Wild Counterpart Research Articles

Epigenetic origin of evolutionary novel centromeres

Most evolutionary new centromeres (ENC) are composed of large arrays of satellite DNA and surrounded by segmental duplications. However, the hypothesis is that ENCs are seeded in an anonymous sequence and only over time have acquired the complexity of “normal” centromeres. Up to now evidence to test this hypothesis was lacking. We recently discovered that the well-known polymorphism of orangutan chromosome 12 was due to the presence of an ENC. We sequenced the genome of an orangutan homozygous for the ENC, and we focused our analysis on the comparison of the ENC domain with respect to its wild type counterpart. No significant variations were found. This finding is the first clear evidence that ENC seedings are epigenetic in nature. The compaction of the ENC domain was found significantly higher than the corresponding WT region and, interestingly, the expression of the only gene embedded in the region was significantly repressed.

Contributions of Glycosaminoglycans to Membrane Adsorption and Conformational Conversion of Peptide Hormones

The membrane catalysis hypothesis advocates that a peptide ligand initially interacts with the plasma membrane and this interaction increases the local concentration at the vicinity of its cognate receptor. Besides, upon membrane adsorption, the peptide undergoes a conformational transition that favors receptor binding. In this mechanism, the lipidic components are the core of the membrane catalysis hypothesis. However, the membrane environment is complex and is constituted of a variety of macromolecules, including glycosaminoglycans (GAGs). Although these long polysulfated heteropolysaccharide chains have be shown to participate in the recruitment of chemokines, nothing is known about the contribution of GAGs in membrane adsorption of cationic peptidic hormones. In this context, we probed the contributions of GAGs in the membrane adsorption of secretin. This 27-residue peptide is secreted by the S cells of duodenum and known for regulating water homeostasis. To investigate the role of GAGs in the cell surface adsorption of secretin, we used CHO pgs-A-745 cells, which lack the first enzyme for GAGs biosynthesis, and their wild type counterpart, CHO K1. By confocal microscopy and flow cytometry, we observed that CHO pgs A-745 cells absorbed less fluorescein-labelled peptides than CHO-K1 cells. Then, we characterized the interaction between GAGs and secretin by affinity chromatography, isothermal titration calorimetry and surface plasmon resonance. A strong interaction between the peptide and GAGs was observed with a Kd in the low μM. Interestingly, heparin stabilizes the helical conformation of secretin. By designing a secretin analog with a restricted conformational ensemble, we observed that the spatial distribution of basic residues constitutes a key element for high affinity binding. Overall, this study redefines the membrane catalysis hypothesis and suggests that cationic peptide hormones initially bind to GAGs allowing a possible key role in receptor binding.

Exposure of airway epithelial cells to Pseudomonas aeruginosa biofilm-derived quorum sensing molecules decrease the activity of the anti-oxidant response element bound by NRF2

Chronic bacterial infections in cystic fibrosis lung disease are often characterized by Pseudomonas aeruginosa biofilms that are regulated by bacterial intercellular signals termed quorum sensing (QS), such as N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). This study reports that biofilm-derived exoproducts decrease the transcriptional activity of the anti-oxidant response element in bronchial epithelial cells. In a live co-culture assay of BEAS-2B cells and P. aeruginosa biofilm, the QS molecule 3OC12-HSL was an important but not sole contributor to the inhibition of basal NRF2 luciferase reporter activity. Moreover, biofilm-derived exoproducts and 3OC12-HSL decrease the expression of endogenous antioxidant response element-regulated genes hemeoxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase-1 (NQO-1) while they increase IL-8 expression. As previously reported, IL-8 expression is partially dependent on p38 MAPK activity, but the inhibitory effect of biofilm QS molecules on HO-1 and NQO-1 expression occurs independently of this protein kinase. Finally, the transfection of CFTRdelF508 but not its wild type counterpart decreases basal, planktonic PsaDM and sulforaphane-driven NRF2 luciferase reporter activity in BEAS-2B cells. Therefore, the presence of quorum sensing molecules derived from bacterial biofilms lowers the transcriptional activity of the anti-oxidant response element, which may contribute to the establishment of chronic bacterial infections, especially in the presence of mutated CFTR. Increasing NRF2 activity may thus be a promising strategy to potentiate anti-biofilm activity in cystic fibrosis lung disease.

Interplay between a cytosolic and a cell surface carbonic anhydrase in pH homeostasis and acid tolerance of Leishmania.

