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The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in pyroptosis. In this study, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell–derived exosomes. In apoptotic human HeLa and HEK293 cells, GSDMA, GSDMC, GSDMD, and GSDME increased the release of apoptotic exosomes. GSDMB and DFNB59, in contrast, negatively affected the release of apoptotic exosomes. GSDME at its full-length and cleaved forms was localized in the exosomes and exosomal membrane. Full-length and cleaved forms of GSDME are suggested to increase Ca2+ influx to the cytosol through endosomal pores and thus increase the biogenesis of apoptotic exosomes. In addition, the GSDME-mediated biogenesis of apoptotic exosomes depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins, that is, annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of apoptotic exosomes begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released apoptotic exosomes to induce inflammatory responses in the in vivo mouse 4T1 orthotropic model of BALB/c breast cancer. The data indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

Sensorineural hearing loss is the most common sensory disorder in humans. Genetic analyses have greatly increased our understanding of the pathogenic mechanisms in play. Thus, characterization of audiologic phenotypes by the genetic etiology may aid elucidation of the etiologies of certain types of inherited hearing loss. Further, delineation of specific audiologic phenotypes based on the genetic etiology aids our understanding of some types of inherited hearing loss in terms of the prediction of clinical course, revelation of genotype-phenotype correlations, and application of appropriate audiologic rehabilitation. Here, we describe the interesting audiologic characteristics of LMX1A -associated deafness, which revealed significant asymmetry between two ears.Among 728 probands of which genomic DNA went through exome sequencing regardless of any specific audiologic phenotypes, probands for which exome sequencing was performed and a causative LMX1A variant was found were all included. Five LMX1A -associated DFNA7 families (approximately 0.7%), the pedigrees of whom indicated autosomal-dominant hearing loss, were identified, and segregation was studied using Sanger sequencing. The affected individuals underwent comprehensive evaluations, including medical history reviews, physical examinations, imaging, and auditory phenotyping. We functionally characterized the novel LMX1A variants via computational structural modeling and luciferase reporter assays.Among 728 probands of which genomic DNA went through exome sequencing, we identified four novel LMX1A heterozygous variants related to DFNA7 (c.622CT:p.Arg208*, c.719AG:p.Gln240Arg, c.721GA:p.Val241Met, and c.887dup:p.Gln297Thrfs*41) and one harboring a de novo heterozygous missense LMX1A variant (c.595AG;p.Arg199Gly) previously reported. It is important to note that asymmetric hearing loss was identified in all probands and most affected individuals, although the extent of asymmetry varied. Structural modeling revealed that the two missense variants, p.Gln240Arg and p.Val241Met, affected conserved residues of the homeodomain, thus attenuating LMX1A-DNA interaction. In addition, Arg208*-induced premature termination of translation destroyed the structure of the LMX1A protein, including the DNA-binding homeodomain, and p.Gln297Thrfs*41 led to the loss of the C-terminal helix involved in LIM2 domain interaction. Compared with the wild-type protein, all mutant LMX1A proteins had significantly reduced transactivation efficiency, indicating that the ability to elicit transcription of the downstream target genes of LMX1A was severely compromised. Thus, in line with the American College of Medical Genetics and Genomics guideline specified to genetic hearing loss, the four novel LMX1A variants were identified as "pathogenic" (p.Arg208* and p.Gln297Thrfs*41), "likely pathogenic" (p.Val241Met), and as a "variant of uncertain significance'' (p.Gln240Arg).For the first time, we suggest that LMX1A is one of the candidate genes which, if altered, could be associated with dominantly inherited asymmetric hearing loss. We also expand the genotypic spectrum of disease-causing variants of LMX1A causing DFNA7 by doubling the number of LMX1A variants reported thus far in the literature.

Novel hearing loss (HL) genes are constantly being discovered, and evidence from independent studies is essential to strengthen their position as causes of hereditary HL. To address this issue, we searched our genetic data of families with autosomal dominant HL (ADHL) who had been tested with high-throughput DNA sequencing methods. For CD164, only one pathogenic variant in one family has so far been reported. For LMX1A, just two previous studies have revealed its involvement in ADHL. In this study we found two families with the same pathogenic variant in CD164 and one family with a novel variant in LMX1A (c.686CA; p.(Ala229Asp)) that impairs its transcriptional activity. Our data show recurrence of the same CD164 variant in two HL families of different geographic origin, which strongly suggests it is a mutational hotspot. We also provide further evidence for haploinsufficiency as the pathogenic mechanism underlying LMX1A-related ADHL.

A challenge for patients with ski-slope hearing loss is that hearing aids do not adequately amplify the mid-to-high frequencies necessary for speech perception and conversely, cochlear implant (CI) may damage low-frequency hearing. We aimed to describe the clinical profile of patients with ski-slope hearing loss, with a special focus on aetiology of such hearing loss and audiological course of low-frequency hearing after CI.We recruited hearing-impaired patients who visited a tertiary referral centre and met the criteria for ski-slope hearing loss patients from 2015 to 2021. Genetic testing was performed in all ski-slope hearing loss patients unless refused. Baseline audiograms of patients who continued to use hearing aids or who finally underwent CIs were reviewed. As for CI patients, outcome and hearing preservation rate were rigorously analysed.Of 46 recruited patients with ski-slope hearing loss, 45 agreed to undergo genetic testing and causative variants were identified in 17 (37.8%) patients. The TMC1, MYO7A, and TMPRSS3 variants were the most common, while LRTOMT was newly identified as a causative gene. Twenty-five patients eventually received CI, while 13 continued to wear the hearing aid and 8 patients did not ever try hearing aids. CI in ski-slope hearing loss led to immediate and sufficient improvement of sentence recognition by as early as 3 months, however, the duration of hearing loss was inversely correlated with the sentence recognition score. The average hearing preservation rate (using the HEARRING classification) after CI was 53.0% (SD 30.0) and 45.6% (SD 31.1) at 1 year. Seventy-nine percent of implantees maintained functional low-frequency hearing (better than 85 dB at 250 and 500 Hz) eligible for electric-acoustic stimulation (EAS). A trend was found that patients with hair cell stereocilia-associated genetic variants may have a slightly better preservation, albeit with no statistical significance.Detection rate of a molecular genetic aetiology of ski-slope hearing loss appears to be lower than other type of hearing loss reported in the literature. Especially with short hearing loss duration, CI in ski-slope hearing loss leads to immediate and sufficient speech improvement, while preserving functional low-frequency hearing eligible for EAS as many as in 79%. A certain genetic aetiology might be associated with a trend towards better low-frequency hearing preservation.

We investigated the changes in subfoveal choroidal thickness and choroidal vascularity index (CVI) and their relationship with the severity of coronary artery stenosis in patients with cardiovascular risk factors and symptoms suggestive of coronary artery disease (CAD). Ninety patients who underwent coronary angiography (CAG) for evaluation of their coronary artery status and cardiac symptoms were included. Forty-two patients showed no evidence of CAD; 31 patients had one to two vessel disease; and 17 had a triple vessel disease. There were no significant differences in the subfoveal choroidal thickness among the three groups; however, the CVI in the triple vessel disease group was lower than those in the other groups. The CVI values were good predictors of the presence of triple-vessel disease (p = 0.020). Multivariate logistic regression analysis results revealed that male sex (odds ratio 5.4, p = 0.049), hypertension (odds ratio 4.9, p = 0.017), and CVI (%, odds ratio 0.8, p = 0.016) were significant factors associated with the presence of triple vessel disease. Although CVI may not be a sensitive marker for detecting early changes in the coronary artery, it may be helpful in indicating severe CAD.

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in the pyroptosis and the release of inflammatory cytokines. Here, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell–derived exosomes. In apoptotic cells, GADMA, GSDMC, GSDMD, and GSDME increased the release of ApoExos, and both their full-length and cleaved forms were localized in the exosomal membrane. GSDMB and DFNB59, on the other hand, negatively affected the release of ApoExos. The caspase-mediated cleavage of gasdermins, especially GSDME, is suggested to increase Ca2+ influx to cytosol through endosomal pores and thus increase the biogenesis of ApoExos. In addition, the GSDME-mediated biogenesis of ApoExos depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins: annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of ApoExos begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released ApoExos to induce inflammatory responses in the in vivo 4T1 orthotropic model of breast cancer. The data presented in this study indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

The etiology of age-related macular degeneration (AMD) is diverse; however, recent evidence suggests that the lipid metabolism-cholesterol pathway might be associated with the pathophysiology of AMD. The ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are essential for the formation of high-density lipoprotein (HDL) and the regulation of macrophage cholesterol efflux. The failure of retinal or retinal pigment epithelium (RPE) cholesterol efflux to remove excess intracellular lipids causes morphological and functional damage to the retina. In this study, we investigated whether treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMP-activated protein kinase (AMPK) activator, improves RPE cholesterol efflux and Bruch's membrane (BM) lipid deposits. The protein and mRNA levels of ABCA1 and ABCG1 in ARPE-19 cells and retinal and RPE/choroid tissue from apolipoprotein E-deficient (ApoE−/-) mice were evaluated after 24 weeks of AICAR treatment. The cholesterol efflux capacity of ARPE-19 cells and the cholesterol-accepting capacity of apoB-depleted serum from mice were measured. The thickness of the BM and the degree of lipid deposition were evaluated using electron microscopy. AICAR treatment increased the phosphorylation of AMPK and the protein and mRNA expression of ABCA1 and ABCG1 in vitro. It promoted cholesterol efflux from ARPE-19 cells and upregulated the protein and mRNA levels of ABCA1 and ABCG1 in the retina and RPE in vivo. ApoB-depleted serum from the AICAR-treated group showed enhanced cholesterol-accepting capacity. Long-term treatment with AICAR reduced BM thickening and lipid deposition in ApoE−/- mice. In conclusion, AICAR treatment increased the expression of lipid transporters in the retina and RPE in vivo, facilitated intracellular cholesterol efflux from the RPE in vitro, and improved the functionality of HDL to accept cholesterol effluxed from the cell, possibly via AMPK activation. Collectively, these effects might contribute to the improvement of early age-related pathologic changes in the BM. Pharmacological improvement of RPE cholesterol efflux via AMPK activation may be a potential treatment strategy for AMD.

This study aimed to determine whether inter-ocular differences in axial length (AL), corneal power (K), and adjusted emmetropic intraocular lens power (EIOLP) and inter-visit differences in these ocular biometric values, measured on different days, are related to refractive outcomes after cataract surgery. We retrospectively reviewed 279 patients who underwent phacoemulsification. Patients underwent ocular biometry twice (1–4 weeks before and on the day of surgery). Patients were divided into three groups: group S (similar inter-ocular biometry in different measurements; n = 201), group P (inter-ocular differences persisted in the second measurement; n = 37), and group D (inter-ocular difference diminished in the second measurement; n = 41). Postoperative refractive outcomes (mean absolute errors [MAEs]) were compared among the groups. Postoperative MAE2, based on second measurement with reduced inter-ocular biometry difference, was smaller than that calculated using the first measurement (MAE1) with borderline significance in group D (MAE1, 0.49 ± 0.45 diopters vs. MAE2, 0.41 ± 0.33 diopters, p = 0.062). Postoperative MAE2 was greater in group P compared to the other two groups (p = 0.034). Large inter-ocular biometry differences were associated with poor refractive outcomes after cataract surgery. These results indicate that measurements with smaller inter-ocular differences were associated with better refractive outcomes in cases with inter-visit biometry differences.

Background and aim The aim of this study was to compare HOXB7 expression level between gastric cancer and non-cancerous gastric tissues. Additionally, the functional effects of HOXB7, including its pro-migration or invasion and anti-apoptosis roles, were evaluated in gastric cancer cells. Methods Both gene and protein expression levels of HOXB7 were examined in gastric cancer cell lines, and HOXB7 expression was compared between primary or metastatic gastric cancer tissues and chronic gastritis or intestinal metaplasia tissues. Functional studies included a wound healing assay, a Matrigel invasion assay, and an Annexin-V assay were performed, and Akt/PTEN activity was measured by western blotting. Results Both gene and protein expression levels of HOXB7 could be clearly detected in various gastric cancer cell lines except MKN-28 cell. HOXB7 expression was significantly higher in primary or metastatic gastric cancer tissues than in chronic gastritis or intestinal metaplasia tissues. HOXB7 knockdown led to inhibition of cell invasion and migration, had an apoptotic effect, downregulated phosphor-Akt, and upregulated PTEN in AGS and SNU-638 cells. Reinforced expression of HOXB7 caused the opposite effects in MKN-28 and MKN-45 cells. Conclusion Our study suggests that HOXB7 has an oncogenic role in gastric cancer, which might be related to the modulation of Akt/PTEN activity to induce cell migration/invasion and anti-apoptotic effects.

