Loss Of Lysine Research Articles

Radiation‐associated sarcomas other than malignant peripheral nerve sheath tumours demonstrate loss of histone H3K27 trimethylation†

Complete loss of histone H3 lysine 27 trimethylation (H3K27me3) has recently emerged as a biomarker for malignant peripheral nerve sheath tumours (MPNST). Loss of H3K27me3 staining has also been reported in post-radiation MPNST; however, it has not been evaluated in a large series of radiation-associated sarcomas of different histological subtypes. The aim of this study was to assess H3K27me3 labelling by immunohistochemistry in radiation-associated sarcomas and to determine the prevalence of H3K27me3 loss in these tumours. Radiation-associated sarcomas (n=119) from two tertiary care referral centres were evaluated for loss of H3K27me3, defined as complete loss of staining within tumour cells in the presence of a positive internal control. Twenty-three cases (19%) showed H3K27me3 loss, including nine of 10 (90%) MPNST, seven of 77 (9%) undifferentiated spindle cell/pleomorphic sarcomas, five of 25 (20%) angiosarcomas, one of five (20%) leiomyosarcomas and one of two (50%) osteosarcomas. Complete H3K27me3 loss was present in 19% of radiation-associated sarcomas in our series. Our findings demonstrate that loss of H3K27me3 is not specific for radiation-associated MPNST and may also occur in other histological subtypes of RAS, including radiation-associated undifferentiated spindle cell/pleomorphic sarcoma, angiosarcoma, leiomyosarcoma and osteosarcoma.

Epigenetic effects of low-level sodium arsenite exposure on human liver HepaRG cells.

Chronic exposure to inorganic arsenic is associated with a variety of adverse health effects, including lung, bladder, kidney, and liver cancer. Several mechanisms have been proposed for arsenic-induced tumorigenesis; however, insufficient knowledge and many unanswered questions remain to explain the integrated molecular pathogenesis of arsenic carcinogenicity. In the present study, using non-tumorigenic human liver HepaRG cells, we investigated epigenetic alterations upon prolonged exposure to a noncytotoxic concentration of sodium arsenite (NaAsO2). We demonstrate that continuous exposure of HepaRG cells to 1µM sodium arsenite (NaAsO2) for 14days resulted in substantial cytosine DNA demethylation and hypermethylation across the genome, among which the claudin 14 (CLDN14) gene was hypermethylated and the most down-regulated gene. Another important finding was a profound loss of histone H3 lysine 36 (H3K36) trimethylation, which was accompanied by increased damage to genomic DNA and an elevated de novo mutation frequency. These results demonstrate that continuous exposure of HepaRG cells to a noncytotoxic concentration of NaAsO2 results in substantial epigenetic abnormalities accompanied by several carcinogenesis-related events, including induction of epithelial-to-mesenchymal transition, damage to DNA, inhibition of DNA repair genes, and induction of de novo mutations. Importantly, this study highlights the intimate mechanistic link and interplay between two fundamental cancer-associated events, epigenetic and genetic alterations, in arsenic-associated carcinogenesis.

Abstract 3722: Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance

Abstract Regional nutrients deprivation, which is frequently encountered in many solid tumors due to poor vascularization, induces metabolic rewiring and genetic dysregulation to support the demands of proliferating cancer cells. Numerous chemotherapies exert their S-phase dependent cytotoxicity by producing deleterious effects on DNA to kill proliferating tumor cells. How regional nutrients deprivation affects the response of cancer cells to chemotherapy, especially DNA damaging genotoxicity, and what is the key nutrient dominates these effects is an active area of interest across tumor biology. Of all the amino acids we examined, only arginine is regional deprived in tumor core and indispensable for S-phase progression in multiple breast cancer cells and non-tumorigenic breast epithelial cells. We found that arginine deprivation stalls DNA replication in an mTORC1 inactivation, insufficient metabolites, or the integrated stress response pathway independent manner. We also found that low levels of newly synthesized Histone H4 marks (H4K5ac and H4K12ac), which were reported to be associated with adverse outcomes in various cancers including basal-like and HER2-positive breast cancer, are positively correlated with arginine bioavailability and could serve as surrogate markers of arginine sufficiency. Mechanistically, extracellular arginine is required for the translation of DNA-synthesis-dependent histone H4, but not H3, to promote DNA replication. Knockdown of arginyl-tRNA-transferase, like arginine deprivation, recapitulates the loss of histone H4 lysine 5 and 12 acetylation marks and compromises replication fork speed. Notably, one round of arginine deprivation and addition did not cause an increase in γH2AX, a hallmark of nuclear DNA double-stand breaks. However, multiple rounds of arginine fluctuation promotes the emergence of ATR-Chk1 checkpoint independent-nuclear p53-binding protein 1 bodies in the subsequent G1-phase of daughter cells and induces DNA damage tolerance and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic response. Citation Format: Yi-Chang Wang, Andrew A. Kelso, Yi-Hsuan Chen, Chi-An Hsieh, Wei-Kai Chen, Jeremy M. Stark, Hsing-Jien Kung, David K. Ann. Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3722.

