Utility of Pre-Hematopoietic Cell Transplantation Sinus CT Screening in Children and Adolescents.

BACKGROUND AND PURPOSE: The clinical benefit of pre-hematopoietic cell transplantation sinus CT screening remains uncertain, while the risks of CT radiation and anesthesia are increasingly evident. We sought to re-assess the impact of screening sinus CT on pretransplantation patient management and prediction of posttransplantation invasive fungal rhinosinusitis. MATERIALS AND METHODS: Pretransplantation noncontrast screening sinus CTs for 100 consecutive patients (mean age, 11.9 ± 5.5 years) were graded for mucosal thickening (Lund-Mackay score) and for signs of noninvasive or invasive fungal rhinosinusitis (sinus calcification, hyperattenuation, bone destruction, extrasinus inflammation, and nasal mucosal ulceration). Posttransplantation sinus CTs performed for sinus-related symptoms were similarly graded. Associations of Lund-Mackay scores, clinical assessments, changes in pretransplantation clinical management (additional antibiotic or fungal therapy, sinonasal surgery, delayed transplantation), and subsequent development of sinus-related symptoms or invasive fungal rhinosinusitis were tested (exact Wilcoxon rank sums, Fisher exact test, significance P < .05). RESULTS: Mean pretransplantation screening Lund-Mackay scores (n = 100) were greater in patients with clinical symptoms (8.07 ± 6.00 versus 2.48 ± 3.51, P < .001) but were not associated with pretransplantation management changes and did not predict posttransplantation sinus symptoms (n = 21, P = .47) or invasive fungal rhinosinusitis symptoms (n = 2, P = .59). CONCLUSIONS: Pre-hematopoietic cell transplantation sinus CT does not meaningfully contribute to pretransplantation patient management or prediction of posttransplantation sinus disease, including invasive fungal rhinosinusitis, in children. The risks associated with CT radiation and possible anesthesia are not warranted in this setting.

Comparison of < 100 cc prostates and > 100 cc prostates undergoing aquablation for benign prostatic hyperplasia.

INTRODUCTION: Surgical options for benign prostatic hyperplasia (BPH) become limited when treating large prostates due to steep learning curves and less effective treatment. Aquablation (AquaBeam System, PROCEPT BioRobotics, Inc., USA) could remedy this. We compare the effectiveness of Aquablation in large prostates between 80 cc and 100 cc and very large prostates > 100 cc. METHODS: WATER II (NCT03123250) is a prospective, multicenter, international clinical trial of Aquablation for the surgical treatment of LUTS/BPH in men of age 45-80 years with prostates between 80 cc and 150 cc. Aquablation was performed using the AquaBeam System. The reported analysis compares the subgroup of patients with a baseline prostate size of < 100 cc versus those with a prostate size of > 100 cc. Students' t test was used for continuous variables and Fisher's test for ordinal/binary variables. RESULTS: Of 114 screened patients, 101 meeting eligibility criteria were enrolled at 13 US and 3 Canadian sites between September and December 2017. Mean operative time was 31.2 ± 8 min in the < 100 cc subgroup and 41.7 ± 14.9 min in the > 100 cc subgroup. The average length of stay following the procedure for the < 100 cc subgroup was 1.5 ± 0.7 days versus 1.7 ± 1.1 days for the > 100 cc subgroup. Mean changes in International Prostate Symptom Score (IPSS), IPSS quality of life, and IPSS voiding and storage subscores were substantial, occurring soon after treatment and averaging (at 3 months) 16.5, 2.8, 10.6, and 5.8 points, respectively (all p < 0.0001). CONCLUSION: Aquablation clinically normalizes outcomes between patients of the < 100 cc and > 100 cc prostate cohorts. It is safe and effective in patients with large prostate glands (> 100 cc) with a smoother learning curve.

A multicentred study to validate a consensus bleeding assessment tool developed by the biomedical excellence for safer transfusion collaborative for use in patients with haematological malignancy.

