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.

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.

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.

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.

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.

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.

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.

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.

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.

A morphometric evaluation of allograft matrix combinations in the treatment of osseous defects in a baboon model.

Recent and ongoing research efforts have been made to increase the efficacy of biomaterials as structural fillers during in vivo bony reconstructions. Although the selection of the possible material choices has grown, a biomaterial that can be physically molded to the defect/void space as well as offer biomimetic tissue regeneration has yet to be made available. With the potential success of demineralized freeze-dried bone allografts (DFDBA) combined with tendonous collagen as an effective filling material, further research should help to elucidate its use. The purpose of this study was to evaluate the regenerative healing response of five allograft mixtures via the morphology of filled, periodontal defects. Critical size mandibular and maxillary osseous defects were surgically created in six adult baboons. The filling response of four combinations of DFDBA and tendon collagen was compared with an all-collagen graft after 3 months of implantation. The overall results indicate that all combinations of DFDBA and collagen provided a better fill response than the all-collagen matrix (P < 0. 05). Statistically, however, all of the combinations were similar (P > 0.05) with a 60:40 collagen to DFDBA mass ratio resulting in the largest defect fill response.

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.

Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam.

The GABA and benzodiazepine binding sites on GABA(A) receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug-naive rats was potentiated approximately 1.6-fold by 100 microM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA potentiation were observed. Diazepam-induced changes in GABA(A) receptor gamma2-subunit gene transcription and alpha1-, beta2-, and gamma2-subunit steady-state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Gynecologic and reproductive services for women with developmental disabilities.

A two-phase study using mail-out questionnaires examined the availability and accessibility of gynecologic and reproductive services for women with developmental disabilities. First, 127 women with developmental disabilities responded to queries about the accessibility and type of available services, the providers of necessary care, and satisfaction with the services. The age range of the women was 18-80 years, with a mean age of 40 years. Of the women queried, 40% indicated that they had not received health education regarding gynecologic and reproductive needs, and 27% indicated that they disliked health education about such matters. In the second phase of the study, agencies that provide services to these women were queried about patterns of the providers, available services, and identified barriers, including financial problems, difficulty in finding a provider, family perceptions, and fear of and distaste for examinations. A surprising finding was that more than one-third of the reporting agencies indicated that questions about the identification and treatment of sexual abuse were not applicable to their agencies. The findings are discussed with an emphasis on identified barriers to care, available services, and the implications for nurse practitioner practice.

Redox control of the catalytic cycle of flavocytochrome P-450 BM3.

Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.

Lased and sandblasted denture base surface preparations affecting resilient liner bonding.

STATEMENT OF PROBLEM: Adhesive failure between the liner and the denture base creates an environment for potential bacterial growth and accelerated breakdown of the soft liner resulting in a deteriorating prosthesis. PURPOSE: This study evaluated the effects of a specific sandblasted or lased preparation on the interfacial bonding of polymethyl methacrylate and silicone and polyethyl methacrylate resilient liners. MATERIAL AND METHODS: Polymethyl methacrylate test specimens were fabricated and received one of three surface treatments: untreated (control), sandblasted (250 microns aluminum oxide particles), and lased (carbon dioxide). Polyethyl methacrylate and silicone resilient lining materials were applied to these surfaces and the peel strengths were determined with the American Society for Testing and Materials peelin-adhesion test. RESULTS: Altering the polymethyl methacrylate surface by sandblasting significantly reduced the peel strengths for the polymethyl methacrylate/polyethyl methacrylate and polymethyl methacrylate/silicone specimens. Altering the polymethyl methacrylate surface by delivering carbon dioxide laser energy to form a grid pattern produced lower peel strengths that were statistically significant from the controls for the polymethyl methacrylate/polyethyl methacrylate specimens, but not so for the polymethyl methacrylate/silicone specimens. Untreated polymethyl methacrylate/polyethyl methacrylate peel strengths were significantly higher than polymethyl methacrylate/silicone. CONCLUSIONS: Results of this study imply that mechanical surface preparation of denture bases before application of a resilient liner may not be warranted.

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.

