Guidelines for Fluorescent Guided Biallelic HDR Targeting Selection With PiggyBac System Removal for Gene Editing

The development of new and easy-to-use nucleases, such as CRISPR/Cas9, made tools for gene editing widely accessible to the scientific community. Cas9-based gene editing protocols are robust for creating knock-out models, but the generation of single nucleotide transitions or transversions remains challenging. This is mainly due to the low frequency of homology directed repair, which leads to the screening of a high number of clones to identify positive events. Moreover, lack of simultaneous biallelic modifications, frequently results in second-allele indels. For example, while one allele might undergo homology directed repair, the second can undergo non-homologous end joining repair. Here we present a step-wise protocol for biallelic gene editing. It uses two donors carrying a combination of fluorescent reporters alongside homology arms directed to the same genomic region for biallelic targeting. These homology arms carry the desired composite of modifications to be introduced (homozygous or heterozygous changes). Plus, the backbone of the plasmid carries a third fluorescent reporter for negative selection (to discard random integration events). Fluorescent selection of non-random biallelic targeted clones can be performed by microscopy guided picking or cell sorting (FACS). The positive selection module (PSM), carrying the fluorescence reporter and an antibiotic resistance, is flanked by inverted terminal repeats (ITR) that are recognized by transposase. Upon purification of the clones correctly modified, transfection of the excision-only transposase allows the removal of the PSM resulting in the integration of only the desired modifications

Phosphatidylethanolamine binding protein 1 enhances sensitivity of gastric cancer cell to 5-fluorouracil via inhibition of cell proliferation, migration and invasion

Purpose: To determine the association between phosphatidylethanolamine binding protein 1, which is an Raf kinase inhibitor protein (RKIP), and 5-fluorouracil (5-FU) via analysis of the association between RKIP and clinical responses in individuals treated using fluorouracil-based chemotherapy.Methods: Human gastric cancer cell lines MGC-803 and SGC-7901 were used in this study. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and migration were determined with flow cytometry and Transwell chamber assays, respectively. The mRNA and protein expressions of apoptosis-related factors were assayed using realtime polymerase chain reaction (RT-PCR) and Western blotting, respectively, while the expression of RKIP was determined by immunohistochemical staining.Results: Chemotherapeutic drug (5-FU) treatment induced low RKIP expression levels in tumorigenic GC cells, thereby sensitizing the cells to apoptosis (8.57 vs 1.25 %, p < 0.01). The highest RKIP level correlated well with initiation of apoptosis (4.20 vs 1.25 %, p < 0.01). Following in vitro downregulation of RKIP, there was increase in the viability and proliferation of RKIP-inhibited cells over time, and these changes were linked to alterations in cell cycle phases and increased optical density in MTT proliferation assay (1.55 vs 1.18, p < 0.01). In vitro Transwell assay measurement revealed an association between RKIP downregulation and enhancement of cell migration potential (652 vs 436, p < 0.01). Ectopic RKIP expression restored the apoptotic sensitivity of resistant cells (14.30 vs 1.36 %, p <0.01). This sensitization was annulled by upregulation of survival routes. Reduction of RKIP by expression of antisense and siRNA conferred resistance on cancer cells sensitive to 5-FU-mediated apoptosis (6.88 vs 2.13 %, p < 0.01).Conclusion: Thus, RKIP is a promising therapeutic strategy for improving the efficacy of clinically relevant chemotherapeutic drugs for GC. Keywords: Gastric cancer, Raf kinase inhibitor protein, Cell proliferation, Invasion, Apoptosis, Chemotherapy,  Phosphatidylethanolamine binding protein

Effect of intramuscular adipose tissue in the skeletal muscle of thigh on glucose metabolism in male patients with obesity

Objective·To investigate the correlation between intramuscular adipose tissue (IMAT) content and glucose metabolism in male patients with obesity.Methods·Eighty male patients with obesity were recruited from the Endocrinology Department of Renji Hospital, Shanghai Jiao Tong University School of Medicine from December 2019 to December 2020. According to the results of oral glucose tolerance test (OGTT), they were divided into normal glucose tolerance (NGT) group and impaired glucose regulation (IGR) group. General data and laboratory test indicators of the two groups were collected and compared. mDixon-Quant technique was used to measure the IMAT content in each skeletal muscle of the thigh in the two groups, and the proton density fat fractions (PDFF) of skeletal muscle in the two groups were compared. The multivariate Logistic regression model was used to analyze the independent influencing factors of IGR occurrence.Results·Compared with the NGT group, patients in the IGR group had a larger waist circumference (P=0.017), higher glutamic-pyruvic transaminase level, glutamic-oxaloacetic transaminase level, γ-glutamyl transferase (GGT) level, triacylglycerol (TAG) level, nonestesterified fatty acid (NEFA) level and sartorius PDFF (all P<0.05). After adjusting for confounding factors such as age, body mass index, GGT, TAG and NEFA, the results of multivariate Logistic regression analysis showed that PDFF of vastus lateralis, semitendinosus and sartorius were the risk factors for IGR (all P<0.05).Conclusion·Higher levels of IMAT content in vastus lateralis, semitendinosus and sartorius will increase the risk of IGR in male patients with obesity

