A combined approach to signal enhancement in fluorescence affinity biosensors and assays is reported. It is based on the compaction of specifically captured target molecules at the sensor surface followed by optical probing with a tightly confined surface plasmon (SP) field. This concept is utilized by using a thermoresponsive hydrogel (HG) binding matrix that is prepared from a terpolymer derived from poly(N-isopropylacrylamide) (pNIPAAm) and attached to a metallic sensor surface. Epi-illumination fluorescence and SP-enhanced total internal reflection fluorescence readouts of affinity binding events are performed to spatially interrogate the fluorescent signal in the direction parallel and perpendicular to the sensor surface. The pNIPAAm-based HG binding matrix is arranged in arrays of sensing spots and employed for the specific detection of human IgG antibodies against the Epstein-Barr virus (EBV). The detection is performed in diluted human plasma or with isolated human IgG by using a set of peptide ligands mapping the epitope of the EBV nuclear antigen. Alkyne-terminated peptides were covalently coupled to the pNIPAAm-based HG carrying azide moieties. Importantly, using such low-molecular-weight ligands allowed preserving the thermoresponsive properties of the pNIPAAm-based architecture, which was not possible for amine coupling of regular antibodies that have a higher molecular weight.
- MeSH
- Acrylic Resins chemistry MeSH
- Biosensing Techniques methods MeSH
- Fluorescence MeSH
- Hydrogels chemistry metabolism MeSH
- Immunoglobulin G analysis immunology MeSH
- Epstein-Barr Virus Infections diagnosis immunology metabolism virology MeSH
- Humans MeSH
- Peptide Fragments immunology metabolism MeSH
- Polymers chemistry MeSH
- Epstein-Barr Virus Nuclear Antigens immunology MeSH
- Herpesvirus 4, Human immunology isolation & purification MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Alzheimer's disease (AD) and sporadic Creutzfeldt-Jakob disease (sCJD) are both characterized by extracellular pathologically conformed aggregates of amyloid proteins-amyloid β-protein (Aβ) and prion protein (PrPSc), respectively. To investigate the potential morphological colocalization of Aβ and PrPSc aggregates, we examined the hippocampal regions (archicortex and neocortex) of 20 subjects with confirmed comorbid AD and sCJD using neurohistopathological analyses, immunohistochemical methods, and confocal fluorescent microscopy. Our data showed that extracellular Aβ and PrPSc aggregates tended to be, in most cases, located separately, and "compound" plaques were relatively rare. We observed PrPSc plaque-like structures in the periphery of the non-compact parts of Aβ plaques, as well as in tau protein-positive dystrophic structures. The AD ABC score according to the NIA-Alzheimer's association guidelines, and prion protein subtype with codon 129 methionine-valine (M/V) polymorphisms in sCJD, while representing key characteristics of these diseases, did not correlate with the morphology of the Aβ/PrPSc co-aggregates. However, our data showed that PrPSc aggregation could dominate during co-aggregation with non-compact Aβ in the periphery of Aβ plaques.
- MeSH
- Alzheimer Disease pathology MeSH
- Amyloid beta-Peptides genetics metabolism MeSH
- Plaque, Amyloid pathology MeSH
- Creutzfeldt-Jakob Syndrome pathology MeSH
- Extracellular Space chemistry MeSH
- Polymorphism, Single Nucleotide genetics MeSH
- Codon genetics MeSH
- Comorbidity MeSH
- Middle Aged MeSH
- Humans MeSH
- Brain pathology MeSH
- Neurons pathology MeSH
- Pilot Projects MeSH
- Protein Aggregates * MeSH
- tau Proteins metabolism MeSH
- PrPSc Proteins genetics metabolism MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Quadrulella (Amoebozoa, Arcellinida, Hyalospheniidae) is a genus of testate amoebae with unmistakable morphology, which secretes characteristic square plates to reinforce the test. They are mainly known from fens and freshwater habitats and have never been documented in deserts. We describe a new species, Quadrulella texcalense, from biological soil crusts in the intertropical desert of Tehuacán (state of Puebla, Mexico). Quadrulella texcalense occurred only at altitudes between 2140 and 2221m.a.s.l., together with the bryophyte genera Pseudocrossidium, Weissia, Bryum, Didymodon, Neohyophyla and Aloina. The soil was extremely dry (moisture of 1.97-2.6%), which contrasts sharply with previous reports for the Quadrulella genus. Single cell mitochondrial cytochrome oxidase I (COI) barcoding of thirteen isolated cells showed an important morphological variability despite having all the same COI barcode sequence. Quadrulella texcalense was placed in a tree containing other Hyalsopheniidae, including a newly barcoded South African species, Q. elegans. Q. texcalense unambiguously branched within genus Quadrulella in a compact clade but with a long branch, suggesting accelerated evolution due to a transition towards a new environment and/or under-sampling.