Leishmania parasites have evolved to endure the acidic phagolysosomal environment within host macrophages. How Leishmania cells maintain near-neutral intracellular pH and proliferate in such a proton-rich mileu remains poorly understood. We report here that, in order to thrive in acidic conditions, Leishmania major relies on a cytosolic and a cell surface carbonic anhydrase, LmCA1 and LmCA2, respectively. Upon exposure to acidic medium, the intracellular pH of the LmCA1+/-, LmCA2+/- and LmCA1+/-:LmCA2+/- mutant strains dropped by varying extents that led to cell cycle delay, growth retardation and morphological abnormalities. Intracellular acidosis and growth defects of the mutant strains could be reverted by genetic complementation or supplementation with bicarbonate. When J774A.1 macrophages were infected with the mutant strains, they exhibited much lower intracellular parasite burdens than their wild-type counterparts. However, these differences in intracellular parasite burden between the wild-type and mutant strains were abrogated if, before infection, the macrophages were treated with chloroquine to alkalize their phagolysosomes. Taken together, our results demonstrate that haploinsufficiency of LmCA1 and/or LmCA2 renders the parasite acid-susceptible, thereby unravelling a carbonic anhydrase-mediated pH homeostatic circuit in Leishmania cells.

Prognostic Significance of IDH1 and IDH2 mutations in Older Patients with Primary Cytogenetically Normal Acute Myeloid Leukemia

IDH1 and IDH2 mutations are frequently identified in older patients with AML.With the development and application of IDH inhibitor, the prognostic significance of these mutations in old patients is of primary importance. To assess the impact on clinic outcome, we analyzed 224 older (> 60 years) patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) similarly treated with intensive induction chemotherapy. Our analysis identified IDH1 and/or IDH2 (IDH1/2), IDH1 or IDH2 mutations in 63(28%), 29(12.9%) and 35(15.6%) patients, respectively. In general, IDH1 and IDH2 mutations were mutually exclusive, with only one patient having both IDH1 R132C and IDH2 R140Q mutations. IDH2 R172 mutations were exclusive with all other mutations analyzed (NPM1, CEBPA, DNMT3A, IDH1 and FLT3-ITD). There was a strong interaction between IDH2 mutation and NPM1 mutational status. Concurrent presence of IDH2 R140 and NPM1 mutations was independently associated with longer overall survival (OS; P=0.038, HR=0.42) and event free survival (EFS; P=0.034, HR=0.46) compared to the wild type counterpart in multivariable analyses. In contrast, IDH1 mutations were independently associated with shorter OS (P=0.004, HR=1.98) and EFS (P=0.014, HR=1.72) in multivariable analyses. Notably, IDH1 R132 mutations harbored higher levels of total 2-hydroxyglutarate compared to IDH2 R140Q mutations. Patients with IDH1 mutations or IDH2 R140 and NPM1 mutations harbored also distinct miRNA-expression signatures. Future studies will establish whether these difference between IDH1 and IDH2 mutations will be relevant with respect to response of the respective subsets of patients to the emerging therapies with IDH1 and IDH2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Correlation Between Somatic Mutation Burden, Neoantigen Load and Progression Free Survival in Multiple Myeloma: Analysis of MMRF CoMMpass Study

IntroductionTumor specific somatic mutations can potentially influence patient response to targeted immunotherapies. Since somatic mutations generate neoantigens, novel tumor-specific peptides can be processed, loaded into MHC class I molecules, and presented to T-cells as potentially immunogenic targets. Indeed, both somatic mutation burden and neoantigen burden have been correlated with responsiveness to checkpoint inhibitor blockade in highly mutagenic advanced melanoma and lung cancer. However, correlation of response with mutation and neoantigen burden has not been reported for cancers of lower characteristic mutational burden such as multiple myeloma (MM). Indeed early results of checkpoint inhibitors in MM have been underwhelming. We therefore wished to better understand the mutation and neoantigen landscape of MM patients as it relates to responsiveness to current and future therapies.Materials/MethodsSomatic mutation burden, neoantigen load, and response to therapy was obtained using interim data from theCoMMpassstudy (NCT01454297), a study by the Multiple Myeloma Research Foundation that collects longitudinal clinical and molecular data on 1,000 newly diagnosed MM patients from baseline through treatment and disease progression. Next-generation exome sequencing data was used to call somatic mutations andbioinformaticallydetermine patient specific HLA type. Neoantigen binding affinity was predicted for all unique patient specific somatic missense mutation and HLA allele combinations using a well validated neural network and machine learning based algorithm. Binding affinity of the corresponding non-mutated peptide was also predicted to identify mutated neoantigens witha high binding affinity while the wild type counterpart has low predicted binding affinity.ResultsFor 627 patients with available sequencing and clinical data, mutation and neoantigen profiles were created. Within this population, the mean overall canonical somatic mutation load was 405.84 (s=608.55) mutations per patient. These mutations included 38,222 unique missense mutations in 12,994 genes. The mean somatic missense mutation load was 64.51(s=95.29) mutations per patient including immunoglobulin genes (52.70, s=94.85 excluding immunoglobulins). There was a positive linear correlation between mutation burden and neoantigen burden. The mean neoantigen load using thresholds of mutant peptide binding affinity (half maximal inhibitory concentration, IC50) less than 500nM was 69.57 (s=124.76) neoantigens (58.53, s=123.58 excluding immunoglobulins). Limiting binding affinity of wild type peptide sequence to greater than 500nM decreased the mean neoantigen load to 23.84 (s=53.55) neoantigens (20.74, s=53.28 excluding immunoglobulins).Progression free survival (PFS) following induction therapy was used to determine correlates of mutation burden excluding immunoglobulin mutations with response. Kaplan-Meier survival analysis revealed significantly shorter PFS time in patients with greater than average tumor somatic missense mutation load (N=126) relative to those with less than average mutation load (49.1% vs. 73.0% PFS at 2yrs, p=0.0055, Figure 1) Similarly, patients with greater than average neoantigen load (N=164) had significantly shorter PFS relative to those with less than average neoantigen load (48.0% vs. 74.2% PFS at 2yrs, p=0.00259, Figure 2). Moreover, patients with both high mutation and neoantigen load (N=85) had significantly shorter PFS relative to those with less than average mutation and neoantigen load (45.3% vs. 72% PFS at 2yrs, p= 0.0168).ConclusionWhile mutation and neoantigen load has been correlated with response to targeted immunotherapies in highly mutated solid tumors, we report here a negative correlation between mutation, neoantigen load and MM patient survival. This indicates that a high mutation andneoantigen load negatively impacts survival of myeloma patients under current standard of care. It is hypothesized that this is because current treatments fail to recruit T cell immunity necessary to exploit tumor neoantigens fundamental to immune-mediated killing of cancer cells. This presents an encouraging basis for pursuing targeted and personalized immunotherapies for multiple myeloma, especially for patients with a higher mutational burden. [Display omitted] [Display omitted] DisclosuresLonial:BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Novartis: Consultancy; BMS: Consultancy; Millenium: Consultancy; Merck: Consultancy.

Genome Sequence of a 5,310-Year-Old Maize Cob Provides Insights into the Early Stages of Maize Domestication

The complex evolutionary history of maize (Zea mays L. ssp. mays) has been clarified with genomic-level data from modern landraces and wild teosinte grasses [1, 2], augmenting archaeological findings that suggest domestication occurred between 10,000 and 6,250 years ago in southern Mexico [3, 4]. Maize rapidly evolved under human selection, leading to conspicuous phenotypic transformations, as well as adaptations to varied environments [5]. Still, many questions about the domestication process remain unanswered because modern specimens do not represent the full range of past diversitydue to abandonment of unproductive lineages, genetic drift, on-going natural selection, and recent breeding activity. To more fully understand the history and spread of maize, we characterized the draft genome of a 5,310-year-old archaeological cob excavated in the Tehuacan Valley of Mexico. We compare this ancient sample against a reference panel of modern landraces and teosinte grasses using D statistics, model-based clustering algorithms, and multidimensional scaling analyses, demonstrating the specimen derives from the same source population that gave rise to modern maize. We find that 5,310 years ago, maize in the Tehuacan Valley was on the whole genetically closer to modern maize than to its wild counterpart. However, many genes associated with key domestication traits existed in the ancestral state, sharply contrasting with the ubiquity of derived alleles in living landraces. These findings suggest much of the evolution during domestication may have been gradual and encourage further paleogenomic research to address provocative questions about the world's most produced cereal.

Comparative study of the nutritional composition and toxic elements of farmed and wild Chanodichthys mongolicus

Information of the difference in quality between farmed and wild fish is central to better ensuring fish products produced in aquaculture meet regulatory and consumer requirements. Proximate composition, amino acid and fatty acid profiles, and toxic elements contents of farmed and wild Chanodichthys mongolicus were established and compared. Significantly higher crude protein content while lower moisture content in farmed fish compared to wild fish were observed (P<0.05). The percentages of total amino acids (TAA), total essential amino acids (TEAA), total non-essential amino acids (TNEAA) and total delicious amino acids (TDAA) in farmed fish were all significantly higher than those in the wild equivalent (P<0.05). Compared to the FAO/WHO recommended reference values, the ratios of TEAA/TAA (39.84%–40.33%) were comparable to 40% and TEAA / TNEAA (66.22%–67.60%) were above 60%. Fatty acid profiles in both farmed and wild C. mongolicus were dominated by monounsaturated fatty acid (MUFA), with farmed fish contained much more MUFA content compared to wild counterpart (P<0.05). Notably, wild fish exhibited significantly higher levels of total polyunsaturated fatty acid (PUFA) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) than farmed fish (P<0.05). The EPA (C20:5n3) and linoleic acid (C18:2n6) were the predominant PUFA in wild and farmed C. mongolicus, respectively. Moreover, farmed fish displayed an overall lower toxic element levels (As, Cd, Pb and Hg) in comparison with wild fish, and both were far lower than the established limit standard. In conclusion, our results suggest that the nutritional quality of farmed C. mongolicus was inferior to their wild counterpart with respect to fatty acids nutrition, and therefore further studies should focus on the improving C. mongolicus diet in order to enhance the overall nutritional composition.

Endothelial Nitric Oxide Synthase Prevents Heparanase Induction and the Development of Proteinuria.

Endothelial nitric oxide synthase (eNOS) deficiency exacerbates proteinuria and renal injury in several glomerular diseases, but the underlying mechanism is not fully understood. We recently showed that heparanase is essential for the development of experimental diabetic nephropathy and glomerulonephritis, and hypothesize that heparanase expression is regulated by eNOS. Here, we demonstrate that induction of adriamycin nephropathy (AN) in C57BL/6 eNOS-deficient mice leads to an increased glomerular heparanase expression accompanied with overt proteinuria, which was not observed in the AN-resistant wild type counterpart. In vitro, the eNOS inhibitor asymmetric dimethylarginine (ADMA) induced heparanase expression in cultured mouse glomerular endothelial cells. Moreover, ADMA enhanced transendothelial albumin passage in a heparanase-dependent manner. We conclude that eNOS prevents heparanase induction and the development of proteinuria.

Abstract PR06: Reciprocal prostate cancer signaling with its microenvironment mediates castrate-resistant disease progression

Abstract Our lab has previously reported that the mouse model knocked out for TGF-beta receptor type II in fibroblasts results in castrate resistance of prostate tissues, mediated by paracrine Wnt signaling. We found that antagonizing androgen signaling leads to Wnt ligand expression by prostatic fibroblasts, interestingly dependent on cell-cell contact. This led us to hypothesize that a juxtacrine action within the stromal compartment was important to the development of castrate resistance. Building upon this observation, we found that Notch signaling was upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs, using DAPT, resulted in decrease in Wnt2b and decreased expression of Notch target genes. Similarly, castration or androgen receptor antagonism (i.e. bicalutamide or enzalutamide) resulted in increased Notch signaling and downstream Wnts 2 and 2b up regulation. The further generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling, derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts having a loss of Notch signaling, from RBPJ-knockout mice, had diminished tumorigenesis, compared to xenografts with the wild type fibroblasts counterpart. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together there is a stromal-derived signaling cascade involving Notch and Wnt ligands, that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer. This abstract is also presented as Poster A44. Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Leland W. K. Chung, Rajeev Mishra, Li Xin, Neil Bhowmick. Reciprocal prostate cancer signaling with its microenvironment mediates castrate-resistant disease progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR06.

