Compressive stress-mediated p38 activation required for ER alpha plus phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK. Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.Peer reviewe

Compressive stress-mediated p38 activation required for ERα + phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.</p

Hapen rooli vehnäleseen raskituksessa elintarvikesovelluksia varten

Wheat bran, which is a good source of fibre, vitamins and minerals, can be used as a supplement in bread-making to ameliorate the nutritional quality. Cereal fermentation prior to bread-making improves stability, safety and nutritional and sensory quality of the product. Oxygen affects this fermentation, but its effects have not been extensively studied. The aim of this study was to investigate the possible effects of oxygen on microbiological and biochemical changes occurring during wheat bran fermentation. The influence of aerated and anaerobic conditions was studied as a function of time in four different types of fermentation, with Kazachstania exigua and Lactobacillus brevis as starter cultures with and without added enzymes. Wheat bran fermentations were carried out in bioreactors. Samples were taken at 0, 12 and 24 hours to analyse the growth of microbes, production of acids and ethanol, sugar consumption, and metabolite profile. In addition samples were subjected to microbial community analysis. Yeasts and moulds grew better when aeration was used, whereas the growth of endogenous heterotrophic bacteria and lactic acid bacteria (LAB) was slightly enhanced under anaerobic conditions. Microbial community analysis revealed the changes in microbial population due to the different oxygen conditions and types of fermentation. In addition to starter strains, the presence of enterobacteria and LAB (Weissella, Lactobacillus and Lactococcus) was shown. After 24 hours, anaerobically fermented bran was far more acidic than bran fermented under aerated conditions. Enhanced hydrolysis of arabinoxylan (AX) occurred when aeration was used, increasing the levels of soluble and free arabinose and xylose. As expected the concentration of glucose, fructose and galactose decreased during fermentation, as they were readily used as energy source. Metabolite profiling revealed the increment in the amount of ferulic acid under aerated conditions. The levels of extracellular amino acids were stable under anaerobic conditions. These findings indicate that the presence of oxygen affects the modification of wheat bran during fermentation due to altered microbial diversity and metabolism, and enzymatic activity of grain and microbial origin. Consequently aeration can be used for adjusting acidity and flavour, enhancing the solubilisation of AX ameliorating bread texture and increasing ferulic acid content.Vehnälesettä, joka on hyvä kuidun, vitamiinien ja mineraalien lähde, voidaan käyttää leivänvalmistuksessa parantamaan ravitsemuksellista laatua. Leivänvalmistuksessa käytetty viljan raskitus eli mikrobikäyminen parantaa tuotteen stabiiliutta, turvallisuutta sekä ravitsemuksellista ja aistinvaraista laatua. Happi vaikuttaa tähän käymiseen, mutta hapen aiheuttamia vaikutuksia ei juurikaan ole tutkittu. Tämän työn tarkoituksena oli tutkia hapen vaikutusta vehnäleseen mahdollisiin mikrobiologisiin ja biokemiallisiin muutoksiin raskituksen aikana. Hapen määrän vaikutusta tutkittiin ajan funktiona käyttämällä neljää erityyppistä raskitusta, joissa starttereina toimivat Kazachstania exigua ja Lactobacillus brevis. Leseraskitukset suoritettiin bioreaktorissa. Näytteet otettiin aikapisteissä 0, 12 ja 24 tuntia, ja niistä tutkittiin mikrobien kasvu, happojen ja etanolin tuotto sekä sokerien kulutus. Lisäksi analysoitiin mikrobipopulaatiota ja tehtiin aineenvaihduntatuotteiden profilointi. Hiivat ja homeet kasvoivat paremmin ilmastuksen avulla, kun taas endogeenisten heterotrofisten bakteerien ja maitohappobakteerien kasvu oli hieman suurempaa hapettomissa olosuhteissa. Erilaiset olosuhteet ja raskitustyypit aiheuttivat muutoksia mikrobipopulaatiossa. Näytteistä tunnistettiin startterikantojen lisäksi enterobakteereita ja maitohappobakteereita (Weissella, Lactobacillus ja Lactococcus). Voimakas hapontuotto aikaansaatiin hapettomissa olosuhteissa. Arabinoksylaanien hydrolyysi lisääntyi ilmastetuissa olosuhteissa, mikä johti sekä liukoisen että vapaan arabinoosin ja ksyloosin määrän kasvuun. Glukoosia, fruktoosia ja galaktoosia käytettiin energianlähteenä, ja niiden pitoisuudet laskivat odotetusti käymisen aikana. Aineenvaihduntatuotteiden profilointi osoitti ferulahapon määrän lisääntyvän ilmastetun raskituksen aikana. Solun ulkopuolisten aminohappojen pitoisuus sitä vastoin pysyi samana hapettomissa olosuhteissa. Tulosten mukaan hapen läsnäololla on merkitystä vehnäleseen muokkautumiseen raskituksen aikana, mikä johtuu oletettavasti muutoksista mikrobipopulaatiossa ja -metaboliassa sekä vilja- ja mikrobiperäisten entsyymien aktiivisuudessa. Näin ollen ilmastusta voidaan käyttää raskin happamuuden ja leivän maun säätämiseen, muun muassa ferulahapon pitoisuuden lisäämiseen, sekä arabinoksylaanien liukoisuuden lisäämiseen, ja sitä kautta leivän rakenteen parantamiseen

