Immune Activation following Irbesartan Treatment in a Colorectal Cancer Patient: A Case Study.

Colorectal cancers are one of the most prevalent tumour types worldwide and, despite the emergence of targeted and biologic therapies, have among the highest mortality rates. The Personalized OncoGenomics (POG) program at BC Cancer performs whole genome and transcriptome analysis (WGTA) to identify specific alterations in an individual's cancer that may be most effectively targeted. Informed using WGTA, a patient with advanced mismatch repair-deficient colorectal cancer was treated with the antihypertensive drug irbesartan and experienced a profound and durable response. We describe the subsequent relapse of this patient and potential mechanisms of response using WGTA and multiplex immunohistochemistry (m-IHC) profiling of biopsies before and after treatment from the same metastatic site of the L3 spine. We did not observe marked differences in the genomic landscape before and after treatment. Analyses revealed an increase in immune signalling and infiltrating immune cells, particularly CD8+ T cells, in the relapsed tumour. These results indicate that the observed anti-tumour response to irbesartan may have been due to an activated immune response. Determining whether there may be other cancer contexts in which irbesartan may be similarly valuable will require additional studies.

Targeting the undruggable: immunotherapy meets personalized oncology in the genomic era.

Owing to recent advances in genomic technologies, personalized oncology is poised to fundamentally alter cancer therapy. In this paradigm, the mutational and transcriptional profiles of tumors are assessed, and personalized treatments are designed based on the specific molecular abnormalities relevant to each patient's cancer. To date, such approaches have yielded impressive clinical responses in some patients. However, a major limitation of this strategy has also been revealed: the vast majority of tumor mutations are not targetable by current pharmacological approaches. Immunotherapy offers a promising alternative to exploit tumor mutations as targets for clinical intervention. Mutated proteins can give rise to novel antigens (called neoantigens) that are recognized with high specificity by patient T cells. Indeed, neoantigen-specific T cells have been shown to underlie clinical responses to many standard treatments and immunotherapeutic interventions. Moreover, studies in mouse models targeting neoantigens, and early results from clinical trials, have established proof of concept for personalized immunotherapies targeting next-generation sequencing identified neoantigens. Here, we review basic immunological principles related to T-cell recognition of neoantigens, and we examine recent studies that use genomic data to design personalized immunotherapies. We discuss the opportunities and challenges that lie ahead on the road to improving patient outcomes by incorporating immunotherapy into the paradigm of personalized oncology.

Effects of hunger on neuronal histone modifications slow aging in Drosophila.

Hunger is an ancient drive, yet the molecular nature of pressures of this sort and how they modulate physiology are unknown. We find that hunger modulates aging in Drosophila. Limitation of branched-chain amino acids (BCAAs) or activation of hunger-promoting neurons induced a hunger state that extended life span despite increased feeding. Alteration of the neuronal histone acetylome was associated with BCAA limitation, and preventing these alterations abrogated the effect of BCAA limitation to increase feeding and extend life span. Hunger acutely increased feeding through usage of the histone variant H3.3, whereas prolonged hunger seemed to decrease a hunger set point, resulting in beneficial consequences for aging. Demonstration of the sufficiency of hunger to extend life span reveals that motivational states alone can be deterministic drivers of aging.

Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus.

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

The genome sequence of Drosophila melanogaster.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Rainbow smelt (Osmerus mordax) genomic library and EST resources.

Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology.

Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis.

Microbial communities reside in healthy tissues but are often disrupted during disease. Bacterial genomes and proteins are detected in brains from humans, nonhuman primates, rodents and other species in the absence of neurological disease. We investigated the composition and abundance of microbiota in frozen and fixed autopsied brain samples from patients with multiple sclerosis (MS) and age- and sex-matched nonMS patients as controls, using neuropathological, molecular and bioinformatics tools. 16s rRNA sequencing revealed Proteobacteria to be the dominant phylum with restricted diversity in cerebral white matter (WM) from MS compared to nonMS patients. Both clinical groups displayed 1,200-1,400 bacterial genomes/cm3 and low bacterial rRNA:rDNA ratios in WM. RNAseq analyses showed a predominance of Proteobacteria in progressive MS patients' WM, associated with increased inflammatory gene expression, relative to a broader range of bacterial phyla in relapsing-remitting MS patients' WM. Although bacterial peptidoglycan (PGN) and RNA polymerase beta subunit immunoreactivities were observed in all patients, PGN immunodetection was correlated with demyelination and neuroinflammation in MS brains. Principal component analysis revealed that demyelination, PGN and inflammatory gene expression accounted for 86% of the observed variance. Thus, inflammatory demyelination is linked to an organ-specific dysbiosis in MS that could contribute to underlying disease mechanisms.

Occurrence of two distinct succinate thiokinases in animal tissues.

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.

Evidence for metabolism of o-xylene by simultaneous ring and methyl group oxidation in a new soil isolate.