Abstract C24: Beyond EMT: Vimentin function in lung cancer metastasis

Abstract Vimentin is an intermediate filament protein that has been used in the clinic as a biomarker for metastatic potential and poor patient prognosis across numerous solid tumor types; however, little is known about how vimentin might contribute to cancer progression and metastasis. Here we created a novel genetically engineered mouse model (GEMM) to study the role of vimentin in lung cancer progression and metastasis. We first re-developed an LSL-KrasG12D,LKB1fl/fl mouse model whereby intranasal administration of lentiviral Cre recombinase causes a KrasG12D mutation and LKB1 knockout in the lungs. In this model Kras/Lkb1 GEMM (termed KLV+/+), approximately half the mice that develop primary lung tumors in 8-10 weeks also develop metastasis to the mediastinal lymph nodes in weeks 12-16. We used this model to test the hypothesis that vimentin loss prevents lung cancer metastasis by crossing this GEMM with a Vim-/- mouse to create a novel LSL-KrasG12D, LKB1fl/fl, Vim-/- (termed here KLV-/-) mouse. Our findings show that in the KLV-/- mouse, primary tumor burden and cell proliferation were not significantly different than wild-type KLV+/+ mice; however, KLV-/- mice exhibit significantly less metastasis to the mediastinal lymph nodes compared to their wild type counterpart indicating that vimentin contributes to the metastatic cascade. Consistent with this finding, histological analysis of the primary tumors show that KLV-/- mice exhibit less focal invasion than KLV+/+ and +/- mice, further confirming that KLV-/- mice have reduced invasiveness. Interestingly, vimentin staining in invasive regions of KLV+/+ mice was not found in the cancer cells but rather in fibroblast-like cells surrounding invasive cell buds that we term collective invasion packs (CIPs). These fibroblast-like, vimentin-positive cells also stain positive for alpha smooth muscle actin, which is consistent with cancer-associated fibroblasts (CAFs). Taken together, these results suggest that in this highly metastatic genetic background, vimentin may play a central role in recruiting CAFs to invading cancer cells but not necessarily in cancer cell epithelial to mesenchymal transition (EMT). Citation Format: Alessandra M. Salgueiro, Melissa Gilbert-Ross, Lauren S. Havel, John Shupe, Allyson E. Koyen, Hans E. Grossniklaus, Gabriel Sica, Adam I. Marcus. Beyond EMT: Vimentin function in lung cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C24.

Abstract 3370: Metabolic profiling of the tumor interstitial fluid using 1H MRS: contribution of breast cancer subtypes and VEGF overexpression

Abstract One of the least examined, and yet critically important factors of the tumor microenvironment (TME) is the tumor interstitial fluid (TIF) that contains the tumor secretome. This fluid surrounds cancer and stromal cells and contains various cytokines, and nutritional and molecular factors that shape the outcome of nearly all aspects of tumor angiogenesis, growth, metastasis, and response to treatment. As mining of targets to treat cancer expands into the TME, the TIF represents an important source of identifying new targets in cancer treatment. Angiogenesis, one of the major hallmarks of cancer, is essential for cancers to establish vasculature for growth and hematogenous metastasis. To further understand the TIF and plasma metabolite content in breast cancer, and the role of VEGF in modifying metabolite concentrations in the TME, here we used 1H MRS to characterize metabolites in TIF collected from the triple negative MDA-MB-231 and the ER-positive MCF-7 human breast cancer xenografts with and without VEGF overexpression implanted in SCID mice. We created a collection chamber to collect IF from the tumors. The chamber was implanted with small tumor pieces in the subcutaneous space until the tumor encompassed the chamber (4 to 12 weeks depending on cell types). The tumor was then excised and the TIF and blood plasma were collected from euthanized mice. We also collected normal subcutaneous IF (SCIF) using the same chamber in healthy mice. 1H spectra showed several differences in metabolites in TIF compared to SCIF, between MDA-MB-231 and MCF-7 TIF, and with VEGF overexpression. We observed a consumption of amino acids in the TME with decreases ranging from 17% (glycine) to 77% (methionine) compared to normal SCIF. Lipids, especially polyunsaturated fatty acids, were markedly increased by VEGF overexpression in both MCF-7 and MDA-MB-231 TIF compared to the wild type TIF. Choline metabolism was also modified by VEGF overexpression and we measured increases of 69% and 20% in free choline in MDA-MB-231_VEGF and MCF-7_VEGF respectively, compared to their wild type counterpart. A stable glucose concentration was associated with a decrease in lactate (30%) and pyruvate (44%) in MDA-MB-231 tumors while an opposite pattern was observed in MDA-MB-231_VEGF TIF with decreased glucose (55%) and increased lactate (109%). Ketonic metabolism was also modified in those tumors. Beta-hydroxybutyrate and acetoacetate were higher (81% and 46% increase vs SCIF, respectively) in MDA-MB-231 TIF while acetone was increased in both VEGF-overexpressing TIF compared to the wild type (+48% for MDA-MB-231_VEGF and +44% for MCF-7_VEGF). The TIF represents an importance source of information to understand mechanisms that drive aggressiveness, and identify new targets for diagnosis and therapy of cancer. Citation Format: Louis Dore-Savard, Santosh K. Bharti, Aleksander S. Popel, Zaver M. Bhujwalla. Metabolic profiling of the tumor interstitial fluid using 1H MRS: contribution of breast cancer subtypes and VEGF overexpression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3370.

