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Supplementary Table 2 from Molecular Evolutionary Analysis of Cancer Cell Lines

Supplementary Tables 3-6 from Molecular Evolutionary Analysis of Cancer Cell Lines

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Supplementary Table 1 from Molecular Evolutionary Analysis of Cancer Cell Lines

Supplementary Figure 1 from Molecular Evolutionary Analysis of Cancer Cell Lines

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Supplementary Methods, Figure Legends 1-8 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 5 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 7 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 3 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 4 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 6 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 1 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Supplementary Figure 2 from Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

Introduction Metformin and glucagon‐like peptide‐1 (GLP‐1) agonists are widely used for treating type two diabetes mellitus (T2DM). While recent studies suggest these drugs might modify the gastrointestinal tract (GIT) microbiome, further confirmation is required from human clinical trials. Materials and methods Here, we compare, in patients with T2DM, the effects of metformin (n = 18 subjects) and liraglutide (n = 19), a GLP‐1 agonist, on their GIT microbiomes over a 42 day period (n = 74 samples) using 16S ribosomal RNA (rRNA) sequencing. Results We found that these drugs had markedly different effects on the microbiome composition. At both baseline and Day 42, subjects taking metformin had a significant increase (Baseline adj. P = .038, Day 42 adj. P = .041) in the relative abundance of the bacterial genus Sutterella, whereas liraglutide dosing is associated with a significant increase (Baseline adj. P = .048, Day 42 adj. P = .003) in the genus Akkermansia, a GIT bacteria positively associated with gut barrier homoeostasis. Bacteroides and Akkermansia relative abundances were also significantly associated with duration of subject diabetes (adj P

Background The amino acid response ( AAR ) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐ tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. Methods and Results Consistent with its ability to inhibit prolyl‐ tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l ‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II /phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/ eIF 2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

The T cell receptor (TCR), a defining structure of T cells is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an immune response. The extreme diversity of the TCR repertoire represents a major analytical challenge. Massively parallel high throughput sequencing is commonly deployed to characterize various key features of T cell receptors including their clonal expansion and repertoire diversity. Genomic DNA is the most widely employed starting material to characterize the TCR repertoire. DNA is highly stable and contains a fixed copy number per cell, which allows for better quantification of TCR clones. However, the inclusiveness is a major limitation of using genomic DNA along with the PCR amplification challenges and sequencing errors due to recombined gene segments within the context of ‘unused’ segments and introns. Here we deploy RNA-based TCR profiling as an alternate strategy to overcome the major limitations associated with the DNA-based approach. Employing RNA with a simplified PCR amplification strategy allows a more comprehensive identification of unique TCR variants with greater sensitivity. It also provides information about expression levels of TCR sequences, and more importantly, excludes the non-productive sequences that are functionally irrelevant. In addition, Unique Molecular Indexing (UMIs) enables the determination of the absolute count of RNA transcripts processed in a sample, allowing straightforward error corrections. Citation Format: Shanker Kalyana-Sundaram, Christopher Traini, Wendy Halsey, Heather Jackson, Sabyasachi Bhattacharya, Niranjan Yanamandra, Ganesh Sathe, George Livi. Comparative analysis between DNA vs RNA-based T cell repertoire profiling for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1208.

With genome-wide cancer studies producing large DNA sequence data sets, novel computational approaches toward better understanding the role of mutations in tumor survival and proliferation are greatly needed. Tumors are widely viewed to be influenced by Darwinian processes, yet molecular evolutionary analysis, invaluable in other DNA sequence studies, has seen little application in cancer biology. Here, we describe the phylogenetic analysis of 353 cancer cell lines based on multiple sequence alignments of 3,252 nucleotides and 1,170 amino acids built from the concatenation of variant codons and residues across 494 and 523 genes, respectively. Reconstructed phylogenetic trees cluster cell lines by shared DNA variant patterns rather than cancer tissue type, suggesting that tumors originating from diverse histologies have similar oncogenic pathways. A well-supported clade of 91 cancer cell lines representing multiple tumor types also had significantly different gene expression profiles from the remaining cell lines according to statistical analyses of mRNA microarray data. This suggests that phylogenetic clustering of tumor cell lines based on DNA variants might reflect functional similarities in cellular pathways. Positive selection analysis revealed specific DNA variants that might be potential driver mutations. Our study shows the potential role of molecular evolutionary analyses in tumor classification and the development of novel anticancer strategies. Mol Cancer Ther; 9(2); 279–91

