Gadd45a is a heterochromatin relaxer that enhances iPS cell generation.

Reprogramming of somatic cells to induced pluripotent stem cells rewrites the code of cell fate at the chromatin level. Yet, little is known about this process physically. Here, we describe a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and show significant heterochromatin loosening at the initial stage of reprogramming. We identify growth arrest and DNA damage-inducible protein a (Gadd45a) as a chromatin relaxer in mouse embryonic fibroblasts, which also enhances somatic cell reprogramming efficiency. We show that residue glycine 39 (G39) in Gadd45a is essential for interacting with core histones, opening chromatin and enhancing reprogramming. We further demonstrate that Gadd45a destabilizes histone-DNA interactions and facilitates the binding of Yamanaka factors to their targets for activation. Our study provides a method to screen factors that impact on chromatin structure in live cells, and identifies Gadd45a as a chromatin relaxer.

Characterization of TsDAF-21/HSP90 protein from the parasitic nematode Trichinella spiralis.

The Tsdaf-21 cDNA was cloned into pET-32a and subsequently expressed in Escherichia coli. The recombinant protein TsDAF-21 was purified and evaluated as an antigen in Western blot. The serum from mouse 14, 21, and 28 days after being infected with Trichinella spiralis recognized rTsDAF-21, but the serum from mouse 7 days post infection of T. spiralis could not react with rTsDAF-21. It implied that the antibody of TsDAF-21 did appear in the host 14 days after infection of T. spiralis. Western blot analysis revealed that the expression of TsDAF-21 in the muscle larvae incubated at room temperature for 8 h and at 37 °C for 4 and 8 h was increased compared to the muscle larvae incubated at 4 °C for 4 and 8 h. The results imply that the expression of TsDAF-21 could be induced at high temperature, not by the cold stress. The expression of TsDAF-21 could be attenuated under the different concentrations of geldanamycin (GA) treatment. Western blot showed that the expression of TsDAF-21 was reduced in the muscle larvae of T. spiralis treated with GA compared to Ampicillin and Gentamycin sulfate treated as control, and this inhibition effect was GA dependent. Other antibiotic treatments did not show any inhibition effects. Immunolocalization showed that TsDAF-21 was expressed in the different stages of T. spiralis including newborn larvae, muscle larvae, and adult worms. TsDAF-21 was mostly localized in nuclei of the cells in the worm. These results suggest that the expression of TsDAF-21 could be induced by the heat stress and attenuated by GA treatment, and TsDAF-21 might be a diagnostic marker for Trichinellosis.

TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis.

Trichinella is an important parasitic nematode of animals worldwide. Heat shock proteins are ubiquitous in nature and allow organisms to quickly respond to environmental stress. A portion of the Tsdaf-21 gene, a Caenorhabditis elegans daf-21 homologue encoding heat-shock protein 90 (Hsp90) was cloned from Trichinella spiralis. The partial nucleotide sequence resided near the 5'-end of the gene and encoded a polypeptide of 254 amino acid residues harboring a HATPase-c superfamily domain and Hsp90 protein domain. Phylogenetic analysis revealed that Tsdaf-21 is highly conserved and formed a monophyletic clade with other nematodes. The partial Tsdaf-21 transcript was subcloned and expressed for antibody production. Results using PCR primers specific for the Tsdaf-21 transcript, and mouse polyclonal antisera specific for the recombinant protein showed that both the RNA transcript and the corresponding protein were ubiquitously and consistently expressed in newborn larvae, muscle larvae and both male and female adult worms in the absence of any external stress or stimulation.

Characterization of TsMRP-L28, a mitochondrial ribosomal protein L28 from the parasitic nematode Trichinella spiralis.

A cDNA encoding mitochondrial ribosomal protein (MRP)-L28 was isolated and cloned from Trichinella spiralis, an economically important parasitic nematode. The predicted TsMRP-L28 protein consists of 276 amino acids. Phylogenetic analysis revealed that TsMRP-L28 was closely related to Caenorhabditis elegans mitochondrial ribosomal protein L28. The TsMRP-L28 transcript was expressed in newborn larvae, muscle larvae and male and female adult worms. Western blot showed that TsMRP-L28 was expressed in muscle larvae and adult worms. Immuno-localization revealed that TsMRP-L28 was ubiquitously distributed in newborn larvae, muscle larvae and adult worms, and that TsMRP-L28 was enriched in cells with higher protein synthesis activity, such as in newborn larvae and the cytoplasm of different developmental stages of embryos. These data suggest that TsMRP-L28 is required for the early development of T. spiralis.

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum.

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.

Expression and functional characterization of a Rho-family small GTPase CDC42 from Trichinella spiralis.

A full-length cDNA encoding a Rho-family small GTPase gene cdc42 of Trichinella spiralis was expressed in E. coli. The recombinant protein of TsCDC42 was purified and used to raise the polyclonal antibodies. The expression of TsCDC42 in different stages of parasite was investigated. The western blot showed that TsCDC42 was expressed in all stages of T. spiralis, including newborn larvae, muscle larvae and adult worms. The immuno-localization revealed that TsCDC42 was ubiquitously distributed in the newborn larvae, muscle larvae and adult worm. Cross-species RNAi was done by knockdown Tscdc42 RNAi in C. elegans. The results revealed that endogenous expression level of CDC42 was decreased by knockdown Tscdc42 RNAi in C. elegans, and this knockdown reduced the progeny of C. elegans. It suggested that Tscdc42 might play the same roles in the early development of T. spiralis.

