Gastroesophageal flap valve (GEFV) endoscopic grading is reported to be associated with gastroesophageal reflux disease (GERD) in adults; however its role in pediatric groups remains unknown. This study aimed to investigate the significance of GEFV grading and the associations to multichannel intraluminal impedance and pH monitoring (MII-pH) in children with GERD.A total of 48 children with GERD symptoms who received esophagogastroduodenoscopy and MII-pH monitoring were enrolled. The degree of GEFV was graded from I to IV according to the Hill classification, and classified into two groups: normal GEFV (Hill grades I and II), and abnormal GEFV (Hill grades III and VI). Endoscopic findings and MII-pH monitoring were analyzed among the groups.p = 0.037, OR 9.84, 95% CI 1.15–84.42). Pathological acidic gastroesophageal reflux (GER) determined by MII-pH was more prevalent in the patients with loosened GEFV geometry . There were significant positive correlations between GEFV Hill grading I to IV and the severity of erosive esophagitis , percentage of supine acid reflux , percentage of total acid reflux , and DeMeester score  detected by pH monitoring. In the impedance study, GEFV Hill grading also positively correlated to median number of acid reflux events .Thirty-six patients had normal GEFV while 12 had abnormal GEFV. The presence of erosive esophagitis was significantly more common in the patients with abnormal GEFV (GEFV dysfunction highly associated with acid GER and severe erosive esophagitis. An abnormal GEFV is a sign of acid GER in children. The gastroesophageal junction acts as a barrier against the retrograde flow from the stomach. Several anti-reflux mechanisms contribute towards its proper function, including the coordination of diaphragm crural fibers, lower esophageal sphincter (LES), and the intra-abdominal portion of the esophagus, all of which combine to maintain an anti-reflux effect. Moreover, the flap valve formed by gastric cardiac sling musculature also plays an important role as a gate against anti-gastric retrograde flow Gastroesophageal reflux disease (GERD) is presented when the reflux of gastric contents causes troublesome symptoms and/or complications. The incidence rate of GERD per1000 person- years is approximately 0.84 in pediatric patients aged 1–17 years in the UK with an increasing trend In adults, the degree of GEFV dysfunction has been reported to be positively related to acid reflux This study was conducted with a retrospective manner in children with GERD at the Department of Pediatrics, National Taiwan University Hospital from January 1, 2010 through January 31, 2014. The patients were referred for evaluations due to either respiratory symptoms/signs  or gastrointestinal diseases . Children who underwent esophagogastroduodenoscopy (EGD) within 3 days before MII-pH monitoring examination, and never received any acid suppression therapy were eligible for inclusion in the study. GEFVs were graded from I to IV by retrograde endoscopic findings The written informed consents were obtained from the parents or guardians of the children. The study protocol was approved by the Ethics Committee of National Taiwan University Hospital’s Institutional Review Board.Each MII-pH study was performed using an ambulatory polyurethane catheter incorporating six impedance amplifying electrodes 2.0 cm apart and a single pH sensor, positioned 5.0 cm above the tip . The catheter was introduced transnasally and placed with the pH sensor 3–5 cm above the LES confirmed by chest radiography. The MII-pH electrodes were connected to an ambulatory recorder. All patients were allowed to maintain their usual activities, regular diet, and sleep routines. This study was carried out for at least 20 hours for each subject. During the study period, the patient or the caregiver was instructed to press event buttons and to keep a diary to record meal times, posture changes, and symptoms. At the end of the measurements, recordings within the device were uploaded to a computer and analyzed manually by the physician using a dedicated software program .While impedance can detect voltage and electrical current changes in consecutive sensors positioned along the catheter, the impedance wave was designed to change inversely to ionic concentration. Air with a relatively low ionic content results in a high impedance wave, and fluid with a higher ionic content results in a low wave. This principle provides an essential advantage in the assessment of the flow direction of bolus passage.The impedance recording was analyzed according to the protocol recently proposed by the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) With regards to pH recording data, catheter acidification by the surrounding contents causing the pH value to drop lower than 4 was classified as an acidic episode, and pH values remaining above 4 were classified as non-acidic episodes. A widely used pH reflux composite score was calculated based on the DeMeester criteria including both percent times in upright and supine acid reflux, total percent of time in acid reflux, duration of the longest acid reflux episode, the number of acid reflux episodes >5 minutes, and total number of reflux events. Pathological acid gastroesophageal reflux in our study population was defined based on the finding of total percent of time in acid reflux >4.2% or DeMeester score >14.7 Flexible EGD was performed in each patient under general or local anesthesia, and the esophagus, stomach, and duodenum were examined with optimal visualization. The severity of erosive esophagitis was graded according to Los Angeles (LA) classification. In addition, we carefully examined the gastroesophageal junction to assess the geometry of the GEFV, using a retroflexed view during gastric inflation. The GEFV was graded from I to IV according to the Hill classification  [6], [8]X2 test and Fisher’s exact test to examine differences in categorical variables between the two patient groups. The Mann-Whitney U test was applied to test the differences in median/25th−75th percentage values in the continuous variables. Linear regression analysis was performed to estimate the correlations between the variables of MII-pH monitoring and Hill classification. All p values were 2-sided, and a p value less than 0.05 were considered to be significant. Statistical calculations were performed with SPSS version 19.0 for Windows software .We used the A total of 48 patients  were enrolled in this study. The median age of these children was 6.2 years. The most common indications for receiving these examinations were vomiting and dysphagia. Demographic data and clinical characteristics were summarized in p<0.001, and p = 0.02, respectively). The median DeMeester score was also significantly higher in the abnormal GEFV group  than that in normal GEFV group . The ratio of subjects fulfilling the criteria of pathological acid gastroesophageal reflux with total acid reflux (%) >4.2% or DeMeester score >14.72 was greater in abnormal GEFV group than normal group  , total acid reflux , and the DeMeester score .For the pH monitoring, the percentage of supine acid reflux and total acid reflux were significantly increased in the abnormal GEFV group (7–27.38) . In addip = 0.1; duration of longest acid reflux, p = 0.07; the number of acid reflux events >5minutes, p = 0.07).Upright acid reflux, duration of the longest acid reflux episode, and the number of acid reflux events >5 minutes also showed higher trends in the abnormal GEFV group than in normal GEFV group. In addition, GEFV gradings also positively correlated to the median number of acid reflux events . However, there were no significant differences between the normal and abnormal GEFV groups in terms of other parameters including nonacid reflux, liquid reflux, mixed liquid-gas reflux, and total impedance events per hour detected.With regards to impedance, the abnormal GEFV group had a significantly higher median number of acid reflux events  .Among the 48 patients, the presence of erosive esophagitis was significantly more prominent in the patients with abnormal GEFV than in those with normal GEFV (5–84.42) . The GEFProton pump inhibitors (PPIs) are the mainstay of treatment of GERD vs. abnormal group: n = 22/22, 100% vs. n = 7/12, 58.3%, p = 0.003, OR 0.58, 95% CI 0.36–0.94). Doubling the PPI dose to a twice-daily manner for the 5 subjects with abnormal GEFV who initially failed to respond to a standard dose of PPIs revealed subsequent therapeutic advantages. None of the treatment subjects in normal GEFV group needed to receive the double dose of PPI. Five of 14 patients in the normal GEFV group who did not use PPIs received prokinetic regimens or mucosal protective agents, and demonstrated symptom relief.The treatment response to standard doses of the PPIs in the normal GEFV group was significantly better than that in the abnormal GEFV group  subsequently alleviated their symptoms. In contrast, none of the subjects with GEFV Hill grade I or II was indicated for a doubling of the PPI dose. This is consistent with previous reports which suggested that an abnormal GEFV configuration could serve as an independent factor predicting a poor response to PPI therapy In conclusion, endoscopic grading of the GEFV had a positive correlation with erosive esophagitis and some parameters of MII- pH monitoring in children. Our findings suggest that evaluation of the GEFV can provide useful information to help predict the presence of acid gastroesophageal reflux and its associated adverse events in children. In addition, loosening GEFVs may correlate with poor responses to PPI regimens for children with GERD.


