SJ Shih - Academia.edu (original) (raw)

Papers by SJ Shih

Journal of Microscopy, Feb 1, 1991

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupl... more Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field-emission gun to a low-aberration immersion lens and the availability of a high-stability cryo-transfer stage, make low-temperature, lowvoltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens. The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room tempera

Journal of Cell Science, Sep 1, 1991

Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their pro... more Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (M r 14-25X10 3), acidic (pi 4.4-5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5-5) showed a relatively wide range of pi values, and existed in xtmx as disuifide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group II proteins (defined by mAb B4-4 and B3-5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electrondense striations with 55 nm intervals. However, these regions were not accessible to antibody B4-4 in ctmx. Group III proteins (defined by mAb B7-4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups III and IV were localized in the core of ctmx, but were distributed uniformly in xtmx. Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx.

Journal of Cell Science, Oct 1, 1992

BioProcessing Journal, Feb 10, 2011

maps obtained from the animal fed with high-fat-diets. (B) MRI sagittal image highlighting the an... more maps obtained from the animal fed with high-fat-diets. (B) MRI sagittal image highlighting the anatomical locations of liver fat imaging slices. ROIs (n=8) sampling was covered across the entire liver, where each ROI has been placed at every liver segment according to the Couinaud liver segmentation: segment 1 located at the caudate lobe and segment 7 and 6 are posterior to the segment 8 and 5, respectively (RPV= right portal vein; LPV= left portal vein. Figure 2 (right) Liver fat fraction (FF) and plasma total cholesterol (TC) (mean ± SD measured from control dietand high fat-diet (HFD)-fed animals. 5431 Evaluation of Hepatic Fat Fraction Measured by MRI and Plasma Lipoprotein Levels in High-Fat Diet Fed Non-Human Primate Ai Leng Liang, Catherine D. G. Hines, Li Chun Huang, Shian-Jiun Shih, Donald S. Williams, Elaine Manigbas, Brian Henry, Jeffrey L. Evelhoch, and ChihLiang Chin Translational Medicine Research Centre, MSD, Singapore, Singapore, Imaging, Merck & Co. Inc., West Point...

Journal of Cell Science, 1992

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unproce... more We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precurso...

BioProcessing Journal, 2011

Proceedings of the National Academy of Sciences, 1994

Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we exam... more Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we examine whether the activation of Xenopus p42 MAP kinase might involve changes in its association with other proteins as well as changes in its phosphorylation state. We find that when p42 MAP kinase is phosphorylated and active, it is monomeric, and that when p42 MAP kinase is nonphosphorylated and inactive, about half of it is monomeric and half is a component of a 110-kDa complex. We identify Rsk, an 82-kDa protein kinase that can be phosphorylated and partially activated by p42 MAP kinase, as being specifically associated with inactive p42 MAP kinase. It is possible that the complex of inactive p42 MAP kinase and inactive Rsk acts as a single signal reception particle and that the activation of the two kinases may be better described as a fork in a bifurcating signal transduction pathway than as successive levels in a kinase cascade.

Journal of Microscopy, 1991

SUMMARYRecent advances in the design of the scanning electron microscope (SEM) column, such as th... more SUMMARYRecent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field‐emission gun to a low‐aberration immersion lens and the availability of a high‐stability cryo‐transfer stage, make low‐temperature, low‐voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens.The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze‐drying. High‐magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze‐substitution and critical‐point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.

Biologicals, 2010

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viabili... more A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-toinstrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13 Â 10 5 to approximately 1.0 Â 10 7 cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3-106.4% recovery) and precision (RSD < 5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.

Biologicals, 2010

GVAX Ò immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer... more GVAX Ò immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-tolot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

Scientific Reports, Feb 24, 2021

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that c... more Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic tran... more Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans

Scientific Reports, 2021

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that c... more Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

Future Science OA, 2017

Aim: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 th... more Aim: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 therapy in multiple cancers. However, obtaining tumor biopsies for PD-L1 interrogation is an invasive procedure and challenging to assess repeatedly as the disease progresses. Materials & methods: Here we assess an alternative, minimally invasive approach to analyze blood samples for circulating tumor cells (CTCs) that have broken away from the tumor and entered the periphery. Our approach uses sized-based microfluidic CTC enrichment and subsequent characterization with microfluidic-based cytometry (chipcytometry). Conclusion: We demonstrate tumor-cell detection and characterization for PD-L1, and other markers, in both spiked and patient samples. This preliminary communication is the first report using chipcytometry for the characterization of CTCs.