BACKGROUND: There continues to be uncertainty about the optimal approach to documenting bleeding data in platelet transfusion trials, with a desire to apply a common assessment tool across all trials. With this in mind, a consensus bleeding assessment tool (BAT) has been developed by the Biomedical Excellence for Safer Transfusion (BEST) collaborative, based on review of data collection forms used in published randomized trials and following content validation with a range of healthcare professionals at seven haematology centres through BEST members. This study aimed to evaluate reliability and reproducibility of the consensus BAT. METHODS: Replicated clinical assessments of bleeding were undertaken by participants with haematological malignancies recruited at four haematology centres in an international, multicentred, observational study. Concordance of repeat assessments was calculated for agreement in site and grade of bleeding observed. RESULTS: Forty patients consented to participate, and 13 trained bleeding assessors collected these data. Bleeding assessments were carried out on 113 separate days. Of all 225 bleeding assessments, 204 were compared for grade concordance, and 160 were compared for site concordance. There was very good grade concordance (83%, 95% confidence interval 74-93%) and good bleeding site concordance (69%, 95% confidence interval 57-79%) in observations of bleeding. Discordance was primarily in relation to assessing skin bleeding. CONCLUSIONS: Alongside a structured training programme, levels of concordance for a consensus BAT were high. Researchers using assessment tools for bleeding need to balance comprehensive data collection against potential loss of accuracy for some types of bleeding, such as skin findings.

Clinical and laboratory correlates of platelet alloimmunization and refractoriness in the PLADO trial.

BACKGROUND AND OBJECTIVES: Platelet alloimmunization and refractoriness to platelet transfusion are complications of platelet transfusion therapy. The platelet dose (PLADO) trial, as the largest prospective randomized trial of prophylactic platelet therapy to date, afforded an opportunity to analyse these two issues. MATERIALS AND METHODS: PLADO patient records were examined for evidence of platelet alloimmunization, defined as an increase in HLA Class I panel-reactive antibodies (PRA) to ≥20%, and clinical refractoriness, defined as two consecutive ≤4 h posttransfusion corrected platelet count increments (CCI) of <5000. Multivariate logistic regression, restricted to platelet-transfused subjects who received exclusively either in-process leucoreduction apheresis or whole blood-derived (WBD) leucocyte-reduced platelets, compared the frequency of these outcomes by platelet unit and patient characteristics. RESULTS: Forty of 816 evaluable platelet-transfused patients (5%) became alloimmunized during the trial. Prior pregnancy, chemotherapy only compared to progenitor cell transplant, and low platelet dose - all were associated with significantly higher rates of alloimmunization. Among 35 alloimmunized patients evaluated for refractoriness, 8 (23%) had two consecutive CCI < 5000/µl. Regardless of alloimmunization status, CCIs < 5000/µl were observed following 17% of platelet transfusions. Among 734 patients receiving at least two platelet transfusions, two consecutive CCIs of ≤5000 occurred in 102 (14%). CONCLUSIONS: The incidence of new platelet alloimmunization was low in the PLADO study, but follow-up was at most 30 days. Alloimmunization was present in only 8 of 102 (8%) of observed cases of refractoriness, suggesting that other causes of poor posttransfusion increments are frequent.

Full Dose-Reduction Potential of Statistical Iterative Reconstruction for Head CT Protocols in a Predominantly Pediatric Population.

BACKGROUND AND PURPOSE: A statistical iterative reconstruction algorithm provides an effective approach to reduce patient dose by compensating for increased image noise in CT due to reduced radiation output. However, after a point, the degree to which a statistical iterative algorithm is used for image reconstruction changes the image appearance. Our aim was to determine the maximum level of statistical iterative reconstruction that can be used to establish dose-reduced head CT protocols in a primarily pediatric population while maintaining similar appearance and level of image noise in the reconstructed image. MATERIALS AND METHODS: Select head examinations (brain, orbits, sinus, maxilla, and temporal bones) were investigated. Dose-reduced head protocols using an adaptive statistical iterative reconstruction were compared for image quality with the original filtered back-projection reconstructed protocols in a phantom by using the following metrics: image noise frequency (change in perceived appearance of noise texture), image noise magnitude, contrast-to-noise ratio, and spatial resolution. Dose-reduction estimates were based on CT dose index values. Patient volume CT dose index and image noise magnitude were assessed in 737 pre- and post-dose-reduced examinations. RESULTS: Image noise texture was acceptable for up to 60% adaptive statistical iterative reconstruction for the soft reconstruction kernel (at both 100 and 120 kV[peak]) and up to 40% adaptive statistical iterative reconstruction for the standard reconstruction kernel. Implementation of 40% and 60% adaptive statistical iterative reconstruction led to an average reduction in the volume CT dose index of 43% for brain, 41% for orbit, 30% for maxilla, 43% for sinus, and 42% for temporal bone protocols for patients between 1 month and 26 years of age, while maintaining an average noise magnitude difference of 0.1% (range, -3% to 5%), improving the contrast-to-noise ratio of low-contrast soft-tissue targets and the spatial resolution of high-contrast bony anatomy, compared with filtered back-projection. CONCLUSIONS: The methodology in this study demonstrates maximizing patient dose reduction and maintaining image quality by using statistical iterative reconstruction for a primarily pediatric population undergoing head CT examinations.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Glucose: Detection and analysis.