[Comparative analysis of three diarrhea-household-surveys in Guangxi].

OBJECTIVE: To study the epidemiological characteristics and the effects of control measurements in Guangxi by comparing the results from diarrhea-household-surveys conducted in the three different periods of time and to develop control strategies. METHODS: Data on the incidence, health care seeking and treatment of diarrhea from three household surveys conducted in 1988, 1995 and 2007 was analyzed using SPSS (version 13.0). RESULTS: The incidence rates of diarrhea over the three periods of time were 0.562, 0.456 and 0.221 per person-year, respectively (P < 0.001). No significant difference was found in the incidence between males and females. The disease mainly attacked young age groups and those with lower educational levels. In recent years, longer durations of disease but less severe were observed in patients with diarrhea. The patients mainly visited first line health services with a health care seeking rate of 28.3%. Antibiotics were used by most of the patients (49.8% - 90.2%), while the rate of using oral rehydration salts (ORS) was only 1.4% - 11.5% but the use of traditional Chinese medicines has increased. Intake of untreated water, contaminated foods and contact with patients were important risk factors on diarrhea. CONCLUSION: The prevalence of diarrhea in Guangxi had declined and the health seeking rate was low in the past two decades. It is necessary to further regulate the treatment, in order to strengthen the health education programs to the general population, in order to improve the accessibility of health services and to increase both the health care seeking rate and effective diagnosis rate.

Molecular cloning and phylogenetic analysis of small GTPase protein Tscdc42 from Trichinella spiralis.

A full-length of complementary deoxyribonucleic acid (Tscdc42) encoding a putative Rho-family small GTPase gene cdc42 was isolated from Trichinella spiralis, an economically important parasitic nematode of zoonosis. The uninterrupted open reading frame of TsCDC42 encodes a predicted protein of 147 amino acids and containing a highly conserved domain of CDC42. Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Danio rerio, Mus musculus, and human showed that Tscdc42 is highly conserved. The highest identity of TsCDC42 with CDC42 from Drosophila is 67%, the similarity is up to 73%, the identity of TsCDC42 with the CDC42 homologue of C. elegans is 62%, and the similarity is up to 71%. Phylogenetic analysis of the amino acid sequence data, using the neighbor-joining and maximum parsimony methods, revealed that TsCDC42 is closely related to the molecule inferred from the cdc-42 gene of C. elegans. The transcript of TsCDC42 was analyzed during different stages of the worm.

Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum.

Hookworm infection is still a public health problem in developing countries. Aspartic protease plays an important role in parasite invasion and migration in the host. Aspartic protease gene from Ancylostoma caninum has been reported (Biochem Biophys Res Commun 227:294-302, 1996), but the activity of Acasp in eggs and larvae stage has not been studied. This paper reported the cloning of Acasp, expression in Escherichia coli, and characterization in eggs and larval stage. The mRNA encoding Acasp was detected using reverse transcriptase polymerase chain reaction in L4 and adult worm. A polyclonal antiserum against E. coli expressed recombinant Acasp was produced and used to detect and localize the Acasp in various developmental stages of A. caninum. Results from Western blot and indirect fluorescent immunoassay showed that the Acasp was present in the embryo, larvae, as well as in the adult worms. The recombinant Acasp exhibited the protease activity by the gelatin gel digestion assay.

[Epidemiologic surveillance for bacterial meningitis in 140 000 children under 5 years of age in Nanning district, Guangxi province].

OBJECTIVE: To characterize the incidence, epidemiologic features, etiologic agents and sequelae of bacterial meningitis in children under 5 years of age in Nanning, Guangxi. METHODS: A population-based surveillance was conducted to evaluate children with signs and symptoms of meningitis. All hospitals, township health centers and village clinics in the surveillance area were structured to participate in the case referral and evaluation. Cerebrospinal fluid (CSF) and blood specimens were obtained and processed using standardized microbiologic methods. RESULTS: During the 26-month surveillance period, among the children under 5 years old, a total of 1272 cases who met the screening criteria of meningitis were studied. 265 of 1272 cases were identified as clinically diagnosed meningitis, with an incidence rate of 86.36 per 100 000 population. The annual incidence rate under the 38 cases of confirmed bacterial meningitis appeared to be 12.38/100 000. Staphylococcus species accounted for the largest proportion of laboratory-confirmed bacterial meningitis, followed by E. coli and S. pneumoniae. The highest attack rate occurred in neonates < 1 month, followed by children aged 1 - 12 months in the confirmed patients. Meningitis caused by Sp and Hi mainly occurred in children aged 1 - 12 months. All cases of meningitis due to Hi and Sp were children aged 1 - 24 months. 13.16% and 0.00% of the cases survived with complications and sequelae, and the case-fatality rate was 18.42%. 40 bacterial isolates were identified from 1193 blood cultures and 23 from 1211 cerebrospinal fluid samples, but no Neisseria meningitidis was found. CONCLUSION: Meningitis due to Hi was first confirmed in Guangxi with the incidence of 0.98 per 100 000 population. The annual incidence rate of confirmed bacterial meningitis was 12.38 per 100 000, which was considered an important public health problem in children. Staphylococci was the predominant pathogen in confirmed bacterial meningitis.