The Great East Japan Earthquake occurred on March 11, 2011. Tokyo and Osaka, which are located 375 km and 750 km, respectively, from the epicenter, experienced tremors of 5.0 lower and 3.0 seismic intensity on the Japan Meteorological Agency scale. The Great East Japan Earthquake was the fourth largest earthquake in the world and was accompanied by a radioactive leak at a nuclear power plant and a tsunami. In the aftermath of a disaster, some affected individuals presented to mental health facilities with acute stress disorder (ASD) and/or post-traumatic stress disorder (PTSD). However, few studies have addressed mental stress problems other than ASD or PTSD among the general public immediately after a disaster. Further, the effects of such a disaster on residents living at considerable distances from the most severely affected area have not been examined.This study aimed to prospectively analyze the effect of a major earthquake on the prevalence of insomnia among residents of Tokyo and Osaka.A prospective online questionnaire study was conducted in Tokyo and Osaka from January 20 to April 30, 2011. An Internet-based questionnaire, intended to be completed daily for a period of 101 days, was used to collect the data. All of the study participants lived in Tokyo or Osaka and were Consumers’ Co-operative Union (CO-OP) members who used an Internet-based food-ordering system. The presence or absence of insomnia was determined before and after the earthquake. These data were compared after stratification for the region and participants’ age. Multivariate analyses were conducted using logistic regression and a generalized estimating equation. This study was conducted with the assistance of the Japanese CO-OP.The prevalence of insomnia among adults and minors in Tokyo and adults in Osaka increased significantly after the earthquake. No such increase was observed among minors in Osaka. The overall adjusted odds ratios for the risk of insomnia post-earthquake versus pre-earthquake were 1.998 (95% CI 1.571–2.542) for Tokyo, 1.558 (95% CI 1.106–2.196) for Osaka, and 1.842  for both areas combined.The prevalence of insomnia increased even in regions that were at a considerable distance from the epicenter. Both adults and minors in Tokyo, where the seismic intensity was greater, experienced stress after the earthquake. In Osaka, where the earthquake impact was milder, disturbing video images may have exacerbated insomnia among adults. On March 11, 2011, the Japanese islands sustained a 9.0-magnitude earthquake. Unlike previous major earthquakes in Japan . More thIn the aftermath of a disaster, people may experience not only physical disorders but also acute stress disorder (ASD), which can persist for up to 4 weeks. Furthermore, chronic post-traumatic stress disorder (PTSD) is common among individuals who have faced such situations . StudiesThe current study made use of a daily health survey that was administered to 3128 participants in Tokyo and 1925 participants in Osaka  from JanThis survey began on January 20, 2011 and continued for 101 days until April 30, 2011. The questionnaire collected data related to the individual’s health status on the day of the survey, and participants were instructed to complete the survey every day for the duration of the study period. The survey was conducted via an Internet-based questionnaire among residents of the Tokyo metropolitan area and Osaka, the largest city in western Japan. Tokyo is located approximately 375 km from the epicenter of the earthquake (N 38°06′ E 142°51′) and approximately 200 km from the Fukushima Daiichi nuclear power plant (N 37°42′ E 141°03′). The seismic intensity of the main shock in the center of Tokyo, as recorded by the Japan Meteorological Agency (JMA), was 5.0 Lower on the JMA scale . The JMAThis study was conducted with the assistance of the Japanese Consumers’ Co-operative Union (CO-OP). All respondents who completed the questionnaire lived in Tokyo or Osaka and resided in households that included CO-OP members who placed food orders via the CO-OP website.This study was conducted with the approval of the Ethics Committee of Nara Medical University (authorization code: 220). The general health condition of the participants, including their sleeping patterns, was investigated using an Internet-based questionnaire. The original aims of this survey were to determine the impact of biological factors, such as infectious diseases, and abiotic factors, such as climate, on the physical condition of residents during the study period. The survey method and data processing methods were described in detail in our previous study .Respondents were recruited through a banner advertisement on the CO-OP’s website. Each participant was rewarded with 500 yen (US $1=91.15 yen on the first day of the survey) upon registration for participating in the survey. No remuneration, in the form of cash, was given for providing answers on a daily basis.The original research plan was to send a reminder email to all the respondents on each day of the survey that would direct them to the website where they could provide their responses. The email was distributed as planned until day 50 of the survey. The Great East Japan Earthquake occurred on day 51 of the survey. The reminder emails were discontinued, as it was decided that the participants, who were recovering from the disaster, should not be burdened. Further responses were left to the participants’ discretion during a hiatus period from March 14 to April 5, 2011, when the reminders were reinstituted. After the earthquake, respondents were able to submit descriptions of their physical condition by voluntarily visiting the website.The daily survey procedure was designed to be simple. After confirming the everyday health condition of the family, participants were asked to access the survey website and answer several questions. The first question asked whether any family member was in poor health. If the participant answered “no”, they were excluded from the survey. If the participant answered “yes”, they were asked to answer additional “yes” or “no” questions on 19 symptoms; these questions pertained to the individual filling out the questionnaire as well as each member of his or her family . The preIn both surveyed areas, the prevalence of insomnia was calculated on a daily basis (the number of people reporting symptoms of insomnia divided by the number of responses per day) among people aged <20 years and those aged ≥20 years. Using a chi-square test, the presence or absence of insomnia before and after the earthquake was investigated for any correlation with region or participant age. A multivariate analysis was carried out using logistic regression analysis and a generalized estimating equation. The presence or absence of insomnia was the dependent variable. The independent variables included insomnia occurring after the earthquake, sex, age, region of each participant, the status of reminder emails (sent or not), and the incidence of pollinosis, which plagued approximately 30% of adults in those urban areas during the spring . The staThe mean (SD) daily response rate during the period when reminder emails were sent was 64.17% (5.78%) for Tokyo and 68.31% (5.18%) for Osaka. The response rate did not decline significantly over the course of the study. The response rate during the period when no reminder emails were sent  was 24.47% (12.97%) for Tokyo and 27.82% (13.55%) for Osaka.Before the earthquake, the average daily prevalence of insomnia in Osaka was 1.25% (0.25%) for adults and 0.092% (0.14%) for minors; after the earthquake, this value increased to 1.83% (0.51%) for adults but remained approximately the same at 0.089% (0.17%) for minors. The maximum seismic intensity of the main earthquake was 3.0 in Osaka .P<.001 for both adults and minors) compared with that before the earthquake. The same findings were reported for adults in Osaka after the earthquake (P<.001). No significant difference was observed among minors in Osaka  for Tokyo, 1.558  for Osaka, and 1.842  for the 2 areas combined. Multivariate analysis was conducted to determine the odds ratios for insomnia . The adjThis study examined the prevalence of insomnia among residents in areas that were at different distances from the epicenter of the Great East Japan Earthquake. This is a unique study in that it analyzes the effect of a great earthquake on the rates of insomnia and includes a pre-event baseline in the same group.Major earthquakes have been common throughout the Asia-Pacific region over the past 2 decades ,13, withThe daily prevalence of insomnia increased among both adults and minors in Tokyo after the Great East Japan Earthquake. Although the daily prevalence of insomnia increased among adults in Osaka, a similar increase was not observed among minors. The adjusted odds ratios for insomnia after versus before the earthquake were 1.998 (95% CI 1.571–2.542) for Tokyo, 1.558 (95% CI 1.106–2.196) for Osaka, and 1.842 (95% CI 1.514–2.242) for the 2 areas combined. These results demonstrate an increased prevalence of insomnia among residents in regions located at considerable distances from the immediate zone of the disaster. In Tokyo, where there was no observable infrastructure damage due to the tsunami, 7 people died as a result of the initial tremor. In addition, many people in Tokyo experienced considerable psychological strain for a prolonged period. Many commuters were stranded because of interrupted transportation services, and there was a high risk of radioactive contamination associated with the nuclear accident. The increased prevalence of insomnia among minors in Tokyo, who are generally less susceptible to stress induced by indirect sources such as media coverage, may be attributable to the effects of the aftershocks. In contrast, the seismic intensity of the main shock in Osaka was 3.0; therefore, direct feelings of fear were likely to be less common, and there was an absence of palpable aftershocks. The prevalence of insomnia among minors in Osaka following the earthquake was not increased, which can be explained by the residents’ exposure to fewer direct and local effects. However, an increased number of adults in Osaka reported insomnia. This may have stemmed from exposure to information reported by the media. Other possible causes of insomnia among these adults include anxiety about their future and memories of the disaster caused by the Great Hanshin Earthquake of 1995.A Web-based questionnaire survey was used in the current study because more data are acquired with Internet-based epidemiological surveys than with conventional, paper-based surveys ,21. ThisImmediately after the earthquake struck, an ethical decision was made to refrain from sending reminder emails. Therefore, the response rate was low during this period. However, no significant difference in the daily prevalence of insomnia correlated with the use of these reminder emails in either Tokyo or Osaka. The chi-square test results were similar between analysis including and excluding this time period. Although the reminder emails were included in the logistic regression analysis as an independent variable, the presence or absence of the reminder emails inevitably remains a limitation of this study and a potential source of bias. However, we believe that this factor had a negligible effect on the results.This study examined the prevalence of insomnia among residents in areas distant from the epicenter of the Great East Japan Earthquake. In Tokyo, where the seismic intensity was higher, both adults and minors experienced increased rates of insomnia as a direct result of the earthquake and its aftershocks. Further, mental stress induced by information broadcast by the media may have influenced the prevalence of insomnia. In Osaka, where the seismic intensity was lower, only adults exhibited an increased prevalence of insomnia. Health care practitioners should be aware that individuals might experience mental stress, including insomnia, even in areas distant from those that are directly affected by a natural disaster.


Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells.MTT 2,5-diphenyltetrazolium bromide) and acid phosphatase cell-viability assays were used to assess NRC-03- and NRC-07-mediated killing of breast carcinoma cells. Erythrocyte lysis was determined with hemolysis assay. NRC-03 and NRC-07 binding to breast cancer cells and normal fibroblasts was assessed with fluorescence microscopy by using biotinylated-NRC-03 and -NRC-07. Lactate dehydrogenase-release assays and scanning electron microscopy were used to evaluate the effect of NRC-03 and NRC-07 on the cell membrane. Flow-cytometric analysis of 3,3'-dihexyloxacarbocyanine iodide- and dihydroethidium-stained breast cancer cells was used to evaluate the effects of NRC-03 and NRC-07 on mitochondrial membrane integrity and reactive oxygen species (ROS) production, respectively. Tumoricidal activity of NRC-03 and NRC-07 was evaluated in NOD SCID mice bearing breast cancer xenografts.50) of cisplatin for breast cancer cells. NRC-03 and NRC-07 bound to breast cancer cells but not fibroblasts, suggesting that killing required peptide binding to target cells. NRC-03- and NRC-07-mediated killing of breast cancer cells correlated with expression of several different anionic cell-surface molecules, suggesting that NRC-03 and NRC-07 bind to a variety of negatively-charged cell-surface molecules. NRC-03 and NRC-07 also caused significant and irreversible cell-membrane damage in breast cancer cells but not in fibroblasts. NRC-03- and NRC-07-mediated cell death involved, but did not require, mitochondrial membrane damage and ROS production. Importantly, intratumoral administration of NRC-03 and NRC-07 killed breast cancer cells grown as xenografts in NOD SCID mice.NRC-03 and NRC-07 killed breast cancer cells, including drug-resistant variants, and human mammary epithelial cells but showed little or no lysis of human dermal fibroblasts, umbilical vein endothelial cells, or erythrocytes. Sublethal doses of NRC-03 and, to a lesser extent, NRC-07 significantly reduced the median effective concentration (ECThese findings warrant the development of stable and targeted forms of NRC-03 and/or NRC-07 that might be used alone or in combination with conventional chemotherapeutic drugs for the treatment of breast cancer. Despite the decline in the incidence and mortality rates of breast cancer from 1990 to 2005, an estimated 192,370 women were expected to be diagnosed with breast cancer in 2009, and 40,170 women were expected to die of the disease, representing nearly 15% of all cancer-related deaths in American women . These sO-sialoglycoproteins, and heparan sulfate proteoglycans  × 100, where E and C denote the optical density of peptide- and medium-treated cells, respectively.Breast cancer cell viability was determined by using the MTT assay. In brief, 2 × 10M sodium acetate, 0.1% Triton X-100 (vol/vol), 4 mg/ml phosphatase substrate) for 90 minutes. The reaction was stopped by the addition of 10 μl 1N NaOH. The optical density (405 nm) was measured, and the percentage cytotoxicity was calculated as described for the MTT assay.The acid phosphatase assay was used instead of the MTT assay to compare NRC-03 and NRC-07 killing of paclitaxel-resistant MCF7-TX400 to wild-type MCF7 cells because drug-efflux pumps interfere with the reduction of MTT . In brieg), and supernatants were transferred to 96-well flat-bottomed tissue-culture plates for analysis. Absorbance (490 nm) was measured, and the percentage hemolysis was calculated by using the equation ((E/S)/(M/S)) × 100, where E and S and M denote experimental, spontaneous, and maximal hemolysis, respectively.The hemolytic activity of NRC-03 and NRC-07 was determined by culturing human erythrocytes (5% (vol/vol) in PBS) in the presence or absence of the indicated concentrations of NRC-03 and NRC-07 for 8 hours in 96-well round-bottomed tissue-culture plates (Sarstedt). Maximum hemolysis was achieved by combining erythrocytes with an equal volume of water. Erythrocytes were then pelleted  plated in six-well flat-bottom tissue-culture plates containing sterile coverslips were incubated for 24 hours to promote cell adhesion to the coverslips. Adherent cells were cultured for 10 minutes in the presence or absence of 50 μM biotinylated-NRC-03 or -NRC-07. Cells on the coverslips were then fixed with paraformaldehyde (4% (wt/vol) in PBS), washed extensively with PBS, and exposed to Texas Red-conjugated streptavidin  for 45 minutes. After extensive washing with PBS, the coverslips were mounted on slides with Dako fluorescent mounting medium . Cells were visualized with phase and fluorescent microscopy at ×400 magnification. Fluorescence per cell was determined by using NIS-Elements software .MDA-MB-231 cells and human dermal fibroblasts (8 × 10M Na2CO3 and 35 mM NaHCO3 buffer (pH 9.2) were incubated overnight in EIA plates at 4°C. Plates were washed with PBS, blocked for 2 hours with 10% FBS, washed again with PBS, and incubated for 2 hours at 23°C in the presence or absence of 50 μM biotinylated-NRC-03 or biotinylated-NRC-07. Plates were then washed with PBS, incubated with avidin-HRP  for 1 h, washed with PBS, and incubated with TMB substrate solution. The reaction was stopped by the addition of 0.3 M H2SO4. Peptide binding to heparan sulfate or chondroitin sulfate was determined by measuring absorbance at 450 nm.Binding of biotinylated-NRC-03 and biotinylated-NRC-07 to plastic-immobilized heparan sulfate or chondroitin sulfate was determined by using a modification of a previously described protocol . In brie5 cells) were plated in 24-well flat-bottom tissue-culture plates (Sarstedt) containing sterile circular coverslips and incubated overnight to promote cellular adhesion. Cells on coverslips were cultured in the presence or absence of 25 or 50 μM NRC-03, NRC-07, or NRC-13 for 10 or 30 minutes. The cells were washed with 0.1 M sodium cacodylate and fixed with glutaraldehyde (2.5% (vol/vol) in sodium cacodylate) for 2 hours. The cells were washed again, fixed with osmium tetroxide (1% (wt/vol) in sodium cacodylate) for 30 minutes, washed again, and dehydrated in increasing concentrations of ethanol until a final concentration of 100% ethanol was achieved. The samples were subsequently dried to their critical point by using a Polaron E3000 Critical Point Dryer , mounted onto stubs, and coated with gold by using a Polaron SC7620 Mini Sputter Coater (Quorum Technologies). The cells were then viewed at the Institute for Research in Materials  on a Hitachi S4700 scanning electron microscope  at ×7,000 and ×40,000 magnification.MDA-MB-231 cells and human dermal fibroblasts (2 × 10E/S)/(M/S)) × 100, where E and S and M denote experimental, spontaneous, and maximal release of LDH, respectively.The LDH-release assay  was used per the manufacturer's instructions to quantify cytolysis and to validate cell-viability measurements obtained with MTT assay. Complete cytolysis was achieved by repeated freeze/thaw cycles. Absorbance (490 nm) was used to calculate cytolysis by using the equation  or DiOC6 (40 nM) 15 minutes before analysis with a FACS Calibur flow cytometer .Flow-cytometric analysis of MDA-MB-231 cells stained with DiOCM NRC-03 or NRC-07 for 10 minutes. Mitochondria-free supernatant (cytosolic fraction) was then collected by centrifugation at 12,000 g for 10 minutes, and the mitochondria pellet was lysed with ice-cold lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 50 mM Na2HPO4, 0.25% sodium deoxycholate (wt/vol), 0.1% Nonidet P-40 (vol/vol), 100 μM Na3VO4, 10 mM NaF, 5 mM EDTA, and 5 mM EGTA) containing freshly added protease inhibitors . Supernatants containing proteins released by lysed mitochondria were collected by centrifugation at 12,000 g. Protein concentrations in cytosolic and mitochondrial fractions were determined with the Bradford assay (Bio-Rad Laboratories), and equal amounts of protein (5 μg) were resolved on a 12% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes, and the resulting blots were blocked for 1 hour in TBS-Tween (0.25 M Tris (pH 7.5), 150 mM NaCl, 0.2% Tween-20 (vol/vol)) containing 5% powdered skim milk (wt/vol) and probed overnight with the desired primary antibody (1:500). Blots were then washed extensively with TBS-Tween and probed for 1 hour with HRP-conjugated goat anti-mouse IgG . After additional washes, cytochrome c or mitochondrial Hsp70 was visualized by using an enhanced chemiluminescence detection system (Bio-Rad Laboratories).Mitochondria were isolated from MDA-MB-231 cells, as previously described , and tre5) were seeded in six-well flat-bottom tissue-culture plates containing sterile coverslips and were incubated overnight to promote cellular adherence. Cells were then cultured in the presence or absence of biotinylated-NRC-03 or biotinylated-NRC-07 for 30 seconds, washed with PBS, fixed with 4% paraformaldehyde, and washed again with PBS. Cells were then stained with 5 U Alexa Fluor 488 phalloidin (in 0.1% Triton-X 100), TO-PRO-3 iodide , or anti-mitochondrial Hsp70 mAb (1:50 in PBS) and FITC-conjugated anti-mouse IgG (1:500 in PBS) for 20 minutes, 10 minutes, and 1 hour, respectively. Stained cells were washed with PBS, incubated in Texas Red-conjugated streptavidin (1.54 μg/ml in PBS) for 45 minutes, washed with PBS, dried, and mounted onto slides by using Dako fluorescent mounting medium. Images  were acquired by using an LSM-510 META laser scanning confocal microscope .NRC-03 and NRC-07 subcellular localization was determined by confocal microscopy. In brief, MDA-MB-231 cells (4 × 105) were seeded into six-well flat-bottom tissue-culture plates containing sterile coverslips, and were incubated overnight to promote cellular adherence. Cells were incubated in the presence or absence of NRC-03 or NRC-07 for 30 minutes, washed with PBS, fixed with 4% paraformaldehyde, washed again with PBS, and incubated at 4°C for 2 minutes in permeabilization solution (0.1% Triton X-100 (vol/vol) in 0.1% sodium citrate (wt/vol)). Cells were then washed with PBS, dried, and stained with TUNEL reaction mixture for 1 hour at 37°C. Stained samples were washed with PBS, dried, mounted on slides by using Dako fluorescent mounting medium, and visualized under visible and UV light (×400).DNA fragmentation was detected by using the TUNEL assay according to the manufacturer's instructions . In brief, MDA-MB-231 breast cancer cells (4 × 10M TEAB buffer were incubated in the presence or absence of trypsin (2 