Growth performance and nutritional composition of Hemifusus ternatanus under artificial culturing conditions

Juvenile Hemifusus ternatanus were cultured in a double-layer culturing system for 186weeks in order to assess growth performance. The growth rate of H. ternatanus under artificial culturing conditions was high during the first 137week post-hatching, followed by a period of relatively slower growth. The shell of the cultured sub-adult whelks was shorter and thicker than that of the wild individuals with same weight. The proximate, amino acid and fatty acid compositions of H. ternatanus foot muscles were analyzed to compare the quality of cultured and wild whelks. Both the cultured and wild H. ternatanus contained high levels of flavor-enhancing amino acids. No significant difference was observed in protein content, moisture content or amino acid composition. The cultured whelks contained significantly higher levels of fat and ash (P<0.05). The total saturated, monounsaturated, and polyunsaturated (PUFA) fatty acids were statistically similar between the cultured and wild groups; however, there were significant differences in the content of individual of fatty acids between the two groups, including 17:0, 16:1, 18:1n-9c, 18:2n-6t, 20:4n-6, 22:5n-3, 22:6n-3, and n-6 PUFAs (P<0.05). The fatty acids 21:0, 15:1, 22:1n-9, 18:2n-6c, and 18:3n-3 were detected in the cultured H. ternatanus only. On the contrary, 18:1n-9t was detected only in the wild H. ternatanus. This study provides novel information regarding the growth and composition of cultured H. ternatanus which will be helpful for the development of aquaculture practices for this species. Statement of RelevanceThis manuscript provides new findings which should be of interest to readers involved in the reproduction and aquaculture of gastropods. H. ternatanus is a marine whelk with agreeable taste and is a potential candidate species for aquaculture. Aquaculture production of H. ternatanus is increasing due to its high market price and the depletion of natural stocks. There is currently no information regarding the nutritional composition of cultured H. ternatanus compared to its wild counterpart. The results of this study will help consumers better understand the nutritional value of cultured whelks and provide aquaculturists with important information for the development of formulated feeds.

Genetic variation for leaf trichome density and its association with sucking insect-pests incidence in Asiatic cotton

Trichomes on the plant body are known to impart resistance/susceptibility to various insect-pests. An intra Gossypium arboreum F2 population between a trichomeless mutant PAUFL 1 and its wild type counterpart was developed to assess the distribution of trichomes on young and mature leaves, and to correlate the trichome density with the sucking insect-pests incidence. Based on pooled data for the years 2011 and 2012, mean trichome count for 552 F2 plants was found to be 47.52 and 90.09 (on 28.27 mm2) for mature and young leaves, respectively. A range of 0–185 and 0–218 trichomes was observed for mature and young leaves, respectively. Significant positive correlation (r = 0.83*) was obtained between trichome density of young and mature leaves. Higher trichome density was observed on young leaves. A positive association between young leaf trichome density and whitefly population was recorded which indicates that higher trichome density is favourable for whitefly incidence and vice versa. However, no correlation between the trichome density of young leaves and jassid population was observed.

Minimal Reduction of PU.1 Is Sufficient to Induce a Preleukemic State and Promote Development of Acute Myeloid Leukemia

