FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis

Accurate and reproducible quantification of the accumulation of proteins into foci in cells is essential for data interpretation and for biological inferences. To improve reproducibility, much emphasis has been placed on the preparation of samples, but less attention has been given to reporting and standardizing the quantification of foci. The current standard to quantitate foci in open-source software is to manually determine a range of parameters based on the outcome of one or a few representative images and then apply the parameter combination to the analysis of a larger dataset. Here, we demonstrate the power and utility of using machine learning to train a new algorithm (FindFoci) to determine optimal parameters. FindFoci closely matches human assignments and allows rapid automated exploration of parameter space. Thus, individuals can train the algorithm to mirror their own assignments and then automate focus counting using the same parameters across a large number of images. Using the training algorithm to match human assignments of foci, we demonstrate that applying an optimal parameter combination from a single image is not broadly applicable to analysis of other images scored by the same experimenter or by other experimenters. Our analysis thus reveals wide variation in human assignment of foci and their quantification. To overcome this, we developed training on multiple images, which reduces the inconsistency of using a single or a few images to set parameters for focus detection. FindFoci is provided as an open-source plugin for ImageJ

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

Characterization of Multi-Functional Properties and Conformational Analysis of MutS2 from Thermotoga maritima MSB8

The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results

Deletion of Genes Implicated in Protecting the Integrity of Male Germ Cells Has Differential Effects on the Incidence of DNA Breaks and Germ Cell Loss

Infertility affects approximately 20% of couples in Europe and in 50% of cases the problem lies with the male partner. The impact of damaged DNA originating in the male germ line on infertility is poorly understood but may increase miscarriage. Mouse models allow us to investigate how deficiencies in DNA repair/damage response pathways impact on formation and function of male germ cells. We have investigated mice with deletions of ERCC1 (excision repair cross-complementing gene 1), MSH2 (MutS homolog 2, involved in mismatch repair pathway), and p53 (tumour suppressor gene implicated in elimination of germ cells with DNA damage).We demonstrate for the first time that depletion of ERCC1 or p53 from germ cells results in an increased incidence of unrepaired DNA breaks in pachytene spermatocytes and increased numbers of caspase-3 positive (apoptotic) germ cells. Sertoli cell-only tubules were detected in testes from mice lacking expression of ERCC1 or MSH2 but not p53. The number of sperm recovered from epididymes was significantly reduced in mice lacking testicular ERCC1 and 40% of sperm contained DNA breaks whereas the numbers of sperm were not different to controls in adult Msh2 -/- or p53 -/- mice nor did they have significantly compromised DNA.These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production. These findings increase our understanding of the ways in which gene mutations can have an impact on male fertility

Mammalian BTBD12 (SLX4) Protects against Genomic Instability during Mammalian Spermatogenesis

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM

Cartelization of the Russian Economy: The Main Causes, Consequences, and Ways of Their Elimination

Abstract—: The preconditions and recent history of the modern anticartel policy in Russia are considered. It is noted that in countries with traditional market economies, a negative attitude to cartels and tools to counter them were gradually formed during the globalization of national economies and changes in attitudes to national and transnational monopolies. Against the background of the semispontaneous destruction of the planned economy of the Soviet Union and the Russian Soviet Federative Socialist Republic in the 1990s, not only voluntary but also compulsory cartel agreements became widespread, the initiators and main beneficiaries of which were local and regional organized criminal groups, which predetermined the deep criminalization of contractual relations for a decade. It is argued that the greatest threat to the successful development of Russia as a welfare state comes from the so-called auction cartels, which are, in fact, a “collusion within a collusion”; up to 90% of cartels are concluded in tenders. Cartel superprofits serve as an incentive to bribe the state apparatus. The main trend of anticartel lawmaking and law enforcement in our country in recent years has been the identification and suppression of the largest cartels, 1/4 of which have signs of criminal acts. It is proposed to solve the problems of decartelization of the Russian economy on the basis of a comprehensive roadmap of anticartel measures, which, in the opinion of the authors of this article, should be included in the new National Plan for the Development of Competition in Russia for 2021–2025, which is currently being developed

QUESTIONS OF INFORMATIONAL SECURITY OF KHERSON STATE UNIVERSITY.

The article deals with problems of informational security of modern organizations and analyzes relevant questions of computer security at Kherson State University

Adenovirus early region 3 transgenes expressed in beta cells prevent autoimmune diabetes in nonobese diabetic mice: effects of deleting the adenovirus death protein 11.6K.

The incidence of type 1 diabetes (T1D) is decreased in nonobese diabetic mice expressing the complete cassette of adenovirus early region 3 (E3) immunomodulating genes in pancreatic beta cells. Embedded among the antiapoptotic E3 genes is one encoding an adenovirus death protein (ADP), which contributes to release of virion particles by promoting cell lysis. Because removal of this proapoptotic protein might have further enhanced the ability of E3 proteins to prevent T1D, an ADP-inactivated E3 construct was tested. Significantly, deletion of ADP did not improve the diabetes-protective effect of an E3 gene cassette

Adenovirus Early Region 3 Transgenes Expressed in β Cells Prevent Autoimmune Diabetes in Nonobese Diabetic Mice: Effects of Deleting the Adenovirus Death Protein 11.6K

The incidence of type 1 diabetes (T1D) is decreased in nonobese diabetic mice expressing the complete cassette of adenovirus early region 3 (E3) immunomodulating genes in pancreatic β cells. Embedded among the antiapoptotic E3 genes is one encoding an adenovirus death protein (ADP), which contributes to release of virion particles by promoting cell lysis. Because removal of this proapoptotic protein might have further enhanced the ability of E3 proteins to prevent T1D, an ADP-inactivated E3 construct was tested. Significantly, deletion of ADP did not improve the diabetes-protective effect of an E3 gene cassette