μg) overnight at 37°C, dried, reconstituted in 0.1% TFA, and diluted 1:1 in Matrix Solution. Samples (500 ng) were spotted on a MALDI plate, dried, and analyzed with a MALDI-TOF mass spectrometer .NRC-03 and NRC-07 reconstituted in 50 m6 MDA-MB-231 cells by subcutaneous injection in one hind flank. Tumor volume was monitored every other day by using the equation (L*P2)/2, where L and P denote the longest diameter and the diameter perpendicular to the longest diameter, respectively. Once the tumors reached a volume greater than 120 mm3 (approximately 33 days after tumor cell implantation), mice were randomized into groups of three and administered 20 μl of the HBSS vehicle or 0.5 mg NRC-03 or NRC-07 in 20 μl of HBSS by intratumoral injection, beginning on day 1 and repeated on days 3 and 5. Tumor-bearing mice were killed 1 week after the last injection. Tumors were excised, photographed, sectioned, and stained with hematoxylin and eosin. Stained tumor sections were visualized under brightfield microscopy (×400 magnification). The experiment was conducted three times.NOD SCID mice were engrafted with 5 × 10t test, or one-way analysis of variance with the Bonferroni multiple comparisons test, as appropriate.All data were analyzed by using the unpaired Student M) and NRC-07 . In contrast, T-47D, MDA-MB-231, and MCF7 cells required 2.5- to 10-fold more NRC-03 and NRC-07 to cause significant cytotoxicity. Nevertheless, MDA-MB-231 cells were chosen as the representative cell line for the remainder of this investigation because these breast cancer cells were susceptible to killing by NRC-03 and NRC-07 and could be grown as xenografts in immune-deficient NOD SCID mice. It is important to note that NRC-13, a noncytotoxic CAP that was used as a control peptide, did not substantially affect the viability of breast cancer cells or mouse mammary carcinoma cells  of NRC-03 or NRC-07 was added to MDA-MB-231 cells 20 minutes before their exposure to increasing concentrations of cisplatin (0 to 16 μg/ml). NRC-03 pretreatment reduced the EC50 of cisplatin by 5.5-fold after 72 and 96 hours, whereas NRC-07 reduced the EC50 of cisplatin by only 1.6- and 1.7-fold after 72 and 96 hours, respectively . Figure O-sialoglycoproteins, MDA-MB-231 cells that had their O-sialoglycoproteins removed by O-sialoglycoprotein endopeptidase were only slightly less sensitive to the cytotoxic action of NRC-03 or NRC-07  than control tumors at day 12. NRC-07-treated tumors showed a similar trend, but the difference did not reach statistical significance. In contrast, tumors that were injected with the NRC-13 control peptide did not exhibit reduced growth , which gives CAPs a completely different mechanism of action than that of conventional chemotherapeutic agents. NRC-03 and NRC-07 exhibited a 56- and 98-fold greater binding capacity, respectively, to breast cancer cells than to normal fibroblasts, even though the doubling time of the neoplastic epithelial cells was approximately the same as that of the untransformed fibroblasts. The potent cytolytic activity of NRC-03 and NRC-07 against slow-growing SKBR3 cells (~36-hour doubling time) suggests that these peptides may be effective against indolent tumors. Peptide binding to anionic heparan sulfate proteoglycans and chondroitin sulfate proteoglycans was involved in NRC-03- and NRC-07-mediated cytotoxicity; however, none of these molecules was exclusively necessary for target-cell death, suggesting that NRC-03 and NRC-07 interact with additional negatively-charged molecules on the surface of susceptible cells. In this regard, increased density of negatively-charged phosphatidylserine on the surface of cancer cells has been suggested to serve as a target for CAPs . BecauseElectrostatic interactions between breast cancer cells and NRC-03 or NRC-07 resulted in severe damage to the cell membrane, as demonstrated with scanning electron microscopy, propidium iodide uptake, and the release of cellular LDH. Integration of bulky hydrophobic amino acids into the hydrophobic core of the target cell membrane and adoption of a stable amphipathic structure is believed to lead to pore formation by CAPs . InteresIt is also conceivable that prolonged exposure to lower concentrations of NRC-03 and/or NRC-07 may cause transient cell-membrane damage and induce cell death by mitochondrial-dependent apoptosis or an inhibition of macromolecular synthesis. This dual effect of pleurocidin has been demonstrated in bacteria ; however50 of cisplatin, leading us to conclude that NRC-03 and NRC-07 possess chemosensitizing properties. A membranolytic mechanism of action likely accounts for the observed ability of NRC-03 and NRC-07 to enhance the killing of breast cancer cells by cisplatin. In addition, nuclear localization of NRC-03 and NRC-07 is predicted to disrupt the nuclear membrane and allow easier access of cisplatin and other DNA-crosslinking agents to the nucleus. However, it is not yet known whether sublethal doses of NRC-03 and/or NRC-07 similarly enhance in vivo cytotoxicity mediated by chemotherapeutic drugs.Sublethal concentrations of NRC-03, and, to a lesser extent, NRC-07, significantly reduced the ECin vitro cytolytic activity of NRC-03 and NRC-07. Importantly, intratumoral delivery of NRC-03 and NRC-07 to mice did not have any noticeable adverse side-effects, indicating that NRC-03 and NRC-07 can be safely administered via intratumoral injection. Interestingly, Berge and colleagues [Unlike control breast cancer xenografts in NOD SCID mice, flank tumors that received intratumoral injections of NRC-03 or NRC-07 did not increase in size once peptide treatment was started, whereas tumors that were injected with a noncytotoxic control peptide grew at the same rate as HBSS-injected tumors. In addition, histologic analysis revealed that the necrotic core of peptide-treated tumors was larger than that of control tumors, which is consistent with the lleagues  recently50 of cisplatin, suggesting the possible use of NRC-03 as a chemosensitizing agent. Importantly, both NRC-03 and NRC-07 killed breast cancer cells grown in NOD SCID mice. These findings indicate that NRC-03 and NRC-07 have several advantages over conventional chemotherapeutic drugs and warrant further investigation as possible novel anticancer agents.Conventional chemotherapeutic drugs are limited by their lack of specificity for cancer cells and their inability to kill multidrug-resistant and slow-growing cancer cells. We have shown for the first time that the pleurocidin-family CAPs NRC-03 and NRC-07 are cytotoxic for multiple breast cancer cell lines, including MCF7-TX400 cells that overexpress P-glycoprotein, and slow-growing SKBR3 cells that contain a 100% ALDEFLUOR-positive breast cancer stem cell population. We established that NRC-03- and NRC-07-mediated cell death is initiated by peptide binding to negatively-charged molecules on the surface of breast cancer cells. NRC-03 also substantially reduces the EC6: 3,3'-dihexyloxacarbocyanine iodide; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; FBS: fetal bovine serum; GSH: glutathione; HBSS: Hank's balanced salt solution; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HMEC: human mammary epithelial cell; HRP: horseradish peroxidise; HUVEC: human umbilical vein endothelial cell; mAb: monoclonal antibody; NOD SCID: non-obese diabetic severe combined immunodeficient; OSGE: O-sialoglycoprotein endopeptidase; ROS: reactive oxygen species.CAP: Cationic antimicrobial peptide; CFU: colony-forming units; DHE: dihydroethidium; DiOCS Douglas has a patent filed in the United States in 2003 and in Europe in 2008.AH participated in study design, conducted the experiments, and drafted the manuscript. CD participated in the animal studies. DP performed MALDI-TOF mass spectrometry. AP and SD provided advice on the study design. DH conceived the study, participated in its design, and finalized the manuscript. All authors have read and approved the manuscript.NRC-03 and NRC-07 are susceptible to degradation by proteases. (a) MDA-MB-231 cells cultured in the presence of 0.5, 2.5, and 5% FBS were exposed to 50 μM NRC-03 or NRC-07. Cell viability was determined with MTT assay after 24 hour. Data shown are statistically significant by ANOVA (p < 0.05) and represent the mean of three independent experiments ± SEM. (b) The 50 μg of NRC-03 or NRC-07 was combined with 1 μg trypsin and incubated overnight at 37°C. Intact and/or fragmented peptides were detected with MALDI-TOF mass spectrometry. Data shown are from one experiment.Click here for fileNRC-03 and NRC-07 interact with mitochondria in breast cancer cells. MDA-MB-231 breast cancer cells were cultured in the presence or absence of 50 μM biotinylated-NRC-03 or biotinylated-NRC-07 for 30 seconds. Biotinylated peptides and mitochondria were visualized with confocal microscopy  by using Texas Red-conjugated streptavidin and anti-mitochondrial Hsp70 mAb, respectively. Arrows point to sites of colocalization. Images shown are from a representative experiment (n = 3).Click here for fileNRC-07, but not NRC-03, causes DNA fragmentation in breast cancer cells. MDA-MB-231 breast cancer cells were cultured in the presence or absence of 50 μM NRC-03 or NRC-07 for 30 minutes. DNA fragmentation was detected with TUNEL staining that was visualized with fluorescence microscopy. Data shown are from a representative experiment (n = 3).Click here for fileNRC-03 and NRC-07 do not cause autophagy-like cell death. ATG5+/+ or ATG5 -/- mouse embryo fibroblasts (MEFs) were cultured in the presence or absence of 50 μM NRC-03 or NRC-07. Cell viability was determined with MTT assay after 24 hours. No statistically significant difference (p > 0.05) was found between peptide-mediated killing of ATG5+/+ or ATG5 -/- mouse embryo fibroblasts, as determined with the Student t test. Data shown are the mean of at least three independent experiments ± SEM.Click here for fileThe noncytotoxic control peptide NRC-13 does not have antitumor activity. MDA-MB-231 breast cancer cells were implanted in the hind flanks of NOD SCID mice. Once tumors reached a volume at least 120 mm3, they were injected with HBSS alone or with 0.5 mg NRC-03 or NRC-13 (in HBSS) on days 1, 3, and 5. Tumor volumes were determined on days 1, 3, 5, 7, 9, 11, and 12 after the start of peptide treatment. Data shown are the mean of five animals ± SD. Statistical significance was determined with the Bonferroni multiple comparisons test; *p < 0.05 compared with HBSS-treated animals.Click here for file


Every year 40,000 people die of malaria in Burkina Faso. In 2010, the Burkinabè authorities implemented a national malaria control program that provides for the distribution of mosquito nets and the home-based treatment of children with fever by community health workers. The objective of this study was to measure the implementation fidelity of this program.We conducted a case study in two comparable districts (Kaya and Zorgho). Data were collected one year after the program’s implementation through field observations (10 weeks), documentary analysis, and individual interviews with stakeholders (n = 48) working at different levels of the program. The analysis framework looked at the fidelity of (i) the intervention’s content, (ii) its coverage, and (iii) its schedule.The program’s implementation was relatively faithful to what was originally planned and was comparable in the two districts. It encountered certain obstacles in terms of the provision of supplies. Coverage fidelity was better in Kaya than in Zorgho, where many community health workers (CHW) experienced problems with the restocking of artemisinin-based combination therapy and with remuneration for periods of training. In both districts, the community was rarely involved in the process of selecting CHWs. The components affected by scheduling all experienced successive implementation delays that pushed nets distribution and the initial provision of artemisinin-based combination therapies to the CHWs past the 2010 malaria season.The activities intended by the program were mostly implemented with good fidelity. However, the implementation was plagued by delays that probably postponed the expected beneficial effects. It has been estimated that malaria causes 40,000 deaths every year in Burkina Faso This is why, in accordance with international recommendations Union des Religions et des Coutumiers du Burkina [Union of Burkina religions and tribal chiefs] in Kaya, and ‘Songtaaba’ in Zorgho.The program received funding of 63 million Euros for five years from the Global Fund to Fight AIDS, Tuberculosis and Malaria. There are two main recipients of this program funding. The first is the State, for the program to support health districts in carrying out the LLIN component, and the second is the non-governmental organization (NGO) PLAN-International, for the community-based home treatment component. The NGO coordinates this component in collaboration with the National Malaria Control Program (NMCP). PLAN has delegated field operations to four national NGOs (considered secondary recipients), as the country’s territory is subdivided into four zones. In the two districts of the present study, the NGOs are the The ultimate aim of these two components is to reduce the incidence of malaria and save lives. The effectiveness of this approach has been widely proven in pilot settings or in experimental clinical trials in which implementation conditions were closely controlled and their efficacy could be monitored Given Burkina Faso’s context, with its public policies and the fact that part of the program was confided to an NGO, there has been some concern that the program’s implementation could deviate quite far from the original plans and that this could generate a so-called implementation gap. The corollary would be the appearance of a sizeable gap between the effectiveness expected of the program based on its efficacy and its practical effectiveness in real-life conditions Even though implementation analysis can, to some extent, explain the success or lack of success of programs that are carried out, it is still rarely done We were interested in examining the fidelity of the implementation, with the assumption that practical effectiveness would more closely approach theoretical effectiveness to the extent that the activities respected the original program plan in terms of content, coverage, and schedule, which are the factors most frequently cited in fidelity studies The evaluation was carried out using a multiple case study with several embedded levels of analysis In the evaluability assessment The empirical data were drawn from field observations (10 weeks), documentary analysis (five project documents and numerous forms from CHWs and CSPSs), and 48 individual interviews with key stakeholders involved in the program’s implementation . The intThe data were analyzed using a framework analysis process ® software. Syntheses by district and by stakeholder groups provided qualitative data to illustrate the proportions quantifying content fidelity.In analyzing the content of the activities carried out, the data were first used to quantify the proportion of activities that fit within each of five possible modalities that could be used to consider how the actors had reinvented the activities To analyze the coverage of activities, we looked at whether the implementation had respected the number of activities planned and the territory involved.To analyze the schedule, we focused on activities for which dates and time periods had been specified in the planning documents, of which there were ultimately very few.® were used to visually present the results related to content fidelity, the details of which are provided in Drawing upon the work of Perez et al. The study was authorized by the health research ethics committees of Burkina Faso and of the CRCHUM in Canada. Written consent was obtained for every participant.On the whole, the results showed good content fidelity of the activities, although better for the LLIN component than for the HMM component . As wellThe detailed results for the 47 activities in both districts are provided in I was trained in one day, and was not remunerated”; this did not happen in Kaya. In addition, some of the census-taking training activities for the NGO organizers were not implemented in three CSPSs because they were absent when the training occurred. In both districts, all the LLIN distribution activities were conducted as planned. Even so, they were not without incident.In general, the 47 activities were not implemented differently in the two districts, except with regard to remuneration. In Zorgho, the major difficulty had to do with remuneration of CHWs and NHMs in four of the five CSPSs. One CHW told us, “that the census was being done for taxation purposes.” Then, even though the plan called for distributing one LLIN to every two persons, it was ultimately decided to provide fewer. “The one thing they didn’t understand was the reduction in the number of LLINs […] people were unhappy about that,” said a CHW in Zorgho. On top of that, only people who were given an LLIN voucher at the time of the census were supposed to receive nets, but under pressure from those who either had not received a voucher or had lost theirs, the decision was taken to give LLINs even to households with no voucher.First, taking the population census was difficult because it was done in a period of intense agricultural activity, when people are not very available and not generally at home, and the census was sometimes perceived with suspicion. According to one CHW in Kaya, people feared “We ran out of drugs […] we went to the CSPS and they didn’t have any drugs,” said a CHW in Zorgho. This problem did not occur in Kaya, because of the multiple interventions going on specifically in that district.The area in which the original plan was least respected was the provision of supplies. In Zorgho, no CHW had received a consultation register. While they had all received the initial stocks of ACT, in three out of four CSPSs the CHWs were unable subsequently to replenish their stocks. “we’re working with BMG ACTs,” said one CHW. This supply of drugs from BMG was considered a stock replenishment, coming not from the NMCP but from another intervention, which explains why the activity was considered to have been modified from the original plan. The CHWs also did not receive any new consultation registers for children, keeping only the one received by BMG. However, these ACTs were limited to this target clientele of children under five years, whereas in 2010, the HMM program was intended for everyone. Then, these BMG stocks either ran out, as had the HMM stock  or expired . Indeed, in the case of one CHW working in a rural setting, for example, 197 of 200 packages of ACT for 2 to 11 month-olds received November 1, 2010, were expired at the time of our observation on August 28, 2011. Finally, in July 2011 a new intervention took everyone by surprise. An NGO decided to make all healthcare free for children under five years in the district, such that “we didn’t put in an order,” said one CHW, since mothers preferred to go to the CSPS, where ACTs were free, even if it required travel, rather than pay to obtain them from the CHW in their village.In fact, in Kaya the situation regarding ACT inputs was more complicated because two other interventions had rendered the implementation more complex, such that even the CHWs sometimes became confused when trying to explain their activities to us. First, Kaya was a Round 7 pilot site, so they had already received ACTs and were therefore supposed to replenish their stocks through the sales of those drugs, without any new funding from the program. However, they did not understand this cost-recovery system, and they waited for a project from the Ministry of Health funded by the Bill & Melinda Gates Foundation (BMG) for children under age five that supplied them with ACTs; “I transport my drugs in a cardboard box, since I didn’t get a bag.” In Zorgho, all the CHWs received from the NGO, not a carrying case as such, but a cloth bag to be used as a carrying case that was stamped with the logos of the program and the Global Fund.On the other hand, in four out of six CSPSs in Kaya, the CHWs did not receive any carrying cases for the ACTs; thus, one CHW told us, “In Zorgho, in only three of the five CSPSs, the CHWs received boxes of images (an added activity) for health education. In Kaya, on the other hand, this component was added without any problem. No NGO organizer in Kaya or Zorgho received the audiovisual material that was in the plan, but they all received motorbikes for their supervision activities.In both districts, the CHWs reported that most of the activities with families were carried out. The same was true of the follow-up done by the organizers from the two NGOs. On the other hand, the