Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis.

Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.

The role of CD8+ T-cell clones in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production.

The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

The mitochondria-targeted peptide SS-31 binds lipid bilayers and modulates surface electrostatics as a key component of its mechanism of action.

Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13 C 3 -pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δ taz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δ crd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13 C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

The 18S rRNA Methyltransferase DIMT-1 Regulates Lifespan in the Germline Later in Life.

Ribosome heterogeneity has emerged as an important regulatory control feature for determining which proteins are synthesized, however, the influence of age on ribosome heterogeneity is not fully understood. Whether mRNA transcripts are selectively translated in young versus old cells and whether dysregulation of this process drives organismal aging is unknown. Here we examined the role of ribosomal RNA (rRNA) methylation in maintaining appropriate translation as organisms age. In a directed RNAi screen, we identified the 18S rRNA N6'-dimethyl adenosine (m6,2A) methyltransferase, dimt-1, as a regulator of C. elegans lifespan and stress resistance. Lifespan extension induced by dimt-1 deficiency required a functional germline and was dependent on the known regulator of protein translation, the Rag GTPase, raga-1, which links amino acid sensing to the mechanistic target of rapamycin complex (mTORC)1. Using an auxin-inducible degron tagged version of dimt-1, we demonstrate that DIMT-1 functions in the germline after mid-life to regulate lifespan. We further found that knock-down of dimt-1 leads to selective translation of transcripts important for stress resistance and lifespan regulation in the C. elegans germline in mid-life including the cytochrome P450 daf-9, which synthesizes a steroid that signals from the germline to the soma to regulate lifespan. We found that dimt-1 induced lifespan extension was dependent on the daf-9 signaling pathway. This finding reveals a new layer of proteome dysfunction, beyond protein synthesis and degradation, as an important regulator of aging. Our findings highlight a new role for ribosome heterogeneity, and specific rRNA modifications, in maintaining appropriate translation later in life to promote healthy aging.

Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation.

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

The 18S rRNA Methyltransferase DIMT-1 Regulates Lifespan in the Germline Later in Life.

Ribosome heterogeneity has emerged as an important regulatory control feature for determining which proteins are synthesized, however, the influence of age on ribosome heterogeneity is not fully understood. Whether mRNA transcripts are selectively translated in young versus old cells and whether dysregulation of this process drives organismal aging is unknown. Here we examined the role of ribosomal RNA (rRNA) methylation in maintaining appropriate translation as organisms age. In a directed RNAi screen, we identified the 18S rRNA N6'-dimethyl adenosine (m6,2A) methyltransferase, dimt-1, as a regulator of C. elegans lifespan and stress resistance. Lifespan extension induced by dimt-1 deficiency required a functional germline and was dependent on the known regulator of protein translation, the Rag GTPase, raga-1, which links amino acid sensing to the mechanistic target of rapamycin complex (mTORC)1. Using an auxin-inducible degron tagged version of dimt-1, we demonstrate that DIMT-1 functions in the germline after mid-life to regulate lifespan. We further found that knock-down of dimt-1 leads to selective translation of transcripts important for stress resistance and lifespan regulation in the C. elegans germline in mid-life including the cytochrome P450 daf-9, which synthesizes a steroid that signals from the germline to the soma to regulate lifespan. We found that dimt-1 induced lifespan extension was dependent on the daf-9 signaling pathway. This finding reveals a new layer of proteome dysfunction, beyond protein synthesis and degradation, as an important regulator of aging. Our findings highlight a new role for ribosome heterogeneity, and specific rRNA modifications, in maintaining appropriate translation later in life to promote healthy aging.

Phylogenomics and genetic analysis of solvent-producing Clostridium species.

The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii, 57 C. saccharobutylicum, 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production.

Retrieval of iatrogenic intravascular foreign bodies.

Malposition, embolization, fracture, and migration of endovascular devices are unfortunate consequences of endovascular intervention and will be encountered at some point by nearly every practitioner. The existing literature on foreign body retrieval consists of large single-institution series and case reports. We provide an overview of this recent literature, clarifying what devices are being lost, what symptoms occur as a result, and how retrieval is being performed. We have identified all case series and case reports since the year 2000, summarized the results, and made some general observations and recommendations that may be useful to the practitioner faced with the prospect of retrieving a fractured medical device, malpositioned coil, or migrated inferior vena cava filter.