Loss of histone H4 lysine 20 trimethylation in osteosarcoma is associated with aberrant expression ofhistone methyltransferase SUV420H2.

Epigenetic modifications of histones have crucial roles in various types of cancers. The aberrant trimethylation of histone H4 at lysine 20 (H4K20) has been implicated in carcinogenesis. At present, the status of trimethylation at H4k20 (H4K20me3) in osteosarcoma (OS), the predominant bone cancer in humans, is unknown. In the present study, a genome-wide decrease was observed in H4K20me3 levels in OS tissues and cell lines. Reduced levels of lysine methyltransferase 5C (SUV420H2), the histone methyltranferase responsible for modification of H4K20me3, was also observed in OS cells with the associated loss of H4K20me3. Furthermore, a total of 507 SUV420H2-regulated genes were identified through RNA-seq and a number of candidate genes were further validated. Bioinformatic analysis revealed an association between SUV420H2 and multiple signaling pathway, including the mitogen-activated protein kinase, P53, transforming growth factor and the ErbB pathways. These results demonstrated that there are aberrant levels of H4K20me3 and SUV420H2 in OS, and highlighted H4K20me3 as a candidate biomarker for the early detection of OS.

BAP1 is a haploinsufficient tumor suppressor linking chronic pancreatitis to pancreatic cancer in mice

Chronic pancreatitis represents a risk factor for the development of pancreatic cancer. We find that heterozygous loss of histone H2A lysine 119 deubiquitinase BAP1 (BRCA1 Associated Protein-1) associates with a history of chronic pancreatitis and occurs in 25% of pancreatic ductal adenocarcinomas and 40% of acinar cell carcinomas. Deletion or heterozygous loss of Bap1 in murine pancreata causes genomic instability, tissue damage, and pancreatitis with full penetrance. Concomitant expression of KrasG12D leads to predominantly intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, while pancreatic intraepithelial neoplasias are rarely detected. These lesions progress to metastatic pancreatic cancer with high frequency. Lesions with histological features mimicking Acinar Cell Carcinomas are also observed in some tumors. Heterozygous mice also develop pancreatic cancer suggesting a haploinsufficient tumor suppressor role for BAP1. Mechanistically, BAP1 regulates genomic stability, in a catalytic independent manner, and its loss confers sensitivity to irradiation and platinum-based chemotherapy in pancreatic cancer.

Infant milk formulae processing: Effect of wet-mix total solids and heat treatment temperature on rheological, emulsifying and nutritional properties

This study investigated the effects of total solids (TS) in the wet-mix and pasteurisation temperature on the whey protein (WP) denaturation, rheological behaviour, emulsifying properties and available lysine content of a model infant milk formula (IMF). IMF with 50% or 60% (w/w) TS were produced by wet-mix process and pasteurised at 75 °C or 100 °C for 18 s. Measurements were taken after dispersion, pasteurisation, homogenisation and spray-drying of wet-mix. Pasteurisation at 100 °C caused extensive WP denaturation (above 80%) and increased the viscosity. Available lysine content was 52% higher in powdered IMF obtained from wet-mixes pasteurised at 75 °C compared to 100 °C. A significant reduction in energy consumption could be achieved by producing IMF from 60% TS wet-mixes pasteurised at 75 °C, while obtaining powders with less than 7% WP denaturation, no loss of available lysine during processing and post-reconstitution stability for 2 h.

Metabolic Availability of Lysine from Oats and Whole Milk Assessed in School-Aged Children

Abstract Objectives The objective of this study was to assess protein quality by determining the metabolic availability (MA) of lysine from commonly consumed plant and animal-based food sources in school-aged children, using the indicator amino acid oxidation (IAAO) method. Methods Using a repeated-measures design, six healthy school-aged children randomly received 10 lysine intakes from either crystalline L-lysine (3, 6, 9, 12 mg/kg/day), moist cooked oats (9, 12 mg/kg/day), oven-baked granola (12 mg/kg/day), or whole milk (6, 9, 12 mg/kg/day). All diets provided protein at 1.5 g/kg/day and calories at 1.7x the participant´s measured resting energy requirement. All test diets were isocaloric and isonitrogenous. Every study day, breath samples were collected at baseline and during isotopic steady state and the ,13C enrichment was measured using an isotope ratio mass spectrometer. MA of lysine from food sources was determined using slope ratio analysis of ,13C-phenylalanine oxidation response to feeding lysine from whole foods compared to L-lysine from a reference crystalline amino acid diet. Results Lysine from whole milk was highly available, (∼100%, similar to our crystalline-lysine reference). The MA of lysine from cooked oats was 92.7%. When the same oats were oven-baked the availability of lysine was reduced by ∼50%. This reduction may be due to lysine loss through the Maillard reaction. Conclusions Lysine is highly metabolically available from milk and from moist cooked oats. However, dry cooking of oats reduces lysine MA considerably. The protein quality of cereal- based diets is dependent in the lysine content and bioavailability of the individual foods. Therefore, it is imperative to study how the human body utilizes this nutrient, especially in growing children. These are the first studies that assess protein quality in commonly consumed foods directly in children. This data will help build a knowledge base that can be further used to develop accurate dietary combinations that fully meet amino acid needs. Funding Sources Supported by: Dairy Farmers of Canada and National Dairy Council.

Coupling arginine with DNA replication and chemoresistance

How cells adapt DNA replication to meet amino acid demands is an active area of interest across biology. Among a broad range of functions, the extracellular arginine is needed for the availability of newly synthesized histones to promote DNA replication fork progression. We show herein that transient deprivation of extracellular arginine, but not glutamine or leucine, causes a reversible S‐phase arrest. This unexpected dynamic S‐phase arrest feature is independent of mTORC1 signaling inhibition, insufficient metabolites, endogenous de novo arginine biosynthesis, and general amino acid control pathway. Moreover, knockdown of arginyl‐tRNA‐transferase recapitulated the loss of histone H4 lysine 5 and 12 acetylation marks and the reduction of replication fork speed without inhibition of origin firing. Importantly, single round of arginine depletion and addition did not induce ATR/Chk1 activation and DNA double‐stand breaks associated γH2AX. The halt of arginine‐dependent histone H4 synthesis is therefore emerging as a “brake” for DNA replication bypassing checkpoint control. However, repeated arginine fluctuation promote the emergence of nuclear p53‐binding protein 1 bodies in the subsequent G1‐phase, creating vulnerabilities of DNA replications and can be exploited by cancer cells that promote DNA repair and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic responses.

Monocyte/Macrophage - like cell differentiation induced by TPA in HL60 cells leads to loss of Histone H4 Lysine 16 Acetylation

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester and induces monocyte/macrophage like cell differentiation in HL60 cells. The levels and patterns of gene expression differ greatly between differentiated and undifferentiated HL60 cells. Epigenetic histone modifications play a crucial role in transcriptional activation and alterations in the modification levels greatly affect the gene expression pattern. Acetylation of Histone H4 Lysine 16 (H4K16ac) is one such modification and has a significant role in transcriptional activation. Changes in its acetylation level either due to a physiological or pathological effect will have a dramatic effect on cellular gene expression. Here, a study was done to see the effect of TPA induced differentiation on H4K16ac levels in HL60 cells. Results obtained from flow cytometric analysis showed expression of macrophage cell surface marker CD11b on TPA differentiated HL60 cells and the western blots revealed a drastic downregulation of H4K16ac in differentiated cells. Immunoblotting and co-immunoprecipitation assay revealed DNA damage dependent enhancement of H4K16 acetylation and its co-localization with γH2AX in undifferentiated cells. Whereas, TPA differentiated cells (CD11B+ve) didn’t show any such enhancement in H4K16acetylation levels in the presence of DNA damage. The present study shows that TPA induced differentiation of HL60 cells into macrophages leads to the downregulation of H4K16 acetylation.