- MeSH
- Species Specificity MeSH
- Phylogeny * MeSH
- Lobosea classification cytology genetics MeSH
- Desert Climate * MeSH
- Soil parasitology MeSH
- Electron Transport Complex IV genetics MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Mexico MeSH
Neopsilotrema n. g. (Digenea: Psilostomidae) and three new species of psilostomid digeneans are described from birds in North America and Europe: Neopsilotrema lakotae n. sp. from Aythya americana (Eyton) in North Dakota, USA, Neopsilotrema affine n. sp. from Aythya affinis (Eyton) in Minnesota, USA and Neopsilotrema lisitsynae n. sp. from Anas crecca L. in Kherson Region, Ukraine. Neopsilotrema n. g. shares a bipartite seminal vesicle with only three genera within the Psilostomidae, Psilotornus Byrd & Prestwood, 1969, Psilostomum Looss, 1899 and Grysoma Byrd, Bogitsh & Maples, 1961. The new genus differs from Psilotornus in the presence of a muscular pharynx and a massive ventral sucker; the location of the cirrus-sac in relation to the ventral sucker and more posterior location of ovary; the nature of the vitellarium (i.e. comprising large, compact follicles with small vitelline cells vs weakly defined follicles with large vitelline cells); a proportionately shorter forebody; and in parasitisation in anseriform (vs passeriform) birds. Differences between the new genus and Psilostomum comprise the shape of the body, the relative size of the suckers, somewhat longer forebody and a more anterior location of the testes. Neopsilotrema n. g. differs from Grysoma in the relative size of the suckers, the degree of development of prostatic cells, the nature of the vitellarium and the size of the eggs in relation to body length. The European species Neopsilotrema lisitsynae n. sp. is distinguished from its congeners in having a longer, narrower and distinctly more elongate body with a longer post-testicular region and anterior limits of the vitelline fields posterior to ventral sucker. The two North American forms, Neopsilotrema lakotae n. sp. and Neopsilotrema affine n. sp., are cryptic species with largely overlapping metrical data; these are distinguished by comparing genetic data. The phylogenetic hypotheses for the Psilostomidae developed from sequence data analyses based on partial 28S rDNA support the erection of the new genus and the distinction of the three new species. Grysoma marilae (Price, 1942) agrees more closely with the generic diagnosis of Neopsilotrema, especially in relation to the size and shape of the body, the relative length of the forebody and post-testicular field, the structure of the vitellarium, the location of the reproductive organs and the sucker ratio. Consequently, it is here transferred to the new genus as Neopsilotrema marilae (Price, 1942) n. comb.
- MeSH
- Species Specificity MeSH
- Phylogeny MeSH
- Ducks parasitology MeSH
- Molecular Sequence Data MeSH
- RNA, Ribosomal, 28S genetics MeSH
- Trematoda anatomy & histology classification genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- North America MeSH
- Ukraine MeSH
Nipponocercyon satoi sp. nov. is described from the mountains of Zhenjiang and Jiangxi Provinces in Southwest China. Although the species bears all synapomorphies of Nipponocercyon Satô, 1963, it differs substantially from the other two species of the genus by its Pacrillum-like habitus (i.e. small, compact and strongly convex body with reduced surface sculpturing). The new species is compared in detail with the other two Nipponocercyon species as well as with Pacrillum Orchymont, 1941 and Megasternum Mulsant, 1844. The generic diagnosis of Nipponocercyon is adapted, and reasons for assigning the new species to Nipponocercyon are discussed.