Background We investigated the effect of arsenic trioxide (ATO) for inhibition of signal transducer and activator of transcription 3 (STAT3) and epithelial-mesenchymal transition (EMT) in gastric cancer cells, and the role of SH2 domain-containing phosphatase-1 (SHP-1) during this process. Methods We used AGS cells, which showed minimal SHP-1 expression and constitutive STAT3 expression. After treatment of ATO, cellular migration and invasion were assessed by using wound closure assay, Matrigel invasion assay and 3-D culture invasion assay. To validate the role of SHP-1, pervanadate, a pharmacologic phosphatase inhibitor, and SHP-1 siRNA were used. Xenograft tumors were produced, and ATO or pervanadate were administered via intraperitoneal (IP) route. Results Treatment of ATO 5 and 10 μM significantly decreased cellular migration and invasion in a dose-dependent manner. Western blot showed that ATO upregulated SHP-1 expression and downregulated STAT3 expression, and immunofluorescence showed upregulation with E-cadherin (epithelial marker) and downregulation of Snail1 (mesenchymal marker) expression by ATO treatment. Anti-migration and invasion effect and modulation of SHP-1/STAT3 axis by ATO were attenuated by pervanadate or SHP-1 siRNA. IP injection of ATO significantly decreased the xenograft tumor volume and upregulated SHP-1 expression, which were attenuated by co-IP injection of pervanadate. Conclusion Our data suggest that ATO inhibits STAT3 activity and EMT process by upregulation of SHP-1 in gastric cancer cells.

A recent study reported that plumbagin downregulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various antitumor effects in multiple myeloma cells. We aimed in this in vitro study to demonstrate the inhibition of JAK2/STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in the MKN-28 gastric cancer cell line. We performed western blot analysis to measure SHP1, phosphor-JAK2/STAT3 level, and observed that plumbagin induced SHP1 expression and simultaneously downregulated phosphor-JAK2/STAT3 in MKN-28 cells, with negative SHP1 expression. This effect was consistent when JAK2/STAT3 signaling was activated by interleukin-6 (IL-6), and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, vascular endothelial growth factor (VEGF)-1, Bcl-xL, survivin and matrix metalloproteinase-9 (MMP-9), known target products of STAT3 activation in gastric carcinogenesis by reverse transcription-polymerase chain reaction (RT-PCR). Several functional studies such as water soluble tetrazolium salt-1 (WST-1) assay, wound closure assay, Matrigel invasion assay and Annexin V assay were also performed, and we validated the functional effect of plumbagin for inhibition of cell proliferation, migration and invasion, and induction of apoptosis. Collectively, our findings suggest that plumbagin is a potential regulator of cellular growth, migration, invasion and apoptosis by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

The influence of gamma irradiation with low doses (0.5, 1.0, and 1.5 kGy) on the meat quality of chicken breast was determined for 6 days of storage at . The pH of irradiated chicken was lower than that of the control at day 1 (p

