Point mutations in the C-terminus of HIV-1 gp160 reduce apoptosis and calmodulin binding without affecting viral replication

AbstractOne hallmark of AIDS progression is a decline in CD4+ T lymphocytes, though the mechanism is poorly defined. There is ample evidence that increased apoptosis is responsible for some, if not all, of the decline. Prior studies have shown that binding of cellular calmodulin to the envelope glycoprotein (Env) of HIV-1 increases sensitivity to fas-mediated apoptosis and that calmodulin antagonists can block this effect. We show that individual mutation of five residues in the C-terminal calmodulin-binding domain of Env is sufficient to significantly reduce fas-mediated apoptosis in transfected cells. The A835W mutation in the cytoplasmic domain of gp41 eliminated co-immunoprecipitation of Env with calmodulin in studies with stably transfected cells. Four point mutations (A835W, A838W, A838I, and I842R) and the corresponding region of HIV-1 HXB2 were cloned into the HIV-1 proviral vector pNL4-3 with no significant effect on viral production or envelope expression, although co-immunoprecipitation of calmodulin and Env was decreased in three of these mutant viruses. Only wild-type envelope-containing virus induced significantly elevated levels of spontaneous apoptosis by day 5 post-infection. Fas-mediated apoptosis levels positively correlated with the degree of calmodulin co-immunoprecipitation, with the lowest apoptosis levels occurring in cells infected with the A835W envelope mutation. While spontaneous apoptosis appears to be at least partially calmodulin-independent, the effects of HIV-1 Env on fas-mediated apoptosis are directly related to calmodulin binding

A Specific IFIH1 Gain-of-Function Mutation Causes Singleton-Merten Syndrome

Singleton-Merten syndrome (SMS) is an infrequently described autosomal-dominant disorder characterized by early and extreme aortic and valvular calcification, dental anomalies (early-onset periodontitis and root resorption), osteopenia, and acro-osteolysis. To determine the molecular etiology of this disease, we performed whole-exome sequencing and targeted Sanger sequencing. We identified a common missense mutation, c.2465G>A (p.Arg822Gln), in interferon induced with helicase C domain 1 (IFIH1, encoding melanoma differentiation-associated protein 5 [MDA5]) in four SMS subjects from two families and a simplex case. IFIH1 has been linked to a number of autoimmune disorders, including Aicardi-Goutières syndrome. Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues. In vitro functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction. Interferon signature genes were upregulated in SMS individuals’ blood and dental cells. Our data identify a gain-of-function IFIH1 mutation as causing SMS and leading to early arterial calcification and dental inflammation and resorption

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Tandemly repeated DNA families in the mouse genome

<p>Abstract</p> <p>Background</p> <p>Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.</p> <p>Results</p> <p>Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites.</p> <p>To confirm our data, we next performed <it>in situ </it>hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to <it>in silico </it>predicted several extra-chromosomes were positive for TR by <it>in situ </it>analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.</p> <p>Conclusions</p> <p>Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified <it>in silico </it>and confirmed <it>in situ </it>3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.</p

STUDYING THE PECULIARITIES OF REPRODUCTION OF HIV-I IN THE INTORTED LINES OF MONOCYTES/MACROCHAGES AND T-LYMPHOCYTES

The purpose of the work: the investigation of the HIV-I reproduction peculiarities in the intorted lines of cells of monocytes/macrophages and T-limphocytes, a comparative analysis of the anti-HIV activity of the azidothymidine and the number of its derivatives on the different models of the HIV-infected cells. The unique chronically infected line of the intorted monocytes of the man GKV-4005 has been received. The antivirus activity of series 5'-phosphonate of azodothymidine and fluorthymidine has been studied. It has been shown, that these compounds less toxic for the uninfected cells, than azidothymidine. The most effective inhibiting action adheres to the 5'-carboxyphosphonate of azidothymidine. The method of cultivating the HIV-I, allowing to receive the antigene with a full set of the virusspecific proteins, has been developed. The method of cultivating of HIV-I on the nutritional media, containing serums of the cattle of pigs processed by PEG, has been developed. The lines of cells GKV-4005 have been used in the institute for the scientific research works. The experimental batches of the test-systems have been made for revealing the antibodies to HIV in the institute. It is purposeful to use the results in the Institutes of the molecular-biological and virusological profiles, in the Institutes of clinic medicineAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Composition of Alcohol Extracts from Poplar Leaves (Populus balsamifera L.)

