Efficient Chemical Synthesis of Multi-Monoubiquitylated and Diubiquitylated Histones by the α-Halogen Ketone-Mediated Strategy.

The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.

The expedient, CAET-assisted synthesis of dual-monoubiquitinated histone H3 enables evaluation of its interaction with DNMT1.

Site-selective conjugation chemistry has proven effective to synthesize homogenously ubiquitinated histones. Recently, a powerful strategy using 2-((2-chloroethyl) amino) ethane-1-thiol (CAET) as a bifunctional handle was developed to generate chemically stable ubiquitin chains without racemization and homodimerization. Herein, we extend this strategy to the expedient synthesis of ubiquitinated histones, exemplifying its utility to not only synthesize single-monoubiquitinated histones, but dual-monoubiquitinated histones as well. The synthetic histones enabled us to evaluate the binding of DNMT1 to ubiquitinated nucleosomes and map the hotspots of this interaction. Our work highlights the potential of modern chemical protein synthesis to synthesize ubiquitinated histones for epigenetic studies.

Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes.

The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe the expedient syntheses of a selection of phosphorylated and ubiquitinated H2AX proteins in a strategy integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation. These modified H2AX proteins were then used to discover that although H2AXS139 phosphorylation can enhance the binding of the DNA damage repair factor 53BP1 to either an unmodified nucleosome or that bearing a single H2AXK15ub or H4K20me2 modification, it augments 53BP1's binding only weakly to nucleosomes bearing both H2AXK15ub and H4K20me2. To better understand why such a trivalent additive effect is lacking, we solved the cryo-EM structure (3.38 Å) of the complex of 53BP1 with the H2AXK15ub/S139ph_H4K20me2 nucleosome, which showed that H2AXS139 phosphorylation distorts the interaction interface between ubiquitin and 53BP1's UDR motif. Our study revealed that there is redundancy in the interplay of multiple histone PTMs, which may be useful for controlling the dynamic distribution of effector proteins onto nucleosomes bearing different histone variants and PTMs in a time-dependent fashion, through specific cellular biochemical events.

Thiirane linkers directed histone H2A diubiquitination suggests plasticity in 53BP1 recognition.

Polyubiquitination with diverse linkages on histones provides another layer of accuracy and complexity for epigenetic regulation, which is rarely studied. Herein, K27 or K48-diubiquitin modified H2A analogues were chemically synthesized using thiirane linkers. These permitted in vitro binding studies suggested the plasticity of ubiquitin chains in 53BP1 recognition.

Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones.

Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones.

Histone ubiquitylation and deubiquitylation processes and the mechanisms of their regulation are closely relevant to the field of epigenetics. Recently, the deubiquitylating enzyme USP51 was reported to selectively cleave ubiquitylation on histone H2A at K13 or K15 (i.e., H2AK13Ub and H2AK15Ub), but not at K119 (i.e., H2AK119Ub), in nucleosomes in vivo. To elucidate the mechanism for the selectivity of USP51, we constructed structurally well-defined in vitro protein systems with a ubiquitin modification at precise sites. A total chemical protein synthesis procedure was developed, wherein hydrazide-based native chemical ligation was used to efficiently generate five ubiquitylated histones (H2AK13Ub, H2AK15Ub, H2AK119Ub, H2BK34Ub, and H2BK120Ub). These synthetic ubiquitylated histones were assembled into nucleosomes and subjected to in vitro USP51 deubiquitylation assays. Surprisingly, USP51 did not show preference between H2AK13/15Ub and H2AK119Ub, in contrast to previous in vivo observations. Accordingly, an understanding of the selectivity of USP51 may require consideration of other factors, such as alternative pre-existing histone modifications, competitive reader proteins, or different nucleosome quality among the in vivo extraction nucleosome and the in vitro reconstitution one. Further experiments established that USP51 in vitro could deubiquitylate a nucleosome carrying H2BK120Ub, but not H2BK34Ub. Molecular dynamics simulations suggested that USP51-catalyzed hydrolysis of ubiquitylated nucleosomes was affected by steric hindrance of the isopeptide bond.

The Study of Pulse Condition of Traditional Chinese Medicine in Information Age.

Using the study of pulse condition in Traditional Chinese Medicine (TCM) as a case example, this paper discusses the characteristics of pulse information obtained by Chinese medicine practitioners, how to correctly understand the relationship between the human body and Chinese medicine in the information age, how to deal with pulse data, and how to study TCM pulse condition . Furthermore, we point out that the application of modern big data processing technology to the pulse of Chinese medicine offers new opportunities.

Research on Ratio of Dosage of Drugs in Traditional Chinese Prescriptions by Data Mining.

Maximizing the effectiveness of prescriptions and minimizing adverse effects of drugs is a key component of the health care of patients. In the practice of traditional Chinese medicine (TCM), it is important to provide clinicians a reference for dosing of prescribed drugs. The traditional Cheng-Church biclustering algorithm (CC) is optimized and the data of TCM prescription dose is analyzed by using the optimization algorithm. Based on an analysis of 212 prescriptions related to TCM treatment of kidney diseases, the study generated 87 prescription dose quantum matrices and each sub-matrix represents the referential value of the doses of drugs in different recipes. The optimized CC algorithm can effectively eliminate the interference of zero in the original dose matrix of TCM prescriptions and avoid zero appearing in output sub-matrix. This results in the ability to effectively analyze the reference value of drugs in different prescriptions related to kidney diseases, so as to provide valuable reference for clinicians to use drugs rationally.

How to Use TCM Informatics to Study Traditional Chinese Medicine in Big Data Age.

This paper introduces the characteristics and complexity of traditional Chinese medicine (TCM) data, considers that modern big data processing technology has brought new opportunities for the research of TCM, and gives some ideas and methods to apply big data technology in TCM.

The characteristics and key issues in electronic medical records (EMR) of traditional Chinese medicine TCM.

This poster briefly introduces the characteristics of TCM data, significances of EMR of TCM, and several key factors in the establishment of EMR of TCM.

Black and green tea consumption and the risk of coronary artery disease: a meta-analysis.

BACKGROUND: Epidemiologic studies are inconsistent regarding the association between tea consumption and the risk of coronary artery disease (CAD). OBJECTIVE: The objective was to perform a meta-analysis to determine whether an association exists between tea consumption and total CAD endpoints in observational studies. DESIGN: We searched PUBMED and EMBASE databases for studies conducted from 1966 through November 2009. Study-specific risk estimates were combined by using a random-effects model. RESULTS: A total of 18 studies were included in the meta-analysis: 13 studies on black tea and 5 studies on green tea. For black tea, no significant association was found through the meta-analysis [highest compared with lowest, summary relative risk (RR): 0.92; 95% CI: 0.82, 1.04; an increment of 1 cup/d, summary RR: 0.98; 95% CI: 0.94, 1.02]. For green tea, the summary RR indicated a significant association between the highest green tea consumption and reduced risk of CAD (summary RR: 0.72; 95% CI: 0.58, 0.89). Furthermore, an increase in green tea consumption of 1 cup/d was associated with a 10% decrease in the risk of developing CAD (summary RR: 0.90; 95% CI: 0.82, 0.99). CONCLUSIONS: Our data do not support a protective role of black tea against CAD. The limited data available on green tea support a tentative association of green tea consumption with a reduced risk of CAD. However, additional studies are needed to make a convincing case for this association.

Association between green tea intake and coronary artery disease in a Chinese population.

BACKGROUND: There is still conflicting evidence that green tea may protect against coronary atherosclerosis therefore the present study investigated the association between green tea consumption and arteriographically determined coronary atherosclerosis in a Chinese population. METHODS AND RESULTS: The study population consisted of 520 consecutive patients (379 men and 141 women) who underwent coronary arteriography for the first time. Patients were divided into 2 groups (Non-coronary artery disease [CAD] and CAD groups) according to the results of coronary arteriography. After adjusting the established and potential confounders, green tea consumption was associated with a reduced risk of CAD in male patients, with an adjusted odds ratio (OR) of 0.62 (95% confidence interval, 0.38-1.01) compared with those who did not drink green tea. Compared to non-tea drinkers, the adjusted ORs were 1.09 (0.61-1.96) in male patients consuming less than 125 g of dried green tea leaves per month, 0.36 (0.19-0.71) for 125-249 g per month and 0.36 (0.17-0.73) for > or =250 g per month, with a statistically significant test for trend (P<0.001). Similar dose-response relationships were also observed for frequency, duration, concentration and starting age of green tea drinking in male patients. In female patients, no inverse association was found between green tea consumption and CAD. CONCLUSIONS: Green tea consumption can protect against the development of coronary atherosclerosis in Chinese male patients.

[How to establish the hospital information system security policies].

It is important to establish the hospital information system security policies. While these security policies are being established, a comprehensive consideration should be given to the acceptable levels of users, IT supporters and hospital managers. We should have a formal policy designing process that is consistently followed by all security policies. Reasons for establishing the security policies and their coverage and applicable objects should be stated clearly. Besides, each policy should define user's responsibilities and penalties of violation. Every organization will need some key policies, such as of information sources usage, remote access, information protection, perimeter security, and baseline host/device security. Security managing procedures are the mechanisms to enforce the policies. An incident-handling procedure is the most important security managing procedure for all organizations.