Antarctic sea ice change based on a new sea ice dataset from 1992 to 2008

The sea ice concentration dataset (covering the period 1992-2008) used in this study is a new dataset based on the Sea Ice Climate Change Initiative (SICCI) algorithm. We investigate whether the SICCI dataset is on a par with other datasets for studying sea ice cover changes in the Southern Ocean. We then examine spatiotemporal variations in sea ice derived from the SICCI dataset over the Southern Ocean, and analyse relationships of sea ice with sea surface temperature (SST). The results indicate that there is no significant difference between the SICCI dataset and the NASA Team dataset, and therefore the former can also be used for studying sea ice changes. Both sea ice extent (SIE) and sea ice area (SIA) derived from the SICCI dataset over the Southern Ocean increased slightly from 1992 to 2008, at rates of (17.75 ± 11.50) × 10^3 and (17.37 ± 9.51) × 10^3 km^2 yr^(–1), respectively. Antarctic sea ice has significant seasonal variations; all seasonally averaged SIE and SIA show an increase, with spring showing the largest positive changing rate. The Weddell Sea, Ross Sea, and Indian Ocean have positive yearly changing rates in SIE and SIA, while the Bellingshausen/Amundsen seas and western Pacific Ocean have negative yearly changing rates. However, overall sea ice over the Southern Ocean has a slight positive trend, which is the same as the sea ice change pattern derived from the NASA Team dataset. This indicates that the contributions to the change in sea ice over the whole Southern Ocean due to the Weddell Sea, Ross Sea, and Indian Ocean dominate over those by the Bellingshausen/Amundsen seas and western Pacific Ocean. Further analysis shows that both SIE and SIA are negatively correlated with SST in the Southern Ocean or each of the 5 longitudinal sectors, and sea ice is more sensitive to SST in spring and autumn

Determination of 16 Selected Trace Elements in Children Plasma from China Economical Developed Rural Areas Using High Resolution Magnetic Sector Inductively Coupled Mass Spectrometry

A rapid, accurate, and high performance method of high resolution sector field inductively coupled plasma mass spectrometry (HR-ICP-MS) combined with a small-size sample (0.1 mL) preparation was established. The method was validated and applied for the determination of 16 selected plasma trace elements (Fe, Cu, Zn, Rb, B, Al, Se, Sr, V, Cr, Mn, Co, As, Mo, Cd, and Pb). The linear working ranges were over three intervals, 0-1 g/L, 0-10 g/L and 0-100 g/L. Correlation coefficients (R 2 ) ranged from 0.9957 to 0.9999 and the limits of quantification (LOQ) ranged from 0.02 g/L (Rb) to 1.89 g/L (Se). The trueness (or recovery) spanned from 89.82% (Al) to 119.15% (Se) and precision expressed by the relative standard deviation (RSD %) for intra-day ranging from 1.1% (Zn) to 9.0% (Se), while ranged from 3.7% (Fe) to 12.7% (Al) for interday. A total of 440 plasma samples were collected from Chinese National Nutrition and Health Survey Project 2002 (CNNHS 2002), which represented the status of plasma trace elements for the children aged 3-12 years from China economical developed rural areas. The concentrations of 16 trace elements were summarized and compared by age groups and gender, which can be used as one of the basic components for the formulation of the baseline reference values of trace elements for the children in 2002

Towards graphane field emitters.

We report on the improved field emission performance of graphene foam (GF) following transient exposure to hydrogen plasma. The enhanced field emission mechanism associated with hydrogenation has been investigated using Fourier transform infrared spectroscopy, plasma spectrophotometry, Raman spectroscopy, and scanning electron microscopy. The observed enhanced electron emissionhas been attributed to an increase in the areal density of lattice defects and the formation of a partially hydrogenated, graphane-like material. The treated GF emitter demonstrated a much reduced macroscopic turn-on field (2.5 V μm-1), with an increased maximum current density from 0.21 mA cm-2 (pristine) to 8.27 mA cm-2 (treated). The treated GFs vertically orientated protrusions, after plasma etching, effectively increased the local electric field resulting in a 2.2-fold reduction in the turn-on electric field. The observed enhancement is further attributed to hydrogenation and the subsequent formation of a partially hydrogenated structured 2D material, which advantageously shifts the emitter work function. Alongside augmentation of the nominal crystallite size of the graphitic superstructure, surface bound species are believed to play a key role in the enhanced emission. The hydrogen plasma treatment was also noted to increase the emission spatial uniformity, with an approximate four times reduction in the per unit area variation in emission current density. Our findings suggest that plasma treatments, and particularly hydrogen and hydrogen-containing precursors, may provide an efficient, simple, and low cost means of realizing enhanced nanocarbon-based field emission devices via the engineered degradation of the nascent lattice, and adjustment of the surface work function.For assistance in ATR FTIR and EDXRF measurements we thank Dr Bob Keighley and Dr Ralph Vokes of Shimadzu Corp; and for plasma optical spectrophotometry analysis, Dr Thomas Schűtte of PLASUS GmbH. This work is supported by National Key Basic Research Program 973(2010CB327705), National Natural Science Foundation Project (51120125001, 51002031, 61101023, 51202028), Foundation of Doctoral Program of Ministry of Education (20100092110015), an EPSRC Impact Acceleration grant, and the Research Fund for International Young Scientists from NSFC (510501101 42, 51350110232). MT Cole thanks the Oppenheimer Trust for their generous financial support.This is the author accepted manuscript. The final version is available from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C5RA20771

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5′UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5′UTR and NS3-3′UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5′UTR and 3′UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated “CH6acc”), releasing HCV of 104.3–104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses