Highly multiplexed label-free imaging sensor for accurate quantification of small-molecule binding kinetics

Investigating the binding interaction of small molecules to large ligands is a compelling task for the field of drug development, as well as agro-biotechnology, since a common trait of drugs and toxins is often a low molecular weight (MW). Here, we improve the limit of detection of the Interferometric Reflectance Imaging Sensor (IRIS), a label-free, highly multiplexed biosensor, to perform small-molecule screening. In this work, characterization of small molecules binding to immobilized probes in a microarray format is demonstrated, with a limit of detection of 1 pg/mm2 in mass density. First, as a proof of concept to show the impact of spatial and temporal averaging on the system noise, detection of biotin (MW = 244.3 Da) binding to a streptavidin-functionalized chip is performed and the parameters are tuned to achieve maximum signal-to-noise ratio (SNR ≈ 34). The optimized system is then applied to the screening of a 20-multiplexed antibody chip against fumonisin B1 (MW = 721.8 Da), a mycotoxin found in cereal grains. The simultaneously recorded binding curves yield an SNR ≈ 8. Five out of twenty antibodies are also screened against the toxin in a lateral flow assay, obtaining consistent results. With the demonstrated noise characteristics, further sensitivity improvements are expected with the advancement of camera sensor technology.Published versio

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Published online in Wiley InterScience, 23 January 2001Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-beta1, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TGF-beta1 and IL-1beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TGF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the notion of distinct functions for these cell-surface proteoglycans.W. Worapamorn, H.R. Haase, H. Li, and P.M. Bartol

Acute molecular response of mouse hindlimb muscles to chronic stimulation

Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 ± 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T3 and I, α-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1β/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-γ coactivator-1α and -β, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1α) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A2, B2, C, and E1 and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs

Liver mTOR Controls IGF-I Bioavailability by Regulation of Protein Kinase CK2 and IGFBP-1 Phosphorylation in Fetal Growth Restriction

Fetal growth restriction (FGR) increases the risk for perinatal complications and predisposes the infant to diabetes and cardiovascular disease later in life. No treatment for FGR is available, and the underlying pathophysiology remains poorly understood. Increased IGFBP-1 phosphorylation has been implicated as an important mechanism by which fetal growth is reduced. However, to what extent circulating IGFBP-1 is phosphorylated in FGR is unknown, and the molecular mechanisms linking FGR to IGFBP-1 phosphorylation have not been established. We used umbilical cord plasma of appropriate for gestational age (AGA) and growth–restricted human fetuses and determined IGFBP-1 and IGF-I concentrations (ELISA) and site-specific IGFBP-1 phosphorylation (Western blotting using IGFBP-1 phospho-site specific antibodies). In addition, we used a baboon model of FGR produced by 30% maternal nutrient restriction and determined mammalian target of rapamycin (mTOR)C1 activity, CK2 expression/activity, IGFBP-1 expression and phosphorylation, and IGF-I levels in baboon fetal liver by Western blot, enzymatic assay, and ELISA. HepG2 cells and primary fetal baboon hepatocytes were used to explore mechanistic links between mTORC1 signaling and IGFBP-1 phosphorylation. IGFBP-1 was hyperphosphorylated at Ser101, Ser119, and Ser169 in umbilical plasma of human FGR fetuses. IGFBP-1 was also hyperphosphorylated at Ser101, Ser119, and Ser169 in the liver of growth–restricted baboon fetus. mTOR signaling was markedly inhibited, whereas expression and activity of CK2 was increased in growth–restricted baboon fetal liver in vivo. Using HepG2 cells and primary fetal baboon hepatocytes, we established a mechanistic link between mTOR inhibition, CK2 activation, IGFBP-1 hyperphosphorylation, and decreased IGF-I–induced IGF-I receptor autophosphorylation. We provide clear evidence for IGFBP-1 hyperphosphorylation in FGR and identified an mTOR and CK2-mediated mechanism for regulation of IGF-I bioavailability. Our findings are consistent with the model that inhibition of mTOR in the fetal liver, resulting in increased CK2 activity and IGFBP-1 hyperphosphorylation, constitutes a novel mechanistic link between nutrient deprivation and restricted fetal growth