Interactions of amyloidogenic proteins with mitochondrial protein import machinery in aging-related neurodegenerative diseases.

Most mitochondrial proteins are targeted to the organelle by N-terminal mitochondrial targeting sequences (MTSs, or "presequences") that are recognized by the import machinery and subsequently cleaved to yield the mature protein. MTSs do not have conserved amino acid compositions, but share common physicochemical properties, including the ability to form amphipathic α-helical structures enriched with basic and hydrophobic residues on alternating faces. The lack of strict sequence conservation implies that some polypeptides can be mistargeted to mitochondria, especially under cellular stress. The pathogenic accumulation of proteins within mitochondria is implicated in many aging-related neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Mechanistically, these diseases may originate in part from mitochondrial interactions with amyloid-ß precursor protein (APP) or its cleavage product amyloid-ß (Aß), α-synuclein (α-syn), and mutant forms of huntingtin (mHtt), respectively, that are mediated in part through their associations with the mitochondrial protein import machinery. Emerging evidence suggests that these amyloidogenic proteins may present cryptic targeting signals that act as MTS mimetics and can be recognized by mitochondrial import receptors and transported into different mitochondrial compartments. Accumulation of these mistargeted proteins could overwhelm the import machinery and its associated quality control mechanisms, thereby contributing to neurological disease progression. Alternatively, the uptake of amyloidogenic proteins into mitochondria may be part of a protein quality control mechanism for clearance of cytotoxic proteins. Here we review the pathomechanisms of these diseases as they relate to mitochondrial protein import and effects on mitochondrial function, what features of APP/Aß, α-syn and mHtt make them suitable substrates for the import machinery, and how this information can be leveraged for the development of therapeutic interventions.

Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation.

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

Development and application of a particle-particle particle-mesh Ewald method for dispersion interactions.

For inhomogeneous systems with interfaces, the inclusion of long-range dispersion interactions is necessary to achieve consistency between molecular simulation calculations and experimental results. For accurate and efficient incorporation of these contributions, we have implemented a particle-particle particle-mesh Ewald solver for dispersion (r(-6)) interactions into the LAMMPS molecular dynamics package. We demonstrate that the solver's O(N log N) scaling behavior allows its application to large-scale simulations. We carefully determine a set of parameters for the solver that provides accurate results and efficient computation. We perform a series of simulations with Lennard-Jones particles, SPC/E water, and hexane to show that with our choice of parameters the dependence of physical results on the chosen cutoff radius is removed. Physical results and computation time of these simulations are compared to results obtained using either a plain cutoff or a traditional Ewald sum for dispersion.

Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds.

Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.

Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon.

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (thlA), CoA-transferase (ctfAB), and acetoacetate decarboxylase (adc) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.

Influenza sequence and epitope database.

Influenza epidemics arise through the acquisition of viral genetic changes to overcome immunity from previous infections. An increasing number of complete genomes of influenza viruses have been sequenced in Asia in recent years. Knowledge about the genomes of the seasonal influenza viruses from different countries in Asia is valuable for monitoring and understanding of the emergence, migration and evolution of strains. In order to make full use of the wealth of information from such data, we have developed an integrated user friendly relational database, Influenza Sequence and Epitope Database (ISED), that catalogs the influenza sequence and epitope information obtained in Asia. ISED currently hosts a total of 13,020 influenza A and 2984 influenza B virus sequence data collected in 17 countries including 9 Asian countries, and a total of approximately 545 amantadine-resistant influenza virus sequences collected in Korea. ISED provides users with prebuilt application tools to analyze sequence alignment and different patterns and allows users to visualize epitope-matching structures, which is freely accessible at http://influenza.korea.ac.kr and http://influenza.cdc.go.kr.

Minimal residual disease is a significant predictor of treatment failure in non T-lineage adult acute lymphoblastic leukaemia: final results of the international trial UKALL XII/ECOG2993.

