First identification of Anaplasma phagocytophilum in both a biting tick Ixodes nipponensis and a patient in Korea: a case report.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum. To date, there have been no reported cases of A. phagocytophilum infection found in both the biting tick and the patient following a tick bite. CASE PRESENTATION: An 81-year-old woman presented with fever following a tick bite, with the tick still intact on her body. The patient was diagnosed with HGA. The tick was identified as Ixodes nipponensis by morphological and molecular biological detection methods targeting the 16S rRNA gene. The patient's blood was cultured after inoculation into the human promyelocytic leukemia cell line HL-60. A. phagocytophilum growth was confirmed via culture and isolation. A. phagocytophilum was identified in both the tick and the patient's blood by Anaplasma-specific groEL- and ankA-based nested polymerase chain reaction followed by sequencing. Moreover, a four-fold elevation in antibodies was observed in the patient's blood. CONCLUSION: We report a case of a patient diagnosed with HGA following admission for fever due to a tick bite. A. phagocytophilum was identified in both the tick and the patient, and A. phagocytophilum was successfully cultured. The present study suggests the need to investigate the possible incrimination of I. nipponensis as a vector for HGA in Korea.

Isolation of Coxiella burnetii in patients with nonspecific febrile illness in South Korea.

BACKGROUND: The number of human Q fever cases in South Korea has been rapidly increasing since 2015. We report the first isolation of Coxiella burnetii in Korea in two patients who initially presented with non-specific febrile illness and were finally diagnosed with acute Q fever in South Korea. CASE PRESENTATION: Two adult patients with fever had serologic tests against C. burnetii initially negative, and polymerase chain reaction against 16S rRNA using whole blood was also negative. After bacterial amplification of C. burnetii in immune-depressed mice, we isolated C. burnetii from patients with acute Q fever. The isolates KZQ2 and KZQ3 were confirmed by polymerase chain reaction, nucleotide sequence analysis, and morphologic observation using a transmission electron microscope. CONCLUSIONS: These results can help us understand the clinical and epidemiologic features of Q fever in South Korea.

Microleakage of different sealing materials in access holes of internal connection implant systems.

STATEMENT OF PROBLEM: Current implant systems cannot completely prevent microleakage from the access holes of screw-retained implant prostheses, which may constitute risks to the clinical success of the implants. PURPOSE: The purpose of this study was to evaluate the levels of microleakage through the access holes of screw-retained implant prostheses sealed with different materials. MATERIAL AND METHODS: An implant with an internal hexagonal configuration was connected to a temporary abutment with an acrylic resin crown. The apical 6.5 mm of the access hole was filled with 1 of the following materials: cotton pellet, silicone sealing material, vinyl polysiloxane, or gutta-percha. The remaining coronal 3 mm was sealed with composite resin. Cyclic loading with 21 N at 1 Hz was applied 16,000 times to the specimens in 0.5% basic fuchsin solution according to the long axis of the tooth. Basic fuchsin dye which penetrated into the internal wall of the abutment through the access hole was dissolved with methyl alcohol. Then the absorbance was measured by a spectrophotometer at 540 nm to evaluate the degree of microleakage. The results were statistically analyzed with 1-way ANOVA and the Tukey HSD test. RESULTS: From greatest to least, the levels of microleakage were in the following order: cotton pellet, silicone sealing material, vinyl polysiloxane, and gutta-percha. The microleakage associated with gutta-percha was not significantly different from that of vinyl polysiloxane. CONCLUSIONS: When sealing the access holes of screw-retained implant prostheses, gutta-percha or vinyl polysiloxane would help reduce microleakage.

Diagnosis and molecular characteristics of human infections caused by Anaplasma phagocytophilum in South Korea.