This study was aimed to investigate the clinical outcome of lumbosacral tuberculosis treatment by one-stage radical debridement with bone allograft reconstruction and anterior instrumentation via a retroperitoneal approach.We retrospectively analyzed a series of 43 patients with lumbosacral tuberculosis in whom the lumbosacral junction was exposed via an anterior midline retroperitoneal approach. After radical debridement, two parallel tricortical iliac crest bone allografts were placed to reconstruct the anterior column, and then anterior fixation was performed.The mean follow-up period was 34 months , during which no obvious loss of correction was observed. No case experienced recurrence, tuberculous peritonitis, erectile dysfunction, or retrograde ejaculation.The midline retroperitoneal approach provides direct and safe access to lesions of lumbosacral tuberculosis. Two parallel structural iliac crest allografts and anterior instrumentation effectively stabilize the lumbosacral junction. Lumbosacral tuberculosis is a rare condition, representing only 2%–3% of all cases of spinal tuberculosis . SurgicaIn this retrospective study, we analyzed outcomes for all 43 consecutive patients treated for lumbosacral tuberculosis at our hospital from July 2001 to December 2011. Patients included 20 males and 23 females with a median age of 40 years . The disease duration prior to treatment ranged from 1 month to 6 years . Among these patients, 27 had complained severe or moderate back pain; 21 reported radiation pain in the lower limb; and 16 had muscle strength loss, pain, and hypoesthesia in the lower limbs. Plain radiography, computed tomography (CT), and/or magnetic resonance imaging (MRI) were performed on all the patients before surgery and showed that the lesions were all located in the L5–S1 segments. Paravertebral abscesses were observed in 40 patients, and vertebral damage was clearly observed in all patients. Intervertebral space was found to be narrower. Overall, 26 cases presented with presacral abscess, 9 cases with fistula from L5 paravertebral abscesses, 16 cases with pulmonary tuberculosis, and 7 cases with sacroiliac joint tuberculosis. Before surgery, the average lumbosacral angle was 13.30°  [Mycobacterium tuberculosis. A curette and rongeur were used to clear the pus, caseous necrotic tissue, granulation tissue, necrotic bone, and intervertebral disc , and the mean blood loss volume was 383.72 mL . No injuries to the large blood vessels, nerves, or ureters occurred in these patients, and none of the 20 male patients experienced erectile dysfunction during the follow-up period. None of the patients developed wound infection or fistula. Two patients suffered abdominal distention, and two patients suffered urinary retention after surgery. The 43 patients were followed up for a mean period of 34.74 months , and no patients experienced recurrence of the disease during follow-up. It should be mentioned that the recurrence rate is 2%–5% in the long term (5–10 years) .Partial absorption of the edge of the bone was observed in two patients. According to the Bridwell criteria , at the L5–S1 is a spinal segment located between the lumbar lordosis and sacral kyphosis. As these vertebrae bear the majority of body weight, the stability of this segment is critical. In addition, the upper surface of S1 vertebra slants forward, and therefore, there is a high risk of L5 spondylolisthesis. The L5–S1 zygopophysis and intervertebral disc are the main structures responsible for resisting L5 spondylolisthesis. In patients with L5–S1 vertebral tuberculosis, the damaged intervertebral discs and vertebrae, as well as the decreased lumbosacral angle, may dislocate the L5–S1 joint, leading to joint capsule laxity. Therefore, the L5–S1 intervertebral disc must be removed during debridement of tuberculosis lesions. However, without the intervertebral disc, the shear stress between L5 and S1 can cause accelerated degradation of the zygopophysis, leading to back pain. Thus, an important goal of treatment is to recover the height of vertebrae and prevent local shear and axial stresses via bone graft fusion. However, the intervertebral bone graft may slip forward during rotation or extension movement of the lumbar spine, and it is essential to use internal fixation for early reconstruction and maintenance of the vertebrae -16. A poThe anterior approach to the lower lumbar spine includes the extraperitoneal or intraperitoneal approaches. The common iliac artery and vein are located anterior-laterally to the L5 vertebra. Although the extraperitoneal approach can clearly expose the lumbosacral segments, this approach can lead to injury of the blood vessels and ureters. On the contrary, the intraperitoneal approach to the L5–S1 anterior interbody fusion has the following advantages: (1) incision through the linea alba can avoid muscular damage; (2) lesions located anteriorly and bilaterally to the sacrum can be fully exposed and debrided, which can facilitate the anterior internal fixation; and (3) a direct view can minimize surgical damage to blood vessels, nerves, and ureters ,18. In oMost patients with spinal tuberculosis will have good outcomes after conservative treatments. However, the surgical approach applied in this study may be considered in patients with the following conditions: large presacral abscess or sequestrum, severely damaged vertebrae that require reconstruction for stability, history of previous multiple surgeries conducted via the transperitoneal approach with local tissue and intestine adhesion, and female.Through 100 years of development, allograft bone transplantation has become a mature technology with minimal risks of immune rejection and infection -21. The Postoperative radiographs and CT images at 3–6 months showed partial absorption of the upper or lower edge of the allografts in two patients. This may have been associated with the replacement of the allogeneic bones and slight loosening of the internal fixation. The two patients achieved grade II bone union at postoperative 9 and 11 months, respectively. The time to bone union in these patients was obviously longer than that achieved with the use of autografts. However, the allografts were satisfactory in terms of deformity correction and stability restoration. In addition, allogeneic iliac crest bone grafting has advantages over autografts including shorter operation time and avoidance of donor site morbidity.In conclusion, the midline retroperitoneal approach provides direct and safe access to lesions of lumbosacral tuberculosis. Moreover, interbody fusion with iliac crest allografts and anterior instrumentation can effectively restore the lumbosacral junction stability.


