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Contact dependent inhibition (CDI) systems facilitate antagonistic toxin exchange between closely-related bacteria in a proximity-dependent manner. CDI+ bacteria use the large (~300 kDa) cell surface protein CdiA to intoxicate their neighbors by binding to an outer membrane (OM) receptor. Receptor binding occurs at a central 200-300 aminoacids (RBD) and initiates cleavage and translocation of the ~200 carboxy-terminal residues of CdiA (CdiA-CT). Delivery of CdiA-CT requires receptor proteins at each stage of membrane transport into the target cell. We have previously identified three OM receptors for CdiA in Enterobacteria: BamA, OmpC/F, and Tsx. Using the CDI locus from E. coli STEC_O31 as a model (cdiBCAI), in Chapter 2 I introduce a fourth class of CdiA (CdiA4) that exclusively binds to lipopolysaccharide to initiate CdiA-CT translocation. CdiA4 is also linked to an accessory gene, cdiC, which encodes a predicted lysine acyltransferase. Site-directed mutagenesis and HPLC-MS reveals CdiC and its close homologues form a subfamily of bacterial lysine acyltransferases within the toxin-activating acyltransferase (TAAT) family that modify their cognate CdiA proteins to promote LPS binding. In Chapter 3, I use this new CDI system to investigate the interaction between CdiA and O-antigen and find that O-antigen presents a barrier to toxin translocation at the outer membrane of target cells. Finally, in Ch. 4, I identify four new inner-membrane (IM) protein receptors that facilitate toxicity by two new members of the CDI ionophore family. I demonstrate that the native activity of these receptors is not required for CDI, but their presence in the IM is necessary for ionophore activity both from the periplasm or from the cytosol. Combined with a primary sequence analysis of these CdiA-CTs, the results in Chapter 4 offer a wealth of opportunities to explore the mechanism of CDI ionophore activity further. Together, the results of this work present a new physiological application for bacterial protein lipidation, introduce a new family of accessory acyltransferases, reveal that O-antigen can be a barrier to CdiA-CT delivery, and advance our understanding of the functional diversity of CDI toxins and how they exploit target cell proteins for activity.

Recent advances in genome sequencing have greatly improved our ability to understand and identify the causes of genetic diseases. However, there remains an urgent need for innovative, safe, and effective treatments for these diseases. CRISPR-based genome editing systems have become important and powerful tools in the laboratory, and efforts are underway to translate these into patient therapies. Therapeutic base editing is one form of genome engineering that has gained much interest because of its simplicity, specificity, and effectiveness. Base editors are a fusion of a partially deactivated Cas9 enzyme with nickase function, together with a base-modifying enzyme. They are capable of precisely targeting and repairing a pathogenic mutation to restore the normal function of a gene, ideally without disturbing the rest of the genome. In the past year, research has identified new safety concerns of base editors and sparked new innovations to improve their safety. In this review, we provide an overview of the recent advances in the safety and effectiveness of therapeutic base editors and prime editing.

As the demand for healthcare workers fluent in foreign languages continues to grow, so does the necessity for successful educational programs that produce providers with the appropriate language skills and cultural humility to serve the target population. Given the interdisciplinary nature of teaching languages for specific purposes, we propose a model of collaboration between experts in the different fields involved to train well-rounded professionals. In the particular case of languages for healthcare, professionals in both the medical and language learning fields should combine their expertise to offer students a well-integrated training that builds on their medical education at the same time that it provides them with meaningful opportunities to practice and develop their language and culture skills. While this paper focuses on Spanish, the main points discussed in it can be applied to teaching any language in the healthcare setting.

Complex socio-economic, political and demographic factors have driven the increased conversion of Europe’s semi-natural grasslands to intensive pastures. This trend is particularly strong in some of the most biodiverse regions of the continent, such as Central and Eastern Europe. Intensive grazing is known to decrease species diversity and alter the composition of plant and insect communities. Comparatively little is known, however, about how intensive grazing influences plant functional traits related to pollination and the structure of plant-pollinator interactions. In traditional hay meadows and intensive pastures in Central Europe, we contrasted the taxonomic and functional group diversity and composition, the structure of plant-pollinator interactions and the roles of individual species in networks. We found mostly lower taxonomic and functional diversity of plants and insects in intensive pastures, as well as strong compositional differences among the two grassland management types. Intensive pastures were dominated by a single plant with a specialized flower structure that is only accessible to a few pollinator groups. As a result, intensive pastures have lower diversity and specificity of interactions, higher amount of resource overlap, more uniform interaction strength and lower network modularity. These findings stand in contrast to studies in which plants with more generalized flower traits dominated pastures. Our results thus highlight the importance of the functional traits of dominant species in mediating the consequences of intensive pasture management on plant-pollinator networks. These findings could further contribute to strategies aimed at mitigating the impact of intensive grazing on plant and pollinator communities.