SAIRAANHOITAJIEN KOKEMUKSIA PITKÄAIKAISSAIRAAN LAPSEN SISARUSTEN HUOMIOIMISESTA

TIIVISTELMÄ Suomi, Katja. Sairaanhoitajien kokemuksia pitkäaikaissairaan lapsen sisarusten huomioimisesta. Oulu, syksy 2010, 48 s. Liitteitä 7. Diakonia-ammattikorkeakoulu, Diak Pohjoinen, Oulu. Hoitotyön koulutusohjelma. Sairaanhoitaja (AMK) Opinnäytetyön tarkoituksena oli kuvailla sairaanhoitajien kokemuksia pitkäaikaissairaan lapsen sisarusten huomioimisesta lastenhoitotyössä. Opinnäytetyön tavoitteena oli lisätä tietoa sisarusten huomioimisen merkityksestä lastenhoitotyössä. Opinnäytetyö oli laadullinen tutkimus, jossa tutkimusaineisto kerättiin lomakekyselyn avulla Oulun yliopistollisen sairaalan Lasten ja nuorten tulosyksikön osastojen 51, 62 sekä lastentautien poliklinikan sairaanhoitajilta huhtikuussa 2010. Aineisto analysoitiin aineistolähtöisen sisällönanalyysin avulla. Aineisto ja tulokset on kuvattu tekstinä ja liitteenä olevien esimerkkien avulla. Opinnäytetyön tulosten mukaan pitkäaikaissairaan lapsen sisarukset huomioidaan lastenhoitotyössä, kun hoitajat työskentelevät perhekeskeisellä työotteella. Perhekeskeisellä työotteella työskentelevä hoitaja on tietoinen perheeseen, yksilöön, vuorovaikutukseen ja hoitosuhteeseen liittyvistä ja niihin vaikuttavista asioista. Opinnäytetyön tuloksena luotiin malli pitkäaikaissairaiden lasten sisarusten huomioimisesta. Mallin mukaan työskennellessään hoitaja työskentelee perhekeskeisellä työotteella. Johtopäätöksenä voidaan sanoa, että pitkäaikaissairaan lapsen sisarusten tilanteen kartoittamiseksi, tulisi hoitotyöstä varata systemaattisesti aikaa kaikille pitkäaikaissairaan lapsen sisaruksille ja vanhemmille, sillä he tarvitsevat apua selviytyäkseen sairauden aiheuttamista muutoksista heidän elämässään. Hoitajille tulisi tarjota tietoa pitkäaikaissairauden vaikutuksista perheeseen ja sisaruksiin sekä antaa heille ”työkaluja” sisarusten huomioimiseen. Opinnäytetyön tuloksista on hyötyä pitkäaikaissairaiden lasten ja heidän perheiden kanssa työskenteleville. Tulokset ohjaavat lasten hoitotyön ammatti-käytäntöjen kehittämistä. Jatkotutkimushaasteena on tutkia millaisin hoitotyön keinoin sisarusten sopeutumista voidaan edistää ja millaisia vaikutuksia systemaattisesti toteutetulla perhekeskeisellä työotteella on sisarusten ja koko perheen terveydelle ja hyvinvoinnille. Asiasanat: krooniset taudit, lapset, sisarukset, perhe, selviytyminen, tukeminen, kvalitatiivinen tutkimusABSTRACT Suomi, Katja. Nurses’ experiences of considering the siblings of children with a chronic disease. Language: Finnish. Oulu, Autumn 2010. 48 pages, 7 appendices. Diaconia University of Applied Sciences, Oulu unit, Oulu. Degree Programme in nursing. Registered nurse. The purpose of the study was to describe nurses’ experiences of considering the siblings of children with a chronic disease in pediatric nursing. The aim of the study was to add to the knowledge of concidering the siblings of children with a chronic disease. This study was a qualitative research. The material was collected with questionnaires and it was analysed with methods of inductive content analysis. The material and the results are described as text and through examples which are attached. According to the results, the siblings of children with a chronic disease were take into consideration when nurses worked in a family-centered manner. A nurse who worked in a family-centered way was conscious of the things related to the family, an individual, interaction and the nursing relationship. As a conclusion it could be said that the situation of the siblings requires mapping. It means that we have to be systematic and reserve time for siblings and parents of the children with a chronic disease because they need help in coping with the changes in their lives caused by the chronic disease. Nurses should be provided information on the effects of children´s chronic disease as well as give them the “tools” to take into account the siblings. The results of this study are useful for nurses who are working with a children with chronical disease and with their families. The results advise to develop the nursing of children and youngsters and guidance of families. In future it is important to examine how we can promote coping skills of siblings and what kinds of implications of siblings’ and the entire family's health and welfare there are when siblings and the family are systematically considered. Keywords: chronic diseases, children, siblings, family, coping, support, qualitative stud

