Chemical approaches for the preparation of ubiquitinated proteins via natural linkages.

Ubiquitination is an important posttranslation modification (PTM) that regulates a variety of cellular processes, including protein degradation, DNA repair, and viral infections. In this process, the C-terminal carboxyl group of ubiquitin (Ub) or poly-Ub is attached to the ε-amine of lysine (Lys) side chain of an acceptor protein through an isopeptide bond. Studying a molecular mechanism of ubiquitination and deubiquitination is fundamental for unraveling its precise role in health and disease and hence crucial for drug development. Enzymatic approaches for protein ubiquitination possess limited ability to selectivity install Ub or Ub chain on the desired position of an acceptor protein and often lead to heterogeneous mixtures. In the past decades, chemical protein (semi)synthesis has been proved to be an efficient tool to facilitate site-specific protein ubiquitination, which significantly contributes to decode the Ub signal at molecular and structural levels. In this review, we summarize the synthetic strategies developed for protein ubiquitination, and the achievements to generate monoubiquitinated, di-ubiquitinated, and tetraubiquitinated proteins with native isopeptide and ester bonds.

The Epidemic Genotypes of Human Adenovirus in Outpatient Children with Adenoviral Conjunctivitis from 2011 to 2012 in Jiangxi, China.

BACKGROUND: Human adenoviruses (HAdV) are a common cause of viral conjunctivitis, making surveillance of them from sporadic cases of conjunctivitis important. METHODS: To acquire a better understanding of the epidemic genotypes of HAdV in outpatient children with adenoviral conjunctivitis in Jiangxi Province, China (2011-2012), 179 samples from cases with a high suspicion of HAdV were analyzed by PCR. Samples confirmed to be HAdV-positive by PCR were cultured in Hep-2 cells to isolate the viruses, which were then identified through hexon gene sequencing. RESULTS: The adenoviral conjunctivitis positivity rate was 74.86% (134/179), from which 71.64% (96/134) were infections in boys, and 92.54% (124/134) were infections in children under 5 years of age. Sixty-nine HAdV strains were isolated from the positive samples and 69 sequences were obtained. Phylogenetic analysis indicated that 33 strains (47.82%) clustered with HAdV-B7, 21 (30.43%) with HAdV-B3, 6 (8.70%) with HAdV-B55, 6 (8.70%) with HAdV-E4, 1 with HAdV-B21 (1.45%), 1 with HAdV-D37 (1.45%), and 1 with HAdV-D64 (1.45%). CONCLUSIONS: This is the first identification of HAdV-B55 relating to adenoviral conjunctivitis in China. These findings provide a firm basis for future surveillance of adenoviral conjunctivitis in China or other East Asian regions.

A rapid point-of-care test for dengue virus-1 based on a lateral flow assay with a near-infrared fluorescent dye.

Dengue fever is caused by the dengue virus (DENV), and DENV1 is the prevalent epidemic serotype in south China. A new lateral flow assay (LFA) based on a near-infrared (NIR) fluorescent dye was developed to detect anti-DENV1 IgG antibodies. DyLight-800 was used as the marker conjugated to goat anti-human IgG antibodies, and recombinant dengue type 1 envelope protein was used as the capture protein on the test line. Twenty samples from patients infected with DENV1 and 160 negative controls were analyzed using this new NIR-LFA. The results of the NIR-LFA were compared with the results of Panbio Dengue IgG ELISA and the Dengue Duo IgM/IgG Cassette. Nineteen confirmed DENV1-positive samples were identified by NIR-LFA, giving 95% (19/20) sensitivity. No significant differences existed in the results when the 20 primary clinical samples were analyzed using NIR-LFA, Panbio ELISA, and the Dengue Duo Cassette. However, NIR-LFA had a lower limit of detection than IgG ELISA and Duo IgM/IgG Cassette did when analyzing a 2-fold dilution series of the 19 samples positively identified by NIR-LFA. When incorporated with an NIR POCT device, the new NIR-LFA was rapid, easy to use, and highly sensitive in detecting DENV1, and has potential for application to clinical diagnosis.

Curing Both Virulent Mega-Plasmids from Bacillus anthracis Wild-Type Strain A16 Simultaneously Using Plasmid Incompatibility.

Plasmid-cured derivative strains of Bacillus anthracis are frequently used in laboratory studies. Plasmid incompatibility, which does not increase the risk of chromosomal mutation, is a useful method for plasmid curing. However, in bacteria containing multiple plasmids, it often requires the sequential introduction of multiple, specific incompatibility plasmids. This lengthy process renders the traditional plasmid incompatibility method inefficient and mutation-prone. In this study, we successfully cured plasmids pXO1 and pXO2 from B. anthracis A16 simultaneously using only one recombinant incompatible plasmid, pKORT, to obtain a plasmid-free strain, designated A16DD. This method may also be useful for the simultaneous, one-step curing of multiple plasmids from other bacteria, including Bacillus thuringiensis and Yersinia pestis.

Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique.

OBJECTIVES: The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing. DESIGN AND METHODS: We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods. RESULTS: The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing. CONCLUSIONS: Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic.

Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

Curing the plasmid pXO2 from Bacillus anthracis A16 using plasmid incompatibility.

Plasmid incompatibility, which has no effect on other plasmids or chromosomal genes, can be used to cure a target plasmid. In this report, we successfully cured the plasmid pXO2 from Bacillus anthracis A16 with a newly constructed, incompatible plasmid pKSV7-oriIV and obtained a new pXO2-cured strain, designated A16PI2. This is the first time that a plasmid was cured from the B. anthracis wild-type strain A16 utilizing this principle, which could be considered as an efficacious method to cure large plasmids.