Comprehensive Analysis of the Complete Mitochondrial Genome of Rehmannia chingii: An Autotrophic Species in the Orobanchaceae Family.

Rehmannia chingii is an important medicinal plant with immense value in scientific research. However, its mitochondrial genome (mitogenome) has not yet been characterized. Herein, based on whole-genome Illumina short reads and PacBio HiFi reads, we obtained the complete mitogenome of R. chingii through a de novo assembly strategy. We carried out comparative genomic analyses and found that, in comparison with the plastid genome (plastome) showing a high degree of structural conservation, the R. chingii mitogenome structure is relatively complex, showing an intricate ring structure with 16 connections, owing to five repetitive sequences. The R. chingii mitogenome was 783,161 bp with a GC content of 44.8% and contained 77 genes, comprising 47 protein-coding genes (CDS), 27 tRNA genes, and 3 rRNA genes. We counted 579 RNA editing events in 47 CDS and 12,828 codons in all CDSs of the R. chingii mitogenome. Furthermore, 24 unique sequence transfer fragments were found between the mitogenome and plastome, comprising 8 mitogenome CDS genes and 16 plastome CDS genes, corresponding to 2.39% of the R. chingii mitogenome. Mitogenomes had shorter but more collinear regions, evidenced by a comparison of the organelles of non-parasitic R. chingii, hemiparasitic Pedicularis chinensis, and holoparasitic Aeginetia indica in the Orobanchaceae family. Moreover, from non-parasitic to holoparasitic species, the genome size in the mitogenomes of Orobanchaceae species did not decrease gradually. Instead, the smallest mitogenome was found in the hemiparasitic species P. chinensis, with a size of 225,612 bp. The findings fill the gap in the mitogenome research of the medicinal plant R. chingii, promote the progress of the organelle genome research of the Orobanchaceae family, and provide clues for molecular breeding.

Loss of CLTRN function produces a neuropsychiatric disorder and a biochemical phenotype that mimics Hartnup disease.

Hartnup disease is an autosomal recessive condition characterized by neutral aminoaciduria and behavioral problems. It is caused by a loss of B0 AT1, a neutral amino acid transporter in the kidney and intestine. CLTRN encodes the protein collectrin that functions in the transportation and activation of B0 AT1 in the renal apical brush bordered epithelium. Collectrin deficient mice have severe aminoaciduria. However, the phenotype associated with collectrin deficiency in humans has not been reported. Here we report two patients, an 11-year-old male who is hemizygous for a small, interstitial deletion on Xp22.2 that encompasses CLTRN and a 22-year-old male with a deletion spanning exons 1 to 3 of CLTRN. Both of them present with neuropsychiatric phenotypes including autistic features, anxiety, depression, compulsions, and motor tics, as well as neutral aminoaciduria leading to a clinical diagnosis of Hartnup disease and treatment with niacin supplementation. Plasma amino acids were normal in both patients. One patient had low 5-hydroxyindoleacetic acid levels, a serotoninergic metabolite. We explored the expression of collectrin in the murine brain and found it to be particularly abundant in the hippocampus, brainstem, and cerebellum. We propose that collectrin deficiency in humans can be associated with aminoaciduria and a clinical picture similar to that seen in Hartnup disease. Further studies are needed to explore the role of collectrin deficiency in the neurological phenotypes.

Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

Detecting useful genetic markers and reconstructing the phylogeny of an important medicinal resource plant, Artemisia selengensis, based on chloroplast genomics.