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation ismediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that inDrosophila the small ubiquitin-like protein SUMOand the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

Hat1 acetylates histone H4 and modulates the transcriptional program in Drosophila embryogenesis

Post-translational modifications of histone proteins play a pivotal role in DNA packaging and regulation of genome functions. Histone acetyltransferase 1 (Hat1) proteins are conserved enzymes that modify histones by acetylating lysine residues. Hat1 is implicated in chromatin assembly and DNA repair but its role in cell functions is not clearly elucidated. We report the generation and characterization of a Hat1 loss-of-function mutant in Drosophila. Hat1 mutants are viable and fertile with a mild sub-lethal phenotype showing that Hat1 is not essential in fruit flies. Lack of Hat1 results in the near complete loss of histone H4 lysine (K) 5 and K12 acetylation in embryos, indicating that Hat1 is the main acetyltransferase specific for these marks in this developmental stage. We found that Hat1 function and the presence of these acetyl marks are not required for the nuclear transport of histone H4 as histone variant His4r retained its nuclear localization both in Hat1 mutants and in His4r-K5R-K12R double point mutants. RNA-seq analysis of embryos indicate that in Hat1 mutants over 2000 genes are dysregulated and the observed transcriptional changes imply a delay in the developmental program of gene expression.

Effect of hydroxyl radicals on biochemical and functional characteristics of myofibrillar protein from large yellow croaker (Pseudosciaena crocea).

The oxidative modification effect of hydroxyl radicals generated by an H2 O2 oxidative solution on the biochemical and functional characteristics of myofibrillar protein (MP) from large yellow croaker (Pseudosciaena crocea) was investigated. The results showed that MP of large yellow croakers was vulnerable to hydroxyl radicals. Incubation of MP with increased concentration of H2 O2 led to the gradual formation of carbonyl derivatives, disulphide bonds and dityrosine, and loss of available lysine, sulphydryl, and free ammonia. Surface hydrophobicity increased, while tryptophan residues decreased, indicating a conformational transition of MP. SDS-PAGE demonstrated that both disulphide and nondisulphide bonds were involved in MP aggregation. The MP functionalities were also affected by hydroxyl radicals, including reduced solubility, gelling, and emulsifying properties. Mild oxidation (0.1 mM H2 O2 ) slightly improved MP gel strength and water-holding capacity, while excessive oxidation caused a reduction in gel properties. The results suggested that the MP oxidation should be controlled. PRACTICAL APPLICATIONS: Protein oxidation mediated by hydroxyl radicals is one of the most crucial reasons affecting the quality of large yellow croaker during processing and storage. Additionally, it is well known that the quality of protein in fish systems directly determines the quality stability and nutritional value of the fish products. Hence, the study explored how the hydroxyl radicals influenced the physicochemical and functional properties of fish MP. The changes in amino acid residues (carbonyl, available lysine, sulphydryl, free ammonia, tryptophan residues, etc.) of MP and its gelling and emulsifying properties after oxidation not only benefit the awareness of quality deterioration and quality control of large yellow croaker, but also provide a basis for the understanding of protein oxidation on the nutritional value of aquatic products and human health.

Glioblastoma initiating cells are sensitive to histone demethylase inhibition due to epigenetic deregulation.

Tumor-initiating cells are a subpopulation of cells that have self-renewal capacity to regenerate a tumor. Here, we identify stem cell-like chromatin features in human glioblastoma initiating cells (GICs) and link them to a loss of the repressive histone H3 lysine 9 trimethylation (H3K9me3) mark. Increasing H3K9me3 levels by histone demethylase inhibition led to cell death in GICs but not in their differentiated counterparts. The induction of apoptosis was accompanied by a loss of the activating H3 lysine 9 acetylation (H3K9ac) modification and accumulation of DNA damage and downregulation of DNA damage response genes. Upon knockdown of histone demethylases, KDM4C and KDM7A both differentiation and DNA damage were induced. Thus, the H3K9me3-H3K9ac equilibrium is crucial for GIC viability and represents a chromatin feature that can be exploited to specifically target this tumor subpopulation.

Beyond "Triton": Malignant Peripheral Nerve Sheath Tumors With Complete Heterologous Rhabdomyoblastic Differentiation Mimicking Spindle Cell Rhabdomyosarcoma.

