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Author(s): Bota, Daniela; Piccioni, David; LaRocca, R; Duma, Christopher; Kesari, Santosh; Carrillo, Jose; O’Donnell, Robert T; Aiken, Robert; Hsu, Frank; Kong, Xiao-Tang; Hsieh, Candace; Langford, Chris; Carta, Krystal; Wang, Adrienne; Langford, James; Taylor, Thomas; Nistor, Gabriel; Dillman, Robert | Abstract: Abstract GBM standard treatment is associated with poor survival. Adjunctive therapy with patient-specific vaccines may improve outcomes by enhancing anti-GBM immune responses. A multi-institutional phase II clinical trial was designed with a primary objective of 75% survival 15 months after intent-to-treat enrollment. IL-4 and GM-CSF were used to generate dendritic cells (DC) from monocytes. DC were incubated with autologous tumor antigens (ATA) from the lysate of cultured GBM cells to produce each patient-specific DC-ATA vaccine. Each dose was admixed with 500 mcg GM-CSF at the time of subcutaneous injections at weeks 1, 2, 3, 8, 12, 16, 20 and 24. Enrollment has been completed in April 2020 (n=60). Three patients withdrew from the study prior to starting treatment leaving 57 patients for whom data is available. So far 57 patients have received 344 doses; 27 have completed all 8 doses, 11 received fewer than 8 doses at the time they discontinued treatment, 19 are currently in treatment. No patient has discontinued treatment because of toxicity. 9 pt had died and the preliminary 12 months overall survival is 74%. In a preliminary serologic analysis 12 of 16 patients (75%) had an increase in markers associated with Th1/NK, Th2/immunoglobulins, and Th2 hypersensitivity (eotaxins, IgE and IL17F) by week-3; 9 of 15 (60%) had a decrease in angiogenesis factors, growth factors, and tumor markers by week-8. Immunologic data for all 55 patients who received at least two injections will be available November 2020. This patient-specific DC-ATA immunotherapy approach is feasible, is associated with changes in serologic markers, and may be increasing intratumor inflammation that may be associated with on-target toxicity and efficacy. A interim survival analysis will be conducted in mid-October 2020, 15 months after the 28th patient was enrolled; results will be available November 2020 [Clinicaltrials.gov NCT03400917].

// Adrienne Wang 1 , Jacob Mouser 1 , Jason Pitt 1 , Daniel Promislow 1,2 and Matt Kaeberlein 1 1 University of Washington, Department of Pathology, Seattle, WA, USA 2 University of Washington, Department of Biology, Seattle, WA, USA Correspondence to: Matt Kaeberlein, email: // Keywords : mitochondria, leigh syndrome, rapamycin, Drosophila, complex I Received : August 08, 2016 Accepted : September 27, 2016 Published : October 11, 2016 Abstract Pediatric mitochondrial disorders are a devastating category of diseases caused by deficiencies in mitochondrial function. Leigh Syndrome (LS) is the most common of these diseases with symptoms typically appearing within the first year of birth and progressing rapidly until death, usually by 6-7 years of age. Our lab has recently shown that genetic inhibition of the mechanistic target of rapamycin (TOR) rescues the short lifespan of yeast mutants with defective mitochondrial function, and that pharmacological inhibition of TOR by administration of rapamycin significantly rescues the shortened lifespan, neurological symptoms, and neurodegeneration in a mouse model of LS. However, the mechanism by which TOR inhibition exerts these effects, and the extent to which these effects can extend to other models of mitochondrial deficiency, are unknown. Here, we probe the effects of TOR inhibition in a Drosophila model of complex I deficiency. Treatment with rapamycin robustly suppresses the lifespan defect in this model of LS, without affecting behavioral phenotypes. Interestingly, this increased lifespan in response to TOR inhibition occurs in an autophagy-independent manner. Further, we identify a fat storage defect in the ND2 mutant flies that is rescued by rapamycin, supporting a model that rapamycin exerts its effects on mitochondrial disease in these animals by altering metabolism.

