抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Integrative analysis of 111 reference human epigenomes

Application of the experimental design of genome-wide association studies (GWASs) is now 10 years old (young), and here we review the remarkable range of discoveries it has facilitated in population and complex-trait genetics, the biology of diseases, and translation toward new therapeutics. We predict the likely discoveries in the next 10 years, when GWASs will be based on millions of samples with array data imputed to a large fully sequenced reference panel and on hundreds of thousands of samples with whole-genome sequencing data.

10 Years of GWAS Discovery: Biology, Function, and Translation

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.

A High-Resolution Map of Human Evolutionary Constraint Using 29 Mammals

Tumors of the mammary gland

The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeable to fully permeable was accelerated at temperatures above 4 degrees C. This sequence of oocyst permeability changes was taken as an indicator of the oocyst inactivation pathway. Using the dye permeability results, inactivation rates of oocysts in two fecal pools stored in the dark at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day-1, respectively. The excystation assay gave similar inactivation rates of 0.0046 and 0.0079 oocyst day-1. These results demonstrate the utility of the dye permeability assay as an indicator of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments.

/pdf/assessment-of-a-dye-permeability-assay-for-determination-of-4aq0etwpoi.pdf

Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

Background: We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects. Methods: RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT 2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes. Results: We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 10 6 )i n luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67. Conclusion: Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

/pdf/identification-of-padi2-as-a-potential-breast-cancer-45kkus17bu.pdf

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target

The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2–8–17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells.

/pdf/potential-role-for-pad2-in-gene-regulation-in-breast-cancer-347s1k5zid.pdf

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

The protozoan parasite Cryptosporidium parvum has been responsible for several recent waterborne disease outbreaks in the United States (26, 31). This gastrointestinal illness is transmitted by an environmentally durable oocyst (15). C. parvum oocysts have been identified in significant amounts in surface waters throughout the United States and Canada (27, 34, 38). Public drinking water supplies derived from filtered surface waters were implicated in all U.S. waterborne cryptosporidiosis outbreaks between 1984 and 1993 (10). Filtration is an important barrier in drinking water purification, because C. parvum oocysts are highly resistant to disinfection with chlorine (25).

Recent studies suggest that the surface properties of C. parvum oocysts may differ from those of bacteria and other microbes. Results reported by Fogel et al. (16) suggest that significant numbers of C. parvum oocysts bypassed a filtration plant that retained smaller coliform bacteria, indicating that oocysts may not adhere to filter media as readily as other microbes. In a series of microscope studies, Anguish and Ghiorse (2) reported that C. parvum oocysts seeded into soil samples and suspended in deionized water (DI), phosphate-buffered saline (PBS), or 0.1% sodium pyrophosphate did not closely associate with inorganic or organic soil particles.

The surface properties of the oocyst wall affect the interactions of the oocysts with filter media and with environmental chemicals and surfaces. Changes in the oocyst wall as they age may affect the adhesion and transport properties of oocysts in natural environments. Changes in the oocyst wall may also affect oocyst survival. For example, Robertson et al. (37) noted that when oocysts were stored in fecal material, the oocyst wall permeability of potentially viable oocysts decreased over time.

Net surface charge and hydrophobicity are important factors mediating microbial adhesion to surfaces (21, 46). Understanding the surface charge and hydrophobicity of C. parvum oocysts will aid the development of optimum filtration media and coagulants to remove oocysts from drinking water and sewage in treatment plants. Such basic knowledge will also help clarify the microscale processes involved in sorption of oocysts onto particle surfaces in natural waters. The identification of noninfective surrogates with similar surface properties will also help facilitate development of treatment strategies and laboratory transport experiments.

Surface charge measurements for C. parvum oocysts have recently been reported by Ongerth and Pecoraro (33), Drozd and Schwartzbrod (14), and Rice et al. (36). Each of these studies utilized different oocyst sources, purification methods, storage solutions, and suspending media, and the reported results varied widely. Some chemicals used for oocyst purification in these studies may damage the oocysts (9) and change oocyst surface properties, including surface charge. A survey of the literature reveals few if any studies of electrophoretic mobilities for C. parvum oocysts in which oocysts were purified and stored under controlled conditions with concern about the use of surface-active chemicals.

Microbial adhesion to hydrophobic surfaces such as polystyrene can be used as a surrogate measurement of microbial adhesion to organic material in the soil. We developed a method for estimating oocyst hydrophobicity that relied on microscopic direct counting of suspended oocyst concentrations after adhesion to a standard polystyrene surface (40, 45, 46). This method was used to measure oocyst hydrophobicity as a function of the ionic strength of the suspending solution. Polystyrene was an ideal substrate for these tests because it is very hydrophobic (1), and the percentage of particles adhering to the polystyrene substrate under the mixing action of a micropipettor provided a reliable qualitative measure of the particle-surface adhesion energy (43, 48).

The objectives of this study were to estimate the electrophoretic mobility of C. parvum oocysts and to determine the effects of purification method and presence of antibiotics on the electrophoretic mobility. We also measured the effects of solution ionic strength on the hydrophobicity of oocysts and polystyrene beads and determined how the electrophoretic mobility and hydrophobic properties of oocysts change as they age.

https://aem.asm.org/content/aem/64/11/4439.full.pdf

Influence of Pretreatment and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of Cryptosporidium parvum Oocysts

Viruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein σNS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, σNS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that σNS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression. IMPORTANCE Viruses lack biosynthetic capabilities and depend upon the host for protein synthesis. This dependence requires viruses to evolve mechanisms to coerce the host translational machinery into synthesizing viral proteins in the face of ongoing cellular stress responses that suppress global protein synthesis. Reoviruses replicate and assemble within cytoplasmic inclusions called viral factories. However, synthesis of viral proteins was thought to occur in the cytosol. To identify the site(s) of viral translation, we undertook a microscopy-based approach using ribopuromycylation to detect active translation. Here, we report that active translation occurs within viral factories and that translational factors are compartmentalized within factories. Furthermore, we find that the reovirus nonstructural protein σNS associates with 43S preinitiation complexes at the factory margins, suggesting a role for σNS in translation. Together, virus-induced compartmentalization of the host translational machinery represents a strategy for viruses to spatiotemporally couple viral protein synthesis with viral replication and assembly.

Lynne J. Anguish

Papers

Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

Influence of Pretreatment and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of Cryptosporidium parvum Oocysts

Virus-mediated compartmentalization of the host translational machinery