Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Sharp change over from compound nuclear fission to shape dependent quasi fission

Fission fragment mass distribution has been measured from the decay of $^{246}$Bk nucleus populating via two entrance channels with slight difference in mass asymmetries but belonging on either side of the Businaro Gallone mass asymmetry parameter. Both the target nuclei were deformed. Near the Coulomb barrier, at similar excitation energies the width of the fission fragment mass distribution was found to be drastically different for the $^{14}$N + $^{232}$Th reaction compared to the $^{11}$B + $^{235}$U reaction. The entrance channel mass asymmetry was found to affect the fusion process sharply.Comment: 4 pages,6 figure

Diazoxide-responsive hyperinsulinemic hypoglycemia caused by HNF4A gene mutations

Objective: The phenotype associated with heterozygous HNF4A gene mutations has recently been extended to include diazoxide responsive neonatal hypoglycemia in addition to maturity-onset diabetes of the young (MODY). To date, mutation screening has been limited to patients with a family history consistent with MODY. In this study, we investigated the prevalence of HNF4A mutations in a large cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH). Subjects and methods: We sequenced the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes in 220 patients with HH responsive to diazoxide. The order of genetic testing was dependent upon the clinical phenotype. Results: A genetic diagnosis was possible for 59/220 (27%) patients. KATP channel mutations were most common (15%) followed by GLUD1 mutations causing hyperinsulinism with hyperammonemia (5.9%), and HNF4A mutations (5%). Seven of the 11 probands with a heterozygous HNF4A mutation did not have a parent affected with diabetes, and four de novo mutations were confirmed. These patients were diagnosed with HI within the first week of life (median age 1 day), and they had increased birth weight (median +2.4 SDS). The duration of diazoxide treatment ranged from 3 months to ongoing at 8 years. Conclusions: In this large series, HNF4A mutations are the third most common cause of diazoxide responsive HH. We recommend that HNF4A sequencing is considered in all patients with diazoxide responsive HH diagnosed in the first week of life irrespective of a family history of diabetes, once KATP channel mutations have been excluded

Combining ability studies in restorer lines of sunflower (Helianthus annuus L.)

The restorer lines are used to get fertile hybrids in hybrid seed production system of sunflower. Improve-ment of R-lines for fertility and oil content of sunflower is required to get desirable hybrids upon crossing. In the pre-sent study, 6 restorer lines were crossed in full diallele and both direct and reciprocal crosses along with parents were evaluated for their combining ability. The analysis of variance revealed higher magnitude of SCA variance than GCA variance for all the characters studied except for days to 50 per cent flowering. Among six parents, GMU -520 and R-GM-41 were best general combiner for plant height, head diameter, test weight and leaf size in the desired direction. However, GMU-520 has advantage of yield per plant and oil content. From 15 direct crosses R-GM-41 x R -GM-49 exhibited significant specific combining ability for viz., days to 50 per cent flowering (-1.08), plant height (13.69), head diameter (4.79), leaf size (0.74), yield per plant (17.14), volume weight (2.54), test weight (1.75) and oil content (0.34) followed by RCR-630 x GMU-520, R-GM-41 x EC-602060, R-GM-49 x GMU-520 and EC-602060 x GMU-520 each exhibiting good specific combining ability for majority of characters. Among reciprocal crosses R-GM -41 x RCR-8 exhibited a high reciprocal effects for head diameter (4.57), yield per plant (15.16), test weight (2.02) and leaf size (0.75)

Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies

Eumelanin Graphene-Like Integration: The Impact on Physical Properties and Electrical Conductivity