Objective To examine the associations between the regular consumption of spicy foods and total and cause specific mortality.Design Population based prospective cohort study.Setting China Kadoorie Biobank in which participants from 10 geographically diverse areas across China were enrolled between 2004 and 2008.Participants 199 293 men and 288 082 women aged 30 to 79 years at baseline after excluding participants with cancer, heart disease, and stroke at baseline.Main exposure measures Consumption frequency of spicy foods, self reported once at baseline.Main outcome measures Total and cause specific mortality.Results During 3 500 004 person years of follow-up between 2004 and 2013 (median 7.2 years), a total of 11 820 men and 8404 women died. Absolute mortality rates according to spicy food consumption categories were 6.1, 4.4, 4.3, and 5.8 deaths per 1000 person years for participants who ate spicy foods less than once a week, 1 or 2, 3 to 5, and 6 or 7 days a week, respectively. Spicy food consumption showed highly consistent inverse associations with total mortality among both men and women after adjustment for other known or potential risk factors. In the whole cohort, compared with those who ate spicy foods less than once a week, the adjusted hazard ratios for death were 0.90 , 0.86 (0.80 to 0.92), and 0.86 (0.82 to 0.90) for those who ate spicy food 1 or 2, 3 to 5, and 6 or 7 days a week, respectively. Compared with those who ate spicy foods less than once a week, those who consumed spicy foods 6 or 7 days a week showed a 14% relative risk reduction in total mortality. The inverse association between spicy food consumption and total mortality was stronger in those who did not consume alcohol than those who did (P=0.033 for interaction). Inverse associations were also observed for deaths due to cancer, ischemic heart diseases, and respiratory diseases.Conclusion In this large prospective study, the habitual consumption of spicy foods was inversely associated with total and certain cause specific mortality, independent of other risk factors of death. Spices have been an integral part of culinary cultures around the world and have a long history of use for flavoring, coloring, and preserving food, as well as for medicinal purposes. The increased use of spices as flavorings in foods is a major trend worldwide.3The beneficial effects of spices and their bioactive ingredients such as capsaicin have long been documented in experimental or small sized population studies. For example, an ecological study showed that populations with a higher consumption of spices have a lower incidence of cancer.We prospectively examined the associations of the regular consumption of spicy foods in a daily diet with total and cause specific mortality in the China Kadoorie Biobank (CKB) study of 0.5 million adults.14The China Kadoorie Biobank is a prospective cohort study of over 0.5 million adults from 10 geographically diverse areas across China; participants were enrolled between 2004 and 2008 and have been followed up ever since for morbidities and mortality. Further details of the China Kadoorie Biobank study have been described elsewhere.In the present study we excluded 2577 people with cancer, 15 472 with heart disease, and 8884 with stroke at baseline, as well as three people with a recorded implausible censoring date for loss to follow-up. The final analyses included 199 293 men and 288 082 women. All participants provided information on spicy food consumption. All gave informed consent before taking part.There was no patient involvement in this study.In the baseline questionnaire we asked the participants “During the past month, about how often did you eat hot spicy foods?”: never or almost never, only occasionally, 1 or 2 days a week, 3 to 5 days a week, or 6 or 7 days a week. The participants who selected the last three categories were further asked “When you eat spicy foods, what are the main sources of spices usually used?” .After completion of the baseline survey in July 2008, about 5% randomly chosen surviving participants in 10 survey sites were resurveyed during August and October of 2008.We obtained covariates from the baseline questionnaire, including sociodemographic characteristics , lifestyle behaviors , personal health and medical history , and information on family members, including biological parents and siblings who had had cancer, heart attack, stroke, or diabetes. The daily level of physical activity was calculated by multiplying the metabolic equivalent tasks (METs) value for a particular type of physical activity by hours spent on that activity per day and summing the MET hours for all activities. Habitual dietary intake in the past year was assessed by a qualitative food frequency questionnaire. A participant was considered as having a family history of a particular disease if they reported at least one first degree relative with the disease.2. A stepwise on-site testing of plasma glucose level was undertaken using the SureStep Plus meter . Prevalent hypertension was defined as a measured systolic blood pressure of 140 mm Hg or more, a measured diastolic blood pressure of 90 mm Hg or more, self reported diagnosis of hypertension, or self reported use of antihypertensive drugs at baseline. Prevalent diabetes was defined as a measured fasting blood glucose concentration of 7.0 mmol/L or more, a measured random blood glucose concentration of 11.1 mmol/L or more, or self reported diagnosis of diabetes.At baseline, trained staff measured body weight, height, and blood pressure using calibrated instruments. Body mass index was calculated as weight (kg)/(height (m)We ascertained vital status by means of linkage with local disease surveillance points system death registriesTrained staff blinded to baseline information classified any deaths occurring among participants by using ICD-10 codes . The deaths were grouped into seven categories: cancer (C00-C97), ischemic heart diseases (I20-I25), cerebrovascular diseases (I60-I69), diabetes mellitus (E10-E14), diseases of the respiratory system (J00-J99), infections (A00-B99), and all other causes. Losses to follow-up in this study refer to participants whose permanent registered residence was no longer in the study area, who could not be contacted after at least three reasonable efforts within one year, or who could be contacted but their new residence was out of the jurisdiction of the regional coordinating center.We measured person years from baseline (2004–08) until the date of death, loss to follow-up, or 31 December 2013, whichever occurred first. Cox proportional hazards regression models were used to estimate the hazard ratios and 95% confidence intervals of mortality for spicy food consumption, with age as the underlying time scale. We accounted for the group specific effect of 10 survey sites on the hazard function by stratifying on the survey site variable in the Cox model.Multivariate models were adjusted for established and potential risk factors for death: age ; sex ; level of education ; marital status ; alcohol consumption ; smoking status ; physical activity in MET hours a day (continuous); body mass index (continuous); intake frequencies of red meat, fresh fruits, and vegetables ; prevalent hypertension and diabetes at baseline (presence or absence); menopausal status ; and status of family history of cancer, heart attack, stroke, or diabetes (presence or absence). We adjusted for the family history variables only in the corresponding analysis of cause specific mortality.To examine the robustness of our findings, we also conducted several sensitivity analyses: additionally adjusting for occupation and household income; additionally adjusting for histories of chronic hepatitis or cirrhosis, peptic ulcer, and gallstone or cholecystitis; adjusting for a 13 level detailed smoking variable, which incorporated the information on amount of smoking in regular smokers and the time since quitting in former smokers, instead of a four level smoking variable; excluding participants dying during the first two years of follow-up; excluding participants who had diabetes at baseline; excluding participants who reported exclusive use of other spices instead of any types of chilli; stratifying analyses by rural or urban residence; and stratifying analyses by follow-up duration (<3 or ≥3 years).Subgroup analyses were conducted separately among participants who did and did not report fresh chilli pepper as one of their commonly used spices. We compared both with those who ate spicy foods less than once a week. We also examined the associations of spicy food consumption with total mortality among prespecified baseline subgroups based on age , smoking status , alcohol consumption , level of physical activity (categorized using tertile cut-offs), and body mass index . The tests for interaction were performed by means of likelihood ratio tests, which involved comparing models with and without cross product terms between the baseline stratifying variable and spicy food consumption as an ordinal variable.The statistical analyses were performed with Stata . All P values were two sided, and we defined statistical significance as P<0.05.Table 1During a median follow-up of 7.2 years , we documented 11 820 deaths among men and 8404 deaths among women. Absolute mortality rates according to spicy food consumption categories were 6.1, 4.4, 4.3, and 5.8 deaths per 1000 person years for participants who ate spicy foods less than once a week and 1 or 2, 3 to 5, and 6 or 7 days a week, respectively. Age adjusted and multivariate adjusted analyses showed a statistically significant inverse association between spicy food consumption and total mortality. In the whole cohort, compared with participants who ate spicy foods less than once a week, the adjusted hazard ratios for death were 0.90  for those who ate spicy foods 1 or 2 days a week, 0.86 (0.80 to 0.92) for 3 to 5 days a week, and 0.86 (0.82 to 0.90) for 6 or 7 days a week . No statistically significant heterogeneity was observed in the associations between spicy food consumption and cause specific mortality by sex . Nevertheless, the associations seemed to be less evident in men than in women (table 3). In addition, more frequent consumption of spicy foods in women was also significantly associated with a reduced risk of death due to infections.In the sensitivity analyses, the associations of spicy food consumption with total and cause specific mortality did not change appreciably with additional adjustment for occupation and household income; or additional adjustment for histories of chronic hepatitis or cirrhosis, peptic ulcer, and gallstone or cholecystitis; or adjustment for a 13 level detailed smoking variable instead of a four level smoking variable; or excluding participants dying during the first two years of follow-up; or excluding participants with prevalent diabetes at baseline; or excluding participants with exclusive use of other spices instead of any types of chilli (data not shown). The associations of spicy food consumption with total and cause specific mortality were consistently observed in participants from both rural and urban areas and for different follow-up durations (<3 or ≥3 years).We further performed stratified analyses according to whether the participants reported using fresh chilli pepper as their predominant spice. We found that the inverse associations of daily spicy food consumption with death due to cancer, ischemic heart diseases, and diabetes seemed stronger in the fresh chilli group than in the non-fresh chilli group in the whole cohort of women and men, and the results were statistically significant in the fresh chilli group   seemed to be stronger in those who consumed fresh chilli pepper than those who consumed non-fresh spicy foods. These data suggest that some of the bioactive ingredients are likely to be effective in driving the observed associations. Interestingly, a statistically significant inverse association between the daily consumption of spicy foods and diabetes, which was not observed in the whole cohort, was found in the subgroup that consumed fresh chilli pepper. This was consistent with previous evidence showing that dietary capsaicin may provide beneficial effects on glucose homeostasis.Our analyses showed significant inverse associations between spicy food consumption and total and certain cause specific mortality . None the less, given the observational nature of this study, it is not possible to make a causal inference. Further prospective studies in other populations would be essential to demonstrate generalizability of these findings. More evidence will lead to updated dietary recommendations and development of functional foods, such as herbal supplements.A beneficial role of spices and their major bioactive components has been reported in a variety of chronic disorders in experimental and small sized population studiesEvidence relating daily consumption of spicy foods to mortality from prospective cohort studies is lackingThe habitual consumption of spicy foods was inversely associated with total and certain cause specific mortality , independent of other risk factors of death


The apparatus had a removal efficiency of over 90% for all concentration ranges of radio–cesium and –iodine tested. The results showing efficient radionuclide removability, together with previous studies on molecular hydrogen and platinum nanoparticles as reactive oxygen species scavengers, strongly suggest that the test apparatus has the potential to offer maximum safety against radionuclide-contaminated foodstuffs, including drinking water.The Fukushima Daiichi Nuclear Power Plant accident on March 11, 2011 attracted people’s attention, with anxiety over possible radiation hazards. Immediate and long-term concerns are around protection from external and internal exposure by the liberated radionuclides. In particular, residents living in the affected regions are most concerned about ingesting contaminated foodstuffs, including drinking water. Efficient removal of radionuclides from rainwater and drinking water has been reported using several pot-type filtration devices. A currently used flow-type test apparatus is expected to simultaneously provide radionuclide elimination prior to ingestion and protection from internal exposure