Glucose is an aldosic monosaccharide that is centrally entrenched in the processes of photosynthesis and respiration, serving as an energy reserve and metabolic fuel in most organisms. As both a monomer and as part of more complex structures such as polysaccharides and glucosides, glucose also plays a major role in modern food products, particularly where flavor and or structure are concerned. Over the years, many diverse methods for detecting and quantifying glucose have been developed; this review presents an overview of the most widely employed and historically significant, including copper iodometry, HPLC, GC, CZE, and enzyme based systems such as glucose meters. The relative strengths and limitations of each method are evaluated, and examples of their recent application in the realm of food chemistry are discussed.

Estimating pediatric entrance skin dose from digital radiography examination using DICOM metadata: A quality assurance tool.

PURPOSE: To develop an automated methodology to estimate patient examination dose in digital radiography (DR) imaging using DICOM metadata as a quality assurance (QA) tool. METHODS: Patient examination and demographical information were gathered from metadata analysis of DICOM header data. The x-ray system radiation output (i.e., air KERMA) was characterized for all filter combinations used for patient examinations. Average patient thicknesses were measured for head, chest, abdomen, knees, and hands using volumetric images from CT. Backscatter factors (BSFs) were calculated from examination kVp. Patient entrance skin air KERMA (ESAK) was calculated by (1) looking up examination technique factors taken from DICOM header metadata (i.e., kVp and mA s) to derive an air KERMA (k air) value based on an x-ray characteristic radiation output curve; (2) scaling k air with a BSF value; and (3) correcting k air for patient thickness. Finally, patient entrance skin dose (ESD) was calculated by multiplying a mass-energy attenuation coefficient ratio by ESAK. Patient ESD calculations were computed for common DR examinations at our institution: dual view chest, anteroposterior (AP) abdomen, lateral (LAT) skull, dual view knee, and bone age (left hand only) examinations. RESULTS: ESD was calculated for a total of 3794 patients; mean age was 11 ± 8 yr (range: 2 months to 55 yr). The mean ESD range was 0.19-0.42 mGy for dual view chest, 0.28-1.2 mGy for AP abdomen, 0.18-0.65 mGy for LAT view skull, 0.15-0.63 mGy for dual view knee, and 0.10-0.12 mGy for bone age (left hand) examinations. CONCLUSIONS: A methodology combining DICOM header metadata and basic x-ray tube characterization curves was demonstrated. In a regulatory era where patient dose reporting has become increasingly in demand, this methodology will allow a knowledgeable user the means to establish an automatable dose reporting program for DR and perform patient dose related QA testing for digital x-ray imaging.