The aim of this study was to compare HOXB7 expression level between gastric cancer and non-cancerous gastric tissues. Additionally, the functional effects of HOXB7, including its pro-migration or invasion and anti-apoptosis roles, were evaluated in gastric cancer cells.Both gene and protein expression levels of HOXB7 were examined in gastric cancer cell lines, and HOXB7 expression was compared between primary or metastatic gastric cancer tissues and chronic gastritis or intestinal metaplasia tissues. Functional studies included a wound healing assay, a Matrigel invasion assay, and an Annexin-V assay were performed, and Akt/PTEN activity was measured by western blotting.Both gene and protein expression levels of HOXB7 could be clearly detected in various gastric cancer cell lines except MKN-28 cell. HOXB7 expression was significantly higher in primary or metastatic gastric cancer tissues than in chronic gastritis or intestinal metaplasia tissues. HOXB7 knockdown led to inhibition of cell invasion and migration, had an apoptotic effect, downregulated phosphor-Akt, and upregulated PTEN in AGS and SNU-638 cells. Reinforced expression of HOXB7 caused the opposite effects in MKN-28 and MKN-45 cells.Our study suggests that HOXB7 has an oncogenic role in gastric cancer, which might be related to the modulation of Akt/PTEN activity to induce cell migration/invasion and anti-apoptotic effects.

SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies have demonstrated that SHP1 promoter methylation is frequently observed in gastric adenocarcinoma tissues. In this in vitro study, we attempted to reveal promoter hypermethylation and to investigate effects of SHP1 in gastric carcinoma cell lines. We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). Methylation-specific PCR (MSP) showed a methylation-specific band only in the 10 gastric cancer lines. Bisulfite pyrosequencing in AGS, MKN-28, and SNU-719 cells indicated that methylation frequency was as high as 94.4, 92.6, and 94.5 %, respectively, in the three cell lines. Treatment of SNU-719, MKN-28, and AGS cells with 5-Aza-2'-deoxycytidine (5-Aza-dc) led to re-expression of SHP1 in these cells. Introduction of exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection substantially downregulated protein expression of constitutive phosphor-Janus kinase 2 (JAK2) (tyrosine 1007/1008) and phosphor-signal transducers and activators of transcription 3 (STAT3) (tyrosine 705), which in turn decreased expression of STAT3 target genes including those encoding cyclin D1, MMP-9, VEGF-1, and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Taken together, epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cells. Overexpression of SHP1 downregulates the JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration, and invasion in gastric cancer cells.

76 Background: Arsenic trioxide (ATO) is known to inhibit epithelial-mesenchymal transition (EMT) in hepatolocellular carcinoma and breast cancer cells, however, little has been reported in gastric cancer cells. In this study, we aimed to investigate the mechanism of ATO to inhibit signal transducer and activator of transcription 3 (STAT3) activity and EMT in gastric cancer cells. Methods: We performed wound closure assay and Matrigel invasion assay for functional studies of EMT, and western blot for measurement of protein markers using AGS gastric cancer cells. Results: Compared with control, 5 and 10 μM of ATO significantly inhibited cellular migration and inhibition in a dose-dependent manner. Furthermore, ATO significantly downregulated snail expression, a mesenchymal marker, and upregulated E-cadherin expression, an epithelial marker. We could observe that ATO induced SH2-containing protein tyrosine phosphatase 1 (SHP1), a non-receptor type protein tyrosine phosphatase, and subsequently downregulated phospho-STAT3 in a dose-dependent manner. To validate the molecular link between ATO and SHP1 to inhibit EMT in gastric cancer cell, we pre-treated 50 μM of pervanadate, a phosphatase inhibitor, before treatment of 10 μM ATO, and this significantly abolished anti-invasive effect by ATO in AGS cells. In xenograft tumor model, intraperitoneal injection of ATO significantly reduced the tumor volume and upregulated SHP-1 expression by immunohistochemistry stain compared with vehicle, which were reversed by ATO with pervanadate injection. Conclusions: Our findings suggest that ATO may show anti-EMT effects via induction of SHP1 and inhibition of STAT3 activity in gastric cancer cells.