Standard treatment of primary glioblastoma (GBM) is associated with poor survival. Adjunctive therapy with patient-specific vaccines may improve outcomes by inducing or enhancing each patient's anti-GBM immune response. A multi-institutional phase II clinical trial was designed with a primary objective of 75% survival 15 months after intent-to-treat enrollment. Key eligibility criteria were: (1) primary GBM diagnosis, (2) age < 70 years at time of tumor resection, (3) successful cell culture of GBM cells in serum-free media, (4) successful monocyte collection by leukapheresis, and (5) KPS > 70 after recovery from surgery. IL-4 and GM-CSF were used to generate dendritic cells (DC) from monocytes. DC were incubated with autologous tumor antigens (ATA) from the lysate of cultured GBM cells to produce each patient-specific DC-ATA vaccine. Each dose was admixed with GM-CSF at the time of subcutaneous injections at weeks 1, 2, 3, 8, 12, 16, 20 and 24. At the time of this analysis, cell line success rate is 61/63 (97%); monocyte collection success rate is 53/55 (96%), but 10 patients required a second apheresis. 50 of a planned 55 patients have enrolled after recovery from surgery, just prior to starting standard concurrent radiation therapy (RT) and temozolomide (TMZ), with intent-to-treat after recovery from RT/TMZ. The 50 patients include 36 men and 14 women; median age is 58 years with a range of 27 to 70. Average KPS is 82.8. MGMT methylation has been documented in 22% of patients and IDH mutation in 14%. 37 patients have started treatment and received 231 doses. 16 have completed all 8 doses, 7 received fewer than 8 doses, and 14 are currently undergoing treatment. No patient has discontinued treatment because of toxicity, but 20 patients have experienced 35 treatment-emergent serious adverse events including hospitalizations for falls and/or increased focal weakness (12 episodes), seizures (10 episodes), or severe headaches or visual changes (3 episodes). 6-month actual survival rate is 96% for the 28 patients at risk 6 months or longer from enrollment. Serologic analyses show that 12 of 16 patients (75%) had an increase in markers associated with Th1/NK, Th2/immunoglobulins, and Th2 hypersensitivity (eotaxins, IgE and IL17F) by week-3; 9 of 15 (60%) had a decrease in angiogenesis factors, growth factors, and tumor markers by week-8. On serial MRI scans, 7 of 13 patients (54%) have exhibited an increase in T2 (tumor) and flare (edema) for several weeks after starting DC-ATA, followed by a slow gradual decrease in both. This patient-specific DC-ATA immunotherapy approach is feasible, is associated with changes in serologic markers, and may be increasing intratumor inflammation that may be associated with on-target toxicity and efficacy. Longer follow up is needed before the survival objective can be assessed. [Clinicaltrials.gov NCT03400917] Citation Format: Daniella A. Bota, Christopher M. Duma, Santosh Kesari, David E. Piccioni, Renato V. LaRocca, Robert T. O'Donnell, Robert D. Aiken, Jose A. Carrillo, Candace Hsieh, Chris J. Langford, Krystal Carta, Adrienne M. Wang, James A. Langford, Thomas H. Taylor, Gabriel I. Nistor, Robert O. Dillman. Adjunctive treatment of primary glioblastoma with patient-specific dendritic cell vaccines pulsed with antigens from self-renewing autologous tumor cells: A phase II trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT182.

Standard treatment of primary ovarian epithelial cancer has improved, but 5-year survival is only about 40% for the cohort of patients who present with stage 3 or 4 disease. Adjunctive therapy with patient-specific vaccines may improve outcomes by enhancing each patient's anti-cancer immune response. A multi-institutional, double- blinded randomized phase II clinical trial was designed with a primary objective of a 50% reduction in the risk of death compared to the control arm. Key eligibility criteria are: (1) primary diagnosis of stage 3 or 4 ovary epithelial cancer, (2) successful cell culture of ovarian cancer cells, (3) successful monocyte collection by leukapheresis, and (4) ECOG of 0 or 1 at the time of randomization. After completing primary systemic therapy, patients are stratified based on whether they have any residual evidence of disease and then randomized 2:1 to vaccine or control arms. Randomization occurs about 7 months after initial surgery. In the active arm IL-4 and GM-CSF are used to differentiate monocytes into dendritic cells (DC) which are incubated with the lysate of cultured ovary cancer cells as the source of autologous tumor antigens (ATA) to produce each patient-specific DC-ATA vaccine. Patients in the control arm receive cryopreserved autologous monocytes from their leukapheresis product. Each dose is admixed with 500 mcg GM-CSF at the time of subcutaneous injections at weeks 1, 2, 3, 8, 12, 16, 20 and 24. DC-ATA may be given alone or in combination with maintenance or salvage therapies. At the time of this analysis, cell line success rate is 35/35 (100%); monocyte collection success rate is 19/20 (95%), and 2 patients required a second apheresis to get a satisfactory product. 14 patients had primary ovarian, 2 fallopian tube, and 1 primary intraperitoneal cancer. 17 of a planned 99 have been randomized. At diagnosis 15 patients had stage 3 disease and two had stage 4; 15 had no evidence of disease at the time of randomization. Primary systemic therapy was adjuvant for 7 and neoadjuvant for 10. Ages of the randomized patients range from 39 to 80 years with a median of 61 years. ECOG status was 0 for 11 subjects, and 1 for 6 subjects. 13 patients have started treatment and received 92 injections; 9 have received all 8 doses and only 2 have stopped before 8 doses. No patient has discontinued treatment because of toxicity, but 1 patient was hospitalized after the 8th dose for refractory urticaria that resolved after high-dose corticosteroids followed by slow tapering. This patient-specific DC-ATA immunotherapy approach is feasible in patients with primary advanced ovarian cancer. The clinical trial is continuing as planned. [Clinicaltrials.gov NCT02033616] Citation Format: Lisa N. Abaid, Bradley R. Corr, James R. Mason, Richard L. Friedman, Ramez N. Eskander, Candace Hsieh, Chris J. Langford, Krystal Carta, Adrienne M. Wang, James A. Langford, Gabriel I. Nistor, Robert O. Dillman. Patient-specific dendritic cell vaccines with tumor antigens from self-renewing autologous tumor cells in the treatment of primary advanced ovarian cancer: A multi-institutional phase II clinical trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT189.

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential, and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex I-associated neurodegeneration.

We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.

A major obstacle for preclinical testing of Alzheimer’s disease (AD) therapies is the availability of translationally relevant AD models. Critical for the validation of such models is the application of the same approaches and techniques used for the neuropathological characterization of AD. Deposition of amyloid-β 42 (Aβ42) plaques and neurofibrillary tangles containing phospho-Tau (pTau) are the pathognomonic features of AD. In the neuropathologic evaluation of AD, immunohistochemistry (IHC) is the current standard method for detection of Aβ42 and pTau. Although IHC is indispensable for determining the distribution of AD pathology, it is of rather limited use for assessment of the quantity of AD pathology. We have recently developed Luminex-based assays for the quantitative assessment of Aβ42 and pTau in AD brains. These assays are based on the same antibodies that are used for the IHC-based diagnosis of AD neuropathologic change. Here we report the application and extension of such quantitative AD neuropathology assays to commonly used genetically engineered AD models and to animals that develop AD neuropathologic change as they age naturally. We believe that identifying AD models that have Aβ42 or pTau levels comparable to those observed in AD will greatly improve the ability to develop AD therapies. Abbreviations: Alzheimer’s disease (AD); amyloid β 42 (Aβ42); phospho-Tau (pTau); immunohistochemistry (IHC)

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

SummaryMany genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

In this paper, we demonstrate that significant performance gains can be achieved in CCG semantic parsing by introducing a linguistically motivated grammar induction scheme. We present a new morpho-syntactic factored lexicon that models systematic variations in morphology, syntax, and semantics across word classes. The grammar uses domain-independent facts about the English language to restrict the number of incorrect parses that must be considered, thereby enabling eective learning from less data. Experiments in benchmark domains match previous models with one quarter of the data and provide new state-of-the-art results with all available data, including up to 45% relative test-error reduction.

Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. Here we show that dietary restriction during chronological aging delays the reduction in subsequent replicative life span up to at least 23 days of chronological age. We further show that among the viable portion of the control population aged 26 days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role for mitochondrial function in this process.

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz–Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.

The quest to slow aging has come far, and what used to be the domain of science fiction writers and snake oil salesmen may soon become science fact. Innovative new approaches, such as the use of the very short-lived African killifish (Harel et al.), are bridging the translational gap and bring the promise of healthy longevity to fruition.