Platelet transfusion: a clinical practice guideline from the AABB

BACKGROUND: The AABB (formerly, the American Association of Blood Banks) developed this guideline on appropriate use of platelet transfusion in adult patients. METHODS: These guidelines are based on a systematic review of randomized, clinical trials and observational studies (1900 to September 2014) that reported clinical outcomes on patients receiving prophylactic or therapeutic platelet transfusions. An expert panel reviewed the data and developed recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework. RECOMMENDATION 1: The AABB recommends that platelets should be transfused prophylactically to reduce the risk for spontaneous bleeding in hospitalized adult patients with therapy-induced hypoproliferative thrombocytopenia. The AABB recommends transfusing hospitalized adult patients with a platelet count of 10 × 109 cells/L or less to reduce the risk for spontaneous bleeding. The AABB recommends transfusing up to a single apheresis unit or equivalent. Greater doses are not more effective, and lower doses equal to one half of a standard apheresis unit are equally effective. (Grade: strong recommendation; moderate-quality evidence). RECOMMENDATION 2: The AABB suggests prophylactic platelet transfusion for patients having elective central venous catheter placement with a platelet count less than 20 × 109 cells/L. (Grade: weak recommendation; low-quality evidence). RECOMMENDATION 3: The AABB suggests prophylactic platelet transfusion for patients having elective diagnostic lumbar puncture with a platelet count less than 50 × 109 cells/L. (Grade: weak recommendation; very-low-quality evidence). RECOMMENDATION 4: The AABB suggests prophylactic platelet transfusion for patients having major elective nonneuraxial surgery with a platelet count less than 50 × 109 cells/L. (Grade: weak recommendation; very-low-quality evidence). RECOMMENDATION 5: The AABB recommends against routine prophylactic platelet transfusion for patients who are nonthrombocytopenic and have cardiac surgery with cardiopulmonary bypass. The AABB suggests platelet transfusion for patients having bypass who exhibit perioperative bleeding with thrombocytopenia and/or evidence of platelet dysfunction. (Grade: weak recommendation; very-low-quality evidence). RECOMMENDATION 6: The AABB cannot recommend for or against platelet transfusion for patients receiving antiplatelet therapy who have intracranial hemorrhage (traumatic or spontaneous). (Grade: uncertain recommendation; very-low-quality evidence).(AU)

Development of a 96-well plate iodine binding assay for amylose content determination.

Cereal starch amylose/amylopectin (AM/AP) is critical in functional properties for food and industrial applications. Conventional methods of AM/AP are time consuming and labor intensive making it difficult to screen the large sample sets necessary for evaluating breeding samples and investigating environmental impact on starch development. The objective was to adapt and optimize the iodine binding assay in a 96-well plate format for measurement at both λ 620 nm and λ 510 nm. The standard curve for amylose content was scaled to a 96-well plate format and demonstrated R(2) values of 0.999 and 0.993 for single and dual wavelengths, respectively. The plate methods were applicable over large ranges of amylose contents: high amylose maize starch at 61.7±2.3%, normal wheat starch at 29.0±0.74%, and a waxy maize starch at 1.2±0.9%. The method exhibited slightly greater amylose content values than the Concanavalin A method for normal type starches; but is consistent with cuvette scale iodine binding assays.

Cantharidins induce ER stress and a terminal unfolded protein response in OSCC.

Mortality and morbidity associated with oral squamous cell carcinoma (OSCC) remain unacceptably high with disfiguring treatment options and a death rate of 1 per hour in the United States. The approval of cituximab for advanced OSCC has been the only new treatment for these patients since the 1970s, although it has not significantly increased overall survival. To address the paucity of effective new therapies, we undertook a high-throughput screen to discover small molecules and natural products that could induce endoplasmic reticulum (ER) stress and enforce a terminal unfolded protein response (UPR) in OSCC. The terpenoid cantharidin (CNT), previously used to treat various malignancies in culture-specific medical practices for over 2,000 y, emerged as a hit. CNT and its analog, cantharidic acid, potently induced protein and gene expression profiles consistent with the activation of ER stress, the UPR, and apoptosis in OSCC cells. Murine embryonic fibroblasts null for the UPR-associated transcription factors Atf4 or Chop were significantly protected from CNT, implicating a key role for the UPR in the death response. These data validate that our high-throughput screen can identify novel modulators of UPR signaling and that such compounds might provide a new therapeutic approach to treating patients with OSCC.

Effect of nitrogen fertilization and cover cropping systems on sorghum grain characteristics.