An o-xylene-utilizing Rhodococcus, strain B3, was isolated from enrichments with o-xylene. The pathway for o-xylene degradation was investigated by simultaneous adaptation experiments, studies of product formation by a mutant and fortuitous oxidation studies using trimethylbenzene isomers as substrates. Two pathways were found to operate simultaneously and both were inducible. The first pathway involved the oxidation of a methyl group to form 2-methylbenzyl alcohol, followed by oxidation via the corresponding acid to 3-methylcatechol. The second pathway involved oxidation of the aromatic ring to form a dimethylcatechol. The bulk of the evidence suggests that the initial reaction was catalysed by a monooxygenase rather than a dioxygenase, and that 2,3-dimethylphenol was produced as an intermediate.

Chronic treatment with diazepam or abecarnil differently affects the expression of GABAA receptor subunit mRNAs in the rat cortex.

Diazepam and abecarnil produce their overt effects by interaction with the GABAA receptor. Chronic treatment with abecarnil, however, does not induce diazepam-like tolerance. This study investigates the effects of chronic diazepam and abecarnil treatment on expression of GABAA receptor alpha 1-6 beta 1-3 and gamma 1-3 subunit isoform mRNAs in rat cortex. Male Sprague-Dawley rats were injected subcutaneously once daily for 7 or 14 days with 15 mg/kg diazepam or 6 mg/kg abecarnil in sesame-oil vehicle, and steady-state levels of GABAA receptor subunit mRNAs were quantified by solution hybridization. The levels of alpha 4- and alpha-, beta 1- and gamma 3-subunit mRNAs were significantly increased after 7 days of diazepam treatment, and this effect was maintained at 14 days. A significant increase in alpha 3-subunit mRNA was apparent only after 14 days of diazepam treatment and a significant decrease in beta 2-subunit mRNA was seen only after 14 days of abecarnil treatment. Gamma 2-Subunit mRNA was significantly decreased after 14 days of either diazepam or abecarnil exposure. A degree of association between a particular drug treatment and changes in the levels of mRNAs arising from a given gene cluster was noted. Our results are consistent with a model of diazepam dependence based on GABAA receptor subunit isoform switching.

Chronic diazepam exposure decreases transcription of the rat GABA(A) receptor gamma2-subunit gene.

The rate of transcription of the GABA(A) receptor gamma2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABA(A) receptor gamma2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABA(A) receptor gene.

Chronic zolpidem treatment alters GABA(A) receptor mRNA levels in the rat cortex.

The effect of chronic zolpidem treatment on the steady-state levels of gamma-aminobutyric acidA alpha1-6, beta1-3 and gamma1-3 subunit mRNAs in rat cortex has been investigated. Male Sprague-Dawley rats were injected once daily, for 7 or 14 days, with 15 mg/kg of zolpidem in sesame oil vehicle. The levels of the alpha4 and beta1 subunit mRNAs were significantly increased after 7 days of treatment and the level of alpha1 subunit mRNA was significantly decreased after 14 days of treatment, as determined by solution hybridization. These results are compared to the previously determined effects of an equivalent schedule of treatment with diazepam.

Instructions for patients who receive immediate dentures.

Adverse physiologic and psychologic effects may occur in patients who receive immediate dentures. These effects may be caused by ill-fitting dentures that result from oral changes that occur after insertion. To improve the comfort of and acceptance by patients with immediate dentures, it is suggested that written instructions be given to each patient before insertion of the dentures.

Contours of the edentulous palate.

Diagnostic casts were made of the palates of 123 patients who had complete dentures. Categorization of all cross-arch palatal forms yielded a distribution in which 93% showed some variation of a U-shaped palatal form, be it angular or mildly curved. Along the midline, 12% had steep anterior inclines, curved midpalatal regions, and curved posterior palates; 69% had moderate anterior inclinations, curved midpalates, and curved posterior palates; 12% had palates that were moderately inclined anteriorly, flat in the midpalatal section, and flat posteriorly. The remaining palates were predominantly flat in two of the three regions measured.

Evaluation of the potential of a polylactic acid barrier for correction of periodontal defects in baboons: a clinical and histologic study.

Created periodontal defects in baboons were treated with one of four possible treatment modes: (1) root preparation and Epi-Guide biodegradable polylactic acid barrier, (2) root preparation and Gore-Tex e-PTFE membrane, (3) root preparation only (no barrier), and (4) no root preparation and no barrier (control). Root preparation consisted of hand instrumentation and use of finishing burs. Measurements of gingival recession were recorded from color photographic slides taken weekly for 6 weeks following barrier placement. Block sections were removed from one animal 6 weeks after barrier placement and prepared for histologic evaluation. Significantly more gingival recession was observed at the Gore-Tex sites than at the Epi-Guide sites. There were no significant differences in gingival recession between the Epi-Guide sites and root preparation-only sites or control sites. Both types of barriers were histologically acceptable. At 6 weeks, the Epi-Guide material was present histologically in a partially resorbed state. There was a mild inflammatory reaction in the surrounding connective tissues.

Retina restored and brain abnormalities ameliorated by single-copy knock-in of human NR2E1 in null mice.

Nr2e1 encodes a stem cell fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal, brain, and behavioral abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial chromosome (BAC). We also documented, for the first time, the expression pattern of the human BAC, using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harboring one functional copy of human NR2E1. These results correlated with an absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatiotemporal brain expression. This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate genomic regions controlling expression in proliferating cells during neurogenesis were identified.

Flavocytochrome P450 BM3: an update on structure and mechanism of a biotechnologically important enzyme.

Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner--a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved.