Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Abstract Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered

Purchase Motives and Factors Shaping Consumer Behaviour on the Ecological Product Market (Poland Case Study)

The growing demand for ecological products is in line with the trend towards the ecologisation of consumption, which has become key in times of striving to achieve sustainable development that aims to satisfy consumer needs while respecting the natural environment and future generations. The shaping of pro-ecological attitudes and behaviours in consumers requires continuous monitoring of such behaviours on the market of ecological products and investigation of the factors that influence consumers’ decisions and market choices. The aim of the article is to present the motives behind the purchase of ecological products, and the factors that shape the purchasing decisions of these products by Polish consumers. The article is based on an in-depth study of the literature and the results of proprietary empirical quantitative research conducted on a national sample of 1032 respondents, of whom 509 had purchased an ecological product within the last 3 months, and 523 had not made such a purchase in this period. Analysis of the results revealed the motives for purchasing ecological products, divided into egotistical motives and altruistic motives. The variation in these motives was also indicated depending on the socio-demographic characteristics of the consumers studied. It was shown that there is a dependency between consumers’ self-assessment of their level of knowledge on the functioning of the natural environment and the effect of humankind on it, and the purchase of ecological products. Analysis was also conducted of the factors perceived by consumers as restricting the purchase of ecological products, as well as the likelihood of a growth in the demand for and consumption of such products. There was shown to be a dependency between the reasons perceived by respondents for restricting the purchase of ecological products or the decision not to make such purchases, and consumer attitudes towards ecology

Purchase Motives and Factors Shaping Consumer Behaviour on the Ecological Product Market (Poland Case Study)

The growing demand for ecological products is in line with the trend towards the ecologisation of consumption, which has become key in times of striving to achieve sustainable development that aims to satisfy consumer needs while respecting the natural environment and future generations. The shaping of pro-ecological attitudes and behaviours in consumers requires continuous monitoring of such behaviours on the market of ecological products and investigation of the factors that influence consumers’ decisions and market choices. The aim of the article is to present the motives behind the purchase of ecological products, and the factors that shape the purchasing decisions of these products by Polish consumers. The article is based on an in-depth study of the literature and the results of proprietary empirical quantitative research conducted on a national sample of 1032 respondents, of whom 509 had purchased an ecological product within the last 3 months, and 523 had not made such a purchase in this period. Analysis of the results revealed the motives for purchasing ecological products, divided into egotistical motives and altruistic motives. The variation in these motives was also indicated depending on the socio-demographic characteristics of the consumers studied. It was shown that there is a dependency between consumers’ self-assessment of their level of knowledge on the functioning of the natural environment and the effect of humankind on it, and the purchase of ecological products. Analysis was also conducted of the factors perceived by consumers as restricting the purchase of ecological products, as well as the likelihood of a growth in the demand for and consumption of such products. There was shown to be a dependency between the reasons perceived by respondents for restricting the purchase of ecological products or the decision not to make such purchases, and consumer attitudes towards ecology

Morphological changes of PC12 cells cultured on: PS (green frame), PMMA (red frame), PMMA-PDMS (blue frame) after 4 days of NGF treatment.

<p>Different types of neurites can be found on all polymeric materials used in the presented study: multiple neurites from the cell body with multiple branches, and multiple neurites from the cell body with a minor branch.</p

Zmiany na rynku polskich produktów ekologicznych pochodzenia rolniczego

This paper attempts to identify changes in the factors influencing the functioning and evolution of the Polish market for organic agricultural products. It brings together the results of surveys of farmers (carried out in 2011, 2019, and 2021), distributors (carried out in 2019 and 2021), and consumers (carried out in 2009 and 2021). Initially, farmers believed that the greatest opportunities for market development lay in demand factors, including in particular consumer environmental awareness. In 2021, their opinions worsened in this regard, which means they had difficulties reaching consumers. Another opportunity that was less popular than before was the EU subsidies. This is due to administrative and bureaucratic burdens, which, along with high production costs and weak links between farmers and distributors, were considered to be the biggest barriers to market development. For distributors, the survey produced similar conclusions. According to consumers, the greatest opportunities for market development result from increasing environmental awareness, increased diversity of products and better promotion. The barriers they highlighted include high prices, limited environmental education, lack of adequate state support, and insufficient information about the offer.Celem artykułu jest określenie zmian czynników wpływających na funkcjonowanie polskiego rynku ekologicznych produktów pochodzenia rolniczego oraz czynników wpływających na przyszły rozwój tego rynku. Zawiera on syntezę wyników badań ankietowych przeprowadzonych wśród rolników (z lat 2011, 2019, 2021), dystrybutorów (z lat 2019 i 2021) i konsumentów (z lat 2009 i 2021). Wyniki badań z 2011 i 2019 wykazały, że według rolników największe szanse rozwoju rynku wiążą się z czynnikami popytowymi, w tym zwłaszcza świadomością ekologiczną konsumentów. W 2021 r. opinie te pogorszyły się, co wskazuje, że rolnicy napotykają trudności w dotarciu ze swoją ofertą do konsumentów. Dotacje unijne również straciły na znaczeniu, co wiąże się z utrudnieniami administracyjnymi i biurokratycznymi. Obok wysokich kosztów produkcji i słabości powiązań rolników z dystrybutorami stanowią one największe bariery rozwoju rynku. Podobne wnioski wynikają z wyników badań dystrybutorów. Według konsumentów największymi szansami rozwoju rynku są rosnąca świadomość ekologiczna, zwiększenie różnorodności oferty i lepsza promocja. Ograniczeniami są wysoka cena, znikoma edukacja ekologiczna, brak odpowiedniego wsparcia ze strony państwa oraz zbyt mało informacji o ofercie

The <i>top</i>10 differentially expressed <i>genes</i> between NGF-differentiated and non-differentiated PC12 cells similarly expressed in all culture conditions: PS, PMMA, PMMA-PDMS.

<p>Notice: the magnitude of the fold-change is similar at all applied culture conditions.</p

Three different experimental PC12 cells culture conditions for DNA microarray study.

<p>(A) laminin-coated PS cell culture dish. (B) laminin-coated 2-mm thick PMMA plate placed inside the cell culture dish. (C) laminin-coated 2-mm thick PMMA plate with through holes placed on the 2-mm thick PDMS layer. (D) A schematic view of a 2 mm thick PMMA plate with through holes placed on the PDMS substrate.</p

Functional classification clustering analysis presenting gene ontology processes associated with the 642 differentially expressed genes in NGF-differentiated PC12 cells cultured on PMMA-PDMS versus PS (Enrichment Score>0.5, Final group member threshold – minimum 10 genes).

<p>a. Terms based on GOTERM_BP_FAT category, DAVID Bioinformatics Resources 6.7.</p><p>b. The overall enrichment score for the group based on the EASE scores of each term members.</p><p>c. P-value (or called EASE score) to determine the significance of gene-term enrichment with a modified Fisher’s exact test (EASE score).</p

A. The overlay histogram for PC12 cells treated with NGF for 24 hours (blank graph) and for 96 hours (solid, grey graph).

<p>The Cyflogic program provided the estimate of percentage of cells in each of the intervals: sub-G1, G1, S, and G2/M. B. PC12 cells were collected after 24, 48, 72, 96 hours from cell seeding (left column, non-differentiated cells), and afterwards 24, 48, 72, 96 hours from NGF treatment (right column, differentiated cells). Samples were analysed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053107#s2" target="_blank"><i>Materials and Methods</i></a>. Error bars show mean ± SD from three independent experiments.</p