The group composition of alcohol extracts from balsamic poplar leaves Populus balsamifera L, growing in the Krasnoyarsk Krai, Krasnoyarsk (Russia) were examined in this work. Leaves selected in different phases of the growing season were used: June, July, August – fresh, September – fallen leaves. Poplar leaves contain up to 56% of extractive substances, alcohol-soluble substances account for up to 67% of the extract amount. Alcohol extracts were fractionated using solvents with increasing polarity: petroleum ether (boiling point 40 to 70 °C), diethyl ether, ethyl acetate, butanol. The results showed that the alcohol extracts of poplar leaves are dominated by substances extracted with ethyl acetate (up to 60%). The study of the component composition of volatile compounds of individual fractions of alcohol extracts was performed on an Agilent chromatography-mass spectrometer. The presence of various groups of compounds was established: hydrocarbons, alcohols, acids, ketones, etc., including biologically active substances such as megasterol acetate, ℽ-sitosterol, sitosterol, 3,7,11,15-tetramethyl-2-hexadecene-1-ol, diethyltoluamide, 4-methoxy-3-nitrobiphenyl, and other compounds that it can be used in different applications

Use of Post-Extraction Fir Wood Greenery Residues by the Bioconversion Method with the Production of Feed Additives

The effectiveness of forest resources depends on the comprehensiveness and rationality of their consumption and processing into finished products. This article discusses the problem of using solid fir wood greenery residues generated during the industrial production of essential oils. Bioconversion is considered to be the most promising use. The objective of this research was to study the chemical composition of bioconversion products of fir wood greenery-based substrates. The PP-3.2 strain of Pleurotus pulmonarius (Fr.) Quél was used as a biodestructor. In the process of bioconversion, the contents of polysaccharides and lignin substances is reduced to 38% and 28%, respectively. Up to 20% of protein accumulates in bioconversion products of fir wood greenery. The amount of nucleic acids is not more than 1.5 g per kg; the contents of heavy metals, such as mercury, cadmium, arsenic, and lead, do not exceed the maximum permissible concentration standards. The substrate weight loss reaches 15%. When fallen leaves and post-extraction poplar bud residues are added to the substrate, the substrate-destroying activity of fungi increases, and the protein content increases by 3%. The digestibility of products as a result of bioconversion increases 1.6–2.8 times depending on the substrate composition. The obtained data support the recommendation of post-fermented substrates based on fir wood greenery and balsam poplar biomass for use as a protein feed additive

The effect of a Trichodermin biopreparation based on poplar litter on the germination of tomatoes and wheat

The paper presents the results of the study of a biological product of the type "Trichodermin", obtained as a result of biodegradation of plant waste - poplar litter - by Siberian strains K6-15 Trichoderma spp. Two methods were used: seed dusting and the introduction of a biological product into the soil with different concentrations. Germination and number of infected seeds of tomato (Irina F1 variety, Geolia) and Novosibirskaya 31 soft spring wheat were evaluated in the work

Normative and economic foundations of high-rise construction in the city of Samara

Every year the number of free land plots for construction of buildings is steadily decreasing in cities. In this regard, the participants in the investment and construction process are increasingly seeking to maximize the use of land. A logical way for this is to use high-rise construction. However, until recently, builders encountered serious obstacles in the form of lack of special norms and rules for the design of high-rise buildings. It led to the need for individual coordination of each high-rise facility, the development and approval of special technical conditions, the passage of numerous administrative approvals. Thus, investment activity regarding the construction of high-rise buildings in the Russian Federation is reduced. In 2016, there were regulatory changes that substantially alleviated these difficulties. In this article, the authors analyze the features of the town-planning normative-legal field of high-rise construction and its development, track the interrelations between the regulatory regulation of the construction of similar facilities, with the real need, the technical feasibility and economic feasibility of their construction in the urban district of Samara. Conclusions and suggestions are also based on the analysis of the norms of urban zoning, the residential real estate market and the value of land plots

Normative and economic foundations of high-rise construction in the city of Samara

Every year the number of free land plots for construction of buildings is steadily decreasing in cities. In this regard, the participants in the investment and construction process are increasingly seeking to maximize the use of land. A logical way for this is to use high-rise construction. However, until recently, builders encountered serious obstacles in the form of lack of special norms and rules for the design of high-rise buildings. It led to the need for individual coordination of each high-rise facility, the development and approval of special technical conditions, the passage of numerous administrative approvals. Thus, investment activity regarding the construction of high-rise buildings in the Russian Federation is reduced. In 2016, there were regulatory changes that substantially alleviated these difficulties. In this article, the authors analyze the features of the town-planning normative-legal field of high-rise construction and its development, track the interrelations between the regulatory regulation of the construction of similar facilities, with the real need, the technical feasibility and economic feasibility of their construction in the urban district of Samara. Conclusions and suggestions are also based on the analysis of the norms of urban zoning, the residential real estate market and the value of land plots