A diminished hydrophobic effect inside the GroEL/ES cavity contributes to protein substrate destabilization.

Confining compartments are ubiquitous in biology, but there have been few experimental studies on the thermodynamics of protein folding in such environments. Recently, we reported that the stability of a model protein substrate in the GroEL/ES chaperonin cage is reduced dramatically by more than 5 kcal mol compared to that in bulk solution, but the origin of this effect remained unclear. Here, we show that this destabilization is caused, at least in part, by a diminished hydrophobic effect in the GroEL/ES cavity.

[Combination of ARE and HRE cis- Regulatory Elements Elevates the Activity of Tumor-Specific hTERT Promoter].

Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (anti-oxidant response elements) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenovi-ruses.

Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization.

The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself.

Xenobiotic Response Elements (XREs) from Human CYP1A1 Gene Enhance the hTERT Promoter Activity.

A hybrid 6XRE-hTERT promoter consisting of the hTERT tumor-specific promoter and six copies of the XRE element from the CYP1A1 human gene promoter was created. Using a human lung cancer cells as a model, we showed that XRE elements in the hybrid promoter greatly increase the activity of the hTERT promoter and ensure the reporter gene transcriptional activation in response to the treatment of the cells with the AhR ligand benzo(a)pyrene. However, similar effects were also observed in normal human bronchial epithelial cells HBEpC, which indicates the loss of the tumor-specific activity by the 6XRE-hTERT hybrid promoter.

[Human Antithrombin III Minigene with an Optimized Splicing Pattern].

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene.

Transcription termination sequences support the expression of transgene product secreted with milk.

Expression of the reporter gene in transgenic animals depends on the surrounding chromatin environment. Recent genome-wide studies have shown that, in mammals, the entire genome is transcribed. Transcription through a transgene often has a negative effect on the expression of a reporter gene.

Neonatal Lethality and Inflammatory Phenotype of the New Transgenic Mice with Overexpression of Human Interleukin-6 in Myeloid Cells.

To model human interleukin-6 (hIL-6) associated diseases, unique mice with transgenic overexpression of human IL-6 and reporter fluorescent protein EGFP in cells of macrophage-monocyte lineage were generated using loxP-Cre system. High level of hIL-6 production by macrophages and monocytes, as confirmed in vitro in primary culture of bone marrow-derived macrophages, in vivo resulted in early postnatal death in vivo, presumably, due to the effect of overexpression of hIL-6 on hematopoiesis.

Production of Recombinant Proteins in the Milk of Transgenic Animals: Current State and Prospects.

The use of transgenic animals as bioreactors for the synthesis of the recombinant proteins secreted into milk is a current trend in the development of biotechnologies. Advances in genetic engineering, in particular the emergence of targeted genome editing technologies, have provided new opportunities and significantly improved efficiency in the generation of animals that produce recombinant proteins in milk, including economically important animals. Here, we present a retrospective review of technologies for generating transgenic animals, with emphasis on the creation of animals that produce recombinant proteins in milk.

[Arterial Hypertension and Methylenetetrahydrofolate Reductase C677T Gene Polymorphism].

Objective: to determine the association of polymorphisms of C677T methylenetetrahydrofolate reductase (MTHFR) gene with essential arterial hypertension (EH) in the group of patients of Belarusian ethnicity.

Methods: The clinical examination and molecular genetic study of the polymorphism C677T of MTHFR gene by polymerase chain reaction and restriction fragment length polymorphism analysis were performed in 423 people, including 315 patients with EH and 108 healthy individuals.

Results: The distribution of C and T alleles of polymorphism C677T of the MTHFR gene in hypertensive patients was 67.

Patient-reported outcomes: A 5-year long study reveals previously unreported therapeutic, demographic, socio-economic, and other correlations in vitiligo.

Vitiligo is a non-lethal, non-communicable, immune-mediated, and generally progressive skin disease, with poorly understood etiopathogenesis and weak evidence base. The aim of the study is to contribute to the scant research on the patient-reported outcomes in vitiligo, and to examine the presence of associations between various inputs for possible use in clinical practice. The study was designed as a web-based questionnaire with 40 inputs across seven dimensions.

[Mutations in the Effector Domain of RhoV GTPase Impair Its Binding to Pak1 Protein Kinase].

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors.

[Insertion of Multiple Artificial Introns of Universal Design into cDNA during Minigene Construction Assures Correct Transgene Splicing].

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing.

A Novel Hybrid Promoter ARE-hTERT for Cancer Gene Therapy.

describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of the human gene promoter (hTERT) and the antioxidant response element (ARE) from the human gene promoter. The hybrid promoter retains the tumor specificity of the basal promoter but is characterized by an enhanced transcriptional activity in cancer cells with abnormal activation of the Nrf2 transcription factor and upon induction of oxidative stress. In the enzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-driven expression of CD : UPRT (yeast cytosine deaminase : uracil phosphoribosyltransferase) chimeric protein induced a more pronounced death of cancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FC was combined with chemotherapeutic drugs as compared to the hTERT promoter.

[Optimal Artificial Mini-Introns for Transgenic Expression in the Cells of Mice and Hamsters].

Introns can frequently enhance transgene expression, and sometimes they are absolutely substantial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to be efficiently spliced in CHO cells from hamster ovaries.

Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle analysis.

The strength and specificity of protein complex formation is crucial for most life processes and is determined by interactions between residues in the binding partners. Double-mutant cycle analysis provides a strategy for studying the energetic coupling between amino acids at the interfaces of such complexes. Here we show that these pairwise interaction energies can be determined from a single high-resolution native mass spectrum by measuring the intensities of the complexes formed by the two wild-type proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant proteins.

A gel-less isolation of untagged plasmid DNA insert from vector backbone in homogeneous format.

Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.

GMOs in Russia: Research, Society and Legislation.

Russian legislation lags behind the rapid developments witnessed in genetic engineering. Only a scientifically based and well-substantiated policy on the place of organisms that are created with the use of genetic engineering technologies and an assessment of the risks associated with them could guarantee that the breakthroughs achieved in modern genetic engineering technologies are effectively put to use in the real economy. A lack of demand for such breakthroughs in the practical field will lead to stagnation in scientific research and to a loss of expertise.

Transient Kinetic Analysis of ATP Hydrolysis by the CCT/TRiC Chaperonin.

The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis.

Genome-wide association studies of autoimmune vitiligo identify 23 new risk loci and highlight key pathways and regulatory variants.

Vitiligo is an autoimmune disease in which depigmented skin results from the destruction of melanocytes, with epidemiological association with other autoimmune diseases. In previous linkage and genome-wide association studies (GWAS1 and GWAS2), we identified 27 vitiligo susceptibility loci in patients of European ancestry. We carried out a third GWAS (GWAS3) in European-ancestry subjects, with augmented GWAS1 and GWAS2 controls, genome-wide imputation, and meta-analysis of all three GWAS, followed by an independent replication.

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine: Brussels, Belgium. 15-18 March 2016.

[This corrects the article DOI: 10.1186/s13054-016-1208-6.].

and gene promoters for cancer gene therapy: A comparative study.

Human telomerase reverse transcriptase () and survivin () gene promoters are frequently used for transcriptional targeting of tumor cells, yet there is no comprehensive comparative analysis allowing rational choice of a promoter for a particular therapy. In the current study, the transcriptional activity of , human and mouse Birc5 promoters and their modifications were compared in 10 human cancer cell lines using the luciferase reporter gene activity assay. The results revealed that - and -based promoters had strikingly different cell specificities with comparable activities in only 40% of cell lines.

Combined therapy for resistant vitiligo lesions: NB-UVB, microneedling, and topical latanoprost, showed no enhanced efficacy compared to topical latanoprost and NB-UVB.

Vitiligo is depigmenting disorder of the skin and mucous membranes but despite various therapeutic options, complete and satisfactory treatment of vitiligo still remains a challenge. Therapeutic success also varies depending on the localization of lesions; hands and bony prominents are considered to be resistant to treatment. We investigated feasibility of treating resistant bilateral symmetrical vitiligo vulgaris and acrofacialis lesions with combination of narrowband UVB and topical prostaglandins (0.