Diversity and Possible Activity of Microorganisms in Underground Gas Storage Aquifers

Microbiology - Underground natural gas storage facilities (UGS) have been recently proposed as sites to store “green” gas containing biogas, synthetic methane, and molecular hydrogen....     

An engineered ultra‐high affinity Fab‐Protein G pair enables a modular antibody platform with multifunctional capability

Engineered recombinant antibody‐based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab‐based affinity reagents in a “plug and play” fashion. This platform exploits the ultra‐tight binding interaction between affinity matured variants of a Fab scaffold (Fab^S) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi‐specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab‐GA1 platform, we applied it to a detection proximity assay based on the β‐lactamase (BL) split enzyme system. We also show the bi‐specific capabilities of the module by using it in context of a Bi‐specific T‐cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T‐cells to cancer cells. We show that GA1‐Fab modules are easily engineered into potent cell‐killing BiTE‐like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer‐related targets in a plug and play fashion.     

The large and small isoforms of human papillomavirus type 16 E6 bind to and differentially affect procaspase 8 stability and activity

Human papillomavirus type 16 (HPV-16) has developed numerous ways to modulate host-initiated immune mechanisms. The HPV-16 E6 oncoprotein, for example, can modulate the cellular level, and consequently the activity, of procaspase 8, thus modifying the cellular response to cytokines of the tumor necrosis factor family. E6 from HPV-16, but not E6 from the low-risk types 6b and 11, alters the cellular level of procaspase 8 in a dose-dependent manner. Both the large and small (E6*) isoforms of E6, which originate by way of alternate splicing, can modulate procaspase 8 stability. Intriguingly, although both isoforms bind to procaspase 8, the large isoform accelerates the degradation of procaspase 8 while the small isoform stabilizes it. Binding leads to a change in the ability of procaspase 8 to bind either to itself or to FADD (Fas-associated death domain), with the large version of E6 able to inhibit this binding while the small isoform does not. Consistent with this model, knockdown of the large version of E6 by small interfering RNA leads to increases in the levels of procaspase 8 and its binding to both itself and FADD. Thus, these alternatively spliced isoforms can modulate both the level and the activity of procaspase 8 in opposite directions.     

Development of a biochip for quantitative determination of two forms of prostate specific antigen using internal calibration curve

Three-dimensional gel-based microchip allowing simultaneous quantitative detection of total (PSAtot) and free (PSAfree) forms of prostate specific antigen in human serum (in a format "one patient-one biochip") was developed. A method, which doesn't require preliminary construction of calibration curves when performing an assay, was applied for quantitative determination of PSAtot and PSAfree. Gel elements with immobilized antigen (PSA) in different concentration, forming an internal calibration curve, were included in a structure of the microchip, in addition to the elements with immobilized antibodies specific against PSAtot and PSAfree. The specialized software "ImaGelAssay" was used for data processing and interpretation. The sensitivity of the assay performed on biochips was 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. Variation coefficient for the measurements inside one series of microchips didn't exceed 10%. Correlation coefficient between the results of measurements in human sera obtained on biochips and by the standard ELISA method was 0.988 for PSAtot and 0.987 for PSAfree.     

Larval identification of species and subspecies of the genus Hyalomma (Acari: Ixodidae) from Russia and neighbouring territories

The following species and subspecies of Hyalomma Koch, 1844 are recorded from Russia and neighbouring territories, including those involved in natural foci of tick-borne diseases: H. (Hyalomma) aegyptium (Linnaeus, 1758), H. (Euhyalomma) dromedarii Koch, 1844, H. (Euh.) asiaticum asiaticum Schulze et Schlottke, 1930, H. (Euh.) asiaticum caucasicum Pomerantzev, 1940, H. (Euh.) asiaticum kozlovi Olenev, 1931, H. (Euh.) anatolicum Koch, 1844, H. (Euh.) excavatum Koch, 1844, H. scupense Schulze, 1918, H. (Euh.) marginatum marginatum Koch, 1844, H. (Euh.) marginatum turanicum Pomerantzev, 1946, and H. (Euh.) marginatum rufipes Koch, 1844. The geographic distribution and host-parasite relationships of each taxon are discussed. Species characters of the larval stage, that can be distinguished using light microscopy, are found to be very few. These characters include shape of scutum, shape and rate of hypostome denticulation, shape and rate of the development of spurs on coxae I to III. Measurements of some morphological structure and their rations show statistically significant differences between some closely related species, even if qualitative discriminating characters are unknown. Only measurements and their ratios can be used for the discrimination of larval H. anatolicum from larval H. excavatum, because qualitative discriminating features have not been found for these species. The complex structure of the subspecific morhological differentiation of all parasitic stages in the polymorphic species H. asiaticum and H. marginatum is revealed. This structure probably reflects some peculiarities of the microevolutionary processes. Identification key for the larval stage of seven Hyalomma species is provided.     

Assessment of the preimaginal stages of the ticks collected from small mammals in Western and Northern Caucasus (Acari: Ixodidae)

The exact identification of the preimaginal stages of the ticks collected from small mammals in Western and Northern Caucasus as a result of ten-year route investigations allowed us to obtain new data on the geographic ranges, altitude and biotopic preferences, and host-parasite relations of 11 species. A wide range of the joint occurrence (in the same locality, on the same host species, on the same host individual) is recorded for the first time for 10 species. The cohabitation of the each of 10 species with other 2-9 species has been found. The number of the cases of joint occurrence of different ixodid species on the same host individual is proportionate to the collection period in one locality. The cohabitation did not observed only for the species Ixodes ghilarovi, a high mountain ixodid species, which is for the first time recorded in 7 localities of Northern and Western Caucasus.     

Investigation of the structure of human coronary vasculature

Some results of a morphometric study of the parameters of coronary arteries are presented. The parameters that characterize the structure of the arterial vasculature as an optimal branching system have been calculated. Statistically reliable correlations between the diameter of the bigger of two daughter vessels in a bifurcation with the diameter of the parent vessel as well as between the diameter of the smaller daughter vessel and the asymmetry coefficient have been obtained. Differences in the structural parameters of the two types of coronary arteries that provide blood delivery and distribution have been revealed. The relationships between the lengths and diameters of the arteries of different subsystems have been obtained. It is shown that asymmetrical branching is characteristic of the coronary vasculature, and self-similar asymmetric tree-like systems may be used for its modeling.     

The role of visceral receptors in the mechanisms of neuroimmune interactions in mammalian small intestine

Exogenous administration of mast cell degranulation products into blood as well as their endogenous release induced by compound 48/80 or repeated egg albumin administration to sensitized rats proved to increase activity of mesenteric afferent nerve fibers in the small intestine. In addition, the primary contact with a foreign protein triggered a cascade of chemical processes mobilizing antioxidant systems and increasing nitric oxide production in the small intestinal tissues. Interleukin-1β or Escherichia coli lipopolysaccharide injections also had a long-term stimulating effect on intestinal receptors. Indometacin inhibited the effects induced by the bacterial endotoxin and interleukin-1β. The obtained data suggest that small intestinal interoceptors can monitor chemical environment and modulate body’s responses to foreign antigens. Interleukins-1β acts as a signal transmitter between immune cells and interoceptors, which is mediated by the production of secondary signal substances, in particular, prostaglandin E2 and histamine.     

Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.