931 research outputs found
Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR
Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired
genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the
kinetic of formation and 5\u2032-end nucleolytic processing of Cas9-induced DSB in different human cells lines.
Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end
joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken
into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of
Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNAPKcs
and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study
DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome
manipulation
Italy-Japan agreement and discrepancies in diagnosis of superficial gastric lesions.
The agreement between Italian and Japanese endoscopists and pathologists on endoscopic and histopathological diagnoses of superficial gastric lesions is verified with the use of Paris and Vienna classifications. The correlations between Paris endoscopic types and Vienna histopathological categories is high in both the independent Italian and Japanese evaluations. However, the agreement between Italian and Japanese endoscopists is moderate due to the difficult evaluation of the height of the lesions, in particular when they are mixed. The agreement on the size of the lesions is fairly good. The probability of the same allocation to the Vienna categories of a single case is 87 per cent, disagreements remaining in dysplasia grading, between dysplasia, not only high-grade but also low-grade, and in situ carcinoma, and on cancer invasion of the lamina propria. The results indicate that use of the Paris and Vienna classifications has reduced the discrepancies between Western and Japanese endoscopists and pathologists in the diagnosis of these lesions
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Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.
The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea
Low dose rituximab shares similar activity but slower timing of response than standard dose in patients with immune thrombocytopenia.
Search for lepton-flavor violation at HERA
A search for lepton-flavor-violating interactions and has been performed with the ZEUS detector using the entire HERA I
data sample, corresponding to an integrated luminosity of 130 pb^{-1}. The data
were taken at center-of-mass energies, , of 300 and 318 GeV. No
evidence of lepton-flavor violation was found, and constraints were derived on
leptoquarks (LQs) that could mediate such interactions. For LQ masses below
, limits were set on , where
is the coupling of the LQ to an electron and a
first-generation quark , and is the branching ratio of
the LQ to the final-state lepton ( or ) and a quark . For
LQ masses much larger than , limits were set on the four-fermion
interaction term for LQs that couple to an electron and a quark
and to a lepton and a quark , where and are
quark generation indices. Some of the limits are also applicable to
lepton-flavor-violating processes mediated by squarks in -Parity-violating
supersymmetric models. In some cases, especially when a higher-generation quark
is involved and for the process , the ZEUS limits are the most
stringent to date.Comment: 37 pages, 10 figures, Accepted by EPJC. References and 1 figure (Fig.
6) adde
Multijet production in neutral current deep inelastic scattering at HERA and determination of alpha_s
Multijet production rates in neutral current deep inelastic scattering have
been measured in the range of exchanged boson virtualities 10 < Q2 < 5000 GeV2.
The data were taken at the ep collider HERA with centre-of-mass energy sqrt(s)
= 318 GeV using the ZEUS detector and correspond to an integrated luminosity of
82.2 pb-1. Jets were identified in the Breit frame using the k_T cluster
algorithm in the longitudinally invariant inclusive mode. Measurements of
differential dijet and trijet cross sections are presented as functions of jet
transverse energy E_{T,B}{jet}, pseudorapidity eta_{LAB}{jet} and Q2 with
E_{T,B}{jet} > 5 GeV and -1 < eta_{LAB}{jet} < 2.5. Next-to-leading-order QCD
calculations describe the data well. The value of the strong coupling constant
alpha_s(M_Z), determined from the ratio of the trijet to dijet cross sections,
is alpha_s(M_Z) = 0.1179 pm 0.0013(stat.) {+0.0028}_{-0.0046}(exp.)
{+0.0064}_{-0.0046}(th.)Comment: 22 pages, 5 figure
Dissociation of virtual photons in events with a leading proton at HERA
The ZEUS detector has been used to study dissociation of virtual photons in
events with a leading proton, gamma^* p -> X p, in e^+p collisions at HERA. The
data cover photon virtualities in two ranges, 0.03<Q^2<0.60 GeV^2 and 2<Q^2<100
GeV^2, with M_X>1.5 GeV, where M_X is the mass of the hadronic final state, X.
Events were required to have a leading proton, detected in the ZEUS leading
proton spectrometer, carrying at least 90% of the incoming proton energy. The
cross section is presented as a function of t, the squared four-momentum
transfer at the proton vertex, Phi, the azimuthal angle between the positron
scattering plane and the proton scattering plane, and Q^2. The data are
presented in terms of the diffractive structure function, F_2^D(3). A
next-to-leading-order QCD fit to the higher-Q^2 data set and to previously
published diffractive charm production data is presented
Inclusive jet cross sections and dijet correlations in photoproduction at HERA
Inclusive jet cross sections in photoproduction for events containing a
meson have been measured with the ZEUS detector at HERA using an integrated
luminosity of . The events were required to have a
virtuality of the incoming photon, , of less than 1 GeV, and a
photon-proton centre-of-mass energy in the range . The measurements are compared with next-to-leading-order (NLO) QCD
calculations. Good agreement is found with the NLO calculations over most of
the measured kinematic region. Requiring a second jet in the event allowed a
more detailed comparison with QCD calculations. The measured dijet cross
sections are also compared to Monte Carlo (MC) models which incorporate
leading-order matrix elements followed by parton showers and hadronisation. The
NLO QCD predictions are in general agreement with the data although differences
have been isolated to regions where contributions from higher orders are
expected to be significant. The MC models give a better description than the
NLO predictions of the shape of the measured cross sections.Comment: 43 pages, 12 figures, charm jets ZEU
Measurement of charm fragmentation ratios and fractions in photoproduction at HERA
The production of D^*+, D^0, D^+, D_s^+ and Lambda_c^+ charm hadrons and
their antiparticles in ep scattering at HERA was measured with the ZEUS
detector using an integrated luminosity of 79 pb^-1. The measurement has been
performed in the photoproduction regime with the exchanged-photon virtuality
Q^2 < 1 GeV^2 and for photon-proton centre-of-mass energies in the range 130 <
W < 300 GeV. The charm hadrons were reconstructed in the range of transverse
momentum p_T(D, Lambda_c) > 3.8 GeV and pseudorapidity |eta(D, Lambda_c)| <
1.6. The production cross sections were used to determine the ratio of neutral
and charged D-meson production rates, R_u/d, the strangeness-suppression
factor, gamma_s, and the fraction of charged D mesons produced in a vector
state, P_v^d. The measured R_u/d and gamma_s values agree with those obtained
in deep inelastic scattering and in e^+e^- annihilations. The measured P_v^d
value is smaller than, but consistent with, the previous measurements. The
fractions of c quarks hadronising as a particular charm hadron, f(c -> D,
Lambda_c), were derived in the given kinematic range. The measured open-charm
fragmentation fractions are consistent with previous results, although the
measured f(c -> D^*+) is smaller and f(c -> Lambda_c^+) is larger than those
obtained in e^+e^- annihilations. These results generally support the
hypothesis that fragmentation proceeds independently of the hard sub-process.Comment: 29 pages, 5 figures, 6 tables; minor text revision
Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation
Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins
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