Artemisia selengenesis is not only a health food, but also a well-known traditional Chinese medicine. Only a fraction of the chloroplast (cp) genome data of Artemisia has been reported and chloroplast genomic materials have been widely used in genomic evolution studies, molecular marker development, and phylogenetic analysis of the genus Artemisia, which makes evolutionary studies, genetic improvement, and phylogenetic identification very difficult. In this study, the complete chloroplast genome of A. selengensis was compared with that of other species within Artemisia and phylogenetic analyses was conducted with other genera in the Asteraceae family. The results showed that A. selengensis is an AT-rich species and has a typical quadripartite structure that is 151,215 bp in length. Comparative genome analyses demonstrated that the available chloroplast genomes of species of Artemisia were well conserved in terms of genomic length, GC contents, and gene organization and order. However, some differences, which may indicate evolutionary events, were found, such as a re-inversion event within the Artemisia genus, an unequal duplicate phenomenon of the ycf1 gene because of the expansion and contraction of the IR region, and the fast-evolving regions. Repeated sequences analysis showed that Artemisia chloroplast genomes presented a highly similar pattern of SSR or LDR distribution. A total of 257 SSRs and 42 LDRs were identified in the A. selengensis chloroplast genome. The phylogenetic analysis showed that A. selengensis was sister to A. gmelinii. The findings of this study will be valuable in further studies to understand the genetic diversity and evolutionary history of Asteraceae.

Integrative analysis of copy number and transcriptional expression profiles in esophageal cancer to identify a novel driver gene for therapy.

An increasing amount of evidence has highlighted the critical roles that copy number variants play in cancer progression. Here, we systematically analyzed the copy number alterations and differentially transcribed genes. Integrative analysis of the association between copy number variants and differential gene expression suggested that copy number variants will lead to aberrant expression of the corresponding genes. We performed a KEGG pathway and GO analysis, which revealed that cell cycle may have an effective role in the progression of esophageal cancer. FAM60A was then screened out as a potential prognostic factor through survival analysis and correlation analysis with clinical-pathological parameters. We subsequently showed that silencing of FAM60A could inhibit esophageal carcinoma tumor cell growth, migration and invasion in vitro. Through the bioinformatic analysis, we predict that FAM60A may act as a transcriptional factor to regulate genes that are correlated with each cell cycle. In summary, we comprehensively analyzed copy number segments and transcriptional expression profiles, which provided a novel approach to identify clinical biomarkers and therapeutic targets of esophageal carcinoma.

The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI.

PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. METHODS: Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. RESULTS: Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. CONCLUSIONS: This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

Rational Selection of Syngeneic Preclinical Tumor Models for Immunotherapeutic Drug Discovery.

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.

Role of genomic factors beyond thymidylate synthase in the prediction of response to 5-fluorouracil.

5-Fluorouracil (5FU) is still a major drug in combinations regimens for the treatment of colorectal cancer (CRC) both in the adjuvant and palliative setting. 5FU or its oral prodrug capecitabine is usually combined with irinotecan/oxaliplatin and the novel agents bevacizumab/cetuximab. Although this improved the outcome, the overall prognosis in patients with metastasized disease is still relatively poor. Although the target for 5FU, thymidylate synthase was shown to have a predictive value, this could only predict response in a subset of patients. Given the heterogeneous and complex nature of CRC, it is likely that other aberrations can affect therapeutic response. As an alternative, we investigated Copy number alterations using oligonucleotide-based high-throughput array-comparative-genomic-hybridization (aCGH) to obtain an unbiased screening of the whole genetic spectrum. Chromosomal aberrations have been identified in 85% of CRC patients and include genomic regions harboring copy number alterations in the DNA. These alterations may change the expression of many genes and might explain the differential response to therapy as shown in recent studies with several 5FU combinations. In order to clarify new predictive parameters for 5FU, we used aCGH in a historical cohort of patients, which received treatment with single agent 5FU, and an unsupervised clustering analysis showed a statistical (p < 0.05) difference between responding and nonresponding patients. We also find that several regions showed differences between responders/non-responders, such as losses in 12p12.3-12q15 and in 18p (where TS is located) in responding patients. Genome-wide analysis may provide an additional tool to discriminate between responders and nonresponders.

Narrowing the FOXF1 distant enhancer region on 16q24.1 critical for ACDMPV.