Methylation rates of the Ras association domain family 1A gene (RASSF1A) have been variously reported as between 7.5 and 66.7% in gastric carcinoma tissues. The role of this gene in gastric cancer also remains to be fully elucidated. The present study aimed to investigate whether promoter hypermethylation of RASSF1A occurs in gastric adenocarcinoma tissues and gastric cancer cell lines, and to determine the effects of RASSF1A in gastric carcinoma cell lines. The results showed a methylation‑specific band only in SNU-719, MKN28 and AGS human gastric cancer cells (indi- cating full methylation), none of which exhibited RASSF1A expression. By contrast, SNU-16, MKN-45 and KATO-III human gastric carcinoma cells exhibited methylation as well as unmethylation‑specific bands (indicating partial methyla - tion), and all displayed positive or weakly positive expression of RASSF1A. Bisulfite sequencing in AGS and SNU‑719 cells revealed that virtually all CpG sites were densely methylated. When SNU-719, MKN-28 and AGS cells were treated with the demethylating agent 5-aza-2'-deoxycytidine, RASSF1A gene expression was restored and the methylation-specific polymerase chain reaction pattern was altered in all three cell lines. Transfection of a plasmid expressing RASSF1A into AGS and SNU-719 cells significantly inhibited cell proliferation. Exogenous RASSF1A also reduced the expres- sion of cyclin D1 and phospho-retinoblastoma protein, and increased that of p27 as demonstrated by western blot analysis. Furthermore, RASSF1A expression was significantly reduced (P=0.048) and the methylation rate was elevated in gastric adenocarcinoma tissues, compared with those in adjacent healthy intestinal metaplasia (34.6 vs. 66.7%, P=0.029). The present study indicated that epigenetic silencing of RASSF1A is frequently caused by promoter hypermethylation in gastric cancer cell lines as well as in gastric adenocarcinoma tissues, which may contribute to gastric carcinogenesis.

To the Editor: Scrub typhus, or tsutsugamushi disease, is a febrile illness caused by the rickettsial bacteria Orientia tsutsugamushi. Scrub typhus is endemic to a geographically distinct region, the so-called tsutsugamushi triangle, which includes Japan, Taiwan, China, and South Korea (1,2). Scrub typhus is a public health issue in Asia, where 1 billion persons may be at risk for the disease (3). In South Korea, scrub typhus is the most common rickettsial disease, and public health authorities are concerned about its increased incidence. Scrub typhus has been a reportable disease in South Korea since 1994. Physicians who diagnose suspected or confirmed cases must report these cases to their local health bureau and the Korea Centers for Disease Control and Prevention (KCDC) through the National Notifiable Disease Surveillance System (NNDSS). For a patient’s illness to meet the case definition for scrub typhus, the clinical signs (acute febrile illness and skin eschar) must be present or there must be laboratory confirmation (4-fold rise in antibody titer, antigen detected in blood, or genetic material detected by PCR). We analyzed NNDSS data confirmed by KCDC and classified all reported cases into 2 groups according to residential area. Cases with rural administrative address codes “eup” or “myun” were defined as rural cases, whereas cases with a city administrative address code of “dong” were defined as urban cases. All case-patients were classified by occupation as farmer or nonfarmer; all agricultural, fishery, and forest workers from rural areas were defined as farmers. In total, 23,929 cases, including 16,199 (67.7%) serologically confirmed cases, were reported between 2001 and 2006, of which 35.5% were male patients and 64.5% female patients. The greatest number of cases was in the age group 50–69 years, in both male (47.2%) and female (51.7%) patients; however, there were 167 boys (2.0%) and 119 girls (0.8%)

46 Background: HOX genes, a subset of the homeobox gene family, are known to be aberrantly expressed in various types of cancers. Among them, HOXB7 recently has been reported to be highly expressed in esophageal or colorectal cancers. We aimed in this study to demonstrate the critical roles of HOXB7 in development and progression of gastric cancer. Methods: We screened gene and protein expression of HOXB7 in various gastric cancer cell lines, and also compared gene expression level of HOXB7 among chronic gastritis, intestinal metaplasia and gastric cancer tissues. To figure out the oncogenic effects of HOXB7 in vitro, we performed annexin-V assay, wound closure assay and Matrigel invasion assay. We performed Western blot analysis to examine the impact of HOXB7 on AKT pathway. Results: Both mRNA and protein was substantially expressed in stomach cancer cell lines (SNU-638, SNU-719, MKN-28, MKN-45, AGS, KATO-III, NCI-N87), however, they were nearly abolished in normal gastric tissues. Gene expression was significantly higher in primary or metastatic stomach cancer, compared with chronic gastritis or intestinal metaplasia. Knockdown of HOXB7 by transfection with siRNA in AGS and SNU-638 cells significantly inhibited migration and invasion, and showed anti-apoptotic effect. Because a previous study demonstrated that enforced expression of HOXB7 could enhance PI3K/AKT pathway activity in colon cancer cells (Liao W.T. et al, Clin Cancer Res; 17(11) June 1, 2011), we investigated the modulation of AKT/PTEN pathway by HOXB7 and observed that knockdown of HOXB7 significantly downregulated phospho-AKT and upregulated PTEN in both cell lines. Furthermore, target gene products of AKT pathway including cyclin D1, survivin, Bcl-xL and MMP-9 were significantly downregulated by siHOXB7. Conclusions: Our findings suggest that HOXB7 might play a crucial role in migration, invasion and anti-apoptotic effect via modulating AKT/PTEN pathway.

