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Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614–642 and 828–854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

The B7 family represents one of the best-studied subgroups within the Ig superfamily, yet new interactions continue to be discovered. However, this binding promiscuity represents a major challenge for defining the biological contribution of each specific interaction. We developed a strategy for addressing these challenges by combining cell microarray and high-throughput FACS methods to screen for promiscuous binding events, map binding interfaces, and generate functionally selective reagents. Applying this approach to the interactions of mPD-L1 with its receptor mPD-1 and its ligand mB7-1, we identified the binding interface of mB7-1 on mPD-L1 and as a result generated mPD-L1 mutants with binding selectivity for mB7-1 or mPD-1. Next, using a panel of mB7-1 mutants, we mapped the binding sites of mCTLA-4, mCD28 and mPD-L1. Surprisingly, the mPD-L1 binding site mapped to the dimer interface surface of mB7-1, placing it distal from the CTLA-4/CD28 recognition surface. Using two independent approaches, we demonstrated that mPD-L1 and mB7-1 bind in cis, consistent with recent reports from Chaudhri A et al. and Sugiura D et al. We further provide evidence that while CTLA-4 and CD28 do not directly compete with PD-L1 for binding to B7-1, they can disrupt the cis PD-L1:B7-1 complex by reorganizing B7-1 on the cell surface. These observations offer new functional insights into the regulatory mechanisms associated with this group of B7 family proteins and provide new tools to elucidate their function in vitro and in vivo.

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample’s response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

Transforming growth factor-beta / bone morphogenetic protein (TGFβ/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFβ ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR-23b cluster miRNAs comprising miR-23b, miR-27b, and miR-24-1 and miR-10a, miR-26a, and miR-30a as up-regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR-23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR-23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFβ signaling molecules, as putative miR-23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3′ UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down-regulation of constructs containing Smad 3, 4, or 5, 3′ UTRs by a mixture of miR-23b cluster mimics. Knockdown of miR-23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC-3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR-23b mimics during bile duct differentiation of HBC-3 cells blocked the process. Conclusion: Our data provide a model in which miR-23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down-regulating Smads and consequently TGFβ signaling. Concomitantly, low levels of the miR-23b miRNAs are needed in cholangiocytes to allow TGFβ signaling and bile duct formation. (HEPATOLOGY 2009.)

Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RnA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RnA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.

Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.

Many phytochemicals possess anti-oxidant and cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. However, in studies to date, most candidate agents are not active in human lung cells. In vitro and in vivo screening studies for discovery are clearly needed for new, more potent chemopreventive agents for lung cancer. Here we applied an mRNA-specific gene expression-based high-throughout screening approach to identify phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBEC). We have screened 800 compounds in the MicroSource Natural Products Library for inducers of GSTP1 and NQO1 expression, and identified 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone, triacetylresveratrol, ivermectin, sanguinarine sulfate, and daunorubicin as new inducers for GSTP1 and NQO1 mRNA expression. Consistent with this effect, the induction of NQO1 protein expression by immunoblot was found for all these agents in both HBEC and NHBE cells at the same concentration that induced NQO1 mRNA expression. However, GSTP1 protein expression did not significantly change with any selected agents. Proliferation/viability assays showed that HBEC and NHBE were significantly inhibited by ivermectin, sanguinarine sulfate, and daunorubicin. These data demonstrate the feasibility of high-throughput screening for inducers of NQO1. Moreover, the data identify 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone and triacetylresveratrol as the most potent and low toxicity inducers [≥3-6-fold, at the protein level], implying they are potential new agents for further in vitro and preclinical in vivo testing for prevention of oxidant-related disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5706.

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. However, in studies to date, most candidate agents are not active in human lung cells. In vitro and in vivo screening studies for discovery are clearly needed for new, more potent chemopreventive agents for lung cancer. Here we applied a gene expression-based high-throughput screening approach to identify phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBEC). We have screened 800 compounds in the MicroSource Natural Products Library for inducers of GSTP1 and NQO1 expression, and identified 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone, triacetylresveratrol, ivermectin, sanguinarine sulfate, and daunorubicin as new inducers for GSTP1 and NQO1 mRNA expression. Consistent with this effect, the induction of NQO1 protein expression by immunoblot was found for all these agents in both HBEC and NHBE cells at the same concentration (1.0 µM, final concentration) that induced NQO1 mRNA expression. However, GSTP1 protein expression did not significantly change with any selected agents. Proliferation/viability assays showed that HBEC and NHBE were significantly inhibited by ivermectin, sanguinarine sulfate, and daunorubicin. These data demonstrate the feasibility of high-throughput screening for inducers of NQO1. Moreover, the data identify 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone and triacetylresveratrol as most potent and low toxicity inducers [12-fold, and 10-fold, respectively, at the protein level], implying they are potential new agents for further in vitro and preclinical in vivo testing for prevention of tobacco-related lung cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A69.

Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods.

We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.

Testicular germ cell tumors (TGCTs) show, for the most part, a characteristic cytogenetic aberration carrying one or more copies of i(12p). Mostly i(12p)-negative TGCTs also show an overrepresentation of 12p11p12 sequences. Critical genes in the amplicon defined by 12p11p12 may be involved in the progression of TGCTs and other tumors. To identify directly the expressed sequence tags (ESTs) involved in the amplicon, we performed comparative genomic hybridization array analysis of five TGCTs demonstrated to exhibit high DNA copy number in this region. The use of arrays spotted with 8,060 IMAGE complementary DNA clones amplified by means of the polymerase chain reaction (PCR) identified 18 amplified ESTs. Most of them were mapped to 12p and the remainder have been mapped to 12. We also identified new testis-associated transcripts by PCR with reverse transcription of the amplified EST sequences. Using combined RNA-array and real-time-beacon PCR analysis, we identified two amplified and overexpressed ESTs. The level of expression in these ESTs was four- to sixfold higher in one tumor sample compared with normal testis. The expression ratios obtained by real-time-beacon PCR analysis correlated with those obtained by RNA-array data analysis, thus validating the techniques used. However, we failed to find an amplified and overexpressed EST commonly overexpressed in all the tumor samples tested, perhaps because the arrays used might only represent approximately 10% of human genes. Therefore we decided to design a 12p array containing 768 ESTs, representing all the 12p-mapped ESTs found in the available databases. Results of comparative genomic hybridization and RNA-array analysis of TGCTs using the 12p array will be presented.

cDNA microarray technology provides a novel approach to identify individual target genes and survey global genetic programs under the control of specific signalling pathways in mammalian systems. Smad2 and Smad3 are highly homologous members of the receptor-regulated subfamily of Smad proteins with a central role in TGF- signalling and target gene regulation.