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Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression. - Biotechnology progress
N-Glycans of human proteins possess both Î±2,6- and Î±2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have Î±2,3-linkage due to the absence of Î±2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds Î±2,6-linked SA. The presence of Î±2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(Â®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of Î±2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have Î±2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.Â© 2015 American Institute of Chemical Engineers.
Overexpression of Serpinb1 in Chinese hamster ovary cells increases recombinant IgG productivity. - Journal of biotechnology
We report the discovery and validation of a novel CHO cell engineering target for improving IgG expression, serpin peptidase inhibitor, clade B, member 1 (Serpinb1). Transcriptomic studies using microarrays revealed that Serpinb1 was up-regulated in cultures with IgG heavy and light chain transcription transiently repressed compared with cultures treated with non-targeting siRNA. As proof of concept, a lentiviral vector was employed to overexpress the Chinese Hamster Serpinb1 in a CHOZN(Â®) Glutamine Synthetase (-/-) recombinant IgG producing CHO line. The lentiviral stable pool demonstrated 4.2-fold SERPINB1 overexpression compared with the non-transduced control. The peak viable cell density (VCD) and peak IgG volumetric productivity of the lentiviral stable pool increased 1.3 and 2.0 fold, respectively, compared with the non-transduced control. For host cell engineering, a plasmid encoding SERPINB1 was transfected into the CHOZN(Â®) GS (-/-) host cell line to create several stable pools. Single-cell clones isolated from the pools were characterized for their SERPINB1 expression levels and growth. The clone (SERPINB1_OE_27) with the highest SERPINB1 expression had decreased peak viable cell density and exponential phase growth rate. Selected SERPINB1 OE clones were subsequently evaluated for their IgG expression capabilities using GS selection. Clone SERPINB1_OE_42 with moderate SERPINB1 overexpression demonstrated increased IgG productivity in "bulk" selection. We conclude that manipulating Serpinb1 expression can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone and by overexpression level. This work represents the ongoing effort in applying "-omics" findings to novel CHO host cell line engineering.Copyright Â© 2014 Elsevier B.V. All rights reserved.
[Formula: see text]evidence of neuropsychological dysfunction in Stevens-Johnson Syndrome and toxic epidermal necrolysis: case report. - The Clinical neuropsychologist
Stevens-Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) is a potentially life-threatening critical illness affecting multiple organ systems including the peripheral and central nervous system. This case report involves a young man who was diagnosed with SJS/TEN at age 16 and underwent neuropsychological assessment at age 21. Results indicate a diffuse pattern of cerebral compromise and represent a decline from premorbid level of functioning. The etiology of the cognitive impairment in this patient is likely multifactorial with possible pathophysiologic mechanisms including hypoxemia, metabolic acid-base perturbations, hyperglycemia, and delirium, as well as sepsis and inflammation.
Use of a morphometric method and body fat index system for estimation of body composition in overweight and obese cats. - Journal of the American Veterinary Medical Association
To develop morphometric equations for prediction of body composition and create a body fat index (BFI) system to estimate body fat percentage in overweight and obese cats.Prospective evaluation study.76 overweight or obese cats â‰¥ 1 year of age.Body condition score (BCS) was determined with a 5-point scale, morphometric measurements were made, and dual-energy x-ray absorptiometry (DEXA) was performed. Visual and palpation-based evaluation of various body regions was conducted, and results were used for development of the BFI system. Best-fit multiple regression models were used to develop equations for predicting lean body mass and fat mass from morphometric measurements. Predicted values for body composition components were compared with DEXA results.For the study population, prediction equations accounted for 85% of the variation in lean body mass and 98% of the variation in fat mass. Values derived from morphometric equations for fat mass and lean mass were within 10% of DEXA values for 55 of 76 (72%) and 66 of 76 (87%) cats, respectively. Body fat as a percentage of total body weight (ie, body fat percentage) predicted with the BCS and BFI was within 10% of the DEXA value for 5 of 39 (13%) and 22 of 39 (56%) cats, respectively.The BFI system and morphometric equations were considered accurate for estimation of body composition components in overweight and obese cats of the study population and appeared to be more useful than BCS for evaluation of these patients. Further research is needed to validate the use of these methods in other feline populations.
Use of a novel morphometric method and body fat index system for estimation of body composition in overweight and obese dogs. - Journal of the American Veterinary Medical Association
To develop morphometric equations for prediction of body composition and create a body fat index (BFI) to estimate body fat percentage in overweight and obese dogs.Prospective evaluation study.83 overweight or obese dogs â‰¥ 1 year of age.Body condition score (BCS) was assessed on a 5-point scale, morphometric measurements were made, and visual and palpation-based assessments and dual-energy x-ray absorptiometry (DEXA) were performed. Equations for predicting lean body mass, fat mass, and body fat as a percentage of total body weight (ie, body fat percentage) on the basis of morphometric measurements were generated with best-fit statistical models. Visual and palpation-based descriptors were used to develop a BFI. Predicted values for body composition components were compared with DEXA-measured values.For the study population, the developed morphometric equations accounted for 98% of the variation in lean body mass and fat mass and 82% of the variation in body fat percentage. The proportion of dogs with predicted values within 10% of the DEXA values was 66 of 83 (80%) for lean body mass, 56 of 83 (68%) for fat mass, and 56 of 83 (67%) for body fat percentage. The BFI accurately predicted body fat percentage in 25 of 47 (53%) dogs, whereas the value predicted with BCS was accurate in 6 of 47 (13%) dogs.Morphometric measurements and the BFI appeared to be more accurate than the 5-point BCS method for estimation of body fat percentage in overweight and obese dogs. Further research is needed to assess the applicability of these findings to other populations of dogs.