The predictive value of molecular minimal residual disease (MRD) monitoring using polymerase chain reaction amplification of clone-specific immunoglobulin or T-cell Receptor rearrangements was analysed in 161 patients with non T-lineage Philadelphia-negative acute lymphoblastic leukaemia (ALL) participating in the UK arm of the international ALL trial UKALL XII/Eastern Cooperative Oncology Group (ECOG) 2993. MRD positivity (> or =10(-4)) in patients treated with chemotherapy alone was associated with significantly shorter relapse-free survival (RFS) at several time-points during the first year of therapy. MRD status best discriminated outcome after phase 2 induction, when the relative risk of relapse was 8.95 (2.85-28.09)-fold higher in MRD-positive (> or =10(-4)) patients and the 5-year RFS 15% [95% confidence interval (CI) 0-40%] compared to 71% (56-85%) in MRD-negative (<10(-4)) patients (P = 0.0002) When MRD was detected prior to autologous stem cell transplantation (SCT), a significantly higher rate of treatment failure was observed [5-year RFS 25% (CI 0-55%) vs. 77% (95% CI 54-100%) in MRD-negative/<10(-4), P = 0.01] whereas in recipients of allogeneic-SCT in first complete remission, MRD positivity pre-transplant did not adversely affect outcome. These data provide a rationale for introducing MRD-based risk stratification in future studies for the delineation of those at significant risk of treatment failure in whom intensification of therapy should be evaluated.

IDBD: infectious disease biomarker database.

Biomarkers enable early diagnosis, guide molecularly targeted therapy and monitor the activity and therapeutic responses across a variety of diseases. Despite intensified interest and research, however, the overall rate of development of novel biomarkers has been falling. Moreover, no solution is yet available that efficiently retrieves and processes biomarker information pertaining to infectious diseases. Infectious Disease Biomarker Database (IDBD) is one of the first efforts to build an easily accessible and comprehensive literature-derived database covering known infectious disease biomarkers. IDBD is a community annotation database, utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It allows users to link infectious diseases or pathogens to protein, gene or carbohydrate biomarkers through the use of search tools. It supports various types of data searches and application tools to analyze sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 611 biomarkers for 66 infectious diseases and 70 pathogens. It is publicly accessible at http://biomarker.cdc.go.kr and http://biomarker.korea.ac.kr.

Reversal of age-associated thymic atrophy: treatments, delivery, and side effects.

Our ability to survive infectious agents depends on making adequate immune responses, but as we get older our thymus atrophies. Production and export of T cells bearing new antigen receptor specificities to the peripheral T cell pool declines and results in shrinkage of the repertoire. Other changes in the peripheral T cell pool include an increase in cells moving closer to their replicative limit. Age related immune dysfunction, evident through the increased susceptibility to infection, follows these changes. Improvement in immune function in the elderly may require us to rejuvenate the immune system starting first with reversing the atrophy seen in the thymus. This has been achieved experimentally with interleukin 7, growth hormone, growth hormone secretagogues, keratinocyte growth factor or through chemical or surgical castration. The widespread use of one or more of these treatments will depend upon their effectiveness, their ease of delivery and the extent of any side effects.

Periodic re-emergence of endemic strains with strong epidemic potential-a proposed explanation for the 2004 Indonesian dengue epidemic.

Indonesia experienced a severe dengue epidemic in the first quarter of 2004 with 58,301 cases and 658 deaths reported to the WHO. All four dengue virus (DENV) serotypes were detected, with DENV-3 the predominant strain. To ascertain the molecular epidemiology of the DENV associated with the epidemic, complete genomes of 15 isolates were sequenced from patient serum collected in Jakarta during the epidemic, and two historical DENV-3 isolates from previous epidemics in 1988 and 1998 were selectively sequenced for comparative studies. Phylogenetic trees for all four serotypes indicate the viruses are endemic strains that have been circulating in Indonesia for a few decades. Whole-genome phylogeny showed the 2004 DENV-3 isolates share high similarity with those isolated in 1998 during a major epidemic in Sumatra. Together these subtype I DENV-3 strains form a Sumatran-Javan clade with demonstrated epidemic potential. No newly-acquired amino acid mutations were found while comparing genomes from the two epidemics. This suggests re-emergence of little-changed endemic strains as causative agents of the epidemic in 2004. Notably, the molecular evidence rules out change in the viral genomes as the trigger of the epidemic.