Human granulocytic anaplasmosis (HGA) is a tick borne infection caused by Anaplasma phagocytophilum. HGA cases in South Korea have been identified since the first report in 2014. In this study, we investigated the serological response in 594 clinical samples of patients with acute febrile illness and molecular characteristics of A. phagocytophilum clinical isolates obtained from HGA patients. In serological test for A. phagocytophilum, 7.91% (47/594 cases) were positive for IgG and Ig M and 13 of 47 cases showed seroconversion. In the detection rate of the 16S rRNA, msp2(p44), and ankA, genes were showed 3.68% (14/380 cases) for A. phagocytophilum-specific 16S rRNA gene. Phylogenetic analysis of three clinical isolates demonstrated high sequence similarity (98.58-100%) with A. phagocytophilum 16S rRNA sequences identified from public databases. Analysis of the msp2(p44) gene showed highly variable similarity rates (7.24-98.85%) even within isolated countries and host ranges. These results provide clues into the bacterial characterization of A. phagocytophilum originating from Korean patients, providing useful guidance for treatment and improving clinical outcomes.

ER stress induces the expression of Jpk, which inhibits cell cycle progression in F9 teratocarcinoma cell.

Jopock (Jpk), a transacting factor associated with the position-specific regulatory element of murine Hoxa-7, has shown to induce cell death in both prokaryotic and eukaryotic cells when introduced and overexpressed. Since Jpk protein harbors a transmembrane domain (TM) and a putative endoplasmic reticulum (ER) -retention signal at the N terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP). Both N-term- (Jpk-EGFP) and C-term-fused Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscope after staining the cells with ER- and Mito-Tracker. Through deletion analysis TM turned out to be important for ER localization of Jpk. When flow cytometric analysis was performed, both cells expressing Jpk-EGFP and EGFP-Jpk led cell cycle arrest and subsequent apoptotic cell death. In order to see whether Jpk is expressed during ER stress-mediated apoptosis, F9 cells were treated with DTT, an ER stress inducer. In the presence of 4 mM of DTT, about 50% of cells died strongly expressing Jpk (sevenfold) as well as Grp78, a molecular chaperone, and CHOP-10, a well-known apoptotic protein. When cells were transfected with both pEGFP-Jpk and pJpk-EGFP, cell cycle progression was interrupted compared to those of control cells. In summary, excess ER stress upregulated the expression of Jpk, which seemed to inhibit the cell cycle progression. These results altogether suggest that Jpk could be a useful cell death-triggering molecule applicable for cancer therapy as well as a useful target molecule for the treatment of certain neurodegenerative diseases caused by ER stress.

Retinoic acid response element in HOXA-7 regulatory region affects the rate, not the formation of anterior boundary expression.

Since it is known that a 307 bp fragment of the position specific regulatory element of human HOXA-7 contains two (DR3 and DR5) retinoic acid response elements (RAREs) at its 3' end, we constructed several deletion constructs containing different numbers of RAREs and examined their effects in vitro and in vivo. The 5' deletion constructs, BM112 and OM213, retaining both RAREs, were highly responsible (about 8 fold induction) for RA in F9 teratocarcinoma cells, versus NM307 (4-5 fold). The construct NS218, with both RAREs deleted but retaining the 5' sequences lost RA responsibility completely, whereas NR271, with one RARE(DR5) deleted retained a 50% inducibility (2.5 fold). In vivo transgenic analysis revealed that the constructs NM307 and NR271, but not OM213 nor BM112, directed the position specific expression pattern. Sequence analysis revealed that HOXA-7 enhancer sequences, including the RARE repeat sequences, were well conserved in human, mouse and chick. Part of the RAREs overlaps with the CDX1 binding site, and sequences of the DR3 RAREs were identical in this species. Two GAGA binding sites were also found to be strictly conserved. Because OM213, which had one GAGA site disrupted but retaining both RAREs, did not direct anterior boundary formation in transgenic mice, these results suggest the importance of the 5' 94 bp region, including the GAGA binding site, in anterior boundary formation and the involvement of the RARE in the rate of expression not in anterior boundary formation.

A novel gene, Jpk, induces apoptosis in F9 murine teratocarcinoma cell through ROS generation.

