Human Spotted Fever Group Rickettsioses Are Underappreciated in Southern Taiwan,Particularly for the Species Closely-Related to Rickettsia felis

Background

Despite increased identification of spotted fever group rickettsioses (SFGR) in animals and arthropods, human SFGR are poorly characterized in Taiwan.

Methods

Patients with suspected Q fever, scrub typhus, murine typhus, leptospirosis, and dengue fever from April 2004 to December 2009 were retrospectively investigated for SFGR antibodies (Abs). Sera were screened for Rickettsia rickettsii Abs by indirect immunofluorescence antibody assay (IFA), and those with positive results were further examined for Abs against R. rickettsii, R. typhi, R. felis, R. conorii, and R. japonica using micro-immunofluorescence (MIF) tests. Polymerase chain reaction (PCR) for detection of SFGR DNA was applied in those indicated acute infections. Case geographic distribution was made by the geographic information system software.

Results

A total of 413 cases with paired serum, including 90 cases of Q fever, 47 cases of scrub typhus, 12 cases of murine typhus, 6 cases of leptospirosis, 3 cases of dengue fever, and 255 cases of unknown febrile diseases were investigated. Using IFA tests, a total of 49 cases with 47 (11.4%) and 4 (1.0%) cases had sera potentially positive for R. rickettsii IgG and IgM, respectively. In the 49 cases screened from IFA, MIF tests revealed that there were 5 cases of acute infections (3 possible R. felis and 2 undetermined SFGR) and 13 cases of past infections (3 possible R. felis and 10 undetermined SFGR). None of the 5 cases of acute infection had detectable SFGR DNA in the blood specimen by PCR. Possible acute infection of R. felis was identified in both one case of Q fever and scrub typhus. The geographic distribution of SFGR cases is similar with that of scrub typhus.

Conclusions

Human SFGR exist and are neglected diseases in southern Taiwan, particularly for the species closely-related to R. felis.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Molecular systematics of threadfin breams and relatives (Teleostei,Nemipteridae)

Threadfin breams and relatives of the family Nemipteridae comprise 69 currently recognized species in five genera. They are found in the tropical and subtropical Indo‐West Pacific and most are commercially important. Using recently developed molecule‐based approaches exploiting DNA sequence variation among species/specimens, this study reconstructed a comprehensive phylogeny of the Nemipteridae, examined the validity of species and explored the cryptic diversity of the family, and tested previous phylogenetic hypotheses. A combined data set (105 taxa from 41 morphospecies) with newly determined sequences from two nuclear genes (RAG1 and RH) and one mitochondrial gene (COI), and a data set with only COI gene sequences (329 newly obtained plus 328 from public databases from a total of 53 morphospecies) were used in the phylogenetic analysis. The latter was further used for species delimitation analyses with two different tools to explore species diversity. Our phylogenetic results showed that all the currently recognized genera were monophyletic. The monotypic genus Scaevius is the sister group of Pentapodus and they together are sister to Nemipterus. These three genera combined to form the sister group of the clade comprising Parascolopsis and Scolopsis. The validity of most of the examined species was confirmed except in some cases. The combined evidence from the results of different analyses revealed a gap in our existing knowledge of species diversity in the Nemipteridae. We found several currently recognized species contain multiple separately evolving metapopulation lineages within species; some lineages should be considered as new species for further assignment. Finally, some problematic sequences deposited in public databases (probably due to misidentification) were also revised in this study to improve the accuracy for prospective DNA barcoding work on nemipterid fishes.     

Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein

The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese encephalitis virus (JEV) envelope protein. The JEV domain III forms a beta-barrel type structure composed of six antiparallel beta-strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. Site-directed mutagenesis experiments are performed to confirm the NMR results. Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses.     

A Robust GWSS Method to Simultaneously Detect Rare and Common Variants for Complex Disease

The rapid advances in sequencing technologies and the resulting next-generation sequencing data provide the opportunity to detect disease-associated variants with a better solution, in particular for low-frequency variants. Although both common and rare variants might exert their independent effects on the risk for the trait of interest, previous methods to detect the association effects rarely consider them simultaneously. We proposed a class of test statistics, the generalized weighted-sum statistic (GWSS), to detect disease associations in the presence of common and rare variants with a case-control study design. Information of rare variants was aggregated using a weighted sum method, while signal directions and strength of the variants were considered at the same time. Permutations were performed to obtain the empirical p-values of the test statistics. Our simulation showed that, compared to the existing methods, the GWSS method had better performance in most of the scenarios. The GWSS (in particular VDWSS-t) method is particularly robust for opposite association directions, association strength, and varying distributions of minor-allele frequencies. It is therefore promising for detecting disease-associated loci. For empirical data application, we also applied our GWSS method to the Genetic Analysis Workshop 17 data, and the results were consistent with the simulation, suggesting good performance of our method. As re-sequencing studies become more popular to identify putative disease loci, we recommend the use of this newly developed GWSS to detect associations with both common and rare variants.     

Apoptosis of Hepatocellular Carcinoma Cells Induced by Nanoencapsulated Polysaccharides Extracted from Antrodia Camphorata

Antrodia camphorata is a well-known medicinal mushroom in Taiwan and has been studied for decades, especially with focus on anti-cancer activity. Polysaccharides are the major bioactive compounds reported with anti-cancer activity, but the debates on how they target cells still remain. Research addressing the encapsulation of polysaccharides from A. camphorata extract (ACE) to enhance anti-cancer activity is rare. In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2). The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group. In apoptosis assays, DNA fragmentation and sub-G₁ phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments. ACE/CS and ACE/S could also increase reactive oxygen species (ROS) generation, induce Fas/APO-1 (apoptosis antigen 1) expression and elevate the proteolytic activities of caspase-3, caspase-8 and caspase-9 in Hep G2 cells. Unsurprisingly, ACE/CS induced a similar apoptosis mechanism at a lower dosage (ACE polysaccharides = 13.2 μg/mL) than those of ACE/S (ACE polysaccharides = 21.2 μg/mL) and ACE polysaccharides (25 μg/mL). Therefore, the encapsulation of ACE polysaccharides by chitosan-silica nanoparticles may provide a viable approach for enhancing anti-tumor efficacy in liver cancer cells.