We developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the identification of potential biomarkers in prostate cancer serum. Protein abundances from 33 prostate cancer and 20 control serum samples were compared to abundances from a common reference pool using a two-color fluorescence assay. Robotically spotted microarrays containing 184 unique antibodies were prepared on two different substrates: polyacrylamide based hydrogels on glass and poly-1-lysine coated glass with a photoreactive cross-linking layer. The hydrogel substrate yielded an average six-fold higher signal-to-noise ratio than the other substrate, and detection of protein binding was possible from a greater number of antibodies using the hydrogels. A statistical filter based on the correlation of data from "reverse-labeled" experiment sets accurately predicted the agreement between the microarray measurements and enzyme-linked immunosorbent assay measurements, showing that this parameter can serve to screen for antibodies that are functional on microarrays. Having defined a set of reliable microarray measurements, we identified five proteins (von Willebrand Factor, immunoglobulinM, Alpha1-antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between the prostate cancer samples and the controls. These developments enable the immediate use of high-density antibody and protein microarrays in biomarker discovery studies.

Quantitative computed tomography analysis for stratifying the severity of Abstract Purpose: To examine the feasibility of using a computer tool for stratifying the severity of Coronavirus Disease 2019 (COVID-19) based on computed tomography (CT) images. Materials and methods: We retrospectively examined 44 confirmed COVID-19 cases. All cases were evaluated separately by radiologists (visually) andthrough an in-house computer software. The degree of lesions was visually scored by the radiologist, as follows, for each of the 5 lung lobes: 0, no lesion present; 1, < 1/3 involvement; 2, >1/3 and <2/3 involvement; and 3, > 2/3 involvement. Lesion density was assessed based on the proportion of ground-glass opacity (GGO), consolidation and fibrosis of the lesions. The parameters obtained using the computer tool included lung volume (mL), lesion volume (mL), lesion percentage (%), and mean lesion density (HU) of the whole lung, right lung, left lung, and each lobe. The scores obtained by the radiologists and quantitative results generated by the computer software were tested for correlation. A Chi-square test was used to test the consistency of radiologist-and computer-derived lesion percentage in the right/left lung, upper/lower lobe, and each of the 5 lobes. Result: The results showed a strong to moderate correlation between lesion percentage scores obtained by radiologists and the computer software (r ranged from 0.7679 to 0.8373, P< 0.05), and a moderate correlation between the proportion of GGO and mean lesion density (r = -0.5894, P < 0.05), and proportion of consolidation and mean lesion density (r = 0.6282, P < 0.05).Computer-aided quantification showed a statistical significant higher lesion percentage for lower lobes than that assessed by the radiologists (χ 2 =8.160, P = 0.004). Conclusions: Our experiments demonstrated that the computer tool could reliably and accurately assess the severity and distribution of pneumonia on CT scans.

The winged helix transcription factor, hepatocyte nuclear factor-3␤ (HNF-3␤), mediates the hepatocytespecific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3␤ in regulating these genes remains unknown because homozygous null HNF3␤ mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3␤, we created transgenic mice in which the ؊3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3␤ protein.Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3␤ on the hepatic expression of the endogenous mouse HNF-3␣,-3␤, -3␥, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3␤ transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.The liver performs essential functions in the body by uniquely expressing both hepatocyte-specific genes encoding plasma proteins and enzymes involved in the detoxification and in the homeostasis of glucose, cholesterol, and bile salts (4). Functional analysis of numerous hepatocyte-specific promoter and enhancer regions reveals that they are composed of multiple cis-acting DNA sequences that bind different families of hepatocyte nuclear factors (HNF) (reviewed in reference 4). These include the HNF-1, HNF-3, HNF-4, CCAAT/enhancer binding protein (C/EBP), HNF-6, and fetoprotein transcription factor families (4,29,15,52,53,57). Although none of these transcriptional regulatory proteins is entirely liver specific, the requirement for combinatorial protein interactions among them in order to achieve abundant transcriptional activity plays an important role in maintaining hepatocyte-specific gene expression.The HNF-3 proteins are members of an extensive family of transcription factors that share homology in the winged helix DNA binding domain and use a modified helix-turn-helix motif to bind DNA as a monomer (8, 37). To date, the winged helix family consists of over 50 members, which play important roles in the differentiat...

The hepatocyte nuclear factor 3/fork head homolog (HFH) proteins are an extensive family of transcription factors which share homology in the winged helix DNA binding domain. Members of the winged helix family have been implicated in cell fate determination during pattern formation, in organogenesis and in cell type-specific gene expression. In this study, we used in situ hybridization to identify the cellular expression pattern of the winged helix transcription factor, HFH-8, during mouse embryonic development. We showed that HFH-8 expression initiates during the primitive streak stage of mouse embryogenesis in the extraembryonic mesoderm and in the lateral mesoderm which gives rise to the somatopleuric and splanchnopleuric mesoderm. During organogenesis, HFH-8 expression is found in the splanchnic mesoderm in close apposition of the gut endoderm, suggesting a role in mesenchymal-epithelial induction of lung and gut morphogenesis. HFH-8 expression continues in lateral mesoderm-derived tissue throughout mouse development. HFH-8 expression is observed in the mesenchymal cells of the oral cavity, esophagus, trachea, lung, intestine, dorsal aorta and intersomitic arteries, but not in the vasculature of the head, liver, kidney or heart. Consistent with these embryonic expression studies, adult HFH-8 expression is restricted to the endothelium and connective fibroblasts of the alveolar sac and in the lamina propria and smooth muscle of the intestine. We also show that several adult endothelial cell lines maintain abundant HFH-8 expression. Furthermore, we used our determined HFH-8 consensus sequence to identify putative target genes expressed in pulmonary and intestinal mesenchymal cells. Cotransfection assays with one of these target promoters, P-selectin, demonstrated that HFH-8 expression was required for IL-6 stimulation of P-selectin promoter activity and suggest that HFH-8 is involved in mediating its cell-specific transcriptional activation in response to cytokines.

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