Quantitative cone-beam computed tomography reconstruction for radiotherapy planning

Radiotherapy planning involves the calculation of dose deposition throughout the patient, based upon quantitative electron density images from computed tomography (CT) scans taken before treatment. Cone beam CT (CBCT), consisting of a point source and flat panel detector, is often built onto radiotherapy delivery machines and used during a treatment session to ensure alignment of the patient to the plan. If the plan could be recalculated throughout the course of treatment, then margins of uncertainty and toxicity to healthy tissues could be reduced. CBCT reconstructions are normally too poor to be used as the basis of planning however, due to their insufficient sampling, beam hardening and high level of scatter. In this work, we investigate reconstruction techniques to enable dose calculation from CBCT. Firstly, we develop an iterative method for directly inferring electron density from the raw X-ray measurements, which is robust to both low doses and polyenergetic artefacts from hard bone and metallic implants. Secondly, we supplement this with a fast integrated scatter model, also able to take into account the polyenergetic nature of the diagnostic X-ray source. Finally, we demonstrate the ability to provide accurate dose calculation using our methodology from numerical and physical experiments. Not only does this unlock the capability to perform CBCT radiotherapy planning, offering more targeted and less toxic treatment, but the developed techniques are also applicable and beneficial for many other CT applications

Letter from Hugh Mason, UK, to GF Story, 9 September 1869

A letter from Hugh Mason, UK, to GF Story 9 September 1869. Mason forwarded Story a copy of a report on the members of the Dennington family.The report details the address and 'status' of each member of the family. From Cotton Family Papers C7/17

Plant-Made Vaccine Antigens and Biopharmaceuticals

Plant cells are ideal bioreactors for the production and oral delivery of vaccines and biopharmaceuticals, eliminating the need for expensive fermentation, purification, cold storage, transportation and sterile delivery. Plant-made vaccines have been developed for two decades but none has advanced beyond Phase I. However, two plant-made biopharmaceuticals are now advancing through Phase II and Phase III human clinical trials. In this review, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear and chloroplast or transient viral) and their current limitations or challenges. We provide suggestions for advancing this valuable concept for clinical applications and conclude that greater research emphasis is needed on large scale production, purification, functional characterization, oral delivery and preclinical evaluation

Vulnerability Assessment and Re-routing of Freight Trains Under Disruptions: A Coal Supply Chain Network Application

In this paper, we present a two-stage mixed integer programming (MIP) interdiction model in which an interdictor chooses a limited amount of elements to attack first on a given network, and then an operator dispatches trains through the residual network. Our MIP model explicitly incorporates discrete unit flows of trains on the rail network with time-variant capacities. A real coal rail transportation network is used in order to generate scenarios to provide tactical and operational level vulnerability assessment analysis including rerouting decisions, travel and delay costs analysis, and the frequency of interdictions of facilities for the dynamic rail system

Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in Nicotiana benthamiana Leaves

Plants are a promising platform to produce biopharmaceutical proteins, however, the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and RepA strongly affects the efficiency of replication. Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death. We find that a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation. A single nucleotide change in the 5′ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies. We also find that replicating vectors achieve optimal expression with lower Agrobacterium concentrations than non-replicating vectors, further reducing cell death. Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5′ UTR does not

Expression of hepatitis B surface antigen in transgenic plants

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems

Determinants of hospital length of stay for people with serious mental illness in England and implications for payment systems: a regression analysis

BackgroundSerious mental illness (SMI), which encompasses a set of chronic conditions such as schizophrenia, bipolar disorder and other psychoses, accounts for 3.4 m (7 %) total bed days in the English NHS. The introduction of prospective payment to reimburse hospitals makes an understanding of the key drivers of length of stay (LOS) imperative. Existing evidence, based on mainly small scale and cross-sectional studies, is mixed. Our study is the first to use large-scale national routine data to track English hospitals’ LOS for patients with a main diagnosis of SMI over time to examine the patient and local area factors influencing LOS and quantify the provider level effects to draw out the implications for payment systems.MethodsWe analysed variation in LOS for all SMI admissions to English hospitals from 2006 to 2010 using Hospital Episodes Statistics (HES). We considered patients with a LOS of up to 180 days and estimated Poisson regression models with hospital fixed effects, separately for admissions with one of three main diagnoses: schizophrenia; psychotic and schizoaffective disorder; and bipolar affective disorder. We analysed the independent contribution of potential determinants of LOS including clinical and socioeconomic characteristics of the patient, access to and quality of primary care, and local area characteristics. We examined the degree of unexplained variation in provider LOS.ResultsMost risk factors did not have a differential effect on LOS for different diagnostic sub-groups, however we did find some heterogeneity in the effects. Shorter LOS in the pooled model was associated with co-morbid substance or alcohol misuse (4 days), and personality disorder (8 days). Longer LOS was associated with older age (up to 19 days), black ethnicity (4 days), and formal detention (16 days). Gender was not a significant predictor. Patients who self-discharged had shorter LOS (20 days). No association was found between higher primary care quality and LOS. We found large differences between providers in unexplained variation in LOS.ConclusionsBy identifying key determinants of LOS our results contribute to a better understanding of the implications of case-mix to ensure prospective payment systems reflect accurately the resource use within sub-groups of patients with SMI

Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

<p>Abstract</p> <p>Background</p> <p>Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.</p> <p>Results</p> <p>The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in <it>N. benthamiana </it>L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated <it>in vitro </it>by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed <it>E. coli </it>enterotoxin B subunit:<it>H. sapiens </it>mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing <it>Y. enterocolitica </it>UDP-GlcNAc 4-epimerase gene and <it>C. elegans </it>UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.</p> <p>Conclusion</p> <p>Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.</p