X-chromosome inactivation mosaicism in the three germ layers and the germ line of the mouse embryo

Electrophoretic variant forms of the X-linked enzyme phosphoglycerate kinase (PGK-1, E.C.2, 7, 2, 3) have been used to examine X-chromosome mosaicism in tissues from 121/2-day post coitum heterozygous female mouse embryos. Samples of yolk-sac endoderm, neural ectoderm, heart (mesoderm), liver (endoderm) and germ cells were analysed from each embryo. In all tissues except yolk-sac endoderm, both PGK-1 isozymes were expressed. The extent of covariance among tissues with respect to the PGK-1 isozyme contribution is consistent with all tissues being derived from the same pool of cells after X-inactivation. The covariance among tissues gives an estimate of the size of this pool (47 cells) and places the earliest time of X-inactivation in epiblast cells between 41/2 and 51/2 days post coitum. From the independent variance among tissues within an individual, the average primordial precursor pool size for the three germ layers and the germ line itself was estimated as 193 cells

Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BackgroundThe mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.MethodsWe utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice.ResultsWe demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.ConclusionsHerein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers

The denatured state of N-PGK is compact and predominantly disordered

The Organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I-b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I-k) and denatured (D)]. The transition to I-b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I-b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I-b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics Of I-b indicate that it forms from the fully denatured state within 100 mu s, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I-b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology. (C) 2008 Elsevier Ltd. All rights reserved

?HUBUNGAN LAMA WAKTU HEMODIALISIS DAN USIA DENGAN MALNUTRISI PADA PENDERITA PENYAKIT GINJAL KRONIK DI INSTALASI DIALISIS RUMAH SAKIT UMUM DAERAH DR. ZAINOEL ABIDIN BANDA ACEH

Malnutrisi berkontribusi terhadap peningkatan risiko kematian pada penderita Penyakit Ginjal Kronik (PGK) yang menjalani hemodialisis. Prosedur dialisis dapat menyebabkan hilangnya nutrisi ke dalam cairan dialisat dan meningkatkan reaksi katabolisme. Pada pasien hemodialisis usia berpengaruh terhadap nilai Indeks Massa Tubuh. Tujuan penelitian ini adalah untuk mengetahui hubungan lama waktu hemodialisis dan usia dengan malnutrisi pada penderita PGK di Instalasi Dialisis RSUDZA Banda Aceh. Penelitian ini merupakan penelitian analitik dengan desain cross sectional. Status gizi diukur melalui wawancara dengan menggunakan Mini Nutritional Assessment. Data lama waktu hemodialisis dan usia didapatkan dari buku register di Instalasi Dialisis RSUDZA Banda Aceh. Sampel pada penelitian ini adalah penderita PGK yang menjalani hemodialisis sebanyak 51 orang. Hasil uji Chi-Square menunjukkan terdapat hubungan lama waktu hemodialisis dengan malnutrisi pada penderita PGK yang menjalani hemodialisis (p value 0,004 ? ? = 0,05) dan hasil uji Two-Sample Kolmogorov-Smirnof menunjukkan terdapat hubungan usia dengan malnutrisi pada penderita PGK yang menjalani hemodialisis (p value 0,027 ? ? = 0,05). Kesimpulan dari penelitian ini adalah terdapat hubungan lama waktu hemodialisis dan usia dengan malnutrisi.Kata kunci: lama waktu hemodialisis, PGK, malnutrisi, usia

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway.

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application

Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumine in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source

Spatially homogeneous solutions of the Vlasov-Nordstr\"om-Fokker-Planck system

The Vlasov-Nordstr\"{o}m-Fokker-Planck system describes the evolution of self-gravitating matter experiencing collisions with a fixed background of particles in the framework of a relativistic scalar theory of gravitation. We study the spatially-homogeneous system and prove global existence and uniqueness of solutions for the corresponding initial value problem in three momentum dimensions. Additionally, we study the long time asymptotic behavior of the system and prove that even in the absence of friction, solutions possess a non-trivial asymptotic profile. An exact formula for the long time limit of the particle density is derived in the ultra-relativistic case.Comment: 25 pages, 1 figure. Several changes from previous version. To appear in J. Diff. Eq