HER2-directed therapies, such as trastuzumab and lapatinib, are important treatments for breast cancer. However, some tumors do not respond or develop resistance to these agents. We isolated and characterized multiple lapatinib-resistant, HER2-positive, estrogen receptor (ER)–positive breast cancer clones derived from lapatinib-sensitive BT474 cells by chronic exposure to lapatinib. We show overexpression of AXL as a novel mechanism of acquired resistance to HER2-targeted agents in these models. GSK1363089 (foretinib), a multikinase inhibitor of AXL, MET, and vascular endothelial growth factor receptor currently in phase II clinical trials, restores lapatinib and trastuzumab sensitivity in these resistant cells that exhibit increased AXL expression. Furthermore, small interfering RNA to AXL, estrogen deprivation, or fulvestrant, an ER antagonist, decreases AXL expression and restores sensitivity to lapatinib in these cells. Taken together, these data provide scientific evidence to assess the expression of AXL in HER2-positive, ER-positive patients who have progressed on either lapatinib or trastuzumab and to test the combination of HER2-targeted agents and GSK1363089 in the clinic. [Cancer Res 2009;69(17):6871–8]

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales , and increases in others, the Betaproteobacteria , Gammaproteobacteria , and Bifidobacteriaceae . Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.)

Importance Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients. Objective To characterize the transcriptomics of peripheral monocytes in patients with ALS. Design, Setting, and Participants Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction. Main Outcomes and Measures The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling. Results The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B , IL8 , FOSB , CXCL1 , and CXCL2 , were confirmed by quantitative reverse transcription polymerase chain reaction ( IL8 , mean [SE], 1.00 [0.18]; P = .002; FOSB , 1.00 [0.21]; P = .009; CXCL1 , 1.00 [0.14]; P = .002; and CXCL2 , 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients. Conclusions and Relevance Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. NCT01381549 for the study of complicated urinary tract infections and registration no. NCT01381562 for the study of complicated intra-abdominal infections.)

Objective To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. Methods Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. Results Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. Conclusion The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.

Objective To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. Methods Poly A + RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. Results Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28–40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4–6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. Conclusion This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv r clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv r mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.