- MeSH
- Coleoptera anatomy & histology classification MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- China MeSH
To gain more complete insight into flexibility and malleability of five forms of human liver cytochrome P450 enzymes, which play major roles in drug metabolism (CYPs 1A2, 2A6, 2C9, 2D6 and 3A4), we employed UV/VIS and resonance Raman spectroscopy in combination with all-atomic molecular dynamics simulations under normal and high pressure conditions (300 MPa). In general, the high pressure reduces the flexibility of CYPs, which become more dense and compact as their radii of gyration and temperature B-factors diminish. The flexibility of CYPs spans the regions, which are localized in solvent exposed loops. A considerable degree of flexibility is also observed at amino-acids making the pw2 and solvent channels, which are suggested to serve for substrate access and/or product release. The number of water molecules as well as the number of protein backbone atoms of the active site in close proximity of heme cofactor generally increases under high pressure. This finding provides new insights regarding the interpretation of pressure-related Soret band red shifts. Presented results also point towards considerable differences between the CYP forms studied: CYP2A6 and CYP1A2 have the least malleable active sites while those of CYP2D6, CYP2C9 and CYP3A4 have considerably greater degrees of flexibility or malleability. In addition, the number of water molecules in the active site cavity of CYP3A4 anomalously decreases under high pressure due to opening of the active site. These results correlate with the known substrate promiscuity of the respective CYP forms, with CYP3A4 displaying the highest substrate promiscuity, corresponding to the most open and malleable active site, whereas CYP1A2 and CYP2A6 show a high substrate-specificity and have a small and rigid active sites.
- MeSH
- Aryl Hydrocarbon Hydroxylases chemistry metabolism MeSH
- Cytochrome P-450 CYP1A2 chemistry metabolism MeSH
- Cytochrome P-450 CYP2D6 chemistry metabolism MeSH
- Cytochrome P-450 CYP3A chemistry metabolism MeSH
- Heme chemistry metabolism MeSH
- Hydrostatic Pressure MeSH
- Isoenzymes chemistry metabolism MeSH
- Liver enzymology MeSH
- Catalytic Domain MeSH
- Protein Conformation MeSH
- Humans MeSH
- Models, Molecular MeSH
- Spectrum Analysis, Raman MeSH
- Molecular Dynamics Simulation MeSH
- Spectrophotometry methods MeSH
- Substrate Specificity MeSH
- Cytochrome P-450 Enzyme System chemistry metabolism MeSH
- Protein Structure, Tertiary MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The X-ray structure of cold-active beta-galactosidase (isoenzyme C-2-2-1) from an Antarctic bacterium Arthrobacter sp. C2-2 was solved at 1.9A resolution. The enzyme forms 660 kDa hexamers with active sites opened to the central cavity of the hexamer and connected by eight channels with exterior solvent. To our best knowledge, this is the first cold-active beta-galactosidase with known structure and also the first known beta-galactosidase structure in the form of compact hexamers. The hexamer organization regulates access of substrates and ligands to six active sites and this unique packing, present also in solution, raises questions about its purpose and function. This enzyme belongs to glycosyl hydrolase family 2, similarly to Escherichia coli beta-galactosidase, forming tetramers necessary for its enzymatic function. However, we discovered significant differences between these two enzymes affecting the ability of tetramer/hexamer formation and complementation of the active site. This structure reveals new insights into the cold-adaptation mechanisms of enzymatic pathways of extremophiles.
- MeSH
- Arthrobacter enzymology MeSH
- Bacterial Proteins genetics chemistry metabolism MeSH
- beta-Galactosidase genetics chemistry metabolism MeSH
- Financing, Organized MeSH
- Ions chemistry MeSH
- Crystallography, X-Ray MeSH
- Protein Structure, Quaternary MeSH
- Humans MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Cold Temperature MeSH
- Solvents chemistry MeSH
- Amino Acid Sequence MeSH
- Sequence Alignment MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH