Antisense suppression of donor splice site mutations in the dystrophin gene transcript

We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis

The Cryogenic Target for the G0^0 Experiment at Jefferson Lab

A cryogenic horizontal single loop target has been designed, built, tested and operated for the G$^0$ experiment in Hall C at Jefferson Lab. The target cell is 20 cm long, the loop volume is 6.5 l and the target operates with the cryogenic pump fully immersed in the fluid. The target has been designed to operate at 30 Hz rotational pump speed with either liquid hydrogen or liquid deuterium. The high power heat exchanger is able to remove 1000 W of heat from the liquid hydrogen, while the nominal electron beam with current of 40 $\mu$A and energy of 3 GeV deposits about 320 W of heat into the liquid. The increase in the systematic uncertainty due to the liquid hydrogen target is negligible on the scale of a parity violation experiment. The global normalized yield reduction for 40 $\mu$A beam is about 1.5 % and the target density fluctuations contribute less than 238 ppm (parts per million) to the total asymmetry width, typically about 1200 ppm, in a Q$^2$ bin.Comment: 27 pages, 14 figure

Clinical and in vitro analysis of Osteopontin as a prognostic indicator and unveil its potential downstream targets in bladder cancer

YesOsteopontin (OPN) plays an important role in cancer progression, however its prognostic significance and its downstream factors are largely elusive. In this study, we have shown that expression of OPN was significantly higher in bladder cancer specimens with higher T-stage or tumor grades. In addition, a high level of OPN was significantly associated with poorer survival in two independent bladder cancer patient cohorts totaling 389 bladder cancer patients with available survival data. We further identified Matrix metallopeptidase 9 (MMP9) and S100 calcium-binding protein A8 (S100A8) were both downstream factors for OPN in bladder cancer specimens and bladder cancer cell lines. Expression of OPN was significantly positively associated with that of MMP9 and S100A8, while overexpression of OPN resulted in upregulation of MMP9 and S100A8, and knockdown of OPN showed consistent downregulation of MMP9 and S100A8 expression levels. Importantly, expression levels of both MMP9 and S100A8 were significantly associated with higher T-stage, higher tumor grade and a shorter survival time in the bladder cancer patients. Interestingly, OPN expression only predicted survival in MMP9-high, but not MMP9-low subgroups, and in S100A8-low but not S100A8-high subgroups. Our results suggest that OPN, MMP9 and S100A8 all play a significant role in bladder cancer progression and are potential prognostic markers and therapeutic targets in bladder cancer. The mechanistic link between these three genes and bladder cancer progression warrants further investigation.University of Macau Multi-Year Research Grant (MYRG2015-00065-FHS

Deletion of dystrophin In-Frame Exon 5 leads to a severe phenotype: Guidance for Exon skipping strategies

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes

The road to deterministic matrices with the restricted isometry property

The restricted isometry property (RIP) is a well-known matrix condition that provides state-of-the-art reconstruction guarantees for compressed sensing. While random matrices are known to satisfy this property with high probability, deterministic constructions have found less success. In this paper, we consider various techniques for demonstrating RIP deterministically, some popular and some novel, and we evaluate their performance. In evaluating some techniques, we apply random matrix theory and inadvertently find a simple alternative proof that certain random matrices are RIP. Later, we propose a particular class of matrices as candidates for being RIP, namely, equiangular tight frames (ETFs). Using the known correspondence between real ETFs and strongly regular graphs, we investigate certain combinatorial implications of a real ETF being RIP. Specifically, we give probabilistic intuition for a new bound on the clique number of Paley graphs of prime order, and we conjecture that the corresponding ETFs are RIP in a manner similar to random matrices.Comment: 24 page

Nonequilibrium relaxation in neutral BCS superconductors: Ginzburg-Landau approach with Landau damping in real time

We present a field-theoretical method to obtain consistently the equations of motion for small amplitude fluctuations of the order parameter directly in real time for a homogeneous, neutral BCS superconductor. This method allows to study the nonequilibrium relaxation of the order parameter as an initial value problem. We obtain the Ward identities and the effective actions for small phase the amplitude fluctuations to one-loop order. Focusing on the long-wavelength, low-frequency limit near the critical point, we obtain the time-dependent Ginzburg-Landau effective action to one-loop order, which is nonlocal as a consequence of Landau damping. The nonequilibrium relaxation of the phase and amplitude fluctuations is studied directly in real time. The long-wavelength phase fluctuation (Bogoliubov-Anderson-Goldstone mode) is overdamped by Landau damping and the relaxation time scale diverges at the critical point, revealing critical slowing down.Comment: 31 pages 14 figs, revised version, to appear in Phys. Rev.

