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Gene replacement

T Koiava, D Gonçalves, J Palmeira, K Arobelidze, M Tediashvili, L Akhvlediani, H Ferreira
Research describing the epidemiology of antibiotic resistant microbes is vital to the proactive development of new antimicrobial agents. In the last years, CTX-M extended-spectrum β-lactamases (ESBLs) have emerged worldwide and have replaced classical TEM and SHV-type ESBLs in many countries. CTX-M-15 is currently the most frequent, with a pandemic distribution, and its rapid spread is facilitated by incorporation of resistance genes in mobile genetic elements. The ESBL is efficacious in Gram-negative bacteria and thus closely associated with nosocomial environments, often colonizing the intestines, particularly in older and dependent patients...
September 2016: Georgian Medical News
Andrey Golubov, Igor Kovalchuk
Hydroxymethylcytosine (hmC or 5-hmC) is a nitrogen base occurring as a result of cytosine methylation followed by replacing a methyl group with a hydroxyl group through active oxidation. 5-hmC is considered to be one of the forms of epigenetic modification and is suggested as an intermediate step in a semi-active loss of DNA methylation mark. 5-hmC plays an important role in the epigenetic regulation of gene expression in animals, although its role in plants remains controversial. Here, we present a colorimetric method of quantification of 5-hmC using Brassica rapa DNA...
2017: Methods in Molecular Biology
Anurati Saha, Rebecca Rolfe, Simon Carroll, Daniel J Kelly, Paula Murphy
Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11...
October 22, 2016: Cell and Tissue Research
Chanakan Tongsook, Johannes Niederhauser, Elena Kronegger, Grit Straganz, Peter Macheroux
The degradation of histamine catalyzed by the SAM-dependent histamine N-methyltransferase (HNMT) is critically important for the maintenance of neurological processes. Recently, two mutations in the encoding human gene were reported to give rise to dysfunctional protein variants (G60D and L208P) leading to intellectual disability. In the present study, we have expressed eight L208 variants with either apolar (L208F and L208V), polar (L208N and L208T) or charged (L208D, L208H, L208K and L208R) amino acids to define the impact of side chain variations on protein structure and function...
October 18, 2016: Biochimica et Biophysica Acta
Charlotte L George, Matthew T Birnie, Benjamin Flynn, Yvonne Kershaw, Stafford L Lightman, Becky L Conway-Campbell
In this paper we report differential decoding of the ultradian corticosterone signal by glucocorticoid target tissues. Pulsatile corticosterone replacement in adrenalectomised rats resulted in different dynamics of Sgk1 mRNA production, with a distinct pulsatile mRNA induction profile observed in the pituitary in contrast to a non-pulsatile induction in the prefrontal cortex. We further report the first evidence for pulsatile transcriptional repression of a glucocorticoid-target gene in vivo, with pulsatile regulation of Pomc transcription in pituitary...
October 18, 2016: Molecular and Cellular Endocrinology
Kristin Bösch, Lamprinos Frantzeskakis, Miroslav Vraneš, Jörg Kämper, Kerstin Schipper, Vera Göhre
Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining...
September 30, 2016: Journal of Visualized Experiments: JoVE
In K Cho, Silun Wang, Hui Mao, Anthony Ws Chan
Recent advances in stem cell-based regenerative medicine, cell replacement therapy, and genome editing technologies (i.e. CRISPR-Cas 9) have sparked great interest in in vivo cell monitoring. Molecular imaging promises a unique approach to noninvasively monitor cellular and molecular phenomena, including cell survival, migration, proliferation, and even differentiation at the whole organismal level. Several imaging modalities and strategies have been explored for monitoring cell grafts in vivo. We begin this review with an introduction describing the progress in stem cell technology, with a perspective toward cell replacement therapy...
2016: American Journal of Nuclear Medicine and Molecular Imaging
Francesca Salamanna, Veronica Borsari, Silvia Brogini, Gianluca Giavaresi, Annapaola Parrilli, Simona Cepollaro, Matteo Cadossi, Lucia Martini, Antonio Mazzotti, Milena Fini
One of the main limitations, when studying cancer-bone metastasis, is the complex nature of the native bone environment and the lack of reliable, simple, inexpensive models that closely mimic the biological processes occurring in patients and allowing the correct translation of results. To enhance the understanding of the mechanisms underlying human bone metastases and in order to find new therapies, we developed an in vitro three-dimensional (3D) cancer-bone metastasis model by culturing human breast or prostate cancer cells with human bone tissue isolated from female and male patients, respectively...
October 19, 2016: Oncotarget
Tae Sub Park, Si Won Kim, Jeong Hyo Lee
Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells...
October 20, 2016: Asian-Australasian Journal of Animal Sciences
Angad P Mehta, Han Li, Sean A Reed, Lubica Supekova, Tsotne Javahishvili, Peter G Schultz
Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E...