by accidental ingestion of radionuclides through the use of a micro-carbon carboxymethyl cartridge unit and an electrochemically reduced water production unit, respectively. However, the removability of radionuclides from contaminated tap water has not been tested to date. Thus, the current research was undertaken to assess the capability of the apparatus to remove radionuclides from artificially contaminated tap water. The results presented here demonstrate that the apparatus can reduce radioactivity levels to below the detection limit in applied tap water containing either 300 Bq/kg of  The earthquake caused a catastrophic tsunami, with the wave height of nearly 40.5 m, which caused failures in the nuclear reactor cooling system in the Fukushima Daiichi Nuclear Power Plant (FDNPP) 131I (1470±26.5 Bq/L), 134Cs (100±25.3 Bq/L) and 137Cs (129±9.47 Bq/L) 131I and 134Cs, respectively 131I in the tap water distributed by these purification plants were 210 Bq/L in Tokyo, 189 Bq/L in Ibaraki, and 220 Bq/L in Chiba, all of which exceeded the upper limit of 131I concentration set as 100 Bq/L for infants under 1 year of age by the Ministry of Health, Labour and Welfare, 1947 Although these technologies are encouraging for removal of various levels of radionuclides and further improvements are expected to arise in the future, securing safe drinking water is also of prime importance. Rainwater samples collected in Fukushima in early April, 2011 have been reported to contain 2O2), the hydroxyl radical (•OH), superoxide anion radicals (•O2−), and other molecular species Ionizing radiation emitted by ingested radionuclides causes water radiolysis by acting on the water molecules, which comprise approximately 80% of body weight in vitro and in vivo studies Considering these requirements, an apparatus designed to produce electrochemically reduced water (ERW) could be thought to fulfill such demands because it contains two functional units; an electrolysis unit for molecular hydrogen enrichment, and a micro-carbon carboxymethyl (CM) cartridge unit for removing various impurities. ERW produced from tap water by this apparatus contains as much as 0.587 ppm dissolved hydrogen . DissolvAs for the second requirement, a micro-carbon CM cartridge unit composed of a nonwoven-fabric filter, several types of activated carbon and an ion-exchange material was present in the current test apparatus to remove particulate matters, microorganisms and 13 designated impurities Cesium chloride (CsCl) and potassium iodide (KI) were purchased from Wako Pure Chemical Industries .137CsCl [0.2021 MBq/g] and Na125 I [12.950 TBq/g] were purchased from Japan Radioisotope Association . We used 125I because Kyushu University Radioisotope Center has an approval to use this radionuclide. Tap water distributed by the Fukuoka City Waterworks Bureau, Fukuoka, Japan was used in all experiments except ultrapure water  for the preparation of standard solutions for inductively coupled plasma-mass spectrometry (ICP-MS) analysis.+ ions in ERW, dependent upon the current intensity. Conversely, negatively charged I ions will be attracted to the anode side, resulting in a decreased concentration of I ions in ERW. The electrolysis currents were increased in the order of levels 1 to 4, where Level 4 represents the strongest current, reflecting the highest dissolved hydrogen (DH) and the lowest oxidation-reduction potential (ORP)  . When thTap water was used as a control. CsCl solutions of 20 liters each with concentrations of 20 and 2,000 ppb were prepared using tap water. Likewise, KI solutions with concentrations of 100 and 4,000 ppb were prepared. These solutions are designated as sample waters. The test system was arranged by placing an adjustable speed pump between the sample waters and the test apparatus to mimic tap water pressure, connected to the inlet of the test apparatus, as shown in Sample waters were passed through the apparatus, and collected filtered waters were quantitated using ICP-MS  in the Radioisotope Center at Kyushu University.137CsCl was diluted with 20 liters of tap water to prepare concentrations of 15,000, 3,000, 300, and 30 Bq/Kg. Likewise, Na125I stock solution was diluted with 20 liters of tap water to prepare concentrations of 15,000, 1,500, and 150 Bq/Kg. All other experimental conditions, such as water flow rate, system equilibration, the electrolysis conditions of the apparatus were carried out as closely as possible to those used for the non-radioisotope experiments, except that 10 ml of each of the sample waters were collected for radioactivity counting.Stock solution of Radioactive sample waters were passed through the apparatus, and collected waters were quantitated using a gamma counter  in the Center of Advanced Instrumental Analysis at Kyushu University. To evaluate the effect of the electrolysis step on radionuclide removal, filtered waters were electrolyzed by a constant current (4.2 A) at level 4 and radioactivities of ERW were quantitated as above.All experiments were performed in triplicate. Data are expressed as means ± SD for each experiment.Prior to radioisotope experiments, CsCl and KI solutions were prepared as described in the Materials and Methods section and their removability was tested. The background Cs concentration in tap water was similar to that for the filtered water , Table 2137CsCl and Na125I. The natural background counts in tap water and filtered water were below the detection limit of the gamma counter (137CsCl as controls showed the expected radioactive counts (2 = 0.999). Control waters were then passed through the micro-carbon CM cartridge and the filtrate radioactivities were measured . Radioactive control tap waters were passed through the micro-carbon CM cartridge, reducing the filtrate radioactivities significantly , hypoiodous acid (HO131I), the iodate ion (131IO3−), iodine molecules (131IO2) and organic 131I. 131I− reacts with chlorine and is transformed mainly into HOI at neutral pH. HOI is further transformed into IO3− by reacting with chlorine 3−) in tap water due to the oxidation by chlorine 131I− removal is increased by water containing 0.1–0.5 mg/L chlorine, with lower concentrations of powdered activated charcoal 131I at about 30–40% efficiency. Additionally, it has been reported that 125I− and 125I3− were prepared from 125I and used to test the removability of these species by a granular type charcoal, which resulted in a small amount of adsorption −) more efficiently than iodate (IO3−) 125I is mostly, if not completely, converted to iodate ions (IO3−) by chlorine in the tap water. In the present results, KI and 125I were efficiently removed from tap waters by the micro-carbon CM cartridge, suggesting that iodide and iodate ions were removed. The micro-carbon CM cartridge is composed of a nonwoven-fabric filter and activated carbons consisting of a coconut shell activated carbon powder, a coconut shell activated carbon conjugated with a silver compound for antimicrobial effect, and an amorphous titanosilicate-based inorganic compound  molded with a fibrous binder for shaping. This cartridge was used in the present test apparatus to remove particulate