Expression and Evolution of the Non-Canonically Translated Yeast Mitochondrial Acetyl-CoA Carboxylase Hfa1p

<div><p>The <i>Saccharomyces cerevisiae</i> genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the <i>HFA1</i> gene and its evolutionary origin have remained unclear. Here, we determined the <i>HFA1</i> transcription initiation sites by 5′ RACE analysis. Using a novel “Stop codon scanning” approach, we mapped the location of the <i>HFA1</i> translation initiation site to an upstream AUU codon at position −372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. <i>In silico</i> analyses of fungal <i>ACC</i> genes revealed conserved “cryptic” upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δ<i>hfa1</i> baker's yeast mutant phenotype rescue studies using the protoploid <i>Kluyveromyces lactis ACC</i> confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in <i>S. cerevisiae</i> have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5′ upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.</p></div

The W1536 8B Δ<i>hfa1</i> strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.

<p>Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The yeast cells were grown on media containing Glucose (SCD) or lactate (Lactate) as the sole carbon source at 33°C. Strains used for this study are W1536 8B, W1536 8B Δ<i>hfa1</i> or W1536 8B Δ<i>htd2</i> (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 <i>HFA1</i>; −381: YCp33 <i>HFA1</i> −381; −372: YCp33 <i>HFA1</i> −372; −363: YCp33<i>HFA1</i> −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114738#pone-0114738-g002" target="_blank">Fig. 2</a> can be found as supplementary data.</p

Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.

<p>All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of <i>HFA1</i>. The underlined region up to position −216 region shows the putative minimum mitochondrial import sequence and upstream position −141 shows the end of the sequence similarity to <i>ACC1</i>. The ORFof <i>HFA1</i> annotated in the <i>Saccharomyces</i> Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu <i>et al</i>. 2013 is located at −273 and 8 more stop codons at −282, −312, −360, −363, −372 −375 −378 and −381 were introduced upstream in the promoter region of <i>HFA1</i>.</p

Identification of the <i>HFA1</i> transcription initiation site by 5′RACE: The <i>HFA1</i> specific primer SP1 (Table 4) was used for the reverse transcription reaction to synthesize a first strand cDNA.

<p>a) Major 5′ RACE product (500 bp), b) Minor 5′RACE product (650 bp) and c) third band produced only occasionally.</p

Mitochondrial targeting prediction of selected proteins in <i>S. cerevisiae.</i>

<p>The whole amino acid sequence and the whole amino acid sequence with the 5′ upstream region of the ORF of each protein was analyzed up to the first in-frame stop codon with MitoProt II. In case of the Hfa1p, the identified upstream start codon with the addition of the methionine for the convenience of the calculation with MitoProt II was used. The whole amino acid sequence with the 5′ upstream region of the ORF of each protein was analyzed up to the first in-frame stop codon with Target P. In case of Acp1p, the whole amino acid sequence was used for the calculation with Target P. <b>mTP, SP</b>: Final NN scores on which the final prediction is based. Note that the scores are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class, see below) may be an indication of how certain the prediction is. <b>Loc: S</b>: Secretory pathway, i.e. the sequence contains SP, a signal peptide <b>M</b>: Mitochondrion, i.e. the sequence contains mTP, a mitochondrial targeting peptide; -: Any other location; <b>RC</b>: Reliability class, from 1 to 5, where 1 indicates the strongest prediction. RC is a measure of the size of the difference ('diff') between the highest (winning) and the second highest output scores. There are 5 reliability classes, defined as follows: 1: diff>0.800, 2: 0.800>diff>0.600, 3: 0.600> diff>0.400, 4: 0.400> diff>0.200, 5: 0.200> diff HFA1 and ACP1 are listed as controls for the prediction calculation of MitoProt II and target P.</p><p>Mitochondrial targeting prediction of selected proteins in <i>S. cerevisiae.</i></p