To characterize binding sites for nonpeptide angiotensin antagonists on the human angiotensin II receptor type 1 (AT1 receptor) we have systematically exchanged segments of the human receptor with corresponding segments from a homologous Xenopus laevis receptor, which does not bind the nonpeptide compounds. Substitution of transmembrane segment VII of the human AT1 receptor dramatically reduced the binding affinity of all of the 11 nonpeptide antagonists tested (55- to > 2000-fold) with no effect on the binding of angiotensin. The affinity for the nonpeptide compounds decreased additionally one order of magnitude when transmembrane segment VI and the connecting extracellular loop 3 from the Xenopus receptor were also introduced into the human AT1 receptor. Exchanges of smaller segments and single residues in transmembrane segments VI and VII and extracellular loop 3 revealed that the binding of nonpeptide antagonists was dependent on nonconserved residues located deep within the transmembrane segments VI and VII, in particular Asn295 in transmembrane segment VII. Surprisingly, all exchanges in transmembrane segment VII, including the Asn295 to Ser substitution, had a more pronounced effect on the binding of the competitive antagonists relative to the insurmountable antagonists. It is concluded that the binding mode for peptide and nonpeptide ligands on the AT1 receptor is rather different and that competitive and insurmountable antagonists presumably bind to overlapping but distinct sites located in transmembrane segments VI and VII.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Antibodies to synthetic peptides of human interleukin 1 beta (IL-1 beta) and to recombinant human IL-1 beta were used to identify epitopes of IL-1 beta associated with the neutralization of its biological activity. Analysis of antisera raised to 17 synthetic peptides derived from the mature IL-1 beta sequence showed that five regions (residues 6-15, 49-80, 58-80, 92-101, and 120-133) were both immunoprecipitating and neutralizing. Using a hexamer epitope mapping method, comparison of the regions recognized by four neutralizing rabbit antisera with those recognized by a rabbit antiserum raised to denatured IL-1 beta suggested two further neutralizing epitopes, residues 39-48 and 83-95. Finally, a neutralizing monoclonal antibody was shown to bind to the peptides 6-11 and 87-95 by peptide binding and mutagenesis. All of these regions appear predominantly on one face of IL-1 beta. The effect of mutations in residues 4-11 and 88-97, which lie within this face, on receptor binding and biological activity was determined. Most of the mutations tested affected both receptor binding and activity, whereas mutations in another face of IL-1 beta (residues 74-80) had no effect. Purification of two of the mutants with reduced bioactivity and receptor binding and analysis by two-dimensional NMR indicated no gross changes in tertiary structure. A third mutant had reduced bioactivity in two different bioassays but no change in receptor binding. Although two-dimensional NMR revealed no gross changes in conformation, small changes did occur at a site distal from that mutated. The data are consistent with other epitope mapping and receptor binding mutagenesis data and suggest that the neutralizing antibodies and receptor recognize different but overlapping regions of IL-1 beta.

A lambda phage expression methodology was adapted to dissect protein/ligand interactions efficiently through the creation and rapid screening of large numbers of mutants. Here we describe the method and its specific application to the interaction between the external envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, and the human cell surface protein CD4. Random substitutions were introduced throughout the gp120 binding region (amino acids 38-62) in the amino-terminal domain of CD4 by oligonucleotide mutagenesis. These mutations were expressed within phage plaques and directly screened for their effect on binding of gp120 using a modified phage plaque lift procedure. Plaques showing increased, decreased, and no effect on binding were identified and mutations were verified by sequence analysis. In this manner, 25 unique mutations were identified that altered CD4 binding to gp120. A new site was identified at which mutations reduced binding to gp120 and several novel amino acid substitutions were defined at sites previously implicated in binding. Of particular interest, this in vitro genetic approach identified a mutation which significantly increased binding to gp120. The phenotypes of several of these mutants were further characterized by quantitative measurement of their binding affinity. The results confirmed the accuracy of the phenotypic selection and demonstrated that the sensitivity of the system allowed detection of a 3-4-fold increase or decrease in affinity. In the context of the recently determined atomic structure of CD4, these results further implicate residues in the CDR2-like region and in an adjacent loop in recognition of gp120. This methodology should be generally applicable to other high affinity protein/ligand interactions that are compatible with expression in Escherichia coli.

Varicella-zoster virus (VZV) is a herpesvirus and is the causative agent of chicken pox (varicella) and shingles (herpes zoster). Active immunization against varicella became possible with the development of live attenuated varicella vaccine. The Oka vaccine strain was isolated in Japan from a child who had typical varicella, and it was then attenuated by serial passages in cell culture. Several manufacturers have obtained this attenuated Oka strain and, following additional passages, have developed their own vaccine strains. Notably, the vaccines Varilrix and Varivax are produced by GlaxoSmithKline Biologicals and Merck & Co., Inc., respectively. Both vaccines have been well studied in terms of safety and immunogenicity. In this study, we report the complete nucleotide sequence of the Varilrix (Oka-V GSK ) and Varivax (Oka-V Merck ) vaccine strain genomes. Their genomes are composed of 124,821 and 124,815 bp, respectively. Full genome annotations covering the features of Oka-derived vaccine genomes have been established for the first time. Sequence analysis indicates 36 nucleotide differences between the two vaccine strains throughout the entire genome, among which only 14 are involved in unique amino acid substitutions. These results demonstrate that, although Oka-V GSK and Oka-V Merck vaccine strains are not identical, they are very similar, which supports the clinical data showing that both vaccines are well tolerated and elicit strong immune responses against varicella.