matters, microorganisms, and for qualified removability of 13 designated impurities, tested according to the standard method set by JIS S 3201, 2004  125I (applicable to Cs+ and 137Cs), even though water was supplied to the apparatus through a pump simulating tap water outlet pressure to attain 1.8–2.0 L/min flow rate, which markedly reduced the contact time of water with the activated carbon surfaces and ion-exchangers compared with those in pot-type water purifiers. It has been reported that the above-mentioned molded activated carbons can replace the hollow fiber membrane filter that is commonly used in other water purifiers to eliminate materials larger than 0.1 µm in size 3−) ions because their radius is 0.326 nm, even when their radius is increased several fold in water 131I by boiling tap water has been reported The FDNPP accident liberated various radionuclides, including +) in water and in soils + could be efficiently removed by the micro-carbon CM cartridge tested here. The mechanism for the removal of Cs+ remains to be investigated. The Cs+ removal efficiency by the apparatus was 87.4% at 20 ppb, which is comparable to that of several pot-type water purifiers that have efficiencies of around 90% for tap water containing 40–50 µg/l (ppb) cesium chloride + appears to be low at the highest concentration (1976.5 ppb) loading. This lower removal efficiency could be explained by the amount of Cs+ getting close to system over loading because this amount is 625.3 times more Cs+ ion loading than the 3.16 ppb Cs+ ion calculated from the highest radioactivity  loading where 96.9% removability was attained  could be a secondary contamination source for vegetation via roots. Uptake of radiocesium from the root is thought to occur via the potassium transport system and is distributed rapidly within the plants Another concern related to radiocesium is its longer half-life and a characteristic of ready transfer to the human diet through plants in vitro and in vivo studies C. elegansin vitro and in vivo studies ERW is regarded as beneficial to health because of its ROS scavenging ability In conclusion, we demonstrated that radio-cesium and -iodine are efficiently removed by an apparatus containing a micro-carbon CM cartridge filter, prior to ingestion. We also suggest that the ERW produced by the test apparatus will provide maximum protection against accidentally and/or unconsciously ingested radionuclides because it contains dissolved hydrogen and Pt nanoparticles. Therefore, the test apparatus is considered to be a potential alternative tool to minimize radiation hazards caused by contaminated foodstuffs.


CD4+ T cells are central to the induction and maintenance of CD8+ T cell and antibody-producing B cell responses, and the latter are essential for the protection against disease in subjects with HIV infection. How to elicit HIV-specific CD4+ T cell responses in a given population using vaccines is one of the major areas of current HIV vaccine research. To design vaccine that targets specifically Chinese, we assembled a database that is comprised of sequences from 821 Chinese HIV isolates and 46 human leukocyte antigen (HLA) DR alleles identified in Chinese population. We then predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and also Chinese HLA-DR types. We suggest that testing this vaccine experimentally will facilitate the development of a CD4+ T cell vaccine especially catered for Chinese. Over 30 million people have died from HIV/AIDS related illnesses since HIV was discovered in the 1980s. There are currently 33 million of HIV carriers . Highly Env, Gag, and Pol) and regulatory  genes. The pol gene encodes for reverse transcriptase which is error prone. This leads to high mutation rate, 15–20% divergence between the nucleic acid sequences of different HIV clades, and 7–12% variability within each clade [HIV genome is comprised of nine structural  and AIDSVAX . These vaccines are theoretically capable of eliciting both CD8+ T cell response and neutralizing antibody response. Despite neither vaccine worked alone, in the combination, they unexpectedly lowered the HIV incidence by 31.2% in vaccine recipients; however, they did not reduce viral load [Due to the extreme sequence diversity and high mutation rate of HIV, it has been difficult to develop an efficacious HIV vaccine. A successful HIV vaccine requires inducing neutralizing antibodies and cytotoxic T cell responses, both of which can only be optimally induced and maintained in the presence of a concurrent CD4+ T helper cell response . Despiteesponses , 10. Desesponses . Later iIn order to stimulate a CD4+ T helper cell response, antigens need to be processed and presented through MHC class II molecules. The form of antigen could be either whole protein or peptide epitopes. A previous study with a subunit vaccine comprised of 18 CD4+ T helper cell epitopes has demonstrated an efficient induction of robust helper T cell response in a Phase I clinical trial in Caucasian population . WhetherTo select antigenic epitopes for a vaccine, one must address several issues. One, HIV exhibits high mutation rates, and thus conserved sequences may be needed to cover a given population. Two, the human leukocyte antigen (HLA) is highly polymorphic, and it restricts the proportion of individuals who will respond to a particular antigen , 15. To α and a highly polymorphic β chain. There are over 400 class II alleles identified, spreading among HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR loci. Among them DRB1 is the most polymorphic gene, consists of 221 alleles; followed by DPB1 and DQB1 that has 84 and 39 alleles, respectively. Whereas other gene loci may have only 1 or 2 alleles [MHC class II is a heterodimer that is comprised of a monomorphic  alleles . Therefo alleles . The aboTo overcome these complex issues of vaccine design, bioinformatics methods may help to determine common features of vaccine antigens that have potential to deal with divergent population and HIV quasispecies. Specifically, bioinformatics-based approach is the most feasible method in screening a large set of peptide epitopes and selection of promising vaccine antigens. In this study, we extracted 821 HIV sequence and 46 Chinese DRB1 alleles from public information and compiled a database. A combination of 7 public available epitope prediction algorithms was used to screen the database and identify CD4+ T cell epitopes as HIV vaccine antigens. We selected a set of 20 epitopes, which in combination could cover more than 98% of our target population.http://www.hiv.lanl.gov/) [http://www.allelefrequencies.net/) [In total, 821 HIV whole genome sequences of Chinese population were retrieved from HIV Database (nl.gov/) , and thees.net/) .Seven existing methods available in Immune Epitope Database (IEDB)  for MHC 50 = 500 nM or below. A selected epitope was removed from the epitope pool before the next prediction. The process is repeated until all epitopes were selected. All calculations of epitope selection process were conducted in INFORSENSE Knowledge Discovery Environment (KDS) software platform [All epitopes are 15 amino acids in length. To