Cover crop treatments and nitrogen (N) fertilization rates were investigated for their impact on sorghum grain quality attributes. Sorghum was planted in field plots treated with differing cover cropping systems and fertilization rates. The size (weight and diameter) and hardness of the kernels were influenced by both the cover crop and N rates. The protein content increased as the N rate increased and also with the addition of cover crops to the system. The protein digestibility values and starch granule size distributions were not affected by N rate or the cover cropping treatments. Soil properties were tested to determine relationships with grain quality attributes. The utilization of cover crops appears to increase the protein content without causing a deleterious effect on protein digestibility. The end-product quality is not hampered by the use of beneficial cropping systems necessary for sustainable agriculture.

ER stress signalling through eIF2α and CHOP, but not IRE1α, attenuates adipogenesis in mice.

AIMS/HYPOTHESIS: Although obesity is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in adipose tissue, it is not known how UPR signalling affects adipogenesis. To test whether signalling through protein kinase RNA-like ER kinase/eukaryotic initiation factor 2 alpha (PERK/eIF2α) or inositol-requiring enzyme 1 alpha/X-box binding protein 1 (IRE1α/XBP1) is required for adipogenesis, we studied the role of UPR signalling in adipocyte differentiation in vitro and in vivo in mice. METHODS: The role of UPR signalling in adipogenesis was investigated using 3T3-L1 cells and primary mouse embryonic fibroblasts (MEFs) by activation or inhibition of PERK-mediated phosphorylation of the eIF2α- and IRE1α-mediated splicing of Xbp1 mRNA. Body weight change, fat mass composition and adipocyte number and size were measured in wild-type and genetically engineered mice fed a control or high-fat diet (HFD). RESULTS: ER stress repressed adipocyte differentiation in 3T3-L1 cells. Impaired eIF2α phosphorylation enhanced adipocyte differentiation in MEFs, as well as in mice. In contrast, increased eIF2α phosphorylation reduced adipocyte differentiation in 3T3-L1 cells. Forced production of CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), a downstream target of eIF2α phosphorylation, inhibited adipogenesis in 3T3-L1 cells. Mice with deletion of Chop (also known as Ddit3) (Chop (-/-)) gained more fat mass than wild-type mice on HFD. In addition, Chop deletion in genetically obese Lepr (db/db) mice increased body fat mass without altering adipocyte size. In contrast to the eIF2α-CHOP pathway, activation or deletion of Ire1a (also known as Ern1) did not alter adipocyte differentiation in 3T3-L1 cells. CONCLUSIONS/INTERPRETATION: These results demonstrate that eIF2α-CHOP suppresses adipogenesis and limits expansion of fat mass in vivo in mice, rendering this pathway a potential therapeutic target.

Characterization of adaptive statistical iterative reconstruction algorithm for dose reduction in CT: A pediatric oncology perspective.

PURPOSE: This study demonstrates a means of implementing an adaptive statistical iterative reconstruction (ASiR™) technique for dose reduction in computed tomography (CT) while maintaining similar noise levels in the reconstructed image. The effects of image quality and noise texture were assessed at all implementation levels of ASiR™. Empirically derived dose reduction limits were established for ASiR™ for imaging of the trunk for a pediatric oncology population ranging from 1 yr old through adolescence∕adulthood. METHODS: Image quality was assessed using metrics established by the American College of Radiology (ACR) CT accreditation program. Each image quality metric was tested using the ACR CT phantom with 0%-100% ASiR™ blended with filtered back projection (FBP) reconstructed images. Additionally, the noise power spectrum (NPS) was calculated for three common reconstruction filters of the trunk. The empirically derived limitations on ASiR™ implementation for dose reduction were assessed using (1, 5, 10) yr old and adolescent∕adult anthropomorphic phantoms. To assess dose reduction limits, the phantoms were scanned in increments of increased noise index (decrementing mA using automatic tube current modulation) balanced with ASiR™ reconstruction to maintain noise equivalence of the 0% ASiR™ image. RESULTS: The ASiR™ algorithm did not produce any unfavorable effects on image quality as assessed by ACR criteria. Conversely, low-contrast resolution was found to improve due to the reduction of noise in the reconstructed images. NPS calculations demonstrated that images with lower frequency noise had lower noise variance and coarser graininess at progressively higher percentages of ASiR™ reconstruction; and in spite of the similar magnitudes of noise, the image reconstructed with 50% or more ASiR™ presented a more smoothed appearance than the pre-ASiR™ 100% FBP image. Finally, relative to non-ASiR™ images with 100% of standard dose across the pediatric phantom age spectrum, similar noise levels were obtained in the images at a dose reduction of 48% with 40% ASIR™ and a dose reduction of 82% with 100% ASIR™. CONCLUSIONS: The authors' work was conducted to identify the dose reduction limits of ASiR™ for a pediatric oncology population using automatic tube current modulation. Improvements in noise levels from ASiR™ reconstruction were adapted to provide lower radiation exposure (i.e., lower mA) instead of improved image quality. We have demonstrated for the image quality standards required at our institution, a maximum dose reduction of 82% can be achieved using 100% ASiR™; however, to negate changes in the appearance of reconstructed images using ASiR™ with a medium to low frequency noise preserving reconstruction filter (i.e., standard), 40% ASiR™ was implemented in our clinic for 42%-48% dose reduction at all pediatric ages without a visually perceptible change in image quality or image noise.

Establishing a standard calibration methodology for MOSFET detectors in computed tomography dosimetry.

PURPOSE: The use of metal-oxide-semiconductor field-effect transistor (MOSFET) detectors for patient dosimetry has increased by ~25% since 2005. Despite this increase, no standard calibration methodology has been identified nor calibration uncertainty quantified for the use of MOSFET dosimetry in CT. This work compares three MOSFET calibration methodologies proposed in the literature, and additionally investigates questions relating to optimal time for signal equilibration and exposure levels for maximum calibration precision. METHODS: The calibration methodologies tested were (1) free in-air (FIA) with radiographic x-ray tube, (2) FIA with stationary CT x-ray tube, and (3) within scatter phantom with rotational CT x-ray tube. Each calibration was performed at absorbed dose levels of 10, 23, and 35 mGy. Times of 0 min or 5 min were investigated for signal equilibration before or after signal read out. RESULTS: Calibration precision was measured to be better than 5%-7%, 3%-5%, and 2%-4% for the 10, 23, and 35 mGy respective dose levels, and independent of calibration methodology. No correlation was demonstrated for precision and signal equilibration time when allowing 5 min before or after signal read out. Differences in average calibration coefficients were demonstrated between the FIA with CT calibration methodology 26.7 ± 1.1 mV cGy(-1) versus the CT scatter phantom 29.2 ± 1.0 mV cGy(-1) and FIA with x-ray 29.9 ± 1.1 mV cGy(-1) methodologies. A decrease in MOSFET sensitivity was seen at an average change in read out voltage of ~3000 mV. CONCLUSIONS: The best measured calibration precision was obtained by exposing the MOSFET detectors to 23 mGy. No signal equilibration time is necessary to improve calibration precision. A significant difference between calibration outcomes was demonstrated for FIA with CT compared to the other two methodologies. If the FIA with a CT calibration methodology was used to create calibration coefficients for the eventual use for phantom dosimetry, a measurement error ~12% will be reflected in the dosimetry results. The calibration process must emulate the eventual CT dosimetry process by matching or excluding scatter when calibrating the MOSFETs. Finally, the authors recommend that the MOSFETs are energy calibrated approximately every 2500-3000 mV.

Defining the concept of 'tick repellency' in veterinary medicine.

Although widely used, the term repellency needs to be employed with care when applied to ticks and other periodic or permanent ectoparasites. Repellency has classically been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from its source. However, for crawling arthropods such as ticks, the term commonly subsumes a range of effects that include arthropod irritation and consequent avoiding or leaving the host, failing to attach, to bite, or to feed. The objective of the present article is to highlight the need for clarity, to propose consensus descriptions and methods for the evaluation of various effects on ticks caused by chemical substances.

Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop.

BACKGROUND: Heterologous expression of factor VIII (FVIII) is about two to three orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels. OBJECTIVE: Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study. METHODS AND RESULTS: Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS-1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2-fold. Finally, the addition of the A1-domain mutation, F309S, in conjunction with the disulfide mutation had an additive effect, resulting in a net improvement in secretion of between 35 and 45-fold higher than wild-type FVIII in CHO cells. CONCLUSION: Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII and contribute toward low-cost factor replacement therapy for hemophilia A.