health education skits and film projections planned by the organizers were not carried out.No, I never received any. Maybe the NGO organizer did. I can tell you that the organizer sends us reports, maybe it’s incorporated into those, but I don’t know.” We also observed on several occasions that the NHMs did not know about activities carried out with and for the CHWs. For example, in Zorgho the NHMs told us the CHWs had not received any case to carry the ACTs, when this was not so. The reason for this was that the cases were distributed directly to the CHWs by the NGO organizers without going through the NHMs. In general, it should be noted that the NHMs had almost no involvement in the program, and no resources appeared to have been planned for them. “You know, often it’s difficult, because we could go out to do supervision, but nothing is planned. Nothing. We asked for fuel, but they said it wasn’t possible. So, it’s hard, even if you wanted to…,” said one NHM in Zorgho. However, particularly in Kaya, the NHMs considered that the NGO organizers’ role was useful and helped fight malaria.Likewise, in two of Kaya’s six CSPSs and four out of five of Zorgho’s, the NHMs did not carry out the activity of approving the CHWs’ monthly activity reports. One NHM confirmed that, “Since I was already working with the health workers, there was no mobilization of people to designate or vote for me,” said one CHW in Zorgho. The CHWs “existed before the HMM,” a nurse in Kaya asserted. The fact that some of the older CHWs were unable to read or write explains why new CHWs had to be selected, sometimes only by a nurse: “I selected them myself.”Half of the CHWs were located in villages that were less than 5 km from a CSPS, which contradicted the plan. Only in 11 of the 26 villages were the communities actually involved in selecting the CHWs. This is largely due to the fact that the recruitment was based on coopting existing health workers. “this system is there to motivate us, since they’re not able to give us a salary,” said one CHW in Kaya. On the whole, the monthly stipends for CHWs and NGO organizers were paid, but always late and covering several months at a time.Problems with restocking ACTs occurred in two of Kaya’s 15 villages, compared with eight of the 13 villages in Zorgho, the difference being due particularly to the existence of multiple interventions in Kaya, as described above. The problem of remuneration remained significant in Zorgho, as the majority of CHWs reported not having been paid during their retraining, and seven out of 14 said they had not received any of the profits from the sales of ACTs. Likewise, many reported not having been paid during training and when distributing LLINs. In Kaya, these difficulties did not arise, except with regard to profits from ACT sales, which was problematic for six out of 15 of the CHWs for the reasons mentioned earlier, even though they had a good understanding of the principle of cost-recovery: “The list of activities showed that few  had explicit implementation schedules that would have allowed us to assess whether they were respected. With regard to the LLIN, we observed a delay of only a few weeks in the recruitment of census-takers, their training, and the conduct of the census itself. While they were all supposed to start in July 2010, their implementation was spread out over July and August 2010 in both districts. The greatest difficulty was in the distribution of LLINs, which was planned for July 2010 and finally only occurred in September (for three CSPSs) and October 2010 (for a fourth one), and in January 2011 in Kaya ; that was between two and six months late, and in each case, after the malaria season. With regard to HMM activities, the situation in Kaya was particular, because this district had been one of the three pilot districts for the NMCP. Thus, all the activities for which start dates had been planned were launched on those specified dates throughout the country. In other words, HMM was already in place in Kaya when the NMCP was launched. On the other hand, in Zorgho all the activities were delayed: recruitment of NGO organizers , training of NHMs (one month), recruitment of CHWs (two months), training of CHWs (two to five months), initial stocking of ACTs .Since this study involved only two districts, the results cannot be extrapolated to the entire country. Moreover, while the two cases are very similar, they are not wholly comparable in terms of their implementation and duration, their operational support, and parallel interventions. However, the use of two case studies where the program was implemented by two different NGOs reinforces the implementation’s replication logic and analytic generalization Stakeholders’ responsiveness and facilitation strategies for program implementation are known to be moderating factors for implementation fidelity On the whole, we must conclude that the program’s implementation was relatively faithful to what was originally intended. No activity essential for the program’s effectiveness appears to have been omitted , and the effectiveness itself will be studied as part of the impact evaluation currently under way. However, from the results of this study, we can foresee a possible time lag in the appearance of the program’s effects. Indeed, the study highlighted difficulties in respecting the program’s coverage and schedule. The late, chaotic and reduced distribution of LLINs, the recurrent problems in restocking ACTs (which appear to have worsened since the data collection period), and regular stock shortages all raise serious concerns about the program’s sustainability and population effectiveness. Considering the combination of these factors and the fact that planned activities were delayed until past the height of the malaria season, it is unlikely that the program was able to achieve its objective of preventing some of the 40,000 malaria-related deaths in its first year of operation, 2010. The impact study currently under way may provide information on this subject.the rapid scale-up of ITN distribution in Africa is an enormous public health achievement, but also presents a formidable challenge for the future in ensuring that the levels of coverage are maintained”It is nothing new that, as happened with the program for disease vector control in Cuba In depending on resources from the Global Fund and relying on NGOs instead of the Ministry of Health for the HMM activities, the program’s chances of achieving sustainability are slim. The LLINs, ACTs and monthly stipends for CHWs are all funded by the Global Fund. The program is not sufficiently integrated into the health system, as was the case elsewhere where NGOs were used Finally, in Kaya District, subsequent interventions authorized by the Ministry of Health interfered with the program. The Ministry of Health, funded by the Bill & Melinda Gates Foundation, and an international NGO funded by the European Union turned the program on its head. The former asked the CHWs, who were sometimes already working in the program, to take on the added job of treating children for diarrhea with rehydration salts and zinc. The second intervention had even more disruptive effects, as it organized a district-wide exemption from user fees for children under age five. Thus, since the program had chosen to subsidize ACTs rather than to eliminate payment, the CHWs’ work disappeared as mothers opted to bring their children to CSPSs for free care. This intervention was clearly not harmful to children’s health–in fact, quite the contrary–but it showed how a lack of cohesion and coordination can be damaging, in this case to the CHWs. Yet coordination of activities at the community level is one of the Ministry of Health’s priority actions Combining the implementation evaluation frameworks suggested by Carroll et al. Finally, our study also shows the need to take contextual dimensions and concurrent interventions more fully into account when studying fidelity of implementation. In fact, even though most FOI analysis frameworks consider context as a dimension to be studied The implementation analysis of this program in Burkina Faso showed that the activities were, on the whole, relatively faithful to the plan. However, delays in the implementation schedule and problems related to LLIN and ACT distribution definitely postponed the potential beneficial effects of this malaria control effort. The impact analysis should help to shed light on this question.Table S1Content fidelity of activities in Kaya District. Source: survey data.(PDF)Click here for additional data file.Table S2Content fidelity of activities in Zorgho District. Source: survey data.(PDF)Click here for additional data file.Table S3Coverage fidelity of activities in Kaya District. Source: survey data.(PDF)Click here for additional data file.Table S4Coverage fidelity of activities in Zorgho District. Source: survey data.(PDF)Click here for additional data file.


