Finding the missing honey bee genes: lessons learned from a genome upgrade

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.Funding for the project was provided by a grant to RG from the National Human Genome Research Institute, National Institutes of Health (NHGRI, NIH) U54 HG003273. Contributions from members of the CGE lab were supported by Agriculture and Food Research Initiative Competitive grant no. 2010- 65205-20407 from the USDA National Institute of Food Agriculture. AKB was supported by a Clare Luce Booth Fellowship at Georgetown University

Toxoplasma Effector MAF1 Mediates Recruitment of Host Mitochondria and Impacts the Host Response

Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria. © 2014 Pernas et al

Balancing repair and tolerance of DNA damage caused by alkylating agents

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity

Influence of O6-benzylguanine on the anti-tumour activity and normal tissue toxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and molecular combinations of 5-fluorouracil and 2-chloroethyl-1-nitrosourea in mice

Previous studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O6-alkylguanine-DNA-alkyltransferase (ATase) depletion by the pseudosubstrate O6-benzylguanine (BG) on the anti-tumour activity and normal tissue toxicity in mice of three such molecular combinations, in comparison with BCNU. When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG. © 1999 Cancer Research Campaig

Potential of Exosomal microRNA-200b as Liquid Biopsy Marker in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor entity, characterized by rapid disease progression, early metastatic dissemination, and late diagnosis at advanced tumor stages. Recently, we explored the clinical impact of several microRNAs (miR) associated with proliferation, epithelial-to-mesenchymal transition (EMT), and chemoresistance in tissue and blood serum specimens of PDAC patients. Here, we evaluated the potential of these miRs as diagnostic and prognostic biomarkers in PDAC in serum exosomes and their respective EpCAM-positive (epithelial cell adhesion molecule) subset. Expression analysis by RT-qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction) revealed an overexpression of miR-200b and miR-200c in serum exosomes of PDAC patients as compared to healthy controls (p < 0.001; p = 0.024) and patients with chronic pancreatitis (p = 0.005; p = 0.19). Receiver operating characteristic (ROC) curve analysis showed that a biomarker panel consisting of miR-200b and miR-200c from total and EpCAM-positive serum exosomes enhanced the diagnostic accuracy of carbohydrate antigen 19-9 (CA.19-9) to 97% (p < 0.0001). Univariate survival analysis revealed a correlation between shorter overall survival (OS) and high expression of miR-200c in total serum exosomes (p = 0.038) and miR-200b in EpCAM-positive serum exosomes (p = 0.032), whereas EpCAM exosomal miR-200b was also indicative of shorter OS in the subgroup of patients treated with curative intent (p = 0.013). Multivariate survival analysis showed that miR-200b derived from EpCAM-positive serum exosomes might serve as an independent prognostic factor in PDAC (p = 0.044). Our findings indicate a potential role of exosomal miR-200 as diagnostic and prognostic liquid biopsy marker in PDAC and call for validation in a larger, multicenter setting

Tumor-Suppressive miR-192-5p Has Prognostic Value in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by fast tumor progression and diagnosis at advanced, inoperable stages. Previous studies could demonstrate an involvement of miR-192-5p in epigenetic regulation of visceral carcinomas. Due to contradictory results, however, the clinical utility of miR-192-5p in PDAC has yet to be determined. MiR-192-5p expression was analyzed by RT-qRT-PCR in human PDAC and benign tissue (n = 78), blood serum (n = 81) and serum exosomes (n = 74), as well as in PDAC cell lines (n = 5), chemoresistant cell clones (n = 2), and pancreatic duct cell line H6c7. Analysis of EMT-associated (epithelial-to-mesenchymal transition) proteins was performed by immunohistochemistry and Western blot. MiR-192-5p was deregulated in PDAC as compared to healthy controls (HCs), with downregulation in macrodissected tissue (p < 0.001) and upregulation in blood serum of PDAC UICC (Union for International Cancer Control) stage IV (p = 0.016) and serum exosomes of PDAC UICC stages II to IV (p < 0.001). MiR-192-5p expression in tumor tissue was significantly lower as compared to corresponding peritumoral tissue (PDAC UICC stage II: p < 0.001; PDAC UICC stage III: p = 0.024), while EMT markers ZEB1 and ZEB2 were more frequently expressed in tumor tissue as compared to peritumoral tissue, HCs, and chronic pancreatitis. Tissue-derived (AUC of 0.86; p < 0.0001) and exosomal (AUC of 0.83; p = 0.0004) miR-192-5p could differentiate between PDAC and HCs with good accuracy. Furthermore, high expression of miR-192-5p in PDAC tissue of curatively resected PDAC patients correlated with prolonged overall and recurrence-free survival in multivariate analysis. In vitro, miR-192-5p was downregulated in gemcitabine-resistant cell clones of AsPC-1 (p = 0.029). Transient transfection of MIA PaCa-2 cells with miR-192-5p mimic resulted in downregulation of ZEB2. MiR-192-5p seems to possess a tumor-suppressive role and high potential as a diagnostic and prognostic marker in PDAC