Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

A Technological Review of Wearable Cueing Devices Addressing Freezing of Gait in Parkinson's Disease.

Freezing of gait is one of the most debilitating symptoms of Parkinson's disease and is an important contributor to falls, leading to it being a major cause of hospitalization and nursing home admissions. When the management of freezing episodes cannot be achieved through medication or surgery, non-pharmacological methods such as cueing have received attention in recent years. Novel cueing systems were developed over the last decade and have been evaluated predominantly in laboratory settings. However, to provide benefit to people with Parkinson's and improve their quality of life, these systems must have the potential to be used at home as a self-administer intervention. This paper aims to provide a technological review of the literature related to wearable cueing systems and it focuses on current auditory, visual and somatosensory cueing systems, which may provide a suitable intervention for use in home-based environments. The paper describes the technical operation and effectiveness of the different cueing systems in overcoming freezing of gait. The "What Works Clearinghouse (WWC)" tool was used to assess the quality of each study described. The paper findings should prove instructive for further researchers looking to enhance the effectiveness of future cueing systems.

Magnetite Biomineralization in Magnetospirillum magneticum Is Regulated by a Switch-like Behavior in the HtrA Protease MamE.

Magnetotactic bacteria are aquatic organisms that produce subcellular magnetic particles in order to orient in the earth's geomagnetic field. MamE, a predicted HtrA protease required to produce magnetite crystals in the magnetotactic bacterium Magnetospirillum magneticum AMB-1, was recently shown to promote the proteolytic processing of itself and two other biomineralization factors in vivo Here, we have analyzed the in vivo processing patterns of three proteolytic targets and used this information to reconstitute proteolysis with a purified form of MamE. MamE cleaves a custom peptide substrate with positive cooperativity, and its autoproteolysis can be stimulated with exogenous substrates or peptides that bind to either of its PDZ domains. A misregulated form of the protease that circumvents specific genetic requirements for proteolysis causes biomineralization defects, showing that proper regulation of its activity is required during magnetite biosynthesis in vivo Our results represent the first reconstitution of the proteolytic activity of MamE and show that its behavior is consistent with the previously proposed checkpoint model for biomineralization.

Effects of soil properties on the transfer of cadmium from soil to wheat in the Yangtze River delta region, China--a typical industry-agriculture transition area.

In order to identify the effects of soil properties on the transfer of Cd from soil to wheat under actual field conditions, 126 pairs of topsoil and wheat samples were collected from the Yangtze River delta region, China. Relevant parameters (Cd, Ca, Mg, Fe, Mn, Zn, N, P, K, S, pH, total organic carbon, and speciation of soil Cd) in soil and wheat tissues were analyzed, and the results were treated by statistical methods. Soil samples (19.8%) and 14.3% of the wheat grain samples exceeded the relevant maximum permissible Cd concentrations in China for agricultural soil and wheat grain, respectively. The major speciations of Cd in soil were exchangeable, bound to carbonates and fulvic and humic acid fraction, and they were readily affected by soil pH, total Ca, Mg, S and P, DTPA-Fe, Ex-Ca, and Ex-Mg. Cadmium showed a strong correlation with Fe, S, and P present in the grain and the soil, whereas there was no significant correlation in the straw or root. Generally, soil pH, Ca, Mg, Mn, P, and slowly available K restricted Cd transfer from soil to wheat, whereas soil S, N, Zn, DTPA-Fe, and total organic carbon enhance Cd uptake by wheat.

RuvBl2 cooperates with Ets2 to transcriptionally regulate hTERT in colon cancer.

Human cancers utilise telomerase to maintain telomeres and prohibit cell senescence. Human telomerase reverse transcriptase (hTERT), an essential component of this complex, is regulated at the level of gene transcription. Using SILAC-proteomic analysis and molecular studies, we identified the AAA+ ATPase, RuvBl2 as a transcriptional regulator of hTERT and established that this regulation is through cooperation with Ets-2. In colon cancer patients, nuclear expression of RuvBl2 associated with nuclear expression of hTERT, pEts2 and advanced nodal disease (P<0.01, P=0.05 and P=0.03 respectively, n=170). These data firmly implicate RuvBl2 in Ets2 mediated regulation of hTERT in colon cancer which has functional and clinical consequences.

Simulation training for vascular access interventions.

