Chemical screen for epigenetic barriers to single allele activation of Oct4

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiA:Oct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiA:Oct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiA:Oct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering

AGS3 antagonizes LGN to balance oriented cell divisions and cell fate choices in mammalian epidermis

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically restricted, evolutionarily conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but why only a subset of cell polarize LGN is not known. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light on how spindle orientation influences epidermal stratification

Inheritance of OCT4 predetermines fate choice in human embryonic stem cells

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single-cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor

Antibiotic-induced accumulation of lipid II synergizes with antimicrobial fatty acids to eradicate bacterial populations

Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is a frontline antibiotic for treating methicillin-resistant Staphylococcus aureus and other Gram-positive bacterial infections. However, vancomycin use has led to the increasing prevalence of bacterial strains with reduced susceptibility to vancomycin. Here, we show that unsaturated fatty acids act as potent vancomycin adjuvants to rapidly kill a range of Gram-positive bacteria, including vancomycin-tolerant and resistant populations. The synergistic bactericidal activity relies on the accumulation of membrane-bound cell wall intermediates that generate large fluid patches in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. Our findings provide a natural therapeutic option that enhances vancomycin activity against difficult-to-treat pathogens, and the underlying mechanism may be further exploited to develop antimicrobials that target recalcitrant infection

Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding

Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition

Analysis of selected chemical parameters in Piemontese wines

Background. Piemontese wines are well known and valued all over the world. The most popular of them are Barolo and Barbaresco wines. However, in Poland, they are still little known and only now are being gradually introduced to a wider range of consumers. Objective. The aim of this study was to evaluate the content of inorganic anions, minerals, sugars and glycerol of Piemontese wines from micro-region Langhe, classified as DOCG („Denominazione di Origine Controllata e Garantita”, ie. controlled designation of origin guaranteed) and DOC („Denominazione di Origine Controllata”, ie. controlled designation of origin) products. Material and Methods. Seven types of red wines and one type of white wine were tested. High Performance Ion Chromatography with conductometric detection (HPLC-CD) was used to measure the content of inorganic anions, ie. fluorides, chlorides, sulfates and phosphates. Flame atomic absorption spectrometry (F-AAS) was used to measure the content of minerals, ie. magnesium, calcium, sodium, copper, potassium, zinc and iron, while High Performance Liquid Chromatography with charged aerosol detection (HPLC-CAD) was used to measure the content of glycerol and sugars, ie. fructose, glucose and sucrose. Results. Our studies show that although Piemontese wines are characterized by a relatively low content of minerals in comparison with the wines from other regions, they contain a lot of ingredients that have beneficial effects for human health. Moreover, we observed that the studied wines contain particularly high concentration of inorganic ions – phosphates and fluorides. Furthermore, all tested red wines show far reaching similarities in their chemical properties, which is possibly a direct consequence of using in their production locally cultivated grape varieties. Conclusions. Analysis of the wines from the Piemont region, classified as DOCG, DOC, confirmed that these are dry wines of a high quality.Wprowadzenie. Wina piemonckie są znane i cenione na całym świecie. Wśród najpopularniejszych wymienia się wina Barolo i Barbaresco. W Polsce wina piemonckie są nadal mało znane i wprowadzane dopiero do dystrybucji. Cel. Celem badań była ocena zawartości anionów nieorganicznych, składników mineralnych oraz cukrów i glicerolu w winach piemonckich, klasyfikowanych jako wina DOCG („Denominazione di Origine Controllata e Garantita”, tj. o kontrolowanym oznaczeniu gwarantowanego pochodzenia) i DOC („Denominazione di Origine Controllata”, tj. o kontrolowanym oznaczeniu pochodzenia) z mikroregionu Langhe. Materiał i metody. W siedmiu czerwonych i jednym białym piemonckich winach oznaczano zawartość: składników mineralnych, tj. magnezu, wapnia, sodu, miedzi, potasu, cynku i żelaza z użyciem metody absorpcyjnej spektrometrii atomowej (ASA), anionów nieorganicznych, tj. fluorków, chlorków, siarczanów (VI) i fosforanów (V) z użyciem metody wysokosprawnej chromatografii jonowej z detekcją konduktometryczną (HPIC-CD) oraz glicerolu i cukrów, tj. fruktozy, glukozy i sacharozy z użyciem metody wysokosprawnej chromatografii cieczowej z detekcją wyładowań koronowych (HPLC-CAD). Wyniki: Badania wykazały, że wina piemonckie mogą być korzystnym źródłem składników prozdrowotnych, choć w porównaniu z innymi winami z całego świata charakteryzują się niską zawartością składników mineralnych. Wykazują także specyfikę pod względem wysokiej zawartości anionów nieorganicznych, tj. fluorków i fosforanów. Odnotowano równocześnie duże podobieństwa w składzie chemicznym win czerwonych, co może być charakterystyczne dla danego rejonu upraw winorośli. Wnioski. Analiza win piemonckich, klasyfikowanych jako DOCG i DOC potwierdziła, że są to wina wysokojakościowe i wytrawne

Mineral content of tree sap from the Subcarpathian region

Tree sap has been used for centuries not only as a source of nutrients available in early spring but also as medicinal substance in folk medicine. Traditionally, it was used to treat various conditions, mostly anaemia and chronic fatigue. This study has been designed to establish the content of metallic elements (sodium, potassium, calcium, magnesium, zinc and copper) in sap collected from eight different species (silver birch, downy birch, hornbeam, Norway maple, boxelder maple, black walnut, black alder and white willow) and to identify which sap has mineral content which would be most beneficial for human health. We measured concentrations of calcium, magnesium, sodium, potassium and zinc on an atomic absorption spectrometer equipped with single-element, hollow cathode lamps and an air/acetylene burner. The content of copper was determined using an atomic absorption spectrometer with Zeeman correction equipped with an electrothermal atomizer and argon as inert gas. White willow sap was determined to have the highest concentrations of magnesium, zinc, calcium and potassium. Moreover, this sap contained the lowest concentration of sodium among all the tree species. The sap with the lowest detected concentration of the metallic elements originated from black alder and boxelder. In conclusion, tree sap can be a valuable source of metallic elements, namely copper, zinc and magnesium, in human diet. Tree sap tapped from white willow is the most valuable in terms of its mineral content. Moreover, the most popular sap of sliver birch has proven to be a rich source of magnesium and zin

Mineral content of tree sap from the Subcarpathian region

Tree sap has been used for centuries not only as a source of nutrients available in early spring but also as medicinal substance in folk medicine. Traditionally, it was used to treat various conditions, mostly anaemia and chronic fatigue. This study has been designed to establish the content of metallic elements (sodium, potassium, calcium, magnesium, zinc and copper) in sap collected from eight different species (silver birch, downy birch, hornbeam, Norway maple, boxelder maple, black walnut, black alder and white willow) and to identify which sap has mineral content which would be most beneficial for human health. We measured concentrations of calcium, magnesium, sodium, potassium and zinc on an atomic absorption spectrometer equipped with single-element, hollow cathode lamps and an air/acetylene burner. The content of copper was determined using an atomic absorption spectrometer with Zeeman correction equipped with an electrothermal atomizer and argon as inert gas. White willow sap was determined to have the highest concentrations of magnesium, zinc, calcium and potassium. Moreover, this sap contained the lowest concentration of sodium among all the tree species. The sap with the lowest detected concentration of the metallic elements originated from black alder and boxelder. In conclusion, tree sap can be a valuable source of metallic elements, namely copper, zinc and magnesium, in human diet. Tree sap tapped from white willow is the most valuable in terms of its mineral content. Moreover, the most popular sap of sliver birch has proven to be a rich source of magnesium and zin

Rapid Remodeling of Invadosomes by Gi-coupled Receptors: DISSECTING THE ROLE OF Rho GTPases

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