BACKGROUND: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disorder caused by heterozygous point mutations or genomic deletions involving FOXF1 or its 60-kb tissue-specific enhancer region mapping 270 kb upstream of FOXF1 and involving fetal lung-expressed long non-coding RNA genes and CpG-enriched sites. Recently, we have proposed that the FOXF1 locus at 16q24.1 may be a subject of genomic imprinting. FINDINGS: Using custom-designed aCGH and Sanger sequencing, we have identified a novel de novo 104 kb genomic deletion upstream of FOXF1 in a patient with histopathologically verified full phenotype of ACDMPV. This deletion allowed us to further narrow the FOXF1 enhancer region and identify its critical 15-kb core interval, essential for lung development. This interval harbors binding sites for lung-expressed transcription factors, including GATA3, ESR1, and YY1, and is flanked by the lncRNA genes and CpG islands. Bisulfite sequencing of one of these CpG islands on the non-deleted allele showed that it is predominantly methylated on the maternal chromosome 16. CONCLUSIONS: Substantial narrowing and bisulfite sequencing of the FOXF1 enhancer region on 16q24.1 provided new insights into its regulatory function and genomic imprinting.

Uniparental disomy causes deficiencies of vitamin K-dependent proteins.

Essentials Vitamin K-dependent coagulant factor deficiency (VKCFD) is a rare autosomal recessive disorder. We describe a case of inherited VKCFD due to uniparental disomy. The homozygous mutation caused the absence of GGCX isoform 1 and overexpression of Δ2GGCX. Hepatic and non-hepatic vitamin K-dependent proteins must be assayed to monitor VKCFD treatment. SUMMARY: Background Inherited deficiency of all vitamin K-dependent coagulant factors (VKCFD) is a rare autosomal recessive disorder caused by mutations in the γ-glutamyl carboxylase gene (GGCX) or the vitamin K epoxide reductase gene (VKORC1), with great heterogeneity in terms of both clinical presentation and response to treatment. Objective To characterize the molecular basis of VKCFD in a Spanish family. Methods and Results Sequencing of candidate genes, comparative genomic hybridization and massive sequencing identified a new mechanism causing VKCFD in the proband. Uniparental disomy (UPD) of chromosome 2 caused homozygosity of a mutation (c.44-1G>A) resulting in aberrant GGCX splicing. This change contributed to absent expression of the mRNA coding for the full-length protein, and to four-fold overexpression of the smaller mRNA isoform lacking exon 2 (Δ2GGCX). Δ2GGCX might be responsible for two unexpected clinical observations in the patient: (i) increased plasma osteocalcin levels following vitamin K1 supplementation; and (ii) a mild non-bleeding phenotype. Conclusions Our study identifies a new autosomal disease, VKCFD1, caused by UPD. These data suggest that the Δ2GGCX isoform may retain enzymatic activity, and strongly encourage the evaluation of both hepatic and non-hepatic vitamin K-dependent proteins to assess differing responses to vitamin K supplementation in VKCFD patients.

Clinicopathological and molecular study of primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma.

BACKGROUND: Primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma (CD4+ PCSM-TCL) is a rare lymphoproliferative disorder with a favorable prognosis. Distinguishing it from other cutaneous lymphomas is often a challenge. METHODS: We retrospectively collected CD4+PCSM-TCL cases from two centers (MD Anderson Cancer Center, USA and University of Milan, Italy) and evaluated their clinicopathological features. Array-comparative genomic hybridization (aCGH) analysis was performed on 11 cases. RESULTS: A total of 62 patients were identified. Single lesions were the most common clinical presentations (79%). Five patients (8%) showed multiple MF-like plaques. All patients' disease had an indolent course. The infiltrate was nodular and diffuse, multinodular or superficial but in all cases, it was characterized by small/medium pleomorphic CD4+/CD279(PD1+) lymphocytes grouped in clusters and 'pseudorosettes' around B-cells. aCGH analysis showed no significant genomic abnormalities. Single lesions were mainly treated with surgical excision (91%) and/or radiotherapy (95%) with low rate of relapse (12%). For multiple lesions, topical steroids, nitrogen mustard and phototherapy were mainly used but the rate of relapse was high (69%). CONCLUSIONS: CD4+PCSM-TCL is characterized by heterogeneous clinical presentations. The arrangement of atypical cells in clusters or pseudorosettes is a useful criterion for diagnosis. The absence of significant genomic alterations is in agreement with its indolent behavior.

Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes.

BACKGROUND: Sugar beet (Beta vulgaris) is a crop cultivated for its high content in sugar, but it is vulnerable to many soil-borne pathogens. One of them is the basidiomycete Rhizoctonia solani. This fungal species has a compatibility system regulating hyphal fusions (anastomosis). Consequently, R. solani species are categorized in anastomosis groups (AGs). AG2-2IIIB isolates are most aggressive on sugar beet. In the present study, we report on the draft genome of R. solani AG2-2IIIB using the Illumina technology. Genome analysis, interpretation and comparative genomics of five sequenced R. solani isolates were carried out. RESULTS: The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb. In addition, two normalized EST libraries were sequenced. In total 20,790 of 21,980 AG2-2IIIB isotigs (transcript isoforms) were mapped on the genome with more than 95 % sequence identity. The genome of R. solani AG2-2IIIB was predicted to harbor 11,897 genes and 4908 were found to be isolate-specific. R. solani AG2-2IIIB was predicted to contain 1142 putatively secreted proteins and 473 of them were found to be unique for this isolate. The R. solani AG2-2IIIB genome encodes a high number of carbohydrate active enzymes. The highest numbers were observed for the polysaccharide lyases family 1 (PL-1), glycoside hydrolase family 43 (GH-43) and carbohydrate estarase family 12 (CE-12). Transcription analysis of selected genes representing different enzyme clades revealed a mixed pattern of up- and down-regulation six days after infection on sugar beets featuring variable levels of resistance compared to mycelia of the fungus grown in vitro. CONCLUSIONS: The established R. solani AG2-2IIIB genome and EST sequences provide important information on the gene content, gene structure and transcriptional activity for this sugar beet pathogen. The enriched genomic platform provides an important platform to enhance our understanding of R. solani biology.

Epileptic Encephalopathy in Childhood: A Stepwise Approach for Identification of Underlying Genetic Causes.

Epilepsy is one of the most common neurological disorders in childhood. Epilepsy associated with global developmental delay and cognitive dysfunction is defined as epileptic encephalopathy. Certain inherited metabolic disorders presenting with epileptic encephalopathy can be treated with disease specific diet, vitamin, amino acid or cofactor supplementations. In those disorders, disease specific therapy is successful to achieve good seizure control and improve long-term neurodevelopmental outcome. For this reason, intractable epilepsy with global developmental delay or history of developmental regression warrants detailed metabolic investigations for the possibility of an underlying treatable inherited metabolic disorder, which should be undertaken as first line investigations. An underlying genetic etiology in epileptic encephalopathy has been supported by recent studies such as array comparative genomic hybridization, targeted next generation sequencing panels, whole exome and whole genome sequencing. These studies report a diagnostic yield up to 70%, depending on the applied genetic testing as well as number of patients enrolled. In patients with epileptic encephalopathy, a stepwise approach for diagnostic work-up will help to diagnose treatable inherited metabolic disorders quickly. Application of detailed genetic investigations such as targeted next generation sequencing as second line and whole exome sequencing as third line testing will diagnose underlying genetic disease which will help for genetic counseling as well as guide for prenatal diagnosis. Knowledge of underlying genetic cause will provide novel insights into the pathogenesis of epileptic encephalopathy and pave the ground towards the development of targeted neuroprotective treatment strategies to improve the health outcome of children with epileptic encephalopathy.

Model Matters: Differences in Orthotopic Rat Hepatocellular Carcinoma Physiology Determine Therapy Response to Sorafenib.