A novel gene Jpk (Jopock) has been originally isolated through yeast 1 hybridization technique as a trans-acting factor interacting with the position-specific regulatory element of a murine Hoxa-7. Northern analysis revealed that the Jpk was expressed at day 7.0 post coitum (p.c.) during early gastrulation. Previously it has been shown that a trace amount of JPK protein led bacterial cells to death. In eukaryotic F9 cells, Jpk also led the cell to death-generating DNA ladder: fewer than 50% of the cells survived after 72-h transfection. Flow cytometric analysis with cells stained with each Annexin V/7-amino-actinomycin D (7-AAD), MitoTracker, and hydroethidine (HE) revealed that Jpk induced apoptotic cell death in a time-dependent manner, reduced mitochondrial membrane potential, and increased ROS (reactive oxygen species) production, respectively. Additionally, Jpk seemed to regulate the Bcl family at the transcriptional level when RT-PCR was performed. Although the precise mechanism is not clear, these results altogether suggest that Jpk is a potent inducer of apoptosis through generation of ROS as well as concomitant reduction of mitochondrial membrane potential.

Lon Mutant of Brucella abortus Induces Tumor Necrosis Factor-Alpha in Murine J774.A1 Macrophage.

OBJECTIVES: The objective of this study was to isolate a Brucella lon mutant and to analyze the cytokine response of B. lon mutant during macrophage infection. METHODS: A wild-type Brucella abortus strain was mutagenized by Tn5 transposition. From the mouse macrophage J774.A1 cells, total RNA was isolated at 0 hours, 6 hours, 12 hours, and 24 hours after infection with Brucella. Using mouse cytokine microarrays, we measured transcriptional levels of the cytokine response, and validated our results with a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to confirm the induction of cytokine messenger RNA (mRNA). RESULTS: In host J774.A1 macrophages, mRNA levels of T helper 1 (Th1)-type cytokines, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), and IL-3, were significantly higher in the lon mutant compared to wild-type Brucella and the negative control. TNF-α levels in cell culture media were induced as high as 2 µg/mL after infection with the lon mutant, a greater than sixfold change. CONCLUSION: In order to understand the role of the lon protein in virulence, we identified and characterized a novel B. lon mutant. We compared the immune response it generates to the wild-type Brucella response in a mouse macrophage cell line. We demonstrated that the B. lon mutants induce TNF-α expression from the host J774.A1 macrophage.

A novel protein Jpk induces bacterial cell death through reactive oxygen species.

Jpk, a trans-acting regulatory factor associating with the position-specific regulatory element of Hoxa-7, has been reported to induce cell death in both prokaryotic and eukaryotic cells upon overexpression. The N- and C-terminal deleted variants of Jpk were constructed and then the toxicity of each construct was analyzed by checking the viability of the cells and the concomitant morphological changes through electron microscopy following the expression. The N-terminus of Jpk harboring transmembrane domain seemed to be more toxic to bacterial cell than C-terminus and the morphology of bacterial cells expressing N-terminal Jpk was similar to that induced by full length Jpk. The toxicity caused by Jpk protein in bacterial cell was through the production of ROS, which was decreased by an antioxidant (DTT) in a concentration dependent manner. The finding described in this study provides valuable clues on the relationship between Jpk toxicity and ROS generation.

Enhanced detection of Coxiella burnetii with a complementary locked primer-based real-time PCR method.

BACKGROUND AND OBJECTIVE: Coxiella burnetii is the bacterial causative agent of Q fever in humans. Because Q fever can establish itself with an initial inoculation of fewer than ten C. burnetii cells, a sensitive detection method for C. burnetii infection is needed for early detection. We aimed to evaluate the effectiveness of a complementary locked primer (CLP)-based real-time PCR method for sensitive detection of C. burnetii infection. METHODS: To evaluate the ability of CLPs to enhance the efficiency of the real-time PCR assay for the C. burnetii IS1111 insertion sequence, the mean threshold cycle values from 20 real-time PCR replicates with either CLPs or conventional primers were determined using tenfold serial dilutions (102-108) of purified C. burnetii Nine Mile genomic DNA. In addition, the cross-reactivity between C. burnetii and 31 non-Coxiella species was examined. RESULTS: The CLP-based real-time PCR allowed specific and reliable detection of as few as 59 copies of the IS1111 element present in the genome of C. burnetii, which represents approximately 2.96 genome equivalents or three cells of C. burnetii. These results demonstrate the effectiveness of CLP-based real-time PCR for sensitive detection of C. burnetii infection. CONCLUSION: It can be concluded that the CLP-based real-time PCR assay is a more appropriate method for sensitive detection and quantification of C. burnetii than previously reported methods.

