Two of the present authors have presented a model for macrofibril template self-assembly which is based on a concerted process of sudden, delayed, short filament formation with immediate phase transfer to a lyotropic liquid crystal (mesophase). In recent years, information on the spatio-temporal patterns of keratin protein expression within wool follicles has come to hand, and we here report some recently acquired important information in this area. If the "mesophase model" is to remain credible it must be compatible with the spatio-temporal patterns of keratin expression. This paper explores the match between the mesophase model and patterns of protein expression, and concludes that they are broadly consistent. At present the model does not extend to keratin associated protein (KAP) expression. The model is also particularly successful in explaining macrofibril structural features in different cell types.

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.

The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte’s Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research’s HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens.

By means of a phenomenon termed “the Warburg effect,” tumor cells shift their energy production by mitochondrial oxidative phosphorylation to aerobic glycolysis, resulting in the upregulation of glucose consumption and increased cellular oxidative and nitrosative stress. To compensate for such toxic levels of ROS/RNS, cancer cells rely heavily on their antioxidant defense mechanisms, which are largely controlled by the NAD(P)/NAD(P)H redox partners. We found that the modulation of NAD metabolism in vivo, specifically via the deletion of the NAD glycohydrolase CD38, resulted in increased production of intrinsic ROS and increased DNA damage following exposure to chemotherapeutics. Furthermore, in vitro experiments showed that CD38 knockdown in CD38-expressing tumor cells prevented the generation of stable transfectants, highlighting a role for CD38 in tumor cell survival. In light of these findings, we hypothesized that pharmacological inhibition of CD38 may be an effective therapy for the treatment of hematological cancers, in particular those which uniformly overexpress CD38, such as MM and chronic lymphocytic leukemia. Indeed, treatment of human MM cell lines LP-1 and KMS-12-PE with CD38 antagonists sensitized the cells to standard ROS-inducing chemotherapeutics. We conducted a high-throughput screening (HTS) campaign of over two hundred thousand unique and non-proprietary lead-like compounds using an optimized and miniaturized HTS based on a luminescent NAD quantitation platform. Five hundred active hits were analyzed for toxicity using a cell-based HTS assay purposely designed with CD38-negative HEK293 cells to avoid elimination of desirable compounds toxic to CD38-positive cells. Hits with non-specific properties, such as PAINS (Pan Assay Interference Compounds), were removed by computational filtering, and the remaining compounds were tested for inhibition of human CD38 activity in cells. The last phase of the compound progression pathway involved testing for non-selective inhibition of other NAD-consuming enzymes, namely Poly(ADP-ribose) polymerase-1, and Sirtuin-1, which led to the identification of two distinct chemical series that exhibit >10-fold selectivity for human CD38. Hit-to-lead chemistry is currently underway to synthesize key analogs by rational drug design. In summary, our data suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing hematological cancers. Our HTS campaign efforts are paving the way for the discovery and development of potent and selective small-molecule inhibitors of CD38. Citation Format: Davide Botta, Tulin Dadali, Betty J. Mousseau, Fen Zhou, Michael D. Schultz, Esther Zumaquero, Anna Manouvakhova, Melinda I. Sosa, Sara N. McKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E. Lucille White, Indira Padmalayam, Wei Zhang, Maaike Everts, Corinne E. Augelli-Szafran, James R. Bostwick, Mark J. Suto, Frances E. Lund. High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-055.

T regulatory cells (Tregs) are a subset of FoxP3+ lymphocytes that maintain immune homeostasis through immunosuppressive signaling. In many helminth infections, one mechanism of immune evasion involves the influx and maintenance of Tregs. Multiple mechanisms can control Treg homeostasis including a Nicotinamide adenine dinucleotide (NAD) dependent apoptotic pathway. In most organisms, the two pathways of NAD synthesis include de novo synthesis from amino acid precursors, and the salvage pathway in which nicotinamide-containing precursors are recycled into NAD. A comparative genome analysis of S. mansoni, the parasite that causes Schistosomiasis, enabled the assembly of a putative NAD biosynthetic pathway. Only orthologues of NAD salvage-specific genes were identified and expression was confirmed by PCR. This suggests that S. mansoni decreases host NAD levels in a salvage-specific manner and rescues Tregs from NAD-dependent cell death. As such, we hypothesized that inhibition of NAD biosynthesis through the salvage pathway would result in impaired NAD uptake by S. mansoni and restoration of NAD-mediated cell death of Tregs. Indeed, co-culture with S. mansoni prevented NAD-induced toxicity and death of Tregs. Blockade of the NAD salvage pathway via the inhibition of Schistosoma mansoni NAD catabolizing enzyme (SmNACE) restored NAD-mediated cell death of Tregs. Furthermore, SmNACE inhibition decreased intracellular NAD levels and reduced metabolism and viability of the parasite. Collectively, our data suggest that inhibition of NAD biosynthesis blocks immune evasion and metabolism of S. mansoni, and that targeting the NAD salvage pathway is a promising therapeutic approach for the treatment of Schistosomiasis.