The recent development of eumelanin pigment-based blends integrating "classical" organic conducting materials is expanding the scope of eumelanin in bioelectronics. Beyond the achievement of high conductivity level, another major goal lays in the knowledge and feasible control of structure/properties relationship. We systematically investigated different hybrid materials prepared by in situ polymerization of the eumelanin precursor 5,6-dihydroxyindole (DHI) in presence of various amounts of graphene-like layers. Spectroscopic studies performed by solid state nuclear magnetic resonance (ss-NMR), x-ray photoemission, and absorption spectroscopies gave a strong indication of the direct impact that the integration of graphene-like layers into the nascent polymerized DHI-based eumelanin has on the structural organization of the pigment itself, while infrared, and photoemission spectroscopies indicated the occurrence of negligible changes as concerns the chemical units. A tighter packing of the constituent units could represent a strong factor responsible for the observed improved electrical conductivity of the hybrid materials, and could be possible exploited as a tool for electrical conductivity tuning

Effect of progressive mandibular advancement on pharyngeal airway size in anesthetized adults.

BACKGROUND: General anesthesia in adult humans is associated with narrowing or complete closure of the pharyngeal airway. The purpose of this study was to determine the effect of progressive mandibular advancement on pharyngeal airway size in normal adults during intravenous infusion of propofol for anesthesia. METHODS: Magnetic resonance imaging was performed in nine normal adults during wakefulness and during propofol anesthesia. A commercially available intraoral appliance was used to manually advance the mandible. Images were obtained during wakefulness without the appliance and during anesthesia with the participants wearing the appliance under three conditions: without mandibular advancement, advancement to 50% maximum voluntary advancement, and maximum advancement. Using computer software, airway area and maximum anteroposterior and lateral airway diameters were measured on the axial images at the level of the soft palate, uvula, tip of the epiglottis, and base of the epiglottis. RESULTS: Airway area across all four airway levels decreased during anesthesia without mandibular advancement compared with airway area during wakefulness (P \u3c 0.007). Across all levels, airway area at 50% advancement during anesthesia was less than that at centric occlusion during wakefulness (P = 0.06), but airway area with maximum advancement during anesthesia was similar to that during wakefulness (P = 0.64). In general, anteroposterior and lateral airway diameters during anesthesia without mandibular advancement were decreased compared with wakefulness and were restored to their wakefulness values with 50% and/or maximal advancement. CONCLUSIONS: Maximum mandibular advancement during propofol anesthesia is required to restore the pharyngeal airway to its size during wakefulness in normal adults

Direct observation of the oxygen isotope effect on the in-plane magnetic field penetration depth in optimally doped YBa2_2Cu3_3O7−δ_{7-\delta}

We report the first direct observation of the oxygen-isotope ($^{16}$O/$^{18}$O) effect on the in-plane penetration depth $\lambda_{ab}$ in a nearly optimally doped YBa$_2$Cu$_3$O$_{7-\delta}$ film using the novel low-energy muon-spin rotation technique. Spin polarized low energy muons are implanted in the film at a known depth $z$ beneath the surface and precess in the local magnetic field $B(z)$. This feature allows us to measure directly the profile $B(z)$ of the magnetic field inside the superconducting film in the Meissner state and to make a model independent determination of $\lambda_{ab}$. A substantial isotope shift $\Delta\lambda_{ab}/\lambda_{ab}=2.8(7)$% at 4 K is observed, implying that the in-plane effective supercarrier mass $m_{ab}^\ast$ is oxygen-isotope dependent with $\Delta m_{ab}^\ast/m_{ab}^\ast = 5.5(1.4)$.Comment: 4 pages, 2 figure

Enhancement of Jc by Hf -Doping in the Superconductor MgB2: A Hyperfine Interaction Study

Measurements of the critical current density (Jc) by magnetization and the upper critical field (Hc2) by magnetoresistance have been performed for hafnium-doped MgB2. There has been a remarkable enhancement of Jc as compared to that by ion irradiation without any appreciable decrease in Tc, which is beneficial from the point of view of applications. The irreversibility line extracted from Jc shows an upward shift. In addition, there has been an increase in the upper critical field which indicates that Hf partially substitutes for Mg. Hyperfine interaction parameters obtained from time differential perturbed angular correlation (TDPAC) measurements revealed the formation of HfB and HfB2 phases along with the substitution of Hf. A possible explanation is given for the role of these species in the enhancement of Jc in MgB2 superconductor