The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin

Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription. This massive chromatin transition from positioned nucleosomes to an extensive hypersensitive site, together with respective transitions at the co-regulated PHO8 and PHO84 promoters, became a prime model for dissecting how remodelers, histone modifiers and chaperones co-operate in nucleosome remodeling upon gene induction. This revealed a surprisingly complex cofactor network at the PHO5 promoter, including five remodeler ATPases (SWI/SNF, RSC, INO80, Isw1, Chd1), and demonstrated for the first time histone eviction in trans as remodeling mode in vivo. Recently, the PHO5 promoter and the whole PHO regulon were harnessed for quantitative analyses and computational modeling of remodeling, transcription factor binding and promoter input-output relations such that this rewarding single-locus model becomes a paradigm also for theoretical and systems approaches to gene regulatory networks

Computer-assisted access to the kidney

OBJECTIVES: The aim of this paper is to introduce the principles of computer-assisted access to the kidney. The system provides the surgeon with a pre-operative 3D planning on computed tomography (CT) images. After a rigid registration with space-localized ultrasound (US) data, preoperative planning can be transferred to the intra-operative conditions and an intuitive man-machine interface allows the user to perform a puncture. MATERIAL AND METHODS: Both CT and US images of informed normal volunteer were obtained to perform calculation on the accuracy of registration and punctures were carried out on a kidney phantom to measure the precision of the whole of the system. RESULTS: We carried out millimetric registrations on real data and guidance experiments on a kidney phantom showed encouraging results of 4.7 mm between planned and reached targets. We noticed that the most significant error was related to the needle deflection during the puncture. CONCLUSION: Preliminary results are encouraging. Further work will be undertaken to improve efficiency and accuracy, and to take breathing into account

The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin

Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription. This massive chromatin transition from positioned nucleosomes to an extensive hypersensitive site, together with respective transitions at the co-regulated PHO8 and PHO84 promoters, became a prime model for dissecting how remodelers, histone modifiers and chaperones co-operate in nucleosome remodeling upon gene induction. This revealed a surprisingly complex cofactor network at the PHO5 promoter, including five remodeler ATPases (SWI/SNF, RSC, INO80, Isw1, Chd1), and demonstrated for the first time histone eviction in trans as remodeling mode in vivo. Recently, the PHO5 promoter and the whole PHO regulon were harnessed for quantitative analyses and computational modeling of remodeling, transcription factor binding and promoter input-output relations such that this rewarding single-locus model becomes a paradigm also for theoretical and systems approaches to gene regulatory networks

Rapid PCR assay for detecting common genetic variants arising in human pluripotent stem cell cultures

Human pluripotent stem cells (hPSCs) are prone to acquiring genetic changes upon prolonged culture. Particularly common are copy number changes, including gains of chromosomes 1q, 12p, 17q, and 20q, and/or loss of chromosomes 10p and 18q. The variant cells harboring common genetic changes display altered behaviors compared to their diploid counterparts, thus potentially impacting upon the validity of experimental results and safety of hPSC-derived cellular therapies. Hence, a critical quality attribute in hPSC maintenance should include frequent monitoring for genetic changes arising in cultures. This in turn places large demands on the genotyping assays for detection of genetic changes. Traditional methods for screening cells entail specialized cytogenetic analyses, but their high costs and a lengthy turnaround time make them impractical for high-throughput analyses and routine laboratory use. Here, we detail a protocol for a rapid, accessible, and affordable PCR-based method for detection of frequently occurring copy number changes in hPSCs

LENGTH HETEROPLASMY IN THE PREDOMINATE MITOCHONDRIAL DNA HAPLOGROUPS IN THE CROATIAN POPULATION

Mitochondrial control region represents the most variable segment of the mitochondrial genome. The frequency and pattern of heteroplasmy has been described in several studies; however, none of the reports documented the Croatian population. In the present study, we screened the control region (1122 bp) of 95 individuals belonging to two predominant mitochondrial phylogenetic branches in the Croatian population, haplogroups H and U. Length heteroplasmy occurred in polycytosine (poly-C) tracts within three hypervariable segments of the control region with the following frequencies: HVSI - 26.3%, HVSII - 52.6% and HVSIII - 7.4%. Furthermore, the association between certain polymorphisms in HVSI and length heteroplasmy was investigated. Our results indicate that only polymorphisms located in the poly-C tract are associated with HVSI length heteroplasmy. The T to C transition at np 16189 is significantly associated with the occurrence of length heteroplasmy (p<0.0001). This effect was even stronger if the C insertion was present in the position 16193. The data support the hypothesis that an uninterrupted poly-C tract of more than eight cytosines leads to length heteroplasmy. Length heteroplasmy associated with the T to C substitution in np 16189 was predominantly found in haplogroup U

LENGTH HETEROPLASMY IN THE PREDOMINATE MITOCHONDRIAL DNA HAPLOGROUPS IN THE CROATIAN POPULATION

Mitochondrial control region represents the most variable segment of the mitochondrial genome. The frequency and pattern of heteroplasmy has been described in several studies; however, none of the reports documented the Croatian population. In the present study, we screened the control region (1122 bp) of 95 individuals belonging to two predominant mitochondrial phylogenetic branches in the Croatian population, haplogroups H and U. Length heteroplasmy occurred in polycytosine (poly-C) tracts within three hypervariable segments of the control region with the following frequencies: HVSI - 26.3%, HVSII - 52.6% and HVSIII - 7.4%. Furthermore, the association between certain polymorphisms in HVSI and length heteroplasmy was investigated. Our results indicate that only polymorphisms located in the poly-C tract are associated with HVSI length heteroplasmy. The T to C transition at np 16189 is significantly associated with the occurrence of length heteroplasmy (p<0.0001). This effect was even stronger if the C insertion was present in the position 16193. The data support the hypothesis that an uninterrupted poly-C tract of more than eight cytosines leads to length heteroplasmy. Length heteroplasmy associated with the T to C substitution in np 16189 was predominantly found in haplogroup U

Human pluripotent stem cells as tools for high-throughput and high-content screening in drug discovery

A significant bottleneck in drug discovery is the lack of suitable models for sensitive, reliable, and rapid assessment of lead molecules in preclinical stages of drug discovery. Human pluripotent stem cells (hPSCs) derived either from early human blastocysts (human embryonic stem cells) or by reprogramming somatic cells to a pluripotent state (human-induced pluripotent stem cells) can be propagated extensively in vitro while retaining the ability to differentiate into any specialized cell type within the body. In this review, we discuss how these unique features of hPSCs could offer a way of producing relevant in vitro models amenable to high-throughput testing for drug discovery. We summarize recent progress in inducing differentiation of hPSCs to specific cell types, and describe the ongoing efforts in applying hPSCs and their differentiated derivatives in disease modeling, drug discovery, and developmental toxicology. Moreover, we review the applications of high-content imaging assays in detecting the changes in the phenotype of hPSCs and their differentiated progeny. Finally, we highlight challenges that need to be overcome in order for the application of hPSC technology to fully benefit drug discovery

Sinteza konjugata fenoprofena i gemfibrozila s kopolimerom stirena i maleinske kiseline

Two types of polymer-drug conjugates were synthesized starting from styrene-maleic acid anhydride copolymer (SMA). Fenoprofen and gemfibrozil were chosen as model drugs because of their short plasma half lives. Both drugs were first converted to their 2-aminoethylamides, which possess free amino groups capable of reacting with SMA anhydride rings. By modifying the degree and type of substitution, lipophilic and hydrophilic conjugates were obtained. Drug loading in the conjugates was between 17 and 47%.U radu je opisana sinteza polimer-lijek konjugata polazeći od kopolimera stirena i anhidrida maleinske kiseline (SMA) i fenoprofena, odnosno gemfibrozila, ljekovitih tvari s kratkim vremenom zadržavanja u plazmi. Fenoprofen i gemfibrozil su prvo prevedeni u 2-aminoetilamide, koji su zbog slobodne amino skupine mogli reagirati s anhidridnim prstenovima u SMA. Modifikacijom tipa i vrste supstitucije pripravljeni su lipofilni i hidrofilni konjugati. Udio vezanog lijeka u konjugatima bio je između 17 and 47%

Assessing cell competition in human pluripotent stem cell (hPSC) cultures

Cell-cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell-cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less-fit cells from mosaic cultures with fitter counterparts