Background: The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer (PCa) randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective: To determine report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, Setting, and Participants: This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention: Two cohorts included 1643 men who agreed to be randomised; 997 declined randomisation and chose treatment. Outcome Measurements and Statistical Analysis: Health-related quality of life impacts on urinary, bowel, and sexual function were assessed using patient-reported outcome measures. Analysis was carried out based on treatment received for each cohort and on pooled estimates using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and Limitations: According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and outdating of the interventions being evaluated during the lengthy follow-up required in trials of screen-detected PCa. Conclusions: Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient Summary: More than 90 out of every 100 men with localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are much better after active monitoring, but the risks of spreading of prostate cancer are more common.

Keywords: multiple myeloma, bone disease, osteogenesis, adipogenesis, adipose-derived stem cells, bone marrow, senescence, Dickkopf-related protein 1, systemic disease

Objectives To present the baseline patient-reported outcome measures (PROMs) in the Prostate Testing for Cancer and Treatment (ProtecT) randomized trial comparing active monitoring, radical prostatectomy and external-beam conformal radiotherapy for localized prostate cancer and to compare results with other populations. Materials and Methods A total of 1643 randomized men, aged 50–69 years and diagnosed with clinically localized disease identified by prostate-specific antigen (PSA) testing, in nine UK cities in the period 1999–2009 were included. Validated PROMs for disease-specific (urinary, bowel and sexual function) and condition-specific impact on quality of life (Expanded Prostate Index Composite [EPIC], 2005 onwards; International Consultation on Incontinence Questionnaire- Urinary Incontinence [ICIQ-UI], 2001 onwards; the International Continence Society short-form male survey [ICSmaleSF]; anxiety and depression (Hospital Anxiety and Depression Scale [HADS]), generic mental and physical health (12-item short-form health survey [SF-12]; EuroQol quality-of-life survey, the EQ-5D-3L) were assessed at prostate biopsy clinics before randomization. Descriptive statistics are presented by treatment allocation and by men’s age at biopsy and PSA testing time points for selected measures. Results A total of 1438 participants completed biopsy questionnaires (88%) and 77–88% of these were analysed for individual PROMs. Fewer than 1% of participants were using pads daily (5/ 754). Storage lower urinary tract symptoms were frequent (e.g. nocturia 22%, 312/1423). Bowel symptoms were rare, except for loose stools (16%, 118/754). One third of participants reported erectile dysfunction (241/735) and for 16% (118/731) this was a moderate or large problem. Depression was infrequent (80/1399, 6%) but 20% of participants (278/1403) reported anxiety. Sexual function and bother were markedly worse in older men (65– 70 years), whilst urinary bother and physical health were somewhat worse than in younger men (49–54 years, all P < 0.001). Bowel health, urinary function and depression were unaltered by age, whilst mental health and anxiety were better in older men (P < 0.001). Only minor differences existed in mental or physical health, anxiety and depression between PSA testing and biopsy assessments. Conclusion The ProtecT trial baseline PROMs response rates were high. Symptom frequencies and generic quality of life were similar to those observed in populations screened for prostate cancer and control subjects without cancer.

The increasing need for red blood cells (RBCs) together with the lack of donors have made the in vitro production of RBCs a major medical challenge.[1][1] We have recently developed a method to produce mature RBCs in vitro , starting from bone marrow, peripheral blood, leukapheresis or cord blood

This case study presents the profile of a female high school English language learner enrolled in an English language literature course. The learner is currently positioned at level B1 on the Common European framework and documents Spanish as her native language. The setting of this case study is a private English language secondary school in Quito, Ecuador. One participant was employed for this study. Qualitative data collection methods were used following a naturalistic inquiry design strategy. This qualitative data was analyzed using phenomenological analysis of narrative data. Study results showed the participant demonstrating an increased ability to process text from a top-down level of textual processing. This result correlates with the finding that visual organizers and semantic maps are of vital use in the ESL/EFL classroom. The TEFL Program Portfolio presents a compilation of work samples demonstrating competency according to the TESOL/NCATE standards. Artifacts and accompanying rationale demonstrate how the program content has been applied to teaching ESL/EFL. By showcasing these artifacts, I have shown my ability to apply the course content to the teaching setting and address the TESOL/NCATE standards.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

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