A Customizable Suite of Methods to Sequence and Annotate Cattle Antibodies.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.

Case report: primary osteonecrosis associated with thrombophilia-hypofibrinolysis and worsened by testosterone therapy.

BACKGROUND: Familial and acquired thrombophilia are often etiologic for idiopathic hip and jaw osteonecrosis (ON), and testosterone therapy (TT) can interact with thrombophilia, worsening ON. CASE PRESENTATION: Case 1: A 62-year-old Caucasian male (previous deep venous thrombosis), on warfarin 1 year for atrial fibrillation (AF), had non-specific right hip-abdominal pain for 2 years. CT scan revealed bilateral femoral head ON without collapse. Coagulation studies revealed Factor V Leiden (FVL) heterozygosity, 4G/4G plasminogen activator inhibitor (PAI) homozygosity, high anti-cardiolipin (ACLA) IgM antibodies, and endothelial nitric oxide (NO) synthase (eNOS) T786C homozygosity (reduced conversion of L-arginine to NO, required for bone health). Apixaban 5 mg twice daily was substituted for warfarin; and L-arginine 9 g/day was started to increase NO. On Apixaban for 8 months, he became asymptomatic. Case 2: A 32-year-old hypogonadal Caucasian male had 10 years of unexplained tooth loss, progressing to primary jaw ON with cavitation 8 months after starting TT gel 50 mg/day. Coagulation studies revealed FVL heterozygosity, PAI 4G/4G homozygosity, and the lupus anticoagulant. TT was discontinued. Jaw pain was sharply reduced within 2 months. CONCLUSIONS: Idiopathic ON, often caused by thrombophilia-hypofibrinolysis, is worsened by TT, and its progression may be slowed or stopped by discontinuation of TT and, thereafter, anticoagulation. Recognition of thrombophilia-hypofibrinolysis before joint collapse facilitates anticoagulation which may stop ON, preserving joints.

Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors.

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSßR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 µM, and CDK9-cyclinT with IC(50)=0.11 µM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 µM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.

Synthesis of isothiazol-3-one derivatives as inhibitors of histone acetyltransferases (HATs).

High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue.

Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity.

Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

Metabolism and pharmacokinetics of the cyclin-dependent kinase inhibitor R-roscovitine in the mouse.

R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 microg/mL (28 micromol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC(infinity) for R-roscovitine (38 micromol/L/h) than for COOH-R-roscovitine (174 micromol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by approximately 24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.

Discovery of genes with highly restricted expression patterns in the Drosophila wing disc using DNA oligonucleotide microarrays.

The Drosophila wing disc is divided along the proximal-distal axis into regions giving rise to the body wall (proximal), wing hinge (central) and wing blade (distal). We applied DNA microarray analysis to discover genes with potential roles in the development of these regions. We identified a set of 94 transcripts enriched (two fold or greater) in the body wall and 56 transcripts enriched in the wing/hinge region. Transcripts that are known to have highly restricted expression patterns, such as pannier, twist and Bar-H1 (body wall) and knot, nubbin and Distal-less (wing/hinge), showed strong differential expression on the arrays. In situ hybridization for 50 previously uncharacterized genes similarly revealed that transcript enrichment identified by the array analysis was consistent with the observed spatial expression. There was a broad spectrum of patterns, in some cases suggesting that the genes could be targets of known signaling pathways. We show that three of these genes respond to wingless signaling. We also discovered genes likely to play specific roles in tracheal and myoblast cell types, as these cells are part of the body wall fragment. In summary, the identification of genes with restricted expression patterns using whole genome profiling suggests that many genes with potential roles in wing disc development remain to be characterized.