To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3′ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3′ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

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Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

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Skeletal muscle atrophy occurs in several pathological conditions, such as cancer, especially during cancer-induced cachexia. This condition is associated with increased morbidity and poor treatment response, decreased quality of life, and increased mortality in cancer patients. A leucine-rich diet could be used as a coadjutant therapy to prevent muscle atrophy in patients suffering from cancer cachexia. Besides muscle atrophy, muscle function loss is even more important to patient quality of life. Therefore, this study aimed to investigate the potential beneficial effects of leucine supplementation on whole-body functional/movement properties, as well as some markers of muscle breakdown and inflammatory status. Adult Wistar rats were randomly distributed into four experimental groups. Two groups were fed with a control diet (18% protein): Control (C) and Walker 256 tumour-bearing (W), and two other groups were fed with a leucine-rich diet (18% protein + 3% leucine): Leucine Control (L) and Leucine Walker 256 tumour-bearing (LW). A functional analysis (walking, behaviour, and strength tests) was performed before and after tumour inoculation. Cachexia parameters such as body weight loss, muscle and fat mass, pro-inflammatory cytokine profile, and molecular and morphological aspects of skeletal muscle were also determined. As expected, Walker 256 tumour growth led to muscle function decline, cachexia manifestation symptoms, muscle fibre cross-section area reduction, and classical muscle protein degradation pathway activation, with upregulation of FoxO1, MuRF-1, and 20S proteins. On the other hand, despite having no effect on the walking test, inflammation status or muscle oxidative capacity, the leucine-rich diet improved muscle strength and behaviour performance, maintained body weight, fat and muscle mass and decreased some protein degradation markers in Walker 256 tumour-bearing rats. Indeed, a leucine-rich diet alone could not completely revert cachexia but could potentially diminish muscle protein degradation, leading to better muscle functional performance in cancer cachexia.

:The herbicide dinitro-o-cresol (DNOC) was evaluated for its e †ects on bioenergetic activities of potato tuber mitochondria to elucidate its mechanism of action and to compare its toxicological properties with those of the chemically related uncoupler dinitrophenol (DNP). DNOC acts as a typical uncoupler, similarly to the classical uncouplers DNP and carbonylcyanide ptriÑuoromethoxyphenyl-hydrazone (FCCP). Low concentrations of DNOC (\100 lM) maximally stimulate succinate-supported respiration of plant mitochondria, with simultaneous collapse of trans-membrane electrical potential, more efficiently than DNP. The herbicide makes the plant mitochondrial membrane more permeable to protons, acting as a protonophore even in nonenergized mitochondria. High concentrations of DNOC ([100 lM) act also more efficiently than DNP simultaneously as a protonophore and inhibitor of respiration, especially when respiration is supported by substrates that are transported to the matrix. The efficiency of DNOC is decreased with increase of mitochondrial protein, BSA and exogenous orthophosphate. Although similar e †ects were observed for animal and plant mitochondria, rat-liver mitochondrial respiration was more sensitive to DNOC than plant mitochondria. Furthermore, in the presence of DNOC, liver mitochondria exhibited a higher state 3 respiratory coupling level than potato tuber mitochondria, as a result of a considerable stimulation (60%) of state 3 respiration. In conclusion, DNOC is a more potent mitochondrial uncoupler and respiratory chain inhibitor than DNP, although their chemical structures are very similar. Apparently, the additional methyl group of DNOC increases its efficiency as an uncoupler and as an inhibitor, as compared to DNP. Plant mitochondria were shown to be as useful as animal mitochondria in evaluating the toxicity of these xenobiotics.1998 Society of ( Chemical Industry.