Altered protein homeostasis underlies degenerative diseases triggered by misfolded proteins, including spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by a CAG/glutamine expansion in the androgen receptor. Here we show that the unfolded protein response (UPR), an ER protein quality control pathway, is induced in skeletal muscle from SBMA patients, AR113Q knock-in male mice, and surgically denervated wild-type mice. To probe the consequence of UPR induction, we deleted CHOP (C/EBP homologous protein), a transcription factor induced following ER stress. CHOP deficiency accentuated atrophy in both AR113Q and surgically denervated muscle through activation of macroautophagy, a lysosomal protein quality control pathway. Conversely, impaired autophagy due to Beclin-1 haploinsufficiency decreased muscle wasting and extended lifespan of AR113Q males, producing a significant and unexpected amelioration of the disease phenotype. Our findings highlight critical cross-talk between the UPR and macroautophagy, and they indicate that autophagy activation accentuates aspects of the SBMA phenotype., Author Summary In many age-dependent neurodegenerative diseases, the accumulation of misfolded or mutant proteins drives pathogenesis. Several protein quality control pathways have emerged as central regulators of the turnover of these toxic proteins and therefore impact phenotypic severity. In spinal and bulbar muscular atrophy (SBMA), the mutant androgen receptor with an expanded glutamine tract undergoes hormone-dependent nuclear translocation, unfolding, and oligomerization—steps that are critical to the development of progressive proximal limb and bulbar muscle weakness in men. Here we show that the unfolded protein response (UPR), an endoplasmic reticulum stress response, is triggered in skeletal muscle from SBMA patients and knock-in mice. We find that disruption of the UPR exacerbates skeletal muscle atrophy through the induction of macroautophagy, a lysosomal protein quality pathway. In contrast, impaired autophagy diminishes muscle wasting and prolongs lifespan of SBMA mice. Our findings highlight cross-talk between the UPR and autophagy, and they suggest that limited activation of the autophagic pathway may be beneficial in certain neuromuscular diseases such as SBMA where the nucleus is the essential site of toxicity.

CHIP (carboxy terminus of Hsc70-interacting protein) an E3 ubiquitin ligase that binds to Hsp70 and Hsp90, promotes degradation of several Hsp90-regulated signaling proteins and disease-causing proteins containing expanded glutamine tracts. In polyglutamine disease models, CHIP has been considered a primary protection factor by promoting degradation of these misfolded proteins. Here, we show that two CHIP substrates, the glucocorticoid receptor (GR), a classic Hsp90-regulated signaling protein, and the expanded glutamine androgen receptor (AR112Q), are degraded at the same rate in CHIP(-/-) and CHIP(+/+) mouse embryonic fibroblasts after treatment with the Hsp90 inhibitor geldanamycin. CHIP(-/-) cytosol has the same ability as CHIP(+/+) cytosol to ubiquitinate purified neuronal nitric oxide synthase (nNOS), another established CHIP substrate. To determine whether other E3 ubiquitin ligases that bind to Hsp70 (Parkin) or Hsp90 (Mdm2) act on CHIP substrates, each E3 ligase was co-expressed with the GR, nNOS, AR112Q or Q78 ataxin-3. CHIP lowered the levels of all four proteins, Parkin acted on nNOS and Q78 ataxin-3 but not on the steroid receptors, and Mdm2 did not affect any of the co-expressed proteins. Moreover, both CHIP and Parkin co-localized to aggregates of the expanded glutamine AR formed in cell culture and in a knock-in mouse model of spinal and bulbar muscular atrophy. These observations establish that CHIP does not play an exclusive role in regulating the turnover of Hsp90 client signaling proteins or expanded glutamine tract proteins, and show that the Hsp70-dependent E3 ligase Parkin acts redundantly to CHIP on some substrates.

The mechanisms underlying biological aging have been extensively studied in the past 20 years with the avail of mainly four model organisms: the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fruitfly Drosophila melanogaster, and the domestic mouse Mus musculus. Extensive research in these four model organisms has identified a few conserved genetic pathways that affect longevity as well as metabolism and development. Here, we review how the mechanistic target of rapamycin (mTOR), sirtuins, adenosine monophosphate-activated protein kinase (AMPK), growth hormone/insulin-like growth factor 1 (IGF-1), and mitochondrial stress-signaling pathways influence aging and life span in the aforementioned models and their possible implications for delaying aging in humans. We also draw some connections between these biochemical pathways and comment on what new developments aging research will likely bring in the near future.

Analysis of conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons., Background It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined. Results We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences. Conclusions Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.

Background It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.