Distinct organization of adaptive immunity in the long-lived rodent Spalax galili.

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

SypHer3s: a genetically encoded fluorescent ratiometric probe with enhanced brightness and an improved dynamic range.

We designed a genetically encoded ratiometric fluorescent probe, SypHer3s, with enhanced brightness and optimized pKa, which responds to pH changes in different cellular compartments. SypHer3s was successfully utilized for imaging the pH dynamics in mitochondria of living neurons and in quantitative pH measurement in zebrafish embryos.

High-quality full-length immunoglobulin profiling with unique molecular barcoding.

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells.

Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines.

Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mechanism of specific degradation of transcripts with a premature stop codon. NMD eliminates aberrant mRNAs arising from mutations, alternative splicing, and other events in cells. In addition, many normal transcripts undergo NMD. Recent studies demonstrated that NMD activity is specifically regulated and that NMD can play a role of global regulator of gene expression. Recently, we developed dual-color fluorescent protein-based reporters for quantification of NMD activity using fluorescence microscopy and flow cytometry (Pereverzev, Gurskaya, et al., 2015). Due to ratiometric fluorescence response, these reporters make it possible to assess NMD activity in live cells at the single-cell level and to reveal otherwise hidden heterogeneity of cells in respect of NMD activity. Here we provide a detailed description of applications of the NMD reporters in mammalian cell lines.

[Differences of Nonsense-Mediated mRNA Degradation Activity in Mammalian Cell Lines Revealed by a Fluorescence Reporter].

Activity of nonsense-mediated mRNA degradation (NMD) was studied in several mammalian cell cultures using recently developed genetically encoded fluorescence sensor [Pereverzev et al., Sci. Rep., 2015, vol. 5, p. 7729]. This NMD reporter enables measurement of NMD activity in single live cells using ratio of green and red fluorescent proteins signals. The following cell lines were analyzed: mouse colon carcinoma CT26, mouse Lewis lung carcinoma LLC, human T-cell leukemia Jurkat, and spontaneously immortalized human keratinocytes HaCaT. These cell lines demonstrated very different NMD activities. In CT26, NMD activity was low, whereas in LLC it was high (8.5-fold higher than in CT26). Jurkat and HaCaT cells possessed strong heterogeneity and consisted of two cell subpopulations with high and low NMD activities. In addition, we detected high NMD activity in primary culture of mouse embryonic hippocampal neurons.

A monomeric red fluorescent protein with low cytotoxicity.

Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.

[Coding region of far-red fluorescent protein katushka contains a strong donor splice site].

Computer analysis predicted a strong donor splice site within the 3'-part of the far-red fluorescent protein Katushka coding region. To test the functional activity of this site a model vector has been constructed. This vector encoded Katushka and green fluorescent protein TagGFP2 with a gene fragment of tafazzin in between. Normal splicing of this pre-mRNA should result in a frameshift between Katushka and TagGFP2. Alternatively, after splicing at internal katushka donor splice site appearance of Katushka-TagGFP2 fusion protein was expected. Expression of this construct in a mammalian cell culture led to bright red and green fluorescence. Therefore, katushka-specific donor splice site is functional. Disruption of this splice site by several silent substitutions resulted in red-only fluorescent cells that corresponded to normal splicing. The mutant katushka can be used for visualization of pre-mRNA splicing at single cell level by fluorescence microscopy and flow cytometry.

[The oligoclonal expansion of T cells: study of its stability over time].

A novel experimental approach to the investigation of the repertoire of peripheral T lymphocytes of patients suffering from ankylosing spondylitis (AS) is proposed. This approach is based on the wide-range sequencing of cDNA of the beta-chain of the T-cellular receptor (TcR). The results of the analysis of the diversity of sequences of the TcR antigen-binding domain (CDR3) inside the total pool of one patient with AS are presented by the example of the second V family (BV2) of TcR. The expansion of six independent TcR-expressing clones of T cells with a similar amino acid sequence of the CDR3 domains was proposed based on the results of the comparative structural analysis of the clone libraries of the cDNA of TcR BV2. The long-time stable expansion of these T clones was demonstrated during the development of the disease by specific monitoring.

Association of ERAP1 Allelic Variants with Risk of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) belongs to a group of autoimmune diseases affecting the axial skeleton. Beside thehla-b*27allele, several other human genes that control the variety processes of immune homeostasis are considered to be associated with AS manifestation in different human populations. Among strong associated non-MHC geneserap1 encodingthe endoplasmic reticulum aminopeptidase 1 isoform was recently identified by single nucleotide polymorphisms (SNPs) meta analysis. In our study we inspected the genetic association of five non-synonymous coding SNPs fromerap1 withAS in Caucasians. We implemented the SSP-PCR system for precise genotyping of 87hla-b*27positive AS patients and 77hla-b*27healthy donors from the Russian population. Considerable differences in allele's frequencies within patients vs control cohort were shown for 3 of 5 SNPs under investigation. Using the EM-algorhitm we reconstructed 3-marker haplotypes that distinguish with high probability two cohorts due to differences in the haplotypes frequencies. In such a way both the sensitive, CCT, haplotype and the protective, TTC, one were predicted. To verify the calculation we determined genuine frequencies of 5-marker haplotypes in AS cohort by haplotyping of individual cDNA samples using improved SSP-PCR primer set. We demonstrated that the frequencies ofin silicareconstucted haplotypes and the frequencies of experimentally detected haplotypes are in a good agreement. Frequency of the risk haplotype CCT (rs17482078/10050860/2287987) detected within AS cohort reaches 88%, as well as the frequency calculated by EM-algorhitm.

Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

Renaturation, activation, and practical use of recombinant duplex-specific nuclease from Kamchatka crab.

We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.

[A natural fluorescent protein that changes its fluorescence during maturation]. / Prirodnyi fluorestsentnyi belok, izmeniaiushchii tsvet fluorestsentsii v khode sozrevaniia.

The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.

[The localization of alpha-fetoprotein and the gap-junction protein Cx32 in the regenerating mouse liver]. / Lokalizatsiia al'fa-fetoproteina i belka shchelevykh kontaktov Cx32 v regeneriruiushchei pecheni myshei.

The data on distribution of alpha-fetoprotein (AFP) and gap junction protein Cx32 in mouse liver regenerating after carbon tetrachloride poisoning are presented. On the second day, when a significant number of AFP-positive cells appear in the liver, Cx32-specific staining was lost from major part of the "intact" parenchyma. On the third day numerous AFP-positive cells could be localized only in the narrow perinecrotic area. By that time, Cx32-specific staining appeared again in major part of liver parenchyma except perinecrotic areas where AFP-containing cells were located. The number of Cx32-negative cells always exceeded that of AFP-positive cells. The latter, however, were always located in Cx32-negative areas. Thus, correlation between appearance of AFP in adult hepatocytes in vivo and disturbance of gap junctions was observed which was described earlier in cultured adult hepatocytes.

[Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro]. / Tormozhenie sinteza al'fa-fetoproteina v gepatotsitakh vzroslykh myshei in vivo i in vitro sul'fatom dekstrana.

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.