be a potential epitope, it must have a MHC binding affinity threshold of ICplatform . The matα represents the epitope; C(j) is the percentage coverage of number j DRB1 allele; δ(α) is the function to indicate whether the epitope exists in the combination of HIV sequence and DR allele, existence scored 1, and absence scored 0. S(α) is the sum of number of times of the binding of HIV sequence i and DR allele j after being standardized to the proportion of DR allele j in all DRB1 alleles. All DRB1 alleles included in the study cover 95.2% all Chinese HLA-DR alleles [In the first equation  alleles .50 was considered. Next, we ranked all epitopes based on the coverage score . After an epitope has been selected, it was removed from the database before next selection. This process was repeated until 20 epitopes were selected. The workflow diagram of this procedure was illustrated in We selected epitopes from a combined pool of epitopes through KDS platform using 7 prediction methods from IEDB with a dataset that consisted of 821 circulating HIV genome sequences in China and 46 Chinese HLA-DRB1 alleles. The epitopes bind to MHC class I molecules that were removed first, and then the value of ICA total of 46 HLA-DR alleles were identified from AFND . The allWith a combination of 7 existing epitope prediction methods in IEDB, using database comprised of 46 different DRB1 alleles and 821 full genome sequences of HIV isolates circulating in China, we then predicted 38,460,402 potential epitopes. After duplicates were removed, 21,007,527 potential epitopes remained. We scored these epitopes based on the allele coverage and total coverage score, which was in general normally distributed. As shown in  gag, env, pol, and nef genes. Six of them were in gag gene, 6 in env, 2 in pol, and 6 in nef.By using the methods described above, we obtained 20 epitopes, which in theory covered all 46 DRB1 allelic genotypes and 821 Chinese HIV sequences . All 20  gag, pol, env, and vpu genes. One published epitope that has a HIA binding IC50 above our threshold of 500, Env 566 IKQFINMWQEVKAMY, was not listed. For these epitopes, HIV coverage is from 2% to 43%, DR coverage is between 35% and 98%, and specific coverage is at highest of 41% and in sum of 69%.We then applied the new method to a previously published HIV vaccine comprised of T helper epitopes and tested in clinical trial . The tabIn this paper, we described a novel method for designing a peptide-based T helper cell vaccine for HIV, which is specific for Chinese HIV strains and Chinese MHC class II genotypes. The current method has several advantages. First, our methodology of epitope prediction is easily accessible to public use. In fact, it is a combination of all seven existing methods publically available in IEDB. The IEDB database comprises a series of most up-to-date and evidence based methods specifically created for the prediction of MHC restricted T cell epitopes. In contrast to other studies that only used one of the methods, we used them all for more accurate prediction of MHC class II restricted T helper epitopes.50 value, to determine epitopes. The other relies on machine learning, and NN-align and NetMHCIIpan methods belong to this category. New sequences are subjected to computer simulated models to predict whether any epitopes can bind to a particular MHC II to high enough affinity. The third type combines several methods together to predict epitopes. These include Consensus method and ComLib method.So far, there are three major types of bioinformatics methods for the prediction of MHC class II restricted T helper cell epitopes. One is called matrix alignment algorithm, and these are SMM, ARB, and Sturniolo methods. This algorithm uses published T cell epitopes and their respective binding affinity to MHC class II, in terms of the ICConsensus method was reported to provide highest true positive rate, followed by NN-align and ARB . NetMHCI50 value as a filter to select T cell epitopes that have the broadest population coverage. Our method did not use all 8 IEDB recommended methods but integrated 7 of the IEDB methods because the 8th IEDB method is an integration of the other seven and thus not an independent measurement. The method we used could be considered as “greedy” algorithm in the bioinformatics field, which predicts the best epitope among all in a pool of potential epitopes. Thus, we believe an integrated method that uses a combination of all seven original algorithms might be the best to predict more accurately MHC class II epitopes.In this study, we used all above seven methods simultaneously, scored the potential epitopes independently, and then used ICAnother unique feature of our study is that we designed candidate helper T cell vaccine targets specifically to the Chinese population. Most common world circulating HIV subtypes are B and C, and recombinant forms are AE and AG. In contrast, the common subtypes are B and recombinant forms are BC and AE in China , 39, 40.In comparison to a previous paper that selected MHC class II binders according to the binding affinity to multiple HLA-DR subtypes , we focuOne limitation in our study, as shown in By using our method, we obtained 20 helper T cell epitopes which covered 98.1% of HIV strains known to have been circulating in China and all Chinese HLA-DR genotypes. There are limited studies that have tested designed peptide T helper vaccine in humans. In a published paper that contains 18 T helper epitopes , our comWIILGLNKIVRMYSP was reported to stimulate strong CD4+ T responses [WIILGLNKIVRMY exhibited superior HLA-DR binding capacity [ nef gene but not vpu gene, whereas Walker's study did not cover nef but vpu. These comparisons suggest that a vaccine designed predominantly for Caucasian may not be optimal for Chinese population. One epitope, for instance, Env 566 (IKQFINMWQEVKAMY) [There was one core epitope WIILGLNKIVRMY, appeared in both studies, showing very high HIV, HLA-DR, and specific coverage. The Gag epitope with two amino acids modification esponses ; anothercapacity . AnotherQEVKAMY) , given iOur method predicted epitopes, in theory, together covered 98.1% of HIV-1 genome and Chinese specific DRB1 alleles. In comparison, Walker's study reported 18 T helper cell epitopes that cover 100% of the global population. By using a prediction algorithm which based mostly on HLA supertypes . HoweverThe allele coverage of DRB1 for Chinese was based on 1704 subjects of whom 1569 were from mainland China and 135 were from Hong Kong and Singapore. All Chinese allele data regarding DRB1 frequencies were extracted from AFND, and all 1704 subjects were Chinese Han ethnics. There is no information on other minor national groups in China available. This may lead to inaccuracy in prediction of helper T cell epitopes for the Chinese. Larger sample size may improve the quality of our prediction.In this study, we report a novel bioinformatics method for designing peptide epitope based T helper vaccine for HIV. We suggest further in vitro and in vivo experiments to be performed to test the immunogenicity of this vaccine and improvement of method of prediction to be made when necessary.


