Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome

Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.Peer reviewe

Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes

Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Cys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.Peer reviewe

Analytical “Bake-Off” of Whole Genome Sequencing Quality for the Genome Russia Project Using a Small Cohort for Autoimmune Hepatitis

A comparative analysis of whole genome sequencing (WGS) and genotype calling was initiated for ten human genome samples sequenced by St. Petersburg State University Peterhof Sequencing Center and by three commercial sequencing centers outside of Russia. The sequence quality, efficiency of DNA variant and genotype calling were compared with each other and with DNA microarrays for each of ten study subjects. We assessed calling of SNPs, indels, copy number variation, and the speed of WGS throughput promised. Twenty separate QC analyses showed high similarities among the sequence quality and called genotypes. The ten genomes tested by the centers included eight American patients afflicted with autoimmune hepatitis (AIH), plus one case’s unaffected parents, in a prelude to discovering genetic influences in this rare disease of unknown etiology. The detailed internal replication and parallel analyses allowed the observation of two of eight AIH cases carrying a rare allele genotype for a previously described AIH-associated gene (FTCD), plus multiple occurrences of known HLA-DRB1 alleles associated with AIH (HLA-DRB1-03:01:01, 13:01:01 and 7:01:01). We also list putative SNVs in other genes as suggestive in AIH influence

Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage

Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are often debated. WES dominated large-scale resequencing projects because of lower cost and easier data storage and processing. Rapid development of 3(rd) generation sequencing methods and novel exome sequencing kits predicate the need for a robust statistical framework allowing informative and easy performance comparison of the emerging methods. In our study we developed a set of statistical tools to systematically assess coverage of coding regions provided by several modern WES platforms, as well as PCR-free WGS. We identified a substantial problem in most previously published comparisons which did not account for mappability limitations of short reads. Using regression analysis and simple machine learning, as well as several novel metrics of coverage evenness, we analyzed the contribution from the major determinants of CDS coverage. Contrary to a common view, most of the observed bias in modern WES stems from mappability limitations of short reads and exome probe design rather than sequence composition. We also identified the similar to 500kb region of human exome that could not be effectively characterized using short read technology and should receive special attention during variant analysis. Using our novel metrics of sequencing coverage, we identified main determinants of WES and WGS performance. Overall, our study points out avenues for improvement of enrichment-based methods and development of novel approaches that would maximize variant discovery at optimal cost

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Healthcare financing in Russia: mechanism and assessment of regional budget opportunities

In the difficult economic conditions in Russia, caused, as in the all countries, by the consequences of the coronavirus pandemic, the search for additional government revenues is becoming especially relevant. All possibilities of cutting budget expenditures are being considered, including the optimization of the number of civil servants. The deputies of the State Duma of the Russia over the past month have been actively discussing the effectiveness of the functioning of state extra-budgetary funds and expressing ideas about the abolition of the Pension Fund of Russia and others, with the simultaneous return of functions back to the federal budget. It is important to reasonably assess the effectiveness of the financial mechanism of each extra-budgetary fund in Russia, including the Federal and Territorial Compulsory Medical Insurance Funds. To this end, it is necessary to comprehensively consider the mechanism of financing health care in the country within the framework of the current budget-insurance model. This article reveals the features of the financial mechanism in the healthcare sector in Russia, primarily in terms of methods for the formation of financial resources. In general, the Russian health care system is financed by the country’s budgets, Compulsory Medical Insurance Funds, voluntary medical insurance and paid medical services. In accordance with this, the budget, insurance and market methods for the formation of financial resources in health care are identified. The article describes each method; the effectiveness of the current financial mechanism was assessed and recommendations for its improvement were proposed

Implementation of Competence Approach with Simulation Technology

The modernization of higher education is mandatory condition for the current stage of Russian society development and the ensuring of the Russian economy competitiveness. One of the priority issues of modernization is the strengthening of the significance of self-students studying. This is due primarily to the considerable reduction in the number of classroom sessions and the increase in hours provided for self-students studying. Self-studying becomes the basis for the professional development of a bachelor, encourage his cognitive interests, promotes the essential objectives of the training, that is, the formation of communicative and professional competences. The nature of the relationship between the teacher and the bachelor alters within the framework of competence-oriented education. The teacher, released from the mere transfer of knowledge, gets freedom in choosing forms of interaction or rather in choosing the methods used in such field of studying as “Foreign Language”. Classical teaching methods are replaced with new ones. The emergence of information and communication technologies led to their rapid introduction into the educational process. The organization of self-students studying through the use of information and communication technologies is of great interest to many educational specialists. The specific nature of information and communication technologies lies in the creation in the educational process of various kinds of relations and conditions of real life, requiring from bachelors not only the knowledge of work-related material, but also their familiarization with a given image, a holistic immersion in the professional environment. The use of simulation technology facilitates the formation of cognitive-search activity of bachelors, causes incentive for learning a foreign language

Distinct effects of guanidine thiocyanate on the structure of superfolder GFP.

Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a β-barrel topology. Here, we studied the unfolding-refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0-0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations of GTC (from 0.7 to 1.7 M), induce a disruption of the sfGFP structure, that is manifested in simultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in which inner alpha-helix and hence green chromophore and Trp57 are still not tuned up to (properly integrated into) the already formed β-barrel scaffold of protein. Incorporation of the chromophore in the β-barrel in the pathway of refolding and restoration of its ability to fluoresce occur in a narrow range of GTC concentrations from 1.0 to 0.7 M, and a correct insertion of Trp 57 occurs at concentrations ranging from 0.7 to 0 M GTC. These two processes determine the hysteresis of protein unfolding and refolding

Flavonoid Accumulation in an Aseptic Culture of Summer Savory (<i>Satureja hortensis</i> L.)

Summer savory (Satureja hortensis L.) is a medicinal and aromatic plant of the Lamiaceae family, a source of valuable secondary metabolites (monoterpenoids, rosmarinic acid, flavonoids). For this paper, flavonoid accumulation in an aseptic culture of summer savory was determined by using a colorimetric method. The organ specificity of flavonoid accumulation in aseptic plants was revealed: In leaves (8.35 ± 0.17 mg/g FW), flower buds (7.55 ± 0.29 mg/g FW), and calyx (5.27 ± 0.28 mg/g FW), flavonoids accumulated in significantly higher amounts than in stems (1.50 ± 0.22 mg/g FW) and corolla (0.78 ± 0.12 mg/g FW). We found that primary callus tissue formed from cotyledon and hypocotyl explants retains the ability to synthesize flavonoids at deficient levels (0.50 ± 0.09 mg/g FW and 0.44 ± 0.11 mg/g FW, respectively), that remained stable throughout six subcultures. Placing the callus tissue in monochrome lighting conditions with blue, green, and red light-emitting diode (LED) lamps leads to morphological changes in the tissue and decreased flavonoid accumulation compared to fluorescent lamps

Role of DNA Polymerases in Repeat-Mediated Genome Instability

Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAA)n repeats in a yeast experimental system. The elementary step of expansion corresponded to ∼160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ∊ strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism