Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412–1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89–905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124–160 and 341–368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1's amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function

Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

<p>Abstract</p> <p>Background</p> <p>The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep.</p> <p>Methods</p> <p>Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries).</p> <p>Results</p> <p>Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries.</p> <p>Conclusion</p> <p>These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.</p

Sonographic features differentiating follicular thyroid cancer from follicular adenoma–a meta-analysis

Certain ultrasound features are associated with an increased risk of thyroid malignancy. However, they were studied mainly in papillary thyroid cancers (PTCs); these results cannot be simply extrapolated for the differentiation of follicular thyroid adenomas and cancers (FTAs and FTCs). The aim of our study was to perform a meta-analysis to identify sonographic features sug-gesting malignancy in the case of follicular lesions, potentially differentiating FTA and FTC. We searched thirteen databases from January 2006 to December 2020 to find all relevant, full-text jour-nal articles written in English. Analyses assessed the accuracy of malignancy detection in case of follicular lesions, potentially differentiating FTA and FTC included the odds ratio (OR), sensitivity, specificity, positive and negative predictive values. A random-effects model was used to summarize collected data. Twenty studies describing sonographic features of 10,215 nodules met the inclusion criteria. The highest overall OR

A designer hyper interleukin 11 (H11) is a biologically active cytokine

<p>Abstract</p> <p>Background</p> <p>Interleukin 11 (IL-11) is a pleiotropic cytokine with anti-apoptotic, anti-inflammatory and hematopoietic potential. The IL-11 activity is determined by the expression of the IL-11R receptor alpha (IL-11Rα) and the signal transducing subunit β (gp130) on the cell membrane. A recombinant soluble form of the IL-11Rα (sIL-11Rα) in combination with IL-11 acts as an agonist on cells expressing the gp130 molecule. We constructed a designer cytokine Hyper IL-11 (H11), which is exclusively composed of naturally existing components. It contains the full length sIL-11Rα connected with the mature IL-11 protein using their natural sequences only. Such a construct has two major advantages: (i) its components are as close as possible to the natural forms of both proteins and (ii) it lacks an artificial linker what should avoid induction of antibody production.</p> <p>Results</p> <p>The H11 construct was generated, the protein was produced in a baculovirus expression system and was then purified by using ion exchange chromatography. The H11 protein displayed activity in three independent bioassays, (i) it induced acute phase proteins production in HepG2 cells expressing IL-11, IL-11Rα and gp130, (ii) it stimulated the proliferation of B9 cells (cells expressing IL-11Rα and gp130) and (iii) proliferation of Baf/3-gp130 cells (cells not expressing IL-11 and IL-11Rα but gp130). Moreover, the preliminary data indicated that H11 was functionally distinct from Hyper-IL-6, a molecule which utilizes the same homodimer of signal transducing receptor (gp130).</p> <p>Conclusions</p> <p>The biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.</p

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al

Abundance and diversity of fungi in oak wood

Deadwood is an important functional and structural component of forest ecosystems since it regulates nutrient cycling, serves as a carbon (C) pool, increases the C content in the soil, and is a source of resources, including water, for a wide range of saproxylic and non-saproxylic organisms. The abundance and diversity of wood-inhabiting fungi in six fallen, horizontally lying logs of Quercus petraea, in the 1st, 2nd and 3rd decay classes, in the Drawa National Park in Poland in 2015, were studied using the Illumina se- quencing technique. The total number of OTUs obtained (264 307) included sequences of culturable fungi (242 369 = 91.70%) and non-culturable fungi (7 056 = 2.66%). The dead oak wood was colonized by 277 taxa. Culturable fungi of Zygomycota, Ascomycota and Basidiomycota comprised 75% of taxa detected and were represented by 14, 139 and 53 taxa respectively. Non-culturable organisms were represented by 70 taxa. Fungi were classified into 18 groups: (i) typical soft rot fungi, (ii) phytopathogens potentially hazard- ous to trees, (iii) epiphytes and endophytes or weak, opportunistic pathogens, (iv) ectomycorrhizal species, (v) ericoid mycorrhizal species, (vi) crust fungi, (vii) resupinate basidiomycetes, (viii) saprotrophs and soil fungi, (ix) opportunistic human pathogens causing superficial or systemic mycoses, (x) pathogens of warm- or cold-blooded animals, (xi) ascomycetous yeasts, (xii) basidiomycetous yeasts, (xiii) antagonists of fungi, (xiv) producers of metabolites effective against pathogens, (xv) hyperparasites, (xvi) lichen-form- ing species, (xvii) partners of other fungi, (xviii) species rare or uncommon in nature. It was shown that: (i) coarse, woody debris of oak is host to abundant and diverse mycobiota, (ii) abundance and diversity of fungi increase with the decay continuum, (iii) oak logs can be a habitat of phytopathogens potentially haz- ardous to forest health, (iv) different phytopathogens favour wood in different decay classes, (v) abundance of pathogens decreases, and of ericoid mycorrhizal (or soil) species increases with the decay continuum. Maintaining different types of deadwood increases fungal diversity, but also the risk of diseases

Investigation of Ophiostoma population infected elms in Poland

Dutch elm disease (DED) still occurs in Poland. Previous studies confirmed occurring O. ulmi and two subspecies of O. novo-ulmi: subsp. novo-ulmi and subsp. amerciana. In this study the population of Ophiostoma occurred in Poland was investigated. The disease incidence was investigated on elms growing in 39 locations. The pathogen’s mycelium was isolated from elm branches and twigs collected from 22 plots. The disease symptoms were noted in 5% to 35% trees. Fungi were identified based on the PCR amplification of the ITS 1/2 rDNA together with phylogenetic analysis of this region. Ophiostoma novo-ulmi was the only agent caused DED on Ulmus glabra, U. minor and U. laevis. There were no genetic diversity of O. novo-ulmi Polish population in analyzed ITS region. All kind of specific symptoms and disease intensity occurred independently on elm species and host age. Ulmus minor was infested most sever among the three elms species

Developing an effective and comprehensive communication curriculum for undergraduate medical education in Poland – the review and recommendations

Abstract Background The recognition of the importance of effective communication in the healthcare system has been growing. Given that communication courses must be adjusted to the specificity of a particular culture, language, and other contextual issues, many countries and communities sharing a common language have proposed their recommendations for a communication curriculum for undergraduate medical education. To date, no recommendations have been developed for either any Central and Eastern Europe countries or for regions where Slavic languages are spoken. Their specificity of post-communist transformation should be acknowledged. This study aims to review communication curriculums and offer recommendations for medical communication training for undergraduate medical students in Poland. Methods The recommendations were developed through an iterative consultation process with lecturers, faculty members of medical schools, and education coordinators. PubMed and Google Scholar databases were searched to identify full text English and Polish language articles on communication curriculum for undergraduate medical education. Additionally, the new Regulation of the Polish Minister of Science and Higher Education, defining educational standards for undergraduate medical education was analysed in search of learning outcomes that could be applied in communication skills teaching. The authors extracted the most relevant communication skill competencies, as determined by the process participants, discussed current challenges, including those of the COVID-19 pandemic era, and indicated best practices. Results A review was conducted, and a set of recommendations was developed pertaining to the scope and methodology of teaching communication skills. The study included: (1) definition, (2) education content, (3) learning outcomes, (4) the recommended teaching methods. The recommendations are in concord with the graduate profile, as well as the current structure of medical studies. The authors listed and discussed the basic communication competencies expected of medical graduates, as well as medical communication course content viewed from different perspectives, including clinical, psychological, sociological, legal, and linguistic. Conclusions Detailed recommendations aimed at integrating best practices into a comprehensive communication curriculum may promote successful teaching, learning, and assessment of medical communication