The gastrointestinal tract microbiome has been suggested as a potential therapeutic target for metabolic diseases such as obesity and Type 2 diabetes mellitus (T2DM). However, the relationship between changes in microbial communities and metabolic disease-phenotypes are still poorly understood. In this study, we used antibiotics with markedly different antibacterial spectra to modulate the gut microbiome in a diet-induced obesity mouse model and then measured relevant biochemical, hormonal and phenotypic biomarkers of obesity and T2DM. Mice fed a high-fat diet were treated with either ceftazidime (a primarily anti-Gram negative bacteria antibiotic) or vancomycin (mainly anti-Gram positive bacteria activity) in an escalating three-dose regimen. We also dosed animals with a well-known prebiotic weight-loss supplement, 10% oligofructose saccharide (10% OFS). Vancomycin treated mice showed little weight change and no improvement in glycemic control while ceftazidime and 10% OFS treatments induced significant weight loss. However, only ceftazidime showed significant, dose dependent improvement in key metabolic variables including glucose, insulin, protein tyrosine tyrosine (PYY) and glucagon-like peptide-1 (GLP-1). Subsequently, we confirmed the positive hyperglycemic control effects of ceftazidime in the Zucker diabetic fatty (ZDF) rat model. Metagenomic DNA sequencing of bacterial 16S rRNA gene regions V1-V3 showed that the microbiomes of ceftazidime dosed mice and rats were enriched for the phylum Firmicutes while 10% OFS treated mice had a greater abundance of Bacteroidetes. We show that specific changes in microbial community composition are associated with obesity and glycemic control phenotypes. More broadly, our study suggests that in vivo modulation of the microbiome warrants further investigation as a potential therapeutic strategy for metabolic diseases.