Training and learning in the field of access for dialysis, including peritoneal and hemodialysis and access for oncologic patients, is well suited for the use of simulators, simulated case learning, and root cause analysis of adverse outcomes and team training. Simulators range over a wide spectrum from simple suture learning devices, inexpensive systems for venous puncture simulation, such as a turkey breast or leg with a pressurized tunneled rubber or graft conduit, to sophisticated computer designed simulators to teach interventional procedures such as vascular access angiogram, balloon angioplasty and stent placing. Team training capitalizes on the principles used in aviation, known as Crew Resource Management (CRM) or Human Factor (HF). The objectives of team training are to improve communication and leadership skills, to use checklists to prevent errors, to promote a change in the attitudes towards vascular access from learning through mistakes in a non-punitive environment, to impacting positively the employee performance and to increase staff retention by making the workplace safer, more efficient and user-friendly.

Can aviation-based team training elicit sustainable behavioral change?

OBJECTIVE: To quantify effects of aviation-based crew resource management training on patient safety-related behaviors and perceived personal empowerment. DESIGN: Prospective study of checklist use, error self-reporting, and a 10-point safety empowerment survey after participation in a crew resource management training intervention. SETTING: Seven hundred twenty-two-bed university hospital; 247-bed affiliated community hospital. PARTICIPANTS: There were 857 participants, the majority of whom were nurses (50%), followed by ancillary personnel (28%) and physicians (22%). MAIN OUTCOME MEASURES: Preoperative checklist use over time; number and type of entries on a Web-based incident reporting system; and measurement of degree of empowerment (1-5 scale) on a 10-point survey of safety attitudes and actions given prior to, immediately after, and a minimum of 2 months after training. RESULTS: Since 2003, 10 courses trained 857 participants in multiple disciplines. Preoperative checklist use rose (75% in 2003, 86% in 2004, 94% in 2005, 98% in 2006, and 100% in 2007). Self-initiated reports increased from 709 per quarter in 2002 to 1481 per quarter in 2008. The percentage of reports related to environment as opposed to actual events increased from 15.9% prior to training to 20.3% subsequently (P < .01). Perceived self-empowerment, creating a culture of safety, rose by an average of 0.5 point in all 10 realms immediately posttraining (mean [SD] rating, 3.0 [0.07] vs 3.5 [0.05]; P < .05). This was maintained after a minimum of 2 months. There was a trend toward a hierarchical effect with participants less comfortable confronting incompetence in a physician (mean [SD] rating, 3.1 [0.8]) than in nurses or technicians (mean [SD] rating, 3.4 [0.7] for both) (P>.05). CONCLUSIONS: Crew resource management programs can influence personal behaviors and empowerment. Effects may take years to be ingrained into the culture.

Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation.

In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and 11.9 min at pH 7.0 and 37 degrees C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR) expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23,000-29,000x selective cytotoxicity toward NTR-expressing V79 cells with an IC(50) as low as 27 nM. They are about as active as and 3-4x more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide mustard ((+/-)-7) was found to be the most active and most selective compound for activation by NTR with 170,000x selective cytotoxicity toward NTR-expressing V79 cells and an IC(50) of 0.4 nM. Compound (+/-)-7also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC(50), high selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.

The B-cell transcription factors BSAP, Oct-2, and BOB.1 and the pan-B-cell markers CD20, CD22, and CD79a are useful in the differential diagnosis of classic Hodgkin lymphoma.

Even with routine immunohistochemical evaluation, distinguishing classic Hodgkin lymphoma (CHL) from diffuse large B-cell lymphoma (DLBCL) or anaplastic large cell lymphoma (ALCL) can be difficult. In these cases, the transcription factors (B cell-specific activator protein [BSAP], octamer-binding transcription factor 2 [Oct-2], and B-cell Oct-binding protein 1 [BOB.1]) and the pan-B-cell markers (CD20, CD22, and CD79a) may aid in clarifying the diagnosis. In 57 cases of CHL, 5 cases of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), and 33 cases of non-Hodgkin lymphoma (25 DLBCL and 8 ALCL) we found the transcription factor phenotype BSAP+ and either Oct-2- or BOB.1- to be predictive of CHL; BSAP+/Oct-2+/BOB.1+ was predictive of NLPHL or DLBCL, while BSAP- was predictive of ALCL. Expression of all 3 pan-B-cell markers was seen only in NLPHL and DLBCL; positivity for a single B-cell marker was present only in CHL. Thus, together, the transcription factors and pan-B-cell markers might be useful in the differential diagnosis of CHL.

Nitroaryl phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: synthesis, stability, enzymatic reduction, and antiproliferative activity in cell culture.

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.