Genomic studies have shown that human cancer is rarely associated with a complete loss of transcripts; instead, acquired DNA alterations often occur within the non-coding part of the genome, are enriched in gene-regulatory regions, and cause only moderate transcriptional changes. It is currently not well understood how such moderate gene expression changes impact normal tissue function and how they contribute to malignant transformation.Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) develop through a multi-step transformation process originating in hematopoietic stem cells (HSCs) and mainly present in the elderly (median age of >65 years at diagnosis). Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces AML in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. At the example of the Ets-family transcription factor PU.1, which is indispensable for HSC function and the differentiation of cells within the myeloid as well as lymphoid lineages, we tested the hypothesis that even moderate gene expression alterations of key regulators can drive malignant transformation. We assessed the effects of minimal PU.1 inhibition on hematopoiesis in a novel mouse model that co-models the genomic context found in aging human individuals and patients with MDS/AML.Mice lacking Msh2, the key component of the MutSα and MutSβ complexes mediating DNA mismatch repair, accumulate elevated numbers of point mutations, in particular C/G>T/A transitions and small insertions/deletions resembling the mutation spectrum acquired in HSCs in aging human individuals and patients with MDS and AML. We crossed Msh2-/- mice with animals carrying a heterozygous deletion of an upstream regulatory element of PU.1 (UREΔ/+). UREΔ/+Msh2-/- mice exhibited a significant, but very modest reduction of PU.1 expression on average by 26-37% in fractionated hematopoietic multipotent stem and myeloid progenitor cells. Strikingly, this minimal reduction of PU.1 led to the emergence of an aggressive, transplantable AML in more than two thirds of UREΔ/+Msh2-/- mice which was never observed in URE+/+Msh2-/- mice. Overt leukemia was preceded by a preleukemic phase hallmarked by an expanded population of multipotent murine hematopoietic stem cell enriched cells (HSPCs) that was myeloid-biased and less quiescent than their wild type counterpart. Longitudinal monitoring of preleukemic UREΔ/+Msh2-/- mice revealed a progressive increase in immature myeloid cells along with a gradual decrease in mature myeloid cells, as well as expansion of phenotypic HSPC compartments and multi-lineage dysplasia resembling human MDS. AML progression was accompanied by additional inhibition of a PU.1-cooperating factor, interferon responsive factor 8 (Irf8). Irf8 expression restoration rescued impaired expression of genes harboring PU.1/IRF consensus binding sites, led to the loss of aberrant self-renewal, promoted myeloid differentiation, and induced apoptosis in leukemic UREhetMsh2-/- cells demonstrating that Irf8 impairment functionally cooperates with minimally reduced PU.1 expression in our model. We also found evidence of disease-relevant joint PU.1/IRF8 inhibition in human myeloid leukemogenesis: (1) patients with MDS with a higher risk for the progression to AML had lower IRF8 levels; (2) lower IRF8 expression was detected specifically in AML patients with reduced PU.1 levels; (3) restoration of IRF8 expression induced differentiation in IRF8 low expressing AML cells, and (4) a positive correlation of PU.1 and IRF8 expression was found in human leukemia stem cells, but not in healthy HSCs. Strikingly, comparative pathway analysis revealed a genome-wide molecular resemblance of preleukemic and leukemic UREΔ/+Msh2-/- mice with gene expression profiles from human MDS and AML patients, respectively.Our study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in MDS and AML. DisclosuresNo relevant conflicts of interest to declare.

Intraspecific variation in desiccation survival time of Aedes aegypti (L.) mosquito eggs of Australian origin.

Aedes aegypti (L.) mosquitoes preferentially oviposit in natural and artificial receptacles where their eggs are able to withstand drying as water levels fluctuate. Desiccation-resistant eggs also increase the potential for establishment in non-native habitats while providing logistical impediments to control programs. Viability and mean survival times of eggs stored under three dryness conditions for up to 367 days were investigated among three field-derived colonies of Australian Ae. aegypti to understand variation in desiccation survival. Further investigations compared egg survival between an established colony and its wild counterpart. Our results confirmed that Ae. aegypti eggs can withstand desiccation for extended periods of time with approximately 2-15% egg viability recorded after one year and viability remaining above 88% under all conditions through 56 days. Intraspecific variations in egg survival times were recorded, suggesting local adaptation while each of the colonies demonstrated a consistent preference for higher humidity. Egg volume varied between the populations, suggesting a relationship between egg volume and survival time, with the marginally larger eggs (Charters Towers and Innisfail) having greater desiccation resistance over the range of conditions. The strong survivorship of Charters Towers eggs in dry, warm conditions demonstrates the adaptive significance of a desiccation-resistant egg.

Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology

Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdhQ111/HdhQ111 cells, a cell model of HD and compared with STHdhQ7/HdhQ7 cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. Biological significanceIn this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock-in CAG repeats in their two alleles. The 111Q containing cell line (STHdhQ111/HdhQ111) mimics diseased condition, whereas the 7Q containing ones (STHdhQ7/HdhQ7), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, HdhQ knock-in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential expressions of proteins in these two cell lines. In this study, we annotated 76 proteins from STHdhQ7/HdhQ7 and STHdhQ111/HdhQ111 cells using 2D-gel/mass spectrometry. Next, by performing 2D-DIGE, we observed differential expressions of 31 proteins (16 upregulated and 15 downregulated) between these two cell lines. We also performed customized qRT-PCR array focused to HD pathway and found differential expressions of 47 genes (8 gene expressions increased and 39 genes were decreased significantly). A total of 77 genes/proteins (Htt downregulated in both the studies) were found to be significantly altered from both the experimental paradigms. We validated the differential expressions of Vim, Hypk, Ran, Dstn, Hspa5 and Sod2 either by qRT-PCR or Western blot analysis or both. Out of these 77, similar trends in alteration of 19 out of 31 and 38 out of 47 proteins/genes were reported in earlier studies. Thus our study confirmed earlier observations on differential gene/protein expressions in HD and are really useful. Additionally, we observed differential expression of some novel genes/proteins. One of this was Hypk, a Htt-interacting chaperone protein with the ability to solubilize mHtt aggregated structures in cell lines. We propose that downregulation of Hypk in STHdhQ111/HdhQ111 has a causal effect towards HD pathogenesis. Thus the novel findings from our study need further research and might be helpful to understand the molecular mechanism behind HD pathogenesis.

Methyl jasmonate inhibits lamina joint inclination by repressing brassinosteroid biosynthesis and signaling in rice

Lamina joint inclination or leaf angle (the angle between the leaf blade and vertical culm) is a major trait of rice plant architecture. The plant hormone brassinosteroid (BR) is the main regulator of this trait, while other plant hormones, including ethylene, gibberellin, and auxin, also influence leaf angle. In this study, we found that methyl jasmonate (MeJA) also participates in regulating lamina joint inclination. MeJA decreased lamina joint inclination and inhibited the BR-induced increase in lamina joint inclination. Furthermore, addition of a BR synthesis inhibitor increased the extent of change in lamina joint inclination in response to treatment with a low concentration of MeJA (0.05 or 0.5mgL−1), but it did not alter the lamina joint inclination of plants treated with a high concentration of MeJA (5mgL−1). Further studies showed that MeJA treatment significantly repressed the expression of BR biosynthesis-related genes and decreased endogenous BRs levels. In addition, the lamina joint inclination in the OsBRI1 mutant d61-1 was less sensitive to MeJA compared with its wild type counterpart, and lithium chloride-induced inactivation of GSK3-like kinase, a negative regulator of BR signaling, partly rescued the MeJA-induced reduction in lamina joint inclination. Further studies showed that MeJA treatment reduced the mRNA levels of BR signaling and target genes. These results indicate that MeJA-inhibition of lamina joint inclination may depend on BR biosynthesis and the BR signaling pathway.

Effects of brain derived neurotrophic factor Val66Met polymorphism in patients with cervical spondylotic myelopathy

Cervical spondylotic myelopathy (CSM) is the leading cause of spinal cord related disability in the elderly. It results from degenerative narrowing of the spinal canal, which causes spinal cord compression. This leads to gait instability, loss of dexterity, weakness, numbness and urinary dysfunction. There has been indirect data that implicates a genetic component to CSM. Such a finding may contribute to the variety in presentation and outcome in this patient population. The Val66Met polymorphism, a mutation in the brain derived neurotrophic factor (BDNF) gene, has been implicated in a number of brain and psychological conditions, and here we investigate its role in CSM. Ten subjects diagnosed with CSM were enrolled in this prospective study. Baseline clinical evaluation using the modified Japanese Orthopaedic Association (mJOA) scale, Nurick and 36-Item Short Form Health Survey (SF-36) were collected. Each subject underwent objective testing with gait kinematics, as well as hand functioning using the Purdue Peg Board. Blood samples were analyzed for the BDNF Val66Met mutation. The prevalence of the Val66Met mutation in this study was 60% amongst CSM patients compared to 32% in the general population. Individuals with abnormal Met allele had worse baseline mJOA and Nurick scores. Moreover, baseline gait kinematics and hand functioning testing were worse compared to their wild type counterpart. BDNF Val66Met mutation has a higher prevalence in CSM compared to the general population. Those with BDNF mutation have a worse clinical presentation compared to the wild type counterpart. These findings suggest implication of the BDNF mutation in the development and severity of CSM.

Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation.

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.