PRAME levels are associated with poor clinical outcome in a variety of cancers. However, the molecular pathways in which PRAME is implicated are not well understood. We recently characterized PRAME as a BC-box subunit of a Cullin2-based E3 ubiquitin ligase. In this study, we mined the PRAME interactome to a deeper level and identified specific interactions with OSGEP and LAGE3, which are human orthologues of the ancient EKC/KEOPS complex. By characterizing biochemically the human EKC complex and its interactions with PRAME, we show that PRAME recruits a Cul2 ubiquitin ligase to EKC. Moreover, EKC subunits associate with PRAME target sites on chromatin. Our data reveal a novel link between the oncoprotein PRAME and the conserved EKC complex and support a role for both complexes in the same pathways.The human tumour antigen PRAME  is frequently overexpressed during oncogenesis, and high  PRAME is expressed at low levels in few normal adult tissues like adrenals, ovaries, and endometrium, and at high levels only in the testis PRAME is frequently found in a wide variety of human cancers, including acute and chronic haematological tumours, synovial sarcoma, lung, breast, and renal carcinoma PRAME levels were found to correlate with advanced stages of disease in melanoma PRAME were found to correlate with good prognosis in leukaemia cases carrying the t PML-RAR translocation (acute promyelocitic leukaemia) The human oncoprotein PRAME  was first identified and cloned as the antigen responsible for an anti-tumour immune response in a melanoma patient Although these findings suggested a role for PRAME in human malignancies, the detailed molecular mechanisms and pathways involved are not yet clear. PRAME was reported to repress retinoic acid signaling in melanoma cell lines Recently, through biochemical characterization of PRAME-containing protein complexes, we established that this oncoprotein is a component of Cullin2-based E3 ubiquitin ligases and belongs to the family of BC-box proteins, associating PRAME to a clear biochemical activity and pathway 6A modification of tRNAs. Yeast EKC comprises four subunits which are also conserved in the human genome (human orthologues are indicated in brackets): Pcc1p , the ATPase Kae1p (OSGEP), the kinase Bud32p , and Cgi121p (TPRKB). In addition, yeast EKC also includes Gon7p , which appears to be fungi-specific Two independent laboratories have identified an ancient and highly conserved multiprotein complex named KEOPS 6A) Intriguingly, the OSGEP subunit is also present in bacteria (YgjD) and eukaryotic genomes express an OSGEP paralogue (Qri7/OSGEPL1) that localizes to mitochondria Human LAGE3 belongs to the NY-ESO gene family together with the closely related LAGE1 and LAGE2 The aim of the present study was to mine the protein-protein interactome of the PRAME oncoprotein to a deeper level by protein complex purification and mass spectrometry. Our experiments revealed that PRAME specifically interacts with the EKC complex in human cells and that it recruits Cul2-based E3 ubiquitin ligases to EKC. We further show that EKC subunits co-localize with PRAME at the promoter regions of transcriptionally active human genes, supporting a common functional role for these complexes on chromatin. Overall, our findings provide novel and important insights in the molecular pathways in which the oncoprotein PRAME is active.We have recently applied biochemical purification strategies and characterized PRAME as a component of Cullin2 E3 ubiquitin ligases Furthermore, a number of additional proteins were specifically detected in eluates of K562-TAG-PRAME and HeLaS3-TAG-PRAME cells, but not in the control parental cell lines. These included OSGEP  and LAGE3 (L antigen family member 3), which are the human orthologues of the Kae1 and Pcc1 subunits of the recently described EKC complex . OSGEP aOther potential interactors identified in PRAME eluates were BAG2 and UBL4A . BAG2 (BThe specificity of the interactions between PRAME and EKC subunits was confirmed by transient transfections in HEK293T cells and coimmunoprecipitation experiments of TAG-PRAME with OSGEP and LAGE3 fused to a TTE (TY1-TY1-ER) epitope tag . We alsoTaken together, our results identified stable interactions between PRAME and human orthologues of the EKC/KEOPS complex.PRAME in normal and cancer cells by querying the GeneSapiens gene expression database PRAME is expressed only in very few normal adult tissues while it is upregulated in several cancers .We next studied the expression patterns of EKC, Cullin2 ligase subunits and  cancers . On the To explore further the protein-protein interactions between PRAME and EKC, we generated K562 cell lines stably expressing epitope-tagged versions of each of the four human predicted human EKC subunits and performed protein complex purifications. As shown in Furthermore, TAG-OSGEP and TAG-LAGE3 (but not TAG-TP53RK or TAG-TPRKB) copurified with significant amounts of endogenous PRAME, consistent with TAG-PRAME purifications. Remarkably, TAG-OSGEP and TAG-LAGE3 associated also with Cullin2 ligase components . The fewInterestingly, peptides matching the putative protein C14orf142 were specifically identified in eluates from OSGEP, LAGE3 and TP53RK . SimilarAlthough we did not identify peptides matching Cul2 by mass spectrometry of TAG-OSGEP eluates, we did detect small amounts of Cul2 by western blotting of the same samples , lane 6.Our proteomic experiments show for the first time that a complete EKC complex is present in human cells, and that the OSGEP and LAGE3 subunits are present in a complex together with PRAME and Cullin2 ligase components.In order to study the topology of the interactions within the human EKC complex, we expressed pairs of EKC subunits in the baculovirus system and performed immunoprecipitations . As showInterestingly, Mao et al. reported that archael Pcc1, the orthologue of human LAGE3, forms highly interdigitated homodimers and hypothesized that dimerization of Pcc1 might support dimerization of the full EKC complex. Moreover, the crystal structures indicated that one moiety of Pcc1 could bind only one moiety of Kae1. We tested the dimerization properties of human EKC using combinations of different epitope tags in transient transfection experiments. As shown in In the same experiments, we noticed that OSGEP levels were significantly lower in the absence of LAGE3  that the human EKC complex is organized in the same way as its archaeal counterpart, (ii) that dimerization of LAGE3 can mediate formation of higher order structures containing at least two molecules of OSGEP, (iii) that formation of these higher order structures is independent of PRAME, and (iv) that the protein levels of OSGEP can be significantly modulated by protein-protein interactions.We next studied the role of the Cullin2 complex in the association of PRAME with EKC. Transient transfection experiments showed that the interaction of PRAME with OSGEP and LAGE3 does not require an intact BC-box, since a BC-box defective PRAME (PRAME M2) coimmunoprecipitated OSGEP and LAGE3 as efficiently as wild type PRAME .Next, we purified TAG-LAGE3 protein complexes from HeLaS3 cells, which express very low to undetectable levels of endogenous PRAME. As expected, all EKC subunits copurified with TAG-LAGE3, while no peptides were identified for PRAME . ImportaThese results, together with the finding that TAG-OSGEP and TAG-LAGE3 associate with endogenous PRAME and Cullin2 ligase components in K562 cells, suggested that PRAME could be involved in recruiting a Cullin2 ligase to the EKC complex.To test this hypothesis, we knocked down endogenous PRAME with stable transfection of retroviral constructs in K562-TAG-OSGEP and K562-TAG-LAGE3 cell lines and performed immunoprecipitations. Taken together, our data reveal that PRAME can bridge Cullin2 ligases to the EKC complex .The yeast orthologues of OSGEP (Kae1p) and LAGE3 (Pcc1p) have been reported to associate with the promoters and transcribed regions of several genes in a transcription-dependent manner In the absence of validated ChIP-grade antibodies against EKC subunits, we generated K562 cell lines stably expressing the proteins of interest fused to the TTE epitope tag (TY1-TY1-ER), which can be efficiently and specifically used in ChIP-qPCR experiments Our genomic experiments extend the biochemical interactions to the chromatin environment, establishing that PRAME and EKC subunits associate with the same promoter regions.We recently reported the first purifications of PRAME-containing protein complexes by epitope-tagged immunoprecipitations and mass spectrometry, which revealed that PRAME is a BC-box substrate receptor component of Cullin2-based E3 ubiquitin ligases 6A modification of tRNAs in yeast EKC is an ancient and extremely conserved complex with orthologues in Archaea and Eukarya. It has been implicated in telomere maintenance, transcriptional regulation, and t6A) 6A modification activity was dependent on Pcc1p, the ATPase activity of Kae1p, and the kinase activity of Bud32p, but not on Cgi121p Consistent with the notion that all subunits contribute to the activity of the complex, yeast EKC was reported to constitute a stable entity. Downey et al. reported that deletion of either Bud32p, Cgi121p, or Gon7p resulted in shorter telomeres. An independent study found that Pcc1p was required for efficient transcription of several yeast genes induced by pheromones or galactose, and that deletion of Pcc1 inhibited the recruitment of the SAGA and Mediator co-activators In our experiments, all human EKC orthologues co-purified together and with similar abundance both in K562 and HeLaS3 cells, demonstrating for the first time that a complete and stable EKC complex is present in human cells. However, we consistently found that PRAME complexes contained primarily OSGEP and LAGE3, and very low or undetectable levels of the other two subunits TP53RK and TPRKB. This might reflect a PRAME-mediated disruption of an otherwise stable EKC complex, or a preferential binding of PRAME to an already existing (and possibly dynamic) EKC submodule that was not previously detected in yeast. It is tempting to speculate that PRAME could mediate ubiquitination of EKC components via recruitment of the Cul2 E3 ligase complex. In an attempt to test this hypothesis, we performed ubiquitination assays upon transient transfections in HEK293 cells in presence or absence of the proteasome inhibitor MG132, and in vitro assays with purified proteins. As expected for BC-box proteins, we detected polyubiquitin chains associated with PRAME itself. However, the high background signals in these assays did not allow us to detect a putative ubiquitination of EKC subunits. Hence, our assays could not rule out that EKC might be ubiquitination targets of Cul2-PRAME, and more efforts are needed to test this hypothesis experimentally.Interestingly, dynamic regulation of EKC components has been reported: the kinase activity of Bud32p was repressed by association with Kae1p The results of our transient transfection experiments and those obtained using the baculovirus system suggested (i) that PRAME is able to interact independently with LAGE3 and OSGEP and (ii) that LAGE3 enhances the association of OSGEP with PRAME. Considering the dimerization properties of the LAGE3-OSGEP submodule, we therefore considered the possibility that multiple PRAME moieties might be present in the same protein complex. In this case, both epitope-tagged PRAME and endogenous PRAME should be present in the same protein complex in K562-TAG-PRAME cells, since the expression level of the two proteins were similar. Contrary to this hypothesis, however, TAG-PRAME did not co-immunoprecipitate endogenous PRAME in our experiments see  lane 4. LAGE3 could not substitute for Pcc1 OSGEP gene was found expressed in all somatic tissues tested and it lies in a head-to-head orientation with the tightly regulated APEX