Spindle cell rhabdomyosarcoma (RMS) is an aggressive sarcoma type with a predilection for the head and neck and frequent transactivating MYOD1 mutations. Malignant peripheral nerve sheath tumors (MPNST) show heterologous (most often rhabdomyoblastic) differentiation in 10% of cases; such tumors have been referred to as malignant "Triton" tumors. MPNST frequently harbors inactivating mutations in SUZ12 or EED, resulting in PRC2 dysfunction and loss of histone H3 lysine 27 trimethylation (H3K27me3), most often seen in sporadic and radiation-associated, high-grade tumors; immunohistochemistry (IHC) for H3K27me3 is a useful diagnostic marker. We recently encountered a tumor showing H3K27me3 loss but with otherwise typical features of spindle cell RMS. The purpose of this study was to evaluate H3K27me3 in spindle cell RMS and further investigate putative spindle cell RMS with loss of H3K27me3. IHC for H3K27me3 was performed on 50 tumors diagnosed as spindle cell RMS. Targeted sequencing of all exonic and selected intronic regions of ~450 genes was performed on the tumors with H3K27me3 loss using hybrid capture with a custom probe set and massively parallel (next-generation) sequencing (NGS). Of the 50 patients, 32 were male and 18 were female with a median age of 33 years (range, 6 wk to 77 y). Tumors most often involved head and neck (N=23), extremities/limb girdles (N=11), and trunk wall (N=5). Three cases (6%) showed loss of H3K27me3; based on all available evidence, we believe at least 2 of these cases in fact represent MPNST with complete heterologous rhabdomyoblastic differentiation: a deep-seated groin mass in a 76-year-old female and a paratesticular mass in a 22-year-old male (neither of whom had a history or signs of type 1 neurofibromatosis). The tumors showed similar histologic appearances: fascicular architecture, marked nuclear atypia, eosinophilic cytoplasm, and a high mitotic rate; rhabdomyoblasts were not apparent. One tumor showed focal areas with scant myxoid stroma and alternating hypocellularity and hypercellularity. By IHC, the tumors showed diffuse staining for desmin, myogenin, and MyoD1, whereas S100 protein and SOX10 were negative. NGS on 2 tumors revealed (1) 2-copy deletion of NF1, CDKN2A, and SUZ12 and a TP53 mutation with arm-level loss of 17p; and (2) 2-copy deletion of CDKN2A and an NF1 mutation with loss of 17q11, findings characteristic of MPNST. NGS on the third tumor showed no distinctive alterations. MPNST may occasionally show complete heterologous rhabdomyoblastic differentiation without histologic evidence of residual conventional MPNST, closely mimicking spindle cell RMS. IHC for H3K27me3 reliably distinguishes MPNST from spindle cell RMS.

Usefulness of some Maillard reaction indicators for monitoring the heat damage of whey powder under conditions applicable to spray drying

The Maillard reaction (MR) rate was observed according to available lysine loss, furosine, hydroxymethylfurfural (HMF), furfural, and brown colour during the heating of freeze-dried nano-filtered whey at 60, 75, and 90 °C and water activities of 0.11, 0.33, 0.43, and 0.73. The physical state of lactose was measured and associated with MR rate. The values obtained for available lysine, furosine, HMF and browning index ranged between, respectively, 11.3 and 1.63 (g 100 g−1 protein), 0.44 and 11.1 (g 100 g−1 protein), not detected and 57.7 (mg 100 g−1 protein) and 0.0104 and 0.1707. The greatest heat damage occurred with low moisture content and high temperature. The MR rate was influenced by the physical state of lactose, heating temperature and the moisture content of the whey. This occurred to a greater extent during the initial and intermediate stages of the MR as opposed to during the formation of coloured compounds.

Malignant Peripheral Nerve Sheath Tumors: From Epigenome to Bedside

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas typically developing in the context of neurofibromatosis type 1 (NF-1). With the exception of surgical resection, these tumors are resistant to all current therapies, and unresectable, recurrent, or metastatic tumors are considered incurable. Preclinical studies have identified several novel candidate molecular targets for therapeutic intervention, but, to date, targeted therapies have proven ineffective. Recent studies have identified recurrent mutations in polycomb repressive complex 2 (PRC2) core components, embryonic ectoderm development protein (EED) and suppressor of zeste 12 homolog (SUZ12), in MPNST. These mutations result in global loss of the histone H3 lysine 27 trimethylation epigenetic mark, normally deposited by PRC2, and subsequent gain in acetylation at this residue. This altered chromatin state has been shown to promote MPNST malignancy; however, acetylation at this residue sensitizes MPNSTs to BRD4 and bromodomain and extra-terminal domain inhibition. Interestingly, the catalytic component of PRC2, enhancer of zeste homolog 2 (EZH2), is not mutated in MPNST, hinting that a noncanonical, PRC2-independent function of EZH2 may play a role in this cancer. This review examines the pathobiology of MPNST, the contribution of PRC2 subunits to this process, and the prospects for PRC2-related therapies for this cancer. IMPLICATIONS: Identification of mutations in the PRC2 components EED and SUZ12 in the majority of MPNSTs may imply noncanonical oncogenic activities of the intact component, EZH2, and provide new opportunities for therapeutic intervention.