Atherosclerosis, 2015

The greater genomic conservation between humans and non-human primates (NHP) enables target valid... more The greater genomic conservation between humans and non-human primates (NHP) enables target validation studies for developing of therapeutic strategies for human diseases. Together with predicting activity and potential adverse clinical signs, the inclusion of NHP testing bequeaths to efficacy models for dose titration and pharmacodynamic effects. We have used lipid nanoparticle encapsulated siRNA to silence ApoB in the liver and assessed the phenotypic effects on serum lipids with various levels of hepatic ApoB mRNA knockdown in healthy lean cynomolgus monkeys. ApoB siRNA dosed animals demonstrated significant reductions of hepatic ApoB mRNA and serum APOB protein, with a substantial lowering of plasma lipid levels without obvious signs of toxicity. Microarray based assessment of ApoB siRNA mediated effects revealed a number of differentially expressed genes which mapped onto biological pathways and processes related to lipid and cholesterol metabolism. Furthermore, we identified potential targets and cellular effects that could be studied for therapeutic benchmarking of APOB mediated effects. The network of ApoB regulated genes should be of significance for the understanding and development of novel hypercholesterolemia therapies.

Atherosclerosis, 2013

To assess the lipid-lowering efficacy of ezetimibe in dyslipidemic cynomolgus monkeys comparing t... more To assess the lipid-lowering efficacy of ezetimibe in dyslipidemic cynomolgus monkeys comparing two dosing methods, and to evaluate PCSK9 plasma levels during dyslipidemia induction by feeding a high-fat/high-cholesterol diet (HFD), ezetimibe (Zetia(®), Ezetrol(®)) treatment, ezetimibe washout, and HFD washout. Twenty dyslipidemic cynomolgus monkeys on HFD for seven months (LDL cholesterol 100-400 mg/dL) were randomized into two groups and treated with ezetimibe for two weeks, either by oral gavage or by using food treats. The lipid-lowering effects of ezetimibe were identical between the two groups. After treatment, mean LDL cholesterol was decreased by 58% (174-72 mg/dL), total cholesterol by 42% (241-138 mg/dL), and PCSK9 levels were increased by 137% (147-314 ng/mL). PCSK9 levels on regular diet before and after HFD were also inversely correlated to LDL cholesterol. In a cynomolgus dyslipidemia model, PCSK9 levels are inversely correlated with LDL cholesterol in the absence of statin treatment, regardless whether lipid changes are modulated by diet or ezetimibe treatment.

Journal of Cell Science, 1991

Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their pro... more Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14–25×103), acidic (pI4.4–5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5–5) showed a relatively wide range of pl values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condense...

Drug metabolism and disposition: the biological fate of chemicals, Jan 26, 2015

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can... more Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys endogenous OATP1B substrates potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the eighteen biomarkers tested, RIF (18mg/kg, PO) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate if cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we dete...

Journal of Microscopy, Feb 1, 1991

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupl... more Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field-emission gun to a low-aberration immersion lens and the availability of a high-stability cryo-transfer stage, make low-temperature, lowvoltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens. The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room tempera

Journal of Cell Science, Sep 1, 1991

Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their pro... more Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (M r 14-25X10 3), acidic (pi 4.4-5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5-5) showed a relatively wide range of pi values, and existed in xtmx as disuifide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group II proteins (defined by mAb B4-4 and B3-5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electrondense striations with 55 nm intervals. However, these regions were not accessible to antibody B4-4 in ctmx. Group III proteins (defined by mAb B7-4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups III and IV were localized in the core of ctmx, but were distributed uniformly in xtmx. Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx.

Journal of Cell Science, Oct 1, 1992

BioProcessing Journal, Feb 10, 2011

maps obtained from the animal fed with high-fat-diets. (B) MRI sagittal image highlighting the an... more maps obtained from the animal fed with high-fat-diets. (B) MRI sagittal image highlighting the anatomical locations of liver fat imaging slices. ROIs (n=8) sampling was covered across the entire liver, where each ROI has been placed at every liver segment according to the Couinaud liver segmentation: segment 1 located at the caudate lobe and segment 7 and 6 are posterior to the segment 8 and 5, respectively (RPV= right portal vein; LPV= left portal vein. Figure 2 (right) Liver fat fraction (FF) and plasma total cholesterol (TC) (mean ± SD measured from control dietand high fat-diet (HFD)-fed animals. 5431 Evaluation of Hepatic Fat Fraction Measured by MRI and Plasma Lipoprotein Levels in High-Fat Diet Fed Non-Human Primate Ai Leng Liang, Catherine D. G. Hines, Li Chun Huang, Shian-Jiun Shih, Donald S. Williams, Elaine Manigbas, Brian Henry, Jeffrey L. Evelhoch, and ChihLiang Chin Translational Medicine Research Centre, MSD, Singapore, Singapore, Imaging, Merck & Co. Inc., West Point...

Journal of Cell Science, 1992

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unproce... more We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precurso...