gene, which encodes a multifunctional DNA repair enzyme OSGEP transcription. Futhermore, OSGEP could partially rescue the growth defects of the yeast kae1Δ mutant TP53RK was first cloned by cDNA subtraction as an interleukin-2 upregulated gene in cytotoxic T-cells, transcripts were detected in a number of normal tissues and cancer cell lines, and it was shown to phosphorylate the human oncoprotein p53 at Serine 15 bone fide p53 homologue. Human TPRKB was first isolated by a two-hybrid screen using TP53RK as a bait, and was suggested to inhibit the binding of TP53RK to p53 TP53RK was detected in the high-PRAME expressing cell line K562 as well as in the low-PRAME expressing cell line KG1 Besides the functional experiments in yeast, no information is available yet about EKC functions in other organisms. At present, only studies focusing on single subunits of the human complex have been reported. LAGE3 appears to be ubiquitously expressed, while the two closely related LAGE1 and LAGE2 are cancer-testis antigens with high expression confined to healthy testis and cancer tissues Considering the extreme conservation of EKC components during evolution, it can be expected that at least some of the functions and pathways already identified for this complex in yeast could be valid for human cells as well. However, it cannot be excluded that human EKC acquired additional, yet to be identified functions during evolution. The absence of efficient knock-down or knock-out models have so far hindered advanced functional analysis of EKC in higher eukaryotes. Future studies will have to address these issues, and in particular to understand the connections between apparently different functions ascribed to this complex, like phosphorylation of the p53 oncogene, chemical modification of tRNAs, and physical association with promoter regions.pcc1-4 mutation prevented activation of GAL1 by impairing recruitment of the SAGA histone acetyltransferase and mediator without affecting recruitment of the activator Gal4p. In contrast, while Pcc1p was recruited to the constitutively active PMA1 and the heat-shock inducible HSP104 genes in a transcription-dependent manner, transcription of these genes was not affected in the pcc1-4 mutant.Kae1p and Pcc1p have been found to associate with active chromatin and to regulate expression of some of the yeast promoters to which they are recruited Our ChIP assays revealed that LAGE3 and OSGEP are also present on chromatin and, in particular, that they colocalize with PRAME at many of its target sites, consistent with our protein-protein interaction data. Since neither PRAME nor EKC proteins possess known DNA-binding domains, it is not clear whether they can bind DNA directly. Genome-wide ChIP-seq experiments have recently revealed that PRAME associates with transcriptionally active NFY-regulated promoters and nearby enhancers PRAME is expressed would a Cul2-PRAME ubiquitin ligase be recruited to chromatin by virtue of specific protein-protein interactions between LAGE3-OSGEP and PRAME.Importantly, EKC proteins have an ancient evolutionary origin and seem to be ubiquitously expressed in adult tissues, while PRAME appeared more recently in evolution, and its expression seems to be tightly confined to particular developmental stages and tissues and to be particularly deregulated in oncogenesis. These characteristics are consistent with a model in which EKC can associate with chromatin independently of PRAME. Only when In an attempt to study the putative function of EKC subunits on target gene transcription, we established K562 cells with stable downregulation of OSGEP or LAGE3 using pRetroSuper vectors and performed genome-wide mRNA-seq analyses. Despite a 50–80% knockdown efficiency for OSGEP and LAGE3, we did not detect genes with significant expression changes when compared to a GFP knockdown control. Our inconclusive data could of course be the result of inefficient downregulation of the target genes. Although the K562 cell line was successfully used to dissect the protein-protein interaction network, we cannot exclude that this cell line might not be suitable for the study of the finer putative chromatin functions and signaling of EKC and PRAME.The development of ChIP-grade antibodies against EKC components will be a crucial step to address important questions as to whether PRAME target sites are a subset of EKC sites (or viceversa), and to address the functional relationships between EKC, Cul2-PRAME ligases and NFY on transcriptional regulation of target genes, and potentially other pathways like the recently described modification of tRNAs.Taken together, our data reveal a novel link between the human oncoprotein PRAME and the ancient EKC complex. Our data clearly show that PRAME can recruit Cullin2 ubiquitin ligases to EKC, and that these complexes associate with the same genomic regions, supporting a functional link in the same pathways. Our results add a new twist to both PRAME and EKC biology and provide an important contribution to understanding the molecular mechanisms in which these proteins are active, both in healthy and cancer cells.2; cell lines were obtained from ATCC. Both media were supplemented with 5% Glutamax, 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin . K562 and HeLaS3 stable cell lines were generated with retroviral constructs as described HEK293T cells were cultured in DMEM and K562 and HeLaS3 in RPMI medium  at 37°C in 5% COMouse monoclonal HA-12CA5 and Myc-9E11 were produced in-house from hybridoma cultures, PRAME antibodies were previously described A modified version of the retroviral vector LZRS(Zeo) with an improved MCS was made by ligation of a synthetic oligo to generate LZRSn(Zeo). The sequence coding for the StrepII-TEV-MYC-3xHA tag was excised with BamHI and NotI from pcDNA5-TAG-PRAME and subcloned in LZRSn(Zeo) to generate LZRSn-TAG, where the EcoRI site is in the same frame as in the pTTE retroviral vector The TTE cassette was subcloned from pTTE Full-length human OSGEP was amplified by PCR from cDNA prepared from K562 cells to generate a NotI-EcoRI-OSGEP-SalI fragment, which was cloned into pFLAG-CMV2 (pFLAG-OSGEP). OSGEP was subsequently subcloned as an EcoRI-SalI fragment into the EcoRI and XhoI sites of pTTE and pcDNA-TTE, to generate pTTE-OSGEP and pcDNA-TTE-OSGEP, respectively. OSGEP was cloned as a NotI-SalI fragment downstream the TAG in LZRSn(Zeo), generating LZRSn-TAG-OSGEP.LAGE3 CDS was obtained from Invitrogen, PCR amplified to introduce EcoRI and XhoI sites, and subcloned in the same sites of pTTE, pcDNA-TTE, and LZRSn-TAG-LAGE3.Full-length TP53RK/PRPK and TPRKB were amplified by PCR from cDNA of K562 cells and subcloned as EcoRI and XhoI fragments to generate all constructs used.Mutagenesis to generate OSGEP and LAGE3 mutants was performed using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.All constructs were checked by sequencing.Protein complex purifications and mass spectrometry were performed from nuclear extracts (unless otherwise indicated) essentially as described For TAG-PRAME purifications with MYC antibody, about 10 ml of protein extracts (∼100 mg) were incubated with 50 µl (MYC1 experiment) or 600 µl (MYC2 experiment) of crosslinked MYC beads and peptide elution was performed. For protein complex purifications with HA antibody, about 5–6 ml of protein extracts were incubated with 600 µl HA-12CA5 crosslinked beads and acidic glycine (pH 2.9) was used for elution.The data analysis steps of peptide-to-protein remapping and emPAI calculation were automated with a PERL script to generate output tables of unique protein IDs with peptide frequencies and emPAI values for each samples . The protein list was manually filtered to discard proteins identified in the control samples .cDNAs encoding wild type or mutant PRAME, OSGEP, LAGE3, TP53RK and TPRKB were subcloned into pBAcPAK 8 and recombinant baculoviruses were generated. SF21 cells were cultured at 27°C in SF-900 medium supplemented with 10% fetal bovine serum. SF21 cells were co-infected with the recombinant baculoviruses indicated in the figures. 50 hours after infection, cells were collected, lysed, and processed as described Chromatin harvests, ChIPs and qPCR analyses were performed as previously described Figure S1Detailed expression profiles of MAG, TBP, CUL2, and PRAME from the GeneSapiens database. Normalized relative gene expression levels in healthy (green) and cancer (red) tissues are plotted as boxplots. MAG and TBP are shown as references: MAG is a known neuronal marker gene and shows an expression profile that is highly specific for the central nervous system. On the contrary, TBP is a ubiquitously expressed gene. Similary to TBP, the expression profile of CUL2 indicates ubiquitous expression in both healthy and cancer tissues. On the contrary, PRAME is expressed mainly in the healthy testis, and is upregulated in a number of cancers.(TIF)Click here for additional data file.Figure S2Detailed expression profiles of EKC subunits from the GeneSapiens database. Normalized relative gene expression levels in healthy (green) and cancer (red) tissues are plotted as boxplots. The expression profiles of LAGE3, OSGEP, TPRKB, and TP53RK indicate expression in all or most healthy and cancer tissues.(TIF)Click here for additional data file.Figure S3Gene expression correlation plots between PRAME and subunits of Cul2 ligases or the EKC complex. Co-expression plots were generated from the GeneSapiens database for PRAME and CUL2 or each of the EKC subunits. Correlations plots are shown for healthy tissues (A), and cancer tissues (B). The plots indicate that the expression of the genes tested do not correlate linearly. In particular, a large number of samples are characterized by low or no PRAME expression, while CUL2 and EKC subunits are expressed at higher levels.(TIF)Click here for additional data file.Figure S4Gene expression correlation plots between subunits of Cul2 ligases and subunits of the EKC complex. Co-expression plots were generated from the GeneSapiens database for CUL2 and TCEB1, and couples of EKC subunits. Correlations plots are shown for healthy tissues (A), and cancer tissues (B).(TIF)Click here for additional data file.Protocol S1Detailed protocol for comparative analyses of mass spectrometry data.(DOC)Click here for additional data file.Analysis Script S1PERL script mapPep2Prot_v0.3.pl for analysis of mass spectrometry data.(PL)Click here for additional data file.