PURPOSE: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology noninvasively and identify differences between the models. EXPERIMENTAL DESIGN: DEN and McA tumor development was monitored by MRI and PET. A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. RESULTS: Histologically and biochemically confirmed liver damage resulted in increased (18)F-fluorodeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1-3 grading compared with uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared with McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. CONCLUSIONS: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies.

Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

BACKGROUND: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing. RESULTS: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes. CONCLUSION: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome, transcriptomes, and proteomes.

BACKGROUND: Microalgae-derived biodiesel is a promising substitute for conventional fossil fuels. In particular, the green alga Chlorella protothecoides sp. 0710 is regarded as one of the best candidates for commercial manufacture of microalgae-derived biofuel. This is due not only to its ability to live autotrophically through photosynthesis, but also to its capacity to produce a large amount of biomass and lipid through fermentation of glucose. However, until the present study, neither its genome sequence nor the platform required for molecular manipulations were available. RESULTS: We generated a draft genome for C. protothecoides, and compared its genome size and gene content with that of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169. This comparison revealed that C. protothecoides has a reduced genome size of 22.9 Mbp, about half that of its close relatives. The C. protothecoides genome encodes a smaller number of genes, fewer multi-copy genes, fewer unique genes, and fewer genome rearrangements compared with its close relatives. In addition, three Chlorella-specific hexose-proton symporter (HUP)-like genes were identified that enable the consumption of glucose and, consequently, heterotrophic growth. Furthermore, through comparative transcriptomic and proteomic studies, we generated a global perspective regarding the changes in metabolic pathways under autotrophic and heterotrophic growth conditions. Under heterotrophic conditions, enzymes involved in photosynthesis and CO2 fixation were almost completely degraded, either as mRNAs or as proteins. Meanwhile, the cells were not only capable of quickly assimilating glucose but also showed accelerated glucose catabolism through the upregulation of glycolysis and the tricarboxylic acid (TCA) cycle. Moreover, the rapid synthesis of pyruvate, upregulation of most enzymes involved in fatty acid synthesis, and downregulation of enzymes involved in fatty acid degradation favor the synthesis of fatty acids within the cell. CONCLUSIONS: Despite similarities to other Chlorella, C. protothecoides has a smaller genome than its close relatives. Genes involved in glucose utilization were identified, and these genes explained its ability to grow heterotrophically. Transcriptomic and proteomic results provided insight into its extraordinary ability to accumulate large amounts of lipid. The C. protothecoides draft genome will promote the use of this species as a research model.

Deletions of the SACPD-C locus elevate seed stearic acid levels but also result in fatty acid and morphological alterations in nitrogen fixing nodules.

BACKGROUND: Soybean (Glycine max) seeds are the primary source of edible oil in the United States. Despite its widespread utility, soybean oil is oxidatively unstable. Until recently, the majority of soybean oil underwent chemical hydrogenation, a process which also generates trans fats. An alternative to chemical hydrogenation is genetic modification of seed oil through identification and introgression of mutant alleles. One target for improvement is the elevation of a saturated fat with no negative cardiovascular impacts, stearic acid, which typically constitutes a minute portion of seed oil (~3%). RESULTS: We examined radiation induced soybean mutants with moderately increased stearic acid (10-15% of seed oil, ~3-5 X the levels in wild-type soybean seeds) via comparative whole genome hybridization and genetic analysis. The deletion of one SACPD isoform encoding gene (SACPD-C) was perfectly correlated with moderate elevation of seed stearic acid content. However, SACPD-C deletion lines were also found to have altered nodule fatty acid composition and grossly altered morphology. Despite these defects, overall nodule accumulation and nitrogen fixation were unaffected, at least under laboratory conditions. CONCLUSIONS: Although no yield penalty has been reported for moderate elevated seed stearic acid content in soybean seeds, our results demonstrate that genetic alteration of seed traits can have unforeseen pleiotropic consequences. We have identified a role for fatty acid biosynthesis, and SACPD activity in particular, in the establishment and maintenance of symbiotic nitrogen fixation.