October 20, 2016: Journal of the American Chemical Society
Natalya M Gashnikova, Darya P Zyryanova, Ekaterina M Astakhova, Vladimir V Ivlev, Maria P Gashnikova, Natalya V Moskaleva, Sergey S Aikin, Tatyana N Bulatova, Sergey V Pustylnikov, Evgeny F Bocharov, Aleksey V Totmenin
Kemerovo Oblast (KO) has had the highest rate of HIV spread in Russia since 2011. The aim of this work was to study the genetic variation of HIV-1 in Kemerovo Oblast. Blood was sampled from a total of 91 HIV-positive antiretroviral-therapy-naïve individuals in 2013 (38) and 2015 (53). HIV-1 subtypes, pol gene drug resistance mutations, and viral tropism were analyzed. In 2013-2015, the prevalence of HIV-1 subtype A decreased in KO from 60.5 to 7.5 %. The samples collected in 2015 from the patients with newly diagnosed HIV demonstrate the current dominance of HIV-1 CRF63_02A1 (71...
October 19, 2016: Archives of Virology
Juan Rodriguez-Paris, Jörg Waldhaus, Jeenal A Gordhandas, Lynn Pique, Iris Schrijver
We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p...
2016: PeerJ
Hye Jin Kim, Hae Min Yoon, Oran Kwon, Won Jun Lee
PURPOSE: Pueraria lobata/rehmannia glutinosa (PR) and exercise have been receiving a lot of attention from postmenopausal women, as a result of the side effects of estrogen replacement therapy. However, the effects of PR and exercise on fatty acid transporters (FATPs), which play essential role in fatty acid transport, have not been studied. In this study, we evaluated the effects of PR and aerobic exercise on FATP1, FABPpm and FAT/CD36 expression in ovariectomized rat skeletal muscles...
September 2016: Journal of Exercise Nutrition & Biochemistry
Jinghua Wu, Shan Jia, Changxi Wang, Wei Zhang, Sixi Liu, Xiaojing Zeng, Huirong Mai, Xiuli Yuan, Yuanping Du, Xiaodong Wang, Xueyu Hong, Xuemei Li, Feiqiu Wen, Xun Xu, Jianhua Pan, Changgang Li, Xiao Liu
Acute B lymphoblastic leukemia (B-ALL) is one of the most common types of childhood cancer worldwide and chemotherapy is the main treatment approach. Despite good response rates to chemotherapy regiments, many patients eventually relapse and minimal residual disease (MRD) is the leading risk factor for relapse. The evolution of leukemic clones during disease development and treatment may have clinical significance. In this study, we performed immunoglobulin heavy chain (IGH) repertoire high throughput sequencing (HTS) on the diagnostic and post-treatment samples of 51 pediatric B-ALL patients...
2016: Frontiers in Immunology
Mohammad Massumi, Farzaneh Pourasgari, Amarnadh Nalla, Battsetseg Batchuluun, Kristina Nagy, Eric Neely, Rida Gull, Andras Nagy, Michael B Wheeler
The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems...
2016: PloS One
Rossella Melchiotti, Filipe Gracio, Shahram Kordasti, Alan K Todd, Emanuele de Rinaldis
Manual gating has been traditionally applied to cytometry data sets to identify cells based on protein expression. The advent of mass cytometry allows for a higher number of proteins to be simultaneously measured on cells, therefore providing a means to define cell clusters in a high dimensional expression space. This enhancement, whilst opening unprecedented opportunities for single cell-level analyses, makes the incremental replacement of manual gating with automated clustering a compelling need. To this aim many methods have been implemented and their successful applications demonstrated in different settings...
October 18, 2016: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Alexandra Proft, Bart Spiesschaert, Satoko Izume, Selina Taferner, Maik J Lehmann, Walid Azab
The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene...
October 12, 2016: Viruses
Solomon Kadiri
Hypertension prevalence rates in most urbanized areas of Africa range from 20-30%, with a recent systematic review reporting 16.2% for sub-Saharan Africa. These rates are lower than those in the West but the age standardized rates are higher than in other regions of the world. The attendant morbidity and mortality are disproportionately high. For example, failure of nocturnal dipping and microalbuminuria, predictive of vascular disease, occur relatively early in the course of hypertension and predispose to early vascular disease...
September 2016: Journal of Hypertension
S Lieb, T Littmann, N Plank, J Felixberger, M Tanaka, T Schäfer, S Krief, S Elz, K Friedland, G Bernhardt, J Wegener, T Ozawa, A Buschauer
A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained...
October 14, 2016: Pharmacological Research: the Official Journal of the Italian Pharmacological Society
Enrique Jurado-Ruiz, Lourdes M Varela, Amparo Luque, Genoveva Berná, Gladys Cahuana, Enrique Martinez-Force, Rocío Gallego-Durán, Bernat Soria, Baukje de Roos, Manuel Romero Gómez, Franz Martín
SCOPE: We evaluated the protective effect of extra virgin olive oil (EVOO) in high-fat diets (HFDs) on the inflammatory response and liver damage in a non-alcoholic fatty liver disease (NAFLD) mouse model. METHODS AND RESULTS: C57BL/6J mice fed a standard diet or a lard-based HFD (HFD-L) for 12 weeks to develop NAFLD. HFD fed mice were then divided into four groups and fed for 24 weeks with: HFD-L; HFD-EVOO; HFD-OL (phenolics-rich EVOO) and R (reversion; standard diet)...
October 17, 2016: Molecular Nutrition & Food Research
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