BioProcessing Journal, 2011

Proceedings of the National Academy of Sciences, 1994

Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we exam... more Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we examine whether the activation of Xenopus p42 MAP kinase might involve changes in its association with other proteins as well as changes in its phosphorylation state. We find that when p42 MAP kinase is phosphorylated and active, it is monomeric, and that when p42 MAP kinase is nonphosphorylated and inactive, about half of it is monomeric and half is a component of a 110-kDa complex. We identify Rsk, an 82-kDa protein kinase that can be phosphorylated and partially activated by p42 MAP kinase, as being specifically associated with inactive p42 MAP kinase. It is possible that the complex of inactive p42 MAP kinase and inactive Rsk acts as a single signal reception particle and that the activation of the two kinases may be better described as a fork in a bifurcating signal transduction pathway than as successive levels in a kinase cascade.

Journal of Microscopy, 1991

SUMMARYRecent advances in the design of the scanning electron microscope (SEM) column, such as th... more SUMMARYRecent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field‐emission gun to a low‐aberration immersion lens and the availability of a high‐stability cryo‐transfer stage, make low‐temperature, low‐voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens.The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze‐drying. High‐magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze‐substitution and critical‐point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.

Biologicals, 2010

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viabili... more A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-toinstrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13 Â 10 5 to approximately 1.0 Â 10 7 cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3-106.4% recovery) and precision (RSD < 5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.

Biologicals, 2010

GVAX Ò immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer... more GVAX Ò immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-tolot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

Scientific Reports, Feb 24, 2021

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that c... more Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic tran... more Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans

Scientific Reports, 2021

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that c... more Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

Future Science OA, 2017

Aim: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 th... more Aim: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 therapy in multiple cancers. However, obtaining tumor biopsies for PD-L1 interrogation is an invasive procedure and challenging to assess repeatedly as the disease progresses. Materials & methods: Here we assess an alternative, minimally invasive approach to analyze blood samples for circulating tumor cells (CTCs) that have broken away from the tumor and entered the periphery. Our approach uses sized-based microfluidic CTC enrichment and subsequent characterization with microfluidic-based cytometry (chipcytometry). Conclusion: We demonstrate tumor-cell detection and characterization for PD-L1, and other markers, in both spiked and patient samples. This preliminary communication is the first report using chipcytometry for the characterization of CTCs.

Atherosclerosis, 2015

The greater genomic conservation between humans and non-human primates (NHP) enables target valid... more The greater genomic conservation between humans and non-human primates (NHP) enables target validation studies for developing of therapeutic strategies for human diseases. Together with predicting activity and potential adverse clinical signs, the inclusion of NHP testing bequeaths to efficacy models for dose titration and pharmacodynamic effects. We have used lipid nanoparticle encapsulated siRNA to silence ApoB in the liver and assessed the phenotypic effects on serum lipids with various levels of hepatic ApoB mRNA knockdown in healthy lean cynomolgus monkeys. ApoB siRNA dosed animals demonstrated significant reductions of hepatic ApoB mRNA and serum APOB protein, with a substantial lowering of plasma lipid levels without obvious signs of toxicity. Microarray based assessment of ApoB siRNA mediated effects revealed a number of differentially expressed genes which mapped onto biological pathways and processes related to lipid and cholesterol metabolism. Furthermore, we identified potential targets and cellular effects that could be studied for therapeutic benchmarking of APOB mediated effects. The network of ApoB regulated genes should be of significance for the understanding and development of novel hypercholesterolemia therapies.

Atherosclerosis, 2013

To assess the lipid-lowering efficacy of ezetimibe in dyslipidemic cynomolgus monkeys comparing t... more To assess the lipid-lowering efficacy of ezetimibe in dyslipidemic cynomolgus monkeys comparing two dosing methods, and to evaluate PCSK9 plasma levels during dyslipidemia induction by feeding a high-fat/high-cholesterol diet (HFD), ezetimibe (Zetia(®), Ezetrol(®)) treatment, ezetimibe washout, and HFD washout. Twenty dyslipidemic cynomolgus monkeys on HFD for seven months (LDL cholesterol 100-400 mg/dL) were randomized into two groups and treated with ezetimibe for two weeks, either by oral gavage or by using food treats. The lipid-lowering effects of ezetimibe were identical between the two groups. After treatment, mean LDL cholesterol was decreased by 58% (174-72 mg/dL), total cholesterol by 42% (241-138 mg/dL), and PCSK9 levels were increased by 137% (147-314 ng/mL). PCSK9 levels on regular diet before and after HFD were also inversely correlated to LDL cholesterol. In a cynomolgus dyslipidemia model, PCSK9 levels are inversely correlated with LDL cholesterol in the absence of statin treatment, regardless whether lipid changes are modulated by diet or ezetimibe treatment.

Journal of Cell Science, 1991

Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their pro... more Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14–25×103), acidic (pI4.4–5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5–5) showed a relatively wide range of pl values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condense...

Drug metabolism and disposition: the biological fate of chemicals, Jan 26, 2015

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can... more Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys endogenous OATP1B substrates potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the eighteen biomarkers tested, RIF (18mg/kg, PO) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate if cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we dete...