Background: GSK2636771 is a potent, orally bioavailable and selective inhibitor of PI3Kβ (apparent Ki = 0.89 nM), with >900-fold selectivity over PI3Kα/PI3Kγ and 10-fold over PI3Kδ. It inhibits AKT phosphorylation and downstream signaling measured as decrease of PRAS40-, GSK3β-, and RPS6-phosphorylation in PTEN deficient cell lines. Methods: An ongoing phase I/IIa FTIH, open label dose escalation study of GSK2636771, once daily (QD), is being conducted to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy in pts with advanced tumors deficient in PTEN. The study comprised 3 parts: starting-dose selection, 3+3 dose escalation, and phase II expansion. PD was assessed in tumor and platelet rich plasma samples. Archival tumor samples were analyzed for alterations in a custom panel of select oncogenes and tumor suppressors by next-generation sequencing and copy number analyses. Results: As of Nov 2014, 62 pts (38 m: 24 f, mean 60 yrs) were enrolled and dosed at 7 dose levels (25 - 500 mg). In total 5 DLTs (3x hypophosphatemia/ 2x hypocalcemia G3) were observed in 3 pts at 500 mg, defining MTD. AEs ≥20% (all grades) included diarrhoea, nausea, vomiting, fatigue, abdominal pain, anemia, decreased appetite and headache. Cmax was reached 4-6 h after single and repeat dosing, with a mean t1/2 of 17.1-38.6 h across cohorts. Increases in mean Cmax and AUC(0-24 h) were dose-proportional up to 350 mg, but less than proportional at 400 and 500 mg. Phospho/total AKT ratio was decreased by >50% in platelet-rich plasma in 21 of 23 pts across various doses. Similarly, phospho/total AKT ratio was decreased (median 35%) in post-treatment tumor biopsies compared to pre-treatment tumors in 4 of 5 pts treated at 400 mg dose. One pt (prostate) had a RECIST PR and 17 pts had SD, 8 of whom were on study for ≥6 months. Exploratory genomic analyses of tumor samples from 10 prostate cancer pts revealed a PIK3CB L1049R mutation in the tumor of one of the pts who remained on study for 33 weeks and an increased PIK3CB gene copy number in the tumor of a pt who achieved PR. Preclinical characterization of PIK3CB L1049R mutation is ongoing, as it is homologous to the oncogenic PIK3CA H1047R mutation. These data suggest potential clinical benefit with GSK2636771 in pts with tumors harboring genetic alterations in PIK3CB. Tumors from other histologies are being analyzed for alterations in key cancer-relevant genes, including PIK3CB. Conclusions: MTD and recommended phase II dose of GSK2636771 is 400 mg QD. Pathway inhibition was observed with limited anti-tumor activity in pts with PTEN deficient tumors. Pts with tumors harboring concomitant genetic alterations in PIK3CB may benefit from GSK2636771 treatment. Recruitment into phase II expansion is ongoing. Citation Format: Johann de Bono, Hendrik-Tobias Arkenau, Joaquin Mateo, Jeffrey R. Infante, Howard A. Burris, Yung-Jue Bang, Joseph Eder, Sunil Sharma, Hyun C. Chung, Shaun Decordova, Karen E. Swales, Michelle D. Garrett, Desamaparados Roda-Perez, Meichun Ding, Mark Russo, Li Yan, Ben Suttle, Jerry M. Tolson, Wendy S. Halsey, Ganji Gopi, Harjeet K. Van Der Keyl, Shanker Kalyana-Sundaram, Ganesh M. Sathe, Monica Motwani, Rakesh Kumar. Exploratory genetic analysis of tumors from a phase I/II dose escalation study of GSK2636771 in patients (pts) with PTEN deficient advanced tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT328. doi:10.1158/1538-7445.AM2015-CT328

Objective: Matrix metalloproteinase-9 (MMP-9) activity is up regulated in the heart subjected to ischemic insult. Whether increased MMP-9 activity contributes to acute myocardial injury after ischemia–reperfusion remains unknown. To investigate the role of MMP-9 in myocardial infarction, we utilized a MMP-9 knockout mouse. Methods and results: Standard homologous recombination in embryonic stem cells was used to generate a mouse lacking MMP-9. The left anterior descending coronary artery was occluded for 30 min followed by 24 h reperfusion, and the ischemic and infarct sizes were determined. Targeted deletion of MMP-9 protected the heart from no-flow ischemia–reperfusion-induced myocardial injury. The myocardial infarct size was reduced by 17.5% in MMP-9 heterozygotes (+/−) ( P

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by cotransforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).

To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at ∼5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression.

Like other members of the Flaviviridae family, the 3' non-translated region (NTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication by interacting with components of the viral replicase complex. To inves-tigate the possibility that host components may also participate in this process, we used UV cross-linking assays to determine if any cellular proteins could bind specifically to the 3'NTR RNA. We demonstrate the specific interaction of two host proteins with the extensive pyrimidine-rich region within the HCV 3'NTR. One host protein migrates as a doublet with a molecular weight of 57 kDa and is immunoreactive with antisera specific for polypyrimidine tract-binding protein (PTB), and the other protein (35 kDa) is recognized by a monoclonal antibody specific for heterogeneous nuclear ribonucleoprotein C (hnRNP C). These results suggest that recognition of the large pyrimidine-rich region by PTB and hnRNP C may play a role in the initiation and/or regulation of HCV RNA replication.

