Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor β-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient’s individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner

Symptomatic hypogammaglobulinemia in infancy and childhood – clinical outcome and in vitro immune responses

BACKGROUND: Symptomatic hypogammaglobulinemia in infancy and childhood (SHIC), may be an early manifestation of a primary immunodeficiency or a maturational delay in the normal production of immunoglobulins (Ig). We aimed to evaluate the natural course of SHIC and correlate in vitro lymphoproliferative and secretory responses with recovery of immunoglobulin values and clinical resolution. METHODS: Children, older than 1 year of age, referred to our specialist clinic because of recurrent infections and serum immunoglobulin (Ig) levels 2 SD below the mean for age, were followed for a period of 8 years. Patient with any known familial, clinical or laboratory evidence of cellular immunodeficiency or other immunodeficiency syndromes were excluded from this cohort. Evaluation at 6- to 12-months intervals continued up to 1 year after resolution of symptoms. In a subgroup of patients, in vitro lymphocyte proliferation and Ig secretion in response to mitogens was performed. RESULTS: 32 children, 24 (75%) males, 8 (25%) females, mean age 3.4 years fulfilled the inclusion criteria. Clinical presentation: ENT infections 69%, respiratory 81%, diarrhea 12.5%. During follow-up, 17 (53%) normalized serum Ig levels and were diagnosed as transient hypogammaglobulinemia of infancy (THGI). THGI patients did not differ clinically or demographically from non-transient patients, both having a benign clinical outcome. In vitro Ig secretory responses, were lower in hypogammaglobulinemic, compared to normal children and did not normalize concomitantly with serum Ig's in THGI patients. CONCLUSIONS: The majority of children with SHIC in the first decade of life have THGI. Resolution of symptoms as well as normalization of Ig values may be delayed, but overall the clinical outcome is good and the clinical course benign

Feasibility study of a clinically-integrated randomized trial of modifications to radical prostatectomy

<p>Abstract</p> <p>Background</p> <p>Numerous technical modifications to radical prostatectomy have been proposed. Such modifications are likely to lead to only slight improvements in outcomes. Although small differences would be worthwhile, an appropriately powered randomized trial would need to be very large, and thus of doubtful feasibility given the expense, complexity and regulatory burden of contemporary clinical trials. We have proposed a novel methodology, the clinically-integrated randomized trial, which dramatically streamlines trial procedures in order to reduce the marginal cost of an additional patient towards zero. We aimed to determine the feasibility of implementing such a trial for radical prostatectomy.</p> <p>Methods</p> <p>Patients undergoing radical prostatectomy as initial treatment for prostate cancer were randomized in a factorial design to involvement of the fascia during placement of the anastomotic sutures, urethral irrigation, both or neither. Endpoint data were obtained from routine clinical documentation. Accrual and compliance rates were monitored to determine the feasibility of the trial.</p> <p>Results</p> <p>From a total of 260 eligible patients, 154 (59%) consented; 56 patients declined to participate, 20 were not approached on recommendation of the treating surgeon, and 30 were not approached for logistical reasons. Although recording by surgeons of the procedure used was incomplete (~80%), compliance with randomization was excellent when it was recorded, with only 6% of procedures inconsistent with allocation. Outcomes data was received from 71% of patients at one year. This improved to 83% as the trial progressed.</p> <p>Conclusions</p> <p>A clinically-integrated randomized trial was conducted at low cost, with excellent accrual, and acceptable compliance with treatment allocation and outcomes reporting. This demonstrates the feasibility of the methodology. Improved methods to ensure documentation of surgical procedures would be required before wider implementation.</p> <p>Trial registration</p> <p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT00928850">NCT00928850</a></p

Pin1-dependent signaling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction

The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2' s ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABA A receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1\ufffd/\ufffd) associated with an increase in amplitude of spontaneous GABA A -mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction. \ufffd 2014 Macmillan Publishers Limited. All rights reserved

Antenatal HIV testing in rural eastern Uganda in 2003: incomplete rollout of the prevention of mother-to-child transmission of HIV programme?

BACKGROUND: Uganda began to implement the prevention of mother-to-child transmission (PMTCT) of HIV programme in 2000, and by the end of 2003 it had expanded to cover 38 of the 56 districts including Mbale District. However, reports from Mbale Hospital showed that less than 10% of pregnant women accepted antenatal HIV testing. We therefore conducted a study to determine the proportion of pregnant women who tested for HIV and the gaps and barriers in PMTCT implementation. METHODS: The study was a cross sectional household survey of women aged 18 years or more, with children aged one year or less, who resided in Mbale Town or in the surrounding Bungokho County. We also conducted in-depth interviews with six health workers in Mbale Hospital. RESULTS: In 2003, we interviewed 457 women with a median age of 24 years. The prevalence of antenatal HIV testing was 10 percent. The barriers to antenatal HIV testing were unavailability of voluntary counselling and testing services (44%), lack of HIV counselling (42%) and perceived lack of benefits for HIV infected women and their infants. Primipara (OR 2.6, 95% CI 1.2–5.8), urban dwellers (OR 2.7, 95% CI 1.3–5.8), women having been counselled on HIV (OR 6.2, 95% CI 2.9–13.2), and women with husbands being their primary confidant (OR 2.3, 95% CI 1.0–5.5) were independently associated with HIV testing. CONCLUSION: The major barriers to PMTCT implementation were unavailability of PMTCT services, particularly in rural clinics, and poor antenatal counselling and HIV testing services. We recommend that the focus of the prevention of mother-to-child transmission of HIV programme should shift to the district and sub-district levels, strengthen community mobilization, improve the quality of antenatal voluntary counselling and HIV testing services, use professional and peer counsellors to augment HIV counselling, and ensure follow-up care and support for HIV positive women and their infants

Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control

Informatics Technology Mimics Ecology: Dense, Mutualistic Collaboration Networks Are Associated with Higher Publication Rates

Information technology (IT) adoption enables biomedical research. Publications are an accepted measure of research output, and network models can describe the collaborative nature of publication. In particular, ecological networks can serve as analogies for publication and technology adoption. We constructed network models of adoption of bioinformatics programming languages and health IT (HIT) from the literature

Effect Sizes in Experimental Pain Produced by Gender, Genetic Variants and Sensitization Procedures

Background: Various effects on pain have been reported with respect to their statistical significance, but a standardized measure of effect size has been rarely added. Such a measure would ease comparison of the magnitude of the effects across studies, for example the effect of gender on heat pain with the effect of a genetic variant on pressure pain. Methodology/Principal Findings: Effect sizes on pain thresholds to stimuli consisting of heat, cold, blunt pressure, punctuate pressure and electrical current, administered to 125 subjects, were analyzed for 29 common variants in eight human genes reportedly modulating pain, gender and sensitization procedures using capsaicin or menthol. The genotype explained 0–5.9% of the total interindividual variance in pain thresholds to various stimuli and produced mainly small effects (Cohen's d 0–1.8). The largest effect had the TRPA1 rs13255063T/rs11988795G haplotype explaining >5% of the variance in electrical pain thresholds and conferring lower pain sensitivity to homozygous carriers. Gender produced larger effect sizes than most variant alleles (1–14.8% explained variance, Cohen's d 0.2–0.8), with higher pain sensitivity in women than in men. Sensitization by capsaicin or menthol explained up to 63% of the total variance (4.7–62.8%) and produced largest effects according to Cohen's d (0.4–2.6), especially heat sensitization by capsaicin (Cohen's d = 2.6). Conclusions: Sensitization, gender and genetic variants produce effects on pain in the mentioned order of effect sizes. The present report may provide a basis for comparative discussions of factors influencing pain