Effect of resin/fluoride and holmium:YAG laser irradiation on the resistance to the formation of caries-like lesions.

PURPOSE: This study compared the effects of a combination of a topical application of resin/NaF mixture and holmium-doped yttrium aluminum garnet (HO:YAG) laser energy with untreated control sites for their resistance to acid destruction/mechanical challenges of root surfaces adjacent to restorative margins. MATERIALS AND METHODS: Forty-eight extracted human teeth were prepared for restorations with coronal margins in enamel and the apical margins on root surfaces. Four types of restorative materials were used. One half of the restorative/root surface margins were treated with an application of nonfilled resin/fluoride mixture and holmium-doped yttrium aluminum garnet laser irradiation; the remaining portion served as the untreated control for each tooth. Acid (10% formic) and mechanical (air sandblaster) challenges were used to test the effects of these treated sites to their controls and to other types of restorative/root surface margins. The depth of root surface loss within 0 to 2 mm apical from the restorative margins was measured and evaluated after 24, 48, and 72 hours of acid exposures. RESULTS: The measurements of tooth loss for the 24- and 48-hour acid-exposure cycles were subjected to statistical analysis (ANOVA) using a paired t test for the variables. The variables, depth of tooth surface loss for the control sites was compared with the depth of loss of the treated sites on opposing sides of each tooth. The depth of tooth surface loss for the control sites had an average mean of 0.35 (0.25) mm for the 24-hour acid exposures, with the opposing treated sides having a mean of 0.053 (0.05) mm (significant at the 0.05 level). The 48-hour acid-exposure results showed significant increased tooth loss for the control sites, but minimum additional loss on the treated sites. The control sites showed an average mean of 1.16 (0.24) mm and a mean of 0.095 (0.103) mm for the treated areas, significant at the 0.05 level. No measurements were made for the 72-hour cycle groups of composite and amalgam alloy restorations because of the loss of most of the filling materials in the control sites. The control groups of crowns showed an average mean loss of 2.06 (0.37) mm with only 0.20 (0.19) mm surface loss for the treated sites. The paired t test for variables indicated no significant differences of tooth surface loss between various types of restorative margins used in this study. CONCLUSIONS: HO:YAG laser energy irradiation after application of resin/NaF to restorative margins and adjacent areas showed a significant increased resistance to acid/mechanical destruction on cementum-dentin root surfaces. The integrity of the restorative/dentin margins were maintained after extended exposures to formic acid and mechanical challenges.

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.

Dimethylsulfide:acceptor oxidoreductase from Rhodobacter sulfidophilus. The purified enzyme contains b-type haem and a pterin molybdenum cofactor.

Dimethylsulfide:receptor oxidoreductase was purified from the purple non-sulfur phototrophic bacterium Rhodobacter sulfidophilus. The native form of the enzyme had a molecular mass of 152 kDa and was composed of three distinct subunits of 94, 38 and 32 kDa. Dimethylsulfide:acceptor oxidoreductase did not oxidise other thioethers which were tested. The enzyme was able to reduce a variety of N-oxides using reduced methylviologen as electron donor but it reduced dimethylsulfoxide at a very low rate. The resting form of dimethylsulfide:acceptor oxidoreductase exhibited a spectrum which was characteristic of a reduced cytochrome with absorbance maxima at 562 nm, 533 nm and 428 nm. Pyridine haemochrome analysis established that the cytochrome contained a b-type haem and a content of 0.65 mol protohaem/mol enzyme was determined. After oxidation of the haem with ferricyanide, the absorbance spectrum of the reduced cytochrome was restored by reduction with dimethylsulfide. Metal analysis revealed that dimethylsulfide:acceptor oxidoreductase contained 0.5 mol Mo and 3.5 mol Fe/mol enzyme. Heat treatment of the enzyme released material with fluorescence excitation and emission spectra which were characteristic of form B of the pterin component of the pterin molybdenum cofactor. From this analysis it is concluded that dimethylsulfide:acceptor oxidoreductase is a molybdenum oxotransferase which may also contain a iron-sulfur cluster. It is suggested that the haem and pterin molybdenum cofactor are associated with the 94-kDa subunit.