Development of the MMPI-2 cognitive complaints scale as an embedded measure of symptom validity. - Brain injury
To empirically-derive a new MMPI-2 sub-scale, the 13-item Cognitive Complaints Scale (CCS), as an embedded measure of symptom validity. This study hypothesized that mild traumatic brain injured subjects with financial incentives who failed performance validity tests (PVTs) would score significantly higher on the CCS compared to mild traumatic brain injured subjects with financial incentives who passed PVTs. Mild traumatic brain injured controls with no financial incentives were predicted to score lowest on the CCS.A known groups design was utilized as this design allows for the accurate classification of criterion groups.One hundred and fifty mild head-injured adults were assigned to one of three groups: the Failed Performance Validity (FPV) group, the Passed Performance Validity (PPV) group or a control group.An ANOVA revealed that the FPV group scored significantly higher on the CCS compared to the PPV group. Controls with no financial incentives scored lowest. A CCS cut-score of â‰¥12 discriminated between the FPV and PPV groups at a high level of specificity (94%).A dissociation between 'performance validity' and 'symptom validity' supports recommendations for the differential application of these descriptive terms. The CCS may be helpful to assess credibility of clinical presentation in situations where PVTs are absent.
Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1). - Journal of biotechnology
While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production.Copyright Â© 2013 Elsevier B.V. All rights reserved.
Use of the color trails test as an embedded measure of performance validity. - The Clinical neuropsychologist
One hundred personal injury litigants and disability claimants referred for a forensic neuropsychological evaluation were administered both portions of the Color Trails Test (CTT) as part of a more comprehensive battery of standardized tests. Subjects who failed two or more free-standing tests of cognitive performance validity formed the Failed Performance Validity (FPV) group, while subjects who passed all free-standing performance validity measures were assigned to the Passed Performance Validity (PPV) group. A cutscore of â‰¥45 seconds to complete Color Trails 1 (CT1) was associated with a classification accuracy of 78%, good sensitivity (66%) and high specificity (90%), while a cutscore of â‰¥84 seconds to complete Color Trails 2 (CT2) was associated with a classification accuracy of 82%, good sensitivity (74%) and high specificity (90%). A CT1 cutscore of â‰¥58 seconds, and a CT2 cutscore â‰¥100 seconds was associated with 100% positive predictive power at base rates from 20 to 50%.
Magnetic resonance imaging of the canine abdomen: effect of pulse sequence on diagnostic quality. - Veterinary radiology & ultrasound : the official journal of the American College of Veterinary Radiology and the International Veterinary Radiology Association
Motion artifact is an important limiting factor for abdominal magnetic resonance imaging (MRI) in veterinary patients. The purpose of this study was to determine the effects of pulse sequence on abdominal MRI diagnostic quality in dogs. Ten normal dogs were each scanned using 16 MRI pulse sequences. Sequences included breath-holding sequences, respiratory navigation sequences, and traditional spin-echo sequences. Four observers independently scored diagnostic quality for each sequence based on the appearance of specific organs, overall diagnostic quality, and degree of artifactual interference. Signal-to-noise ratio and contrast-to-noise ratio were also calculated for each sequence. The sequence with the highest overall mean diagnostic quality score was the dorsal T2 turbo spin echo (TSE) with fat saturation and breath-holding. The sequence with the lowest mean diagnostic quality score was the dorsal T2 fast spin echo. The sequence with the highest signal-to-noise ratio for all evaluated organs was the sagittal T1 spin echo. Signal-to-noise and contrast-to-noise ratios did not correlate with subjective assessment of overall diagnostic quality for the majority of the sequences evaluated (P < 0.05). The three sequences considered to have the highest diagnostic quality for the cranial abdomen were the dorsal T2 TSE with fat saturation and breath-hold, transverse T1 turbo fast low-angle shot gradient echo with breath-hold, and dorsal T2 half-Fourier acquisition single shot TSE with respiratory navigation. These sequences had short acquisition times, yielded studies of similar diagnostic quality, provided complementary information, and are therefore recommended for routine canine abdominal MRI protocols.Â© 2013 Veterinary Radiology & Ultrasound.
Derivation of the MMPI-2-RF Henry-Heilbronner Index-r (HHI-r) scale. - The Clinical neuropsychologist
The 15-item Henry-Heilbronner Index (HHI) was published in 2006 as an MMPI-2 embedded measure of psychological response validity. When the MMPI-2 was revised in 2008 only 11 of the 15 original HHI items were retained on the MMPI-2-RF, prohibiting use of the HHI as an embedded validity indicator on the MMPI-2-RF. Using the original HHI sample an 11-item version of the HHI, the HHI-r, was evaluated for use as an embedded measure of psychological response validity for the MMPI-2-RF. The 11-item HHI-r was very similar to the HHI in classification accuracy. An HHI-r cutoff score of â‰¥7 was associated with a classification accuracy rate of 84.0%, good sensitivity (68.9%), and high specificity (93.2%) in identifying symptom exaggeration in personal injury and disability litigants versus non-litigating head-injured patients. These preliminary results suggest the HHI-r functions in a manner similar to the original HHI as a measure of psychological response validity, and may be used by psychologists and neuropsychologists as an MMPI-2-RF embedded validity indicator.
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