Genomic patterns resembling BRCA1- and BRCA2-mutated breast cancers predict benefit of intensified carboplatin-based chemotherapy.

INTRODUCTION: BRCA-mutated breast cancer cells lack the DNA-repair mechanism homologous recombination that is required for error-free DNA double-strand break (DSB) repair. Homologous recombination deficiency (HRD) may cause hypersensitivity to DNA DSB-inducing agents, such as bifunctional alkylating agents and platinum salts. HRD can be caused by BRCA mutations, and by other mechanisms. To identify HRD, studies have focused on triple-negative (TN) breast cancers as these resemble BRCA1-mutated breast cancer closely and might also share this hypersensitivity. However, ways to identify HRD in non-BRCA-mutated, estrogen receptor (ER)-positive breast cancers have remained elusive. The current study provides evidence that genomic patterns resembling BRCA1- or BRCA2-mutated breast cancers can identify breast cancer patients with TN as well as ER-positive, HER2-negative tumors that are sensitive to intensified, DSB-inducing chemotherapy. METHODS: Array comparative genomic hybridization (aCGH) was used to classify breast cancers. Patients with tumors with similar aCGH patterns as BRCA1- and/or BRCA2-mutated breast cancers were defined as having a BRCA-likeCGH status, others as non-BCRA-likeCGH. Stage-III patients (n = 249) had participated in a randomized controlled trial of adjuvant high-dose (HD) cyclophosphamide-thiotepa-carboplatin (CTC) versus 5-fluorouracil-epirubicin-cyclophosphamide (FE90C) chemotherapy. RESULTS: Among patients with BRCA-likeCGH tumors (81/249, 32%), a significant benefit of HD-CTC compared to FE90C was observed regarding overall survival (adjusted hazard ratio 0.19, 95% CI: 0.08 to 0.48) that was not seen for patients with non-BRCA-likeCGH tumors (adjusted hazard ratio 0.90, 95% CI: 0.53 to 1.54) (P = 0.004). Half of all BRCA-likeCGH tumors were ER-positive. CONCLUSIONS: Distinct aCGH patterns differentiated between HER2-negative patients with a markedly improved outcome after adjuvant treatment with an intensified DNA-DSB-inducing regimen (BRCA-likeCGH patients) and those without benefit (non-BRCA-likeCGH patients).

Modified array-based comparative genomic hybridization detects cryptic and variant PML-RARA rearrangements in acute promyelocytic leukemia lacking classic translocations.

Acute promyelocytic leukemia (APL) is typically defined at the molecular level by a reciprocal translocation of the promyelocytic leukemia (PML) and retinoic acid receptor α (RARA) genes. An accurate diagnosis of APL is critical for appropriate choice of therapy and prognostic assessment. Cryptic and variant rearrangements in APL are discoverable by a variety of molecular methods including fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction, or gene sequencing. Rare reports of FISH-negative APL harboring cryptic rearrangements of PML-RARA detected by reverse transcriptase polymerase chain reaction or sequencing have been described. Here, we describe the detection of cryptic or variant PML-RARA rearrangements by translocation-based comparative genomic hybridization (tCGH), a recently described modification of traditional CGH technology that facilitates the detection of balanced translocations by means of the linear amplification of a potential translocation breakpoint region(s), in 2 unusual cases of APL. One tumor lacked detectable t(15;17) by karyotype and FISH, and the other tumor lacked the typical morphologic and immunophenotypic features of APL and had a variant 3-way translocation involving PML and RARA. PML-RARA translocations were identified by tCGH in both cases providing confirmation of the diagnosis of APL. These data emphasize the benefit of using complementary molecular methods including tCGH for detecting cryptic and variant PML-RARA translocations in unusual cases of APL.