Simultaneous precise editing of multiple genes in human cells

Abstract. When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results i

Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile

In industry Escherichia coli is the preferred host system for the heterologous biosynthesis of therapeutic proteins that do not need posttranslational modifications. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of a therapeutic hormone is described. The strategy is to guide the process along a predefined profile of the total biomass that was derived from a given specific growth rate profile. This profile might have been built upon experience or derived from numerical process optimization. A surprisingly simple adaptive procedure correcting for deviations from the desired path was developed. In this way the batch-to-batch reproducibility can be drastically improved as compared to the process control strategies typically applied in industry. This applies not only to the biomass but, as the results clearly show, to the product titer also

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS

Hydroxyurea and sickle cell anemia: effect on quality of life

BACKGROUND: The Multicenter Study of Hydroxyurea (HU) in Sickle Cell Anemia (MSH) previously showed that daily oral HU reduces painful sickle cell (SS) crises by 50% in patients with moderate to severe disease. The morbidity associated with this disease is known to have serious negative impact on the overall quality of life(QOL) of affected individuals. METHODS: The data in this report were collected from the 299 patients enrolled in the MSH. Health quality of llife (HQOL) measures were assessed in the MSH as a secondary endpoint to determine if the clinical benefit of HU could translate into a measurable benefit perceptible to the patients. HQOL was assessed with the Profile of Mood States, the Health Status Short Form 36 (SF-36), including 4-week pain recall, and the Ladder of Life, self-administered twice 2-weeks apart pre-treatment and every 6 months during the two-year, randomized, double-blind, treatment phase. The effects of factors including randomized treatment, age, gender, pre-treatment crises frequency, Hb-F level mean, daily pain from 4-week pre-treatment diaries, and 2-year Hb-F response level (low or high) were investigated. RESULTS: Over two years of treatment, the benefit of HU treatment on QOL, other than pain scales, was limited to those patients taking HU who maintained a high HbF response, compared to those with low HbF response or on placebo. These restricted benefits occurred in social function, pain recall and general health perception. Stratification according to average daily pain prior to treatment showed that responders to HU whose average daily pain score was 5–9 (substantial pain) achieved significant reduction in the tension scale compared to the placebo group and to non-responders. HU had no apparent effect on other QOL measures. CONCLUSION: Treatment of SS with HU improves some aspects of QOL in adult patients who already suffer from moderate-to-severe SS

Exploring the tensions of being and becoming a medical educator

BackgroundPrevious studies have identified tensions medical faculty encounter in their roles but not specifically those with a qualification in medical education. It is likely that those with postgraduate qualifications may face additional tensions (i.e., internal or external conflicts or concerns) from differentiation by others, greater responsibilities and translational work against the status quo. This study explores the complex and multi-faceted tensions of educators with qualifications in medical education at various stages in their career.MethodsThe data described were collected in 2013–14 as part of a larger, three-phase mixed-methods research study employing a constructivist grounded theory analytic approach to understand identity formation among medical educators. The over-arching theoretical framework for the study was Communities of Practice. Thirty-six educators who had undertaken or were undertaking a postgraduate qualification in medical education took part in semi-structured interviews.ResultsParticipants expressed multiple tensions associated with both becoming and being a healthcare educator. Educational roles had to be juggled with clinical work, challenging their work-life balance. Medical education was regarded as having lower prestige, and therefore pay, than other healthcare career tracks. Medical education is a vast speciality, making it difficult as a generalist to keep up-to-date in all its areas. Interestingly, the graduates with extensive experience in education reported no fears, rather asserting that the qualification gave them job variety.ConclusionThis is the first detailed study exploring the tensions of educators with postgraduate qualifications in medical education. It complements and extends the findings of the previous studies by identifying tensions common as well as specific to active students and graduates. These tensions may lead to detachment, cynicism and a weak sense of identity among healthcare educators. Postgraduate programmes in medical education can help their students identify these tensions in becoming and develop coping strategies. Separate career routes, specific job descriptions and academic workload models for medical educators are recommended to further the professionalisation of medical education

EpCAM an immunotherapeutic target for gastrointestinal malignancy: current experience and future challenges

Despite advances in surgery and adjuvant regimes, gastrointestinal malignancy remains a major cause of neoplastic mortality. Immunotherapy is an emerging and now successful treatment modality for numerous cancers that relies on the manipulation of the immune system and its effector functions to eradicate tumour cells. The discovery that the pan-epithelial homotypic cell adhesion molecule EpCAM is differentially expressed on gastrointestinal tumours has made this a viable target for immunotherapy. Clinical trials using naked anti EpCAM antibody, immunoconjugates, anti-idiotypic and dendritic cell vaccines have met variable success. The murine IgG2a Edrecolomab was shown to reduce mortality and morbidity at a level slightly lower than treatment with 5FU and Levamisole when administered to patients with advanced colorectal carcinoma in a large randomised controlled trial. Fully human and trifunctional antibodies that specifically recruit CD3-positive lymphocytes are now being tested clinically in the treatment of minimal residual disease and ascites. Although clinical trials are in their infancy, the future may bring forth an EpCAM mediated approach for the effective activation and harnessing of the immune system to destroy a pathological aberrance that has otherwise largely escaped its attention

Genome-wide association identifies ATOH7 as a major gene determining human optic disc size

Optic nerve assessment is important for many blinding diseases, with cup-to-disc ratio (CDR) assessments commonly used in both diagnosis and progression monitoring of glaucoma patients. Optic disc, cup, rim area and CDR measurements all show substantial variation between human populations and high heritability estimates within populations. To identify loci underlying these quantitative traits, we performed a genome-wide association study in two Australian twin cohorts and identified rs3858145, P = 6.2 × 10−10, near the ATOH7 gene as associated with the mean disc area. ATOH7 is known from studies in model organisms to play a key role in retinal ganglion cell formation. The association with rs3858145 was replicated in a cohort of UK twins, with a meta-analysis of the combined data yielding P = 3.4 × 10−10. Imputation further increased the evidence for association for several SNPs in and around ATOH7 (P = 1.3 × 10−10 to 4.3 × 10−11, top SNP rs1900004). The meta-analysis also provided suggestive evidence for association for the cup area at rs690037, P = 1.5 × 10−7, in the gene RFTN1. Direct sequencing of ATOH7 in 12 patients with optic nerve hypoplasia, one of the leading causes of blindness in children, revealed two novel non-synonymous mutations (Arg65Gly, Ala47Thr) which were not found in 90 unrelated controls (combined Fisher's exact P = 0.0136). Furthermore, the Arg65Gly variant was found to have very low frequency (0.00066) in an additional set of 672 controls

Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models

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