Heart failure drug proscillaridin A targets MYC overexpressing leukemia through global loss of lysine acetylation

BackgroundCardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation.MethodsProscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry.ResultsAt a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression.ConclusionOverall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.

Experimental and Computational Study of the Group 1 Metal Cation Chelates with Lysine: Bond Dissociation Energies, Structures, and Structural Trends.

The kinetic energy dependence of the collision-induced dissociation (CID) of Group 1 metal cations (M+ = Li+, Na+, K+, Rb+, and Cs+) chelated to the amino acid lysine (Lys) was measured by threshold CID using a guided ion beam tandem mass spectrometer. The simple loss of neutral lysine is the only dissociation channel observed with the heavier alkali metal cations, whereas CID of Li+(Lys) yields other competing channels including loss of NH3 (the dominant channel at low energy) and eight other reactions. Analysis of the kinetic energy-dependent cross sections yields experimental M+(Lys) bond dissociation energies (BDEs) of 376 ± 30, 219 ± 13, 160 ± 10, 141 ± 6, and 128 ± 4 kJ/mol for Li+, Na+, K+, Rb+, and Cs+, respectively. Computational searches yielded 18 distinct, low-energy structural families related to sites of M+ binding in M+(Lys) complexes and 10 distinct, low-energy structural families for neutral lysine. Among the four levels of theory and three basis sets used, four different ground conformers of M+(Lys) and four different ground conformers of lysine were found, including a ground conformer of K+(Lys) and Cs+(Lys), [Nε,CO(OH)], and its higher energy zwitterionic analogue, [Nε,CO2-], that better explains recent infrared multiple photon dissociation action spectroscopy results. Computational results for predicted ground structures of M+(Lys) complexes yielded computed BDEs in reasonable agreement with experiment.

Heating-Aided pH Shifting Modifies Hemp Seed Protein Structure, Cross-Linking, and Emulsifying Properties.

Alkaline pH12 shift treatment performed at varying temperatures (20-80 °C for 1, 5, and 60 min) was applied to structurally modify hemp seed protein isolate (HPI). The solubility of HPI (∼20%) was remarkably improved ( p < 0.05) with elevating the temperature and prolonging the holding time, reaching 97.5% at 80 °C for 60 min. The treated HPI exhibited a strong tendency of forming soluble large aggregates. To limit lysinoalanine (LAL) production, heating was methodically controlled to 60 °C and 5 min, where the LAL content never exceeded 100 mg/100 g of protein and the loss of cysteine and lysine was also minimal. The emulsifying activity of HPI was improved by this mild pH shift-heating combination treatment as a result of the dissociation of protein subunits, unraveling of the tertiary structure, and exposure of hydrophobic groups. Moreover, the emulsion formed by the treated protein maintained a superior stability in particle size and distribution during storage.

Perturbed maintenance of transcriptional repression on the inactive X-chromosome in the mouse brain after Xist deletion

BackgroundThe long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo.ResultsBy using an inactive X-chromosome-linked MeCP2-GFP reporter, which allowed us to enumerate reactivation events in the mouse brain even when they occur in very few cells, we found that deletion of Xist in the brain after establishment of X-chromosome inactivation leads to reactivation in 2–5% of neurons and in a smaller fraction of astrocytes. In contrast to global loss of both H3 lysine 27 trimethylation (H3K27m3) and histone H2A lysine 119 monoubiquitylation (H2AK119ub1) we observed upon Xist deletion, alterations in CpG methylation were subtle, and this was mirrored by only minor alterations in X-chromosome-wide gene expression levels, with highly expressed genes more prone to both derepression and demethylation compared to genes with low expression level.ConclusionOur results demonstrate that Xist plays a role in the maintenance of histone repressive marks, DNA methylation and transcriptional repression on the inactive X-chromosome, but that partial loss of X-dosage compensation in the absence of Xist in the brain is well tolerated.