First-in-class Microbial Ecosystem Therapeutic 4 (MET4) in combination with immune checkpoint inhibitors in patients with advanced solid tumors (MET4-IO trial)

Background: The intestinal microbiome has been associated with response to immune checkpoint inhibitors (ICIs) in humans and causally implicated in ICI responsiveness in animal models. Two recent human trials demonstrated that fecal microbiota transplant (FMT) from ICI responders can rescue ICI responses in refractory melanoma, but FMT has specific limitations to scaled use.Patients and methods: We conducted an early-phase clinical trial of a cultivated, orally delivered 30-species microbial consortium (Microbial Ecosystem Therapeutic 4, MET4) designed for co-administration with ICIs as an alternative to FMT and assessed safety, tolerability and ecological responses in patients with advanced solid tumors.Results: The trial achieved its primary safety and tolerability outcomes. There were no statistically significant differences in the primary ecological outcomes; however, differences in MET4 species relative abundance were evident after randomization that varied by patient and species. Increases in the relative abundance of several MET4 taxa, including Enterococcus and Bifidobacterium, taxa previously associated with ICI responsiveness, were observed and MET4 engraftment was associated with decreases in plasma and stool primary bile acids.Conclusions: This trial is the first report of the use of a microbial consortium as an alternative to FMT in advanced cancer patients receiving ICI and the results justify the further development of microbial consortia as a therapeutic co-intervention for ICI treatment in cancer

Cultivar governs plant response to inoculation with single isolates and the microbiome associated with arbuscular mycorrhizal fungi

Plant Growth-Promoting Microbes (PGPM) have the potential to enhance sustainable agriculture, but there is still a limited understanding of how the complex interplay between plant genetic variability, the native soil community, and soil nutrients affects PGPM recruitment. To address this challenge, we investigated the impact of bacteria isolates and arbuscular mycorrhizal fungi (AMF) along with their accompany microbiome (AMFc) derived from a wild chrysanthemum on the growth of five different commercial chrysanthemum cultivars (Chic, Chic 45, Chic Cream, Haydar and Barolo), as well as their rhizosphere microbiomes, within a nutrient-rich complex substrate environment. We found 23 bacterial strains capable of producing siderophore, 14 strains capable of producing Indole-3-acetic acid, and 18 strains capable of solubilizing phosphate. The AMFc had six AMF species, and the bacterial and fungal communities associated with AMF belonged to different phyla. Using generalized joint models, we investigated the impact of the three most effective bacterial strains and the AMFc on plant growth (shoot and root dry mass) while integrating information on plant genotype, environment, and microbes. The impact of PGPM inoculation varied from positive to negative effects depending on the cultivar, with Chic Cream showing the highest increase in root biomass after inoculation with both bacterial strain SMF006 (57 %) and AMFc inoculation (79 %). Our study demonstrates that PGPM from wild relative can impact the growth and assembly of the chrysanthemum root microbiome, but this impact is cultivar-dependent. Furthermore, inoculation with a complex AMF containing community (AMFc) induced greater changes in the rhizosphere microbiome than with a single bacterial isolate. Our study shows that inoculation of a complex community of beneficial microbes results in more effective plant growth promotion

Elliptic flow in Pb+Pb collisions at sqrt{s_{NN}} = 2.76 TeV: hybrid model assessment of the first data

We analyze the elliptic flow parameter v_2 in Pb+Pb collisions at sqrt{s_{NN}} = 2.76 TeV and in Au+Au collisions at sqrt{s_{NN}} =200 GeV using a hybrid model in which the evolution of the quark gluon plasma is described by ideal hydrodynamics with a state-of-the-art lattice QCD equation of state, and the subsequent hadronic stage by a hadron cascade model. For initial conditions, we employ Monte-Carlo versions of the Glauber and the Kharzeev-Levin-Nardi models and compare results with each other. We demonstrate that the differential elliptic flow v_2(p_T) hardly changes when the collision energy increases, whereas the integrated v_2 increases due to the enhancement of mean transverse momentum. The amount of increase of both v_2 and mean p_T depends significantly on the model of initialization.Comment: 5 pages, 5 figure