Single-cell genome-wide concurrent haplotyping and copy-number profiling through genotyping-by-sequencing

Single-cell whole-genome haplotyping allows simultaneous detection of haplotypes associated with monogenic diseases, chromosome copy-numbering and subsequently, has revealed mosaicism in embryos and embryonic stem cells. Methods, such as karyomapping and haplarithmisis, were deployed as a generic and genome-wide approach for preimplantation genetic testing (PGT) and are replacing traditional PGT methods. While current methods primarily rely on single-nucleotide polymorphism (SNP) array, we envision sequencing-based methods to become more accessible and cost-efficient. Here, we developed a novel sequencing-based methodology to haplotype and copy-number profile single cells. Following DNA amplification, genomic size and complexity is reduced through restriction enzyme digestion and DNA is genotyped through sequencing. This single-cell genotyping-by-sequencing (scGBS) is the input for haplarithmisis, an algorithm we previously developed for SNP array-based single-cell haplotyping. We established technical parameters and developed an analysis pipeline enabling accurate concurrent haplotyping and copy-number profiling of single cells. We demonstrate its value in human blastomere and trophectoderm samples as application for PGT for monogenic disorders. Furthermore, we demonstrate the method to work in other species through analyzing blastomeres of bovine embryos. Our scGBS method opens up the path for single-cell haplotyping of any species with diploid genomes and could make its way into the clinic as a PGT application.     

The Akt Inhibitor ISC-4 Synergizes with Cetuximab in 5-FU-Resistant Colon Cancer

Phenylbutyl isoselenocyanate (ISC-4) is an Akt inhibitor with demonstrated preclinical efficacy against melanoma and colon cancer. In this study, we sought to improve the clinical utility of ISC-4 by identifying a synergistic combination with FDA-approved anti-cancer therapies, a relevant and appropriate disease setting for testing, and biomarkers of response. We tested the activity of ISC-4 and 19 FDA-approved anticancer agents, alone or in combination, against the SW480 and RKO human colon cancer cell lines. A synergistic interaction with cetuximab was identified and validated in a panel of additional colon cancer cell lines, as well as the kinetics of synergy. ISC-4 in combination with cetuximab synergistically reduced the viability of human colon cancer cells with wild-type but not mutant KRAS genes. Further analysis revealed that the combination therapy cooperatively decreased cell cycle progression, increased caspase-dependent apoptosis, and decreased phospho-Akt in responsive tumor cells. The synergism between ISC-4 and cetuximab was retained independently of acquired resistance to 5-FU in human colon cancer cells. The combination demonstrated synergistic anti-tumor effects in vivo without toxicity and in the face of resistance to 5-FU. These results suggest that combining ISC-4 and cetuximab should be explored in patients with 5-FU-resistant colon cancer harboring wild-type KRAS.     

Population dynamics of the citrus nematode,Tylenchulus semipenetrans,on navel orange as affected by some plant residues,an organic manure and a biocide

The population dynamics of the citrus nematode, Tylenchulus semipenetrans, on navel orange trees was studied from January 2012 to September 2012. The highest population of the citrus nematode appeared in May 2012 in the soil of navel orange trees, and the highest nematode population in roots appeared in August in the same year. Control of the citrus nematode by using smashed garlic cloves, powders of olive leaves and orange peels, an organic manure, chicken litter, either alone or in combination with a biocide, and sincocin compared to two nematicides, fenamiphos 10%G and oxamyl 24%L, was carried out in April 2012 .The best results for controlling the citrus nematode were obtained four months after the addition of the tested materials in soil; the highest nematode percentages reduction obtained were 90.9%, for smashed garlic cloves, and 72.8%, for chicken litter. On roots, the best results were 92.3% for garlic cloves and 92.0% for oxamyl, one month after application. The concomitant treatments of sincocin plus garlic clove or sicocin plus chicken litter were most effective in managing T. semipenetrans on navel orange trees after four and five months of application.     

Age,growth and length–weight relationships of Pinna bicolor Gmelin (Bivalvia: Pinnidae) in the seagrass beds of Sungai Pulai Estuary,Johor, Peninsular Malaysia

Age and growth of Pinna bicolor were examined in the seagrass beds of Merambong shoal (N 1°19′55.62″; E 103°35′57.75″) off the south‐western coast of Johor, Peninsular Malaysia between May 2006 and April 2007. Monthly growth increment data of P. bicolor were analyzed using FiSAT software (FAO‐ICLARM Stock Assessment Tools) to estimate the asymptotic length (L_∞) and growth coefficient (K). Average growth rate of P. bicolor was 1.42 (±0.01) cm per month; the estimated asymptotic length (L_∞) and growth coefficient (K) were 34.66 cm and 0.88 per year, respectively. In their natural habitat, P. bicolor attain shell heights of approximately 17, 25 and 30 cm at the end of their first, second and third years of growth. The length–weight relationship was estimated as Log W = ?5.397 + 3.111Log L, and in exponential form the equation was W = 0.000004L^3.111 (r² = 0.99, P < 0.01). Habitat temperature and salinity ranged between 27.47 and 29.66°C and 28.66–33.00 ppt with a mean of 29.10 (±0.66) m°C and 30.52 (±1.41) ppt, respectively.     

<Emphasis Type="Italic">Nigella sativa</Emphasis> Oil and Chromium Picolinate Ameliorate Fructose-Induced Hyperinsulinemia by Enhancing Insulin Signaling and Suppressing Insulin-Degrading Enzyme in Male Rats

Lead (Pb) pollution has become one of the most serious global ecological problems. In animals, Pb ingestion induces apoptosis in many tissues. However, the mechanisms by which Pb induces apoptosis in chicken splenic lymphocytes in vitro via the PI3K/Akt pathway and the antagonistic effect of selenium (Se) on Pb remain unclear. Therefore, we established the in vitro Se-Pb interaction model in chicken splenic lymphocytes and examined the frequency of apoptotic cells using acridine orange/ethidium bromide (AO/EB) staining and the TdT-mediated dUTP nick end labeling assay and detected the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The expression of PI3K/Akt pathway-related genes was also examined by qRT-PCR and western blotting. MDA and ROS levels were markedly increased, whereas the activities of GPx, SOD, and CAT were significantly decreased; the levels of the PI3K, Akt, and Bcl-2 messenger RNAs (mRNAs) and proteins were decreased; and the levels of the p53, Bax, cytochrome c (Cyt-c), caspase 3, and caspase 9 mRNAs and proteins were increased in the Pb group. In addition, the frequency of apoptotic cells was also significantly increased by the Pb treatment. However, Se supplementation during Pb exposure observably attenuated Pb-induced apoptosis; increased the levels of the PI3K, Akt, and Bcl-2 mRNAs and proteins; and decrease the levels of the p53, Bax, Cyt-c, caspase 3, and caspase 9 mRNAs and proteins in the chicken spleen. In conclusion, Pb exposure causes oxidative stress, inhibits the PI3K/Akt pathway, and subsequently induces apoptosis in chicken splenic lymphocytes in vitro, and these effects are partially attenuated by Se supplementation. To the best of our knowledge, this study is the first to reveal the antagonistic effect of Se on Pb-induced apoptosis of chicken splenic lymphocytes in vitro via the activation of the PI3K/Akt pathway.     

Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis.

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.