Phylogenetic clustering of 4 prevalent virulence genes in Orientia tsutsugamushi isolates from human patients.

The pathogenicity of microbes is involved in many kinds of virulence genes. The relationships between these virulence genes and strains are not clear in Orientia tsutsugamushi yet. In this study, we confirmed the presence of the virulence genes and classified into O. tsutsugamushi isolates using phylogenetic analysis of the virulence genes. We also compared the fatality rates of every isolate via an infection experiment in BALB/c mice using the O. tsutsugamushi isolates, Deajeon03-01, Wonju03-01, and Muju03-01. Moreover, we compared the phylogenetic analysis, in basis with 56 kDa protein sequence which determined from serotype, and virulence genes of O. tsutsugamushi. Our results showed remarkably different fatality rates between Deajeon03-01 and Muju03-01, which are both Boryong strains of O. tsutsugamushi. Also, clustering analyses including these two isolates gave slightly different results depending on whether they were clustered based on virulence genes or on the 56 kDa protein sequences. Consequently, we conclude that fatality rates in O. tsutsugamushi are correlated with differences in both serotypes and virulence genes. We identified some variations within the virulence genes dnaA, virB8, tolR, and trxA among the isolates.

Microleakage and fracture patterns of teeth restored with different posts under dynamic loading.

STATEMENT OF PROBLEM: Many studies concerned with the microleakage of endodontically treated teeth restored with posts and cores and subjected to loading can be found in the literature. However, no studies have investigated microleakage under dynamic loading with simultaneous dye penetration, which is more relevant to clinical situations. PURPOSE: The purpose of this study was to compare microleakage and to classify fracture patterns of endodontically treated teeth restored with various post systems under dynamic loading. MATERIAL AND METHODS: The crown portions of 40 human mandibular incisors were sectioned at the cementoenamel junction, and the teeth were endodontically treated. Teeth were divided into 4 groups (n=10): teeth restored with a cast post and core, prefabricated metal post (ParaPost), fiber-reinforced composite resin post (FRC Postec), and ceramic post (Cosmopost). After preparing the post space, each post was cemented with dual-polymerized resin cement (DuoLink). With the exception of the cast post-and-core group, the cores were formed directly using a light-polymerized composite resin (Light-Core). An intermittent load of 98 N at 1 Hz was applied for 50,000 cycles at an angle of 135 degrees to the long axis of the restored teeth, which were immersed in a 0.5% basic fuchsin solution. The ratio of the dyed surface area to the total area of the sectioned root surface was determined using an image analysis program. The data were analyzed by a 1-way ANOVA and Duncan's multiple range test (alpha =.05). The fracture patterns of the teeth were classified according to their fracture propagation lines. RESULTS: The cast post group showed a significantly higher level of microleakage compared to the other groups (P=.001). Regarding the failure mode, the FRC Postec and Cosmopost groups showed fracture patterns that would favor retreatment. The number of cycles of repeated loading was not significantly different among the groups (P=.161). CONCLUSIONS: Both FRC Postec and Cosmopost groups showed less microleakage under dynamic loading and fracture patterns favoring a retreatment of fractured specimens.

A novel factor associating with the upstream regulatory element of murine Hoxa-7 induces bacterial cell death.

In order to understand the function of a cDNA (c171) associated with the upstream regulatory region of the Hoxa-7, the cDNA was cloned into the pGEX-4T-1 vector to produce it as a GST fusion protein. The size of the fusion protein was determined to be 48-kilodalton (kDa). Sequence analysis revealed that a protein C171 contained one hydrophobic transmembrane domain in the N-terminal region and several putative phosphorylation and glycosylation sites. C171 protein inhibited the bacterial growth within 30 min after induction. The transmission electron microscopic examination revealed that the morphology of the cells expressing C171 was changed dramatically: i.e., unusually elongated phenotype compared with those of controls, and finally leading to a cell death. These results altogether indicate that a trace amount of C171 induces bacterial cell death.