porowatą mikrostrukturę
o stosunkowo dużych porach, natomiast mikrostruktura twarogów uzyskanych z mleka zagęszczonego
wyparnie oraz koncentratu UF była bardziej zwarta.
Słowa kluczowe : mikrostruktura, tekstura, twaróg kwasowy, ultrafiltracja (UF), zagęszczanie
wyparn
A biosensor based on electroactive dipyrromethene-Cu(II) layer deposited onto gold electrodes for the detection of antibodies against avian influenza virus type H5N1 in hen sera.
Get PDFThis paper describes the development of a biosensor for the detection of anti-hemagglutinin antibodies against the influenza virus hemagglutinin. The steps of biosensor fabrications are as follows: (i) creation of a mixed layer containing the thiol derivative of dipyrromethene and 4-mercapto-1-butanol, (ii) complexation of Cu(II) ions, (iii) oriented immobilization of the recombinant histidine-tagged hemagglutinin, and (iv) filling free spaces with bovine serum albumin. The interactions between recombinants hemagglutinin from the highly pathogenic avian influenza virus type H5N1 and anti-hemagglutinin H5 monoclonal antibodies were explored with Osteryoung square-wave voltammetry. The biosensor displayed a good detection limit of 2.4 pg/mL, quantification limit of 7.2 pg/mL, and dynamic range from 4.0 to 100.0 pg/mL in buffer. In addition, this analytical device was applied for the detection of antibodies in hen sera from individuals vaccinated and non-vaccinated against the avian influenza virus type H5N1. The limit of detection for the assay was the dilution of sera 1: 7 × 10(6), which is about 200 times better than the enzyme-linked immunosorbent assay. Graphical Abstract Scheme of immunosensor based on dipyrromethene-Cu(II)-Histidine-tagged hemagglutinin deposited on gold electrode
Mycophenolic Acid Metabolites Acyl-Glucuronide and Glucoside Affect the Occurrence of Infectious Complications and Bone Marrow Dysfunction in Liver Transplant Recipients.
Get PDFBACKGROUND Mycophenolic acid (MPA) prodrugs are anti-proliferative immunosuppressive agents commonly used after organ transplantation. Although they are generally well tolerated by patients, adverse effects may occur. It is postulated that MPA metabolites could also contribute to these adverse effects. MATERIAL AND METHODS The objective of this study was the assessment of concentrations of total MPA and its metabolites, phenyl glucuronide (MPAG), acyl glucuronide (AcMPAG) and glucoside (GluMPA), using liquid chromatography combined with mass spectrometry (LC/MS/MS) in two groups: kidney transplant recipients and liver transplant patients. Associations of MPA and its metabolites with adverse effects were analyzed. RESULTS The study group consisted of 211 recipients of liver or kidney transplants who received immunosuppressive therapy, including MPA prodrugs. Multivariant analysis showed a positive influence of MPA on gastroenterotoxicity in kidney transplant recipients. In liver patients, gastroenterotoxicity was associated with lower MPAG concentrations. A positive influence of AcMPAG on bacterial infections in liver transplant patients was observed. In liver transplant recipients, a positive influence of MPA and a negative influence of GluMPA levels on the PLT count were revealed. MPA and its metabolites did not influence the hemoglobin levels in both groups. There were no significant relationships among MPA, its metabolites and WBC counts. CONCLUSIONS In kidney transplant recipients, total MPA trough concentration is associated with gastroenterotoxicity and its monitoring could have important role in management of gastrointestinal complications. The quantification of AcMPAG in liver recipients receiving MPA may be helpful in avoiding bacterial infections. GluMPA seems to have a toxic effect on thrombopoiesis
Constrained nucleoside analogues – crystal and molecular structure of 6,5’ - O-anhydrouridines fixed in the anti conformation.
Get PDFA series of analogues of anhydrouridine have been synthesized and their crystal structures established using X-ray diffraction. For all cases, the ribose ring has O(4')-exo, C(4')-endo pucker and the pyrimidine base is in the anti conformation. Investigated compounds crystallise in different crystal systems (monoclinic, orthorhombic), have different space group symmetry (P21, P212121) and exhibit different intermolecular interactions (halogen and hydrogen bonds) among molecules in their crystal lattices. Moreover, in the case of the 5-benzyl-6,5'-O-anhydrouridine a significant positional disorder is present with the phenyl rings existing in two orientations
Response of Poa annua to extreme conditions: comparison of morphological traits between populations from cold and temperate climate conditions
Get PDFAbstract Poa annua is an expansive species that has
developed a stable breeding population on the west shore
of Admiralty Bay, King George Island (Antarctica). We
investigated whether the colonization success of this species
in extreme climatic conditions is associated with
morphological variability. We compared the differences in
12 traits among P. annua populations thriving in Admiralty
Bay, Tatra Mountains and Warsaw. Our expectations that
plants occurring in maritime Antarctic and mountain conditions
should exhibit similar morphological characteristics
were not confirmed. Comparison of individual morphometric
traits indicated high variability within as well as
between the studied populations. Plants from the Admiralty
Bay population differed significantly from plants from the
Warsaw and Tatra populations in 9 of the 12 studied traits.
We discovered more similarities between the Polish populations
(Warsaw and Tatra) than between the populations
from harsh environments (maritime Antarctic and Tatra).
The Tatra population exhibited intermediate morphological
characteristics in relation to plants from the other twostudied populations. In parallel, the climatic conditions
expressed in mean monthly air temperature were intermediate
in the Tatra location. Four traits analyzed by other
authors in the sub-Antarctic populations and by us in the
maritime Antarctic population were consistently lower than
for the Tatra and Warsaw populations. This finding is in
accordance with our working hypothesis (i.e., plants
growing in harsh cold conditions exhibit similar morphological
characteristics). Our results might suggest that the
morphological response to environmental stress of plants
occurring in mountain and polar conditions may be similar