The extracellular domain of the type I Interleukin-1 receptor (sIL-1R) was expressed in Drosophila S2 cells as a secreted 43 kDa glycoprotein, as evidenced by its binding to Concanavalin A and enzymatic deglycosylation. sIL-1R bound IL-1 beta with a K(D) of 2 nM as determined by competition ELISA. N-Glycanase treated sIL-1R had a C. 100 fold lower affinity than glycosylated sIL-1R for IL-1 beta, suggesting that glycosylation is a key component of the IL-1 beta/IL-1 receptor interaction. Crosslinking of sIL-1R to (125)I-IL-1 beta could be competed with unlabelled IL-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), and a mutant of IL-1 (Th9Gly) which has reduced bioactivity but wild type receptor binding affinity. Limited proteolysis of sIL-1R in the presence of IL-1 alpha, IL-1 beta, IL-1ra, and Thr9Gly IL-1 beta with several different proteases followed by analysis of sIL-1R by Western blot was used to assess the effect of binding on sIL-1R conformation. While some proteases showed no differences in cleavage patterns or sensitivity between free and bound sIL-1R, others showed differences in either cleavage sites or sensitivity with different ligands. This implies that upon ligand binding there is a conformational change in the receptor which is sensitive to the particular ligand bound, and hence has implications for the ability of different ligands to trigger responses after binding to receptor.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent Rmrrbp1 mutants and screened them for both RB1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm. Analysis of [3H]Rm binding in rbp1 mutant vs. wild-type yeast cell extracts indicated that the Tyr89 → Asp (Y89D) change results in an approx. 95% loss of binding activity. These data confirm the assignment of this Tyr residue as being involved in Rm binding.

In 404 Lepob/ob F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lepob. The phenotypes of B6.DBA congenic mice include reduced β-cell replication rates accompanied by reduced β-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated “Lisch-like” (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646–amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes., Author Summary Type 2 diabetes (T2D) accounts for over 90% of instances of diabetes and is a leading cause of medical morbidity and mortality. Twin studies indicate a strong polygenic contribution to susceptibility within the context of obesity. Although approximately ten genes making important contributions to individual risk have been identified, it is clear that others remain to be identified. In this study, we intercrossed obese, diabetes-resistant and diabetes-prone mouse strains to implicate a genetic interval on mouse Chr1 associated with reduced β-cell numbers and elevated blood glucose. We narrowed the region using molecular genetics and computational approaches to identify a novel gene we designated “Lisch-like” (Ll). The orthologous human genetic interval has been repeatedly implicated in T2D. Mice with an induced mutation that reduces Ll expression are impaired in both β-cell development and glucose metabolism, and reduced expression of the homologous gene in zebrafish disrupts islet development. Ll is expressed in organs implicated in the pathophysiology of T2D (hypothalamus, islets, liver, and skeletal muscle) and is predicted to encode a transmembrane protein that could mediate cholesterol transport and/or convey signals related to cell division. Either mechanism could mediate effects on β-cell mass that would predispose to T2D.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

The stereospecific total synthesis of the pentacyclic tetrasubstituted intermediate 39 is described. The conversion of this material to the delphinine type alkaloid chasmanine IV by the method worked out recently by the same group (1) is envisaged.

The DNA/RNA Synthesizer provides a complete and automated procedure for the synthesis of DNA sequences. Each base unit is added in a 30-minute cycle, permitting a tetradecamer to be constructed in 6 1/2 hours. The complete procedure is described, including a practical procedure for isolation and purification of the desired DNA sequence.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

Transient transfection of simian COS cells with a recombinant plasmid encoding the human transforming growth factor TGF-beta 2 precursor protein results in the production of a latent, biologically inactive protein. Upon acidification, recombinant TGF-beta 2 exhibits full biological activity, including inhibition of mink lung epithelial cell growth, stimulation of anchorage-independent growth of murine embryonic fibroblasts, and competition for TGF-beta receptor binding. Further analysis of conditioned media with antiserum to either a pro- [amino acid (aa) residues 1-220] or mature [aa 297-414] peptide of the TGF-beta 2 precursor suggests that TGF-beta 2, similar to TGF-beta 1 production in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol. 7 (1987) 3418-3427], is initially synthesized as a larger precursor protein which is proteolytically cleaved to yield the mature 112-aa transforming growth factor.

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.