Obesity, physical inactivity, and poor nutrition have been linked to many chronic diseases. Research indicates that interventions in community-based settings such as houses of worship can build on attendees’ trust to address health issues and help them make behavioral changes.New Brunswick, New Jersey, has low rates of physical activity and a high prevalence of obesity. An adapted community-based intervention was implemented there to improve nutrition and physical activity among people who attend houses of worship and expand and enhance the network of partners working with Rutgers Cancer Institute of New Jersey.An adapted version of Body & Soul: A Celebration of Healthy Living and Eating was created using a 3-phase model to 1) educate lay members on nutrition and physical activity, 2) provide sustainable change through the development of physical activity programming, and 3) increase access to local produce through collaborations with community partners.Nineteen houses of worship were selected for participation in this program. Houses of worship provided a questionnaire to a convenience sample of its congregation to assess congregants’ physical activity levels and produce consumption behaviors at baseline using questions from the Health Information National Trends Survey instrument. This information was also used to inform future program activities.Community-based health education can be a promising approach when appropriate partnerships are identified, funding is adequate, ongoing information is extracted to inform future action, and there is an expectation from all parties of long-term engagement and capacity building. Obesity, lack of physical activity, and poor nutrition have been linked to chronic diseases such as heart disease, stroke, diabetes, and some cancers . An estiNew Brunswick is a diverse community centrally located in Middlesex County, New Jersey. Nicknamed “The Healthcare City,” New Brunswick is home to 2 academic institutions, 2 hospitals, a transit hub, and various pharmaceutical companies.2 or higher, slightly less than the New Jersey and US benchmarks of 25%  to establish a comprehensive program to improve nutrition and physical activity among people who attend houses of worship and 2) enhance the network of partners working with Rutgers Cancer Institute of New Jersey to provide community education. This case study highlights the successes and challenges through the development and recruitment periods of our program.Body & Soul: A Celebration of Healthy Living and Eating (B&S) is an evidence-based program developed by the National Cancer Institute in collaboration with the American Cancer Society, the University of North Carolina, and the University of Michigan –11. The The original B&S program was adapted for implementation in our diverse community in a few ways. The original B&S Program was designed for implementation in African American churches. Houses of worship in the New Brunswick area reflect the diversity of the neighborhoods in which they are located. Our goal was to create community change by implementing a program that would affect a broader community and not restrict implementation to African American churches. In addition, the original program had a strong focus on fruit and vegetable consumption. Although program staff and advisors agreed that this component should remain a focus of the program, they also felt it was important to include education on broader nutrition topics and general healthful living strategies of interest to participants. Therefore, we added education on other nutrition topics such as sodium, fat, calcium, sugar-sweetened beverages, and whole grains. We also included a stronger focus on physical activity. Experience with B&S and other research suggests that ongoing technical assistance and training would help engage lay members of the houses of worship (ambassadors) and continue their participation ,13,14. OOur adapted program used a “train the trainer” model in which consultants with expertise in nutrition and physical activity content areas provided education to ambassadors, who were then charged with delivering health messages to their congregation using the strategies that they felt would be successful in their respective groups. Before the full launch in 2012, elements from phases I and II were piloted using 2 houses of worship that agreed to serve as a prelaunch pilot. Feedback from this pilot was used to design reporting forms, adjust the program schedule, and frame strategies for the nutrition and physical activity consultant to consider during site visits. Conversations with community partners and key informants with expertise in the content areas were used to enhance session offerings and program support in an effort to increase the likelihood of continued participation among houses of worship after the program was formally launched.A program staff member was selected to participate in the National Cancer Institute’s Research to Reality Mentorship Program (R2R). R2R is “an online community of practice that links cancer control practitioners and researchers and provides opportunities for discussion, learning, and enhanced collaboration on moving research into practice” . ThroughBecause this project was conducted in the context of a community philanthropic project where 1) data were collected for program evaluation purposes, not to evaluate individual outcomes, and 2) the goal was not to produce generalizable results, the project was determined not to require approval by the Rutgers University Institutional Review Board. No identifiers  are used in the reporting of the results of this program evaluation.www.njsnap-ed.org/fit), the faith-based arm of our state’s Supplemental Nutrition Assistance Program (SNAP) educational unit, because of our strong relationship with New Brunswick Tomorrow (www.nbtomorrow.org), a citywide nonprofit that works to bring like-minded partners together to address community issues. Faithfully Fit provides education on these topics in limited-resource communities, and it had varied pretested materials available for our use at low or no cost. New Brunswick is in its catchment area, and we were able to combine our efforts with Faithfully Fit’s efforts to reach shared goals. In addition, B&S staff joined networks such as Shaping NJ  to learn about new organizations  that could be tapped for support and guidance, as well as best practices that could be incorporated into program planning.Community engagement is an important piece of any community-based initiative. A committed effort was made to engage and develop relationships with local community partners, both public and private, at the onset of the program. Potential community partners were identified by using existing relationships, and an emphasis was made to connect to those who had expertise in nutrition or physical activity or who were working with houses of worship. Existing relationships were also used to establish relationships with new partners. For example, we were connected to Faithfully Fit  at baseline. In addition, it provided an opportunity to introduce ambassadors to data collection activities they would be participating in throughout the program. Through this process, we were able to reflect on their previous activities and organizational climate regarding physical activity and nutrition at baseline. In addition, more than 1 congregant was invited to serve as an ambassador to increase the ability for the house of worship to remain engaged throughout the program; this strategy has been suggested in other research . To submApplications were received through January 2012 and reviewed for completeness; applicants were contacted when necessary to obtain additional information. Follow-up phone calls and e-mails were sent to any house of worship that previously expressed an interest but did not submit an application. Additional outreach and phone calls from 2 community partners (Faithfully Fit and New Brunswick Tomorrow) were provided to houses of worship in the catchment area to confirm our credibility and encourage participation in the program.Nineteen houses of worship in the Central New Jersey area submitted applications, and all were selected to participate in our adapted program. This number represents approximately 10% of those invited who participated. There is great racial and ethnic diversity among these houses of worship, reflecting members of African American, Hispanic/Latino, white, East Asian, South Asian, African, Middle Eastern, and Afro-Caribbean communities, and representing various faiths and denominations. Many identified multiple races and ethnicities in their congregations. Questions were included on the application to identify a house of worship’s current activities related to physical activity and nutrition. Results indicated that many had conducted health-related activities before application, including health ministries (50%), soup kitchens (17%), food pantries (50%), physical activity programs (61%), and food policies (22%). We used this information to help us develop program activities for the following phases.Ambassadors were provided with surveys to assess their congregation’s physical activity level and fruit and vegetable consumption before the start of the program. Questions related to physical activity behaviors and fruit and vegetable consumption were extracted from the Health Information National Trends Survey, a nationally representative biennial survey developed by the National Cancer Institute and designed to learn how Americans find, use, and understand health information . QuestioAmbassadors used convenience sampling methods to obtain assessments from any member of their congregation. Congregation members used worship services and other events  to obtain completed instruments. The final response rate was 97%; houses of worship submitted most of their assessment cards by April 2012. Data were analyzed by using SPSS version 19.0 . Each house of worship received a data report , which wRecent research has highlighted various community and institutional factors that can determine the success or failure of program implementation ,18. ProvThe use of informal open houses and a formal orientation session allowed program staff to reach lay members in person and explain the components of the program and how it complements the existing mission(s) of their faith-based organizations. Elements of program phases were designed to help build ambassadors’ skills and their self-efficacy toward the design of activities that would be successful and well received by their congregations. In-depth training of ambassadors throughout program delivery was helpful to build their data collection and reporting skills, as well as to help them understand why providing data is so important. Ambassadors were given skills and tools through monthly training sessions and individualized technical assistance, but the program was purposely designed not to be prescriptive. This design allowed ambassadors to use their creativity and take ownership of the activities they were conducting within their houses of worship.This project underscored the importance of community engagement and partnership from the onset, which has been identified as a factor to successful program implementation . These fSimilar to the experience of other health education programs delivered in community settings, a limitation of our program was the inability to reach as many houses of worship as we wanted. Even with a large mailing, informal open houses, and follow-up contacts from trusted community partners, we attracted a less-than-ideal sample of houses of worship to apply for participation in the program. We learned through our recruitment process that some houses of worship were experiencing various issues, such as dwindling memberships or worship service attendance, reduced revenue, growing urgent needs from their congregations , and changes in or absence of leadership during the recruitment or promotion period. In some cases, our introductory letters may have been misplaced or discarded. Some of these issues have been highlighted as barriers in other implementation research, and we recognize that participating in our program may not have been a priority for houses of worship with larger institutional concerns .Through implementing this program, we learned lessons and would make some changes if it were to be replicated. This program was implemented with only 1 full-time staff member, supported by a host of community partners and volunteers. Additional staff would have allowed more in-person visits to houses of worship during the recruitment phase. Research has shown that such visits may increase program interest among clergy leadership, because people often prefer face-to-face interaction, especially when encountering organizations with which they are not familiar. Although we were fortunate to have a consistent staff member, additional staff may have increased our reach and thus increased the number of houses of worship who applied for participation .In future implementation, we would provide incentives to both the houses of worship and ambassadors for their commitment. Research suggests that financial incentives can be helpful in providing additional support and keeping organizations engaged ,19,20. ODespite these limitations, our results contribute to the body of knowledge regarding the use of faith-based organizations to provide health education. This case study highlights the successes and challenges of recruiting volunteer lay members for program implementation and helps identify elements that should be incorporated to maximize participant recruitment in future programming.


To the Editor: The earthquake that occurred in Japan on March 11, 2011, triggered an extremely destructive tsunami  who had visited the hospital. We examined clinical and bacteriologic data for these patients. We excluded from the study patients without sputum culture and patients with other conditions, such as lung cancer, pulmonary infarction, or cardiac failure.To determine the characteristics of pneumonia after the earthquake, we conducted a retrospective study of patients who had pneumonia during the 6 weeks before the earthquake and the first 9 weeks after the earthquake. To identify patients with pneumonia, we checked all chest radiographs and computed tomography scans of adult patients , and within the 9 weeks after the earthquake, community-acquired or health care–associated pneumonia was newly diagnosed for 172 adults. Patient data from 2 pre-earthquake periods and 3 postearthquake periods are shown in the Haemophilus influenzae and Moraxella catarrhalis were more predominant than Streptococcus pneumoniae; most strains were isolated from purulent sputum specimens. In contrast, pneumonia caused by enterobacteria, staphylococci, or atypical pathogens did not increase after earthquake.Chest radiographs were taken and hematologic examinations were performed for all patients; computed tomography of the chest and rapid diagnostic tests for influenza were performed for 42.2% and 54.2% of 83 patients, respectively, who had pneumonia in the early postearthquake period. During the first 3 weeks after the earthquake, H. influenzae remained constant at 15.4% (4/26 patients); before the earthquake the rate was 17.4% (4/23), and soon after the earthquake it increased to 32.5% (27/83). Detection rates of M. catarrhalis increased from 0–3.8% before the earthquake to 31.3% (26/83) after the earthquake (p<0.01). These bacterial strains were isolated widely from refugees at shelters and from persons living at home without running water and/or electricity. Soon after the earthquake, it was thought that infections with these strains were not part of a localized outbreak but were widespread in the region. Most patients from whom M. catarrhalis was isolated were located throughout the area flooded by the tsunami. In contrast, many patients with H. influenzae were mainly located outside the flooded area. There was no regional imbalance in isolation of S. pneumoniae.Detection rates of M. catarrhalis , might have largely affected the etiology of pneumonia. We found no increase in cases of severe pneumonia caused by resistant bacteria or aspiration pneumonia in elderly patients. We conclude that multiple localized small community outbreaks might have occurred widely in this area after earthquake.