61 Background: The SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies demonstrated that promoter methylation of SHP1 is frequently observed in gastric adenocarcinoma tissues. We tried in this in vitrostudy to reveal promoter hypermethylation and to investigate the effects of SHP1 in gastric carcinoma cell lines. Methods: We performed RT-PCR, Western blot, methylation specific PCR (MSP) and bisulfite pyrosequencing to demonstrate promoter hypermethylation of SHP1. To evaluate functional roles of SHP1, we transfected SHP1 plasmid and analyzed WST-1 assay, wound closure assay and Matrigel invasion assay. Results: We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). MSP showed methylation-specific band only in all 10 gastric cancer lines. Bisulfite pyrosequencing revealed 96.5% and 97.3% of methylation frequency in AGS and SNU-719 cells. When treating SNU-719, MKN-28 and AGS cells with 5-Aza-2’-deoxycytidine (5-Aza-dc), SHP1 was re-expressed in all three cells. SHP1 expression is known to be correlated with Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signaling pathway in epithelial cells. When introducing exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection, protein expression of constitutive phospho-JAK2 (Tyr 1007/1008) and phospho-STAT3 (Tyr 705) were substantially down-regulated both, which in turn decreased target gene expression of STAT3, including cyclin D1, MMP-9, VEGF and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Conclusions: Epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cell lines. Overexpression of SHP1 down-regulates JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration and invasion in gastric cancer cells.

74 Background: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a plant-drived natural agent extracted from the root of Plumbago zeylanic. A recent study reported that plumbagin down-regulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various anti-tumor effects. We aimed in this in vitrostudy to demonstrate the inhibition of JAK2-STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in gastric cancer cell line. Methods: We performed Wetern blot to measure SHP1, phospho-JAK2/STAT3 level, and reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate target gene expression of STAT3. Several functional studies such as water soluble tetrazolium-1 (WST-1) assay, wound closure assay and matrigel invasion assay were also performed. Results: Plumbagin induced SHP1 expression and simultaneously down-regulated phospho-JAK2/STAT3 level via dose-and time-dependant manner in MKN28 cell, a gastric carcinoma cell line which has negative SHP1 expression. This effect was consistent when JAK2-STAT3 signaling was activated by interleukin-6, and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, VEGF1, survivin, MMP9, known target products of STAT3 activation in gastric cancinogenesis. The functional effect of plumbagin could be validated as inhibition of cell proliferation, migration and invasion, which are the results of activation of JAK2-STAT3 pathway in gastric cancer cells. Conclusions: Plumbagin is a potential negative regulator of cellular growth, migration and invasion by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

This report describes the pattern of the spread of the pandemic H1N1 2009 and compares 3 monitoring tools until the 57th week or January 31, 2010. The 1st week was from December 28th, 2008 to January 3rd, 2009. A total of 740,835 patients were reported to be infected with pandemic H1N1 2009 and 225 patients were reported to have died of pandemic H1N1 2009. The number of patients aged from 7 to 12 was the largest (183,363 patients in total) but the virus spread and then was suppressed most quickly among the children between 13 and 18. The region-determinant incidence of patients showed diverse patterns according to regions. The peak of the ILI per thousand was at the 45th week, the number of antiviral prescriptions reached its peak at the 44th week, and the peak based on reported patients was the 46th week. As of February 3 2010, the outbreak passed through the peak and has gradually subsided. Now it is time for the government and the academic world to review this outbreak, efficacy of vaccination, and further preparation and response for the next pandemic.