The combination of habitat loss, climate change, direct persecution, introduced species and other components of the global environmental crisis has resulted in a rapid loss of biodiversity, including species, population and genetic diversity. Birds, which inhabit a wide spectrum of different habitat types, are particularly sensitive to and indicative of environmental changes. The Caucasus endemic bird area, part of which covers northeastern Turkey, is one of the world’s key regions harboring a unique bird community threatened with habitat loss. More than 75% of all bird species native to Turkey have been recorded in this region, in particular along the Kars-Iğdır migratory corridor, stopover, wintering and breeding sites along the Aras River, whose wetlands harbor at least 264 bird species. In this study, DNA barcoding technique was used for evaluating the genetic diversity of land bird species of Aras River Bird Paradise at the confluence of Aras River and Iğdır Plains key biodiversity areas. Seventy three COI sequences from 33 common species and 26 different genera were newly generated and used along with 301 sequences that were retrieved from the Barcoding of Life Database (BOLD). Using the sequences obtained in this study, we made global phylogeographic comparisons to define four categories of species, based on barcoding suitability, intraspecific divergence and taxonomy. Our findings indicate that the landbird community of northeastern Turkey has a genetical signature mostly typical of northern Palearctic bird communities while harboring some unique variations. The study also provides a good example of how DNA barcoding can build upon its primary mission of species identification and use available data to integrate genetic variation investigated at the local scale into a global framework. However, the rich bird community of the Aras River wetlands is highly threatened with the imminent construction of the Tuzluca Dam by the government. Birds are found in all habitat types and are sensitive to environmental changes. Therefore, they are good indicator species for environmental monitoring . Main caDNA sequences are significant sources of information for improving our understanding of the evolutionary and genetic relationships of organisms . In the Mitochondrial gene cytochrome c oxidase I  is frequently used for DNA barcoding in animals. Studies show that more than 95% of species possess unique COI barcode sequences, so species-level identifications can generally be done successfully , 8. In eUntil now, using DNA barcoding to document avian diversity has been limited primarily to North America, Korea, Argentina and Scandinavia , 10, 11.™ 1016 series mist nets designed for safe bird and bat sampling, with 16x16mm mesh made of D110 netting (DENIER 110/2) that capture birds without harming them. At the station, wetlands along the Aras River are sampled every year with mist nets, 40–60 of which are open every day between mid-March to early June and mid-August to early November. Nets are checked every half hour, birds are taken out, brought to the processing center, identified, banded, measured, and released after the collection of the blood sample. The bird community of Aras River wetlands is mostly typical of eastern Europe and the species examined for this study are common and well-known European species that were identified by licensed and experienced bird banders, using detailed field guides and identification books when necessary [The study was undertaken at the Aras River Bird Research and Education Center , the first bird banding station in eastern Turkey . At the ecessary , 15, 16.TTCTCCAACCACAAAGACATT GGCAC), Bird R1 (ACGTGGGAGATAATTCCAAATCCTG), and Bird R2 (ACTACATGTGAGATGATTCCGAATCCAG) were used [For extracting DNA from the blood samples, Roche High Pure PCR Template Preparation Kit (Germany) and Invitrogen Pure Ink Genomic DNA Extraction Kit (USA) were used, and the manufacturer protocols were applied. Cytochrome oxidase I gene (COI) was used as the molecular marker of choice, as it is accepted to be the unique DNA barcoding marker by the Consortium for the Barcode of Life (CBOL) and the All Birds Barcoding Initiative (ABBI) . For PCRere used .2 (25 mM), 1.5 μl of 10 mM deoxyribonucleotide triphosphate (dNTPs), 2.5 μl of each primer (20 μM), 0.3 μl of Taq DNA polymerase (5U/μl) and 32.7 μl H2O. Cycling parameters consisted of an initial denaturation step of 1 min at 94°C followed by 35 cycles of 1 min at 95°C, 1.5 min at 51°C and 1.5 min at 72°C, with a final extension step of 5 min at 72°C.Protocols for COI amplification followed Hebert et al. . For eacPCR products were cleaned up for further use in the sequencing reactions using Roche High Pure PCR Product Purification Kit . After the clean-ups, PCR products were sent to the Macrogen Inc. (South Korea) for commercial sequencing. The obtained base sequences were cleaned with the software Sequencher v. 4.1 (Gene Codes Corp.), and Clustal X  was usedA neighbor-joining tree for each species was prepared with the samples sequenced in this study, combined with the sequences obtained from BOLD for each species, using Kimura 2-parameter distances in the software MEGA 4.0 . TCS 1.2Sylvia curruca 3.2% (based on 12 sequences), Saxicola maurus 2.8% (based on eight sequences), Phoenicurus phoenicurus 2.6% (based on 13 sequences), Parus major 1.7% (based on 21 sequences), and Caprimulgus europaeus 1.7% (based on six sequences). When these clades were removed, the mean intraspecific divergence dropped to 0.3%. The minimum interspecific distance was 6.8%  formed a separate clade, apart from the rest of the samples. Six different haplotypes were observed for this species, which clustered into three main groups in the haplotype network . Group II is composed of five species  which have designated subspecies in their ranges, show high intraspecific divergence and DNA barcodes that were different from those found in other species  were analyzed, along with two samples collected from Aras  in one of these groups  clustered in the same clade  were analyzed for the fourth species in group IV, rom Aras . Again, rom Aras . Hap 6, rom Aras .Phoenicurus phoenicurus , were differentiated from the Swedish samples (L. l. luscinioides). Similarly, the DNA barcode differentiation of Phylloscopus trochilus samples we analyzed was in concordance with the accepted subspecies designations. According to our study, samples from both countries had similar DNA barcodes.A subset of the third group consists of Passer domesticus samples from Russia, Turkey, Argentina, Norway, Kazakhstan, Canada, and Sweden analyzed in this study, a maximum of six base pair differences were observed, although 12 subspecies are recognized for this species. Being an introduced species might be the main reason behind the similarity in the DNA barcodes for the North American samples. Although the native range of the house sparrows contains most of Europe and Asia, this species is now found on every continent except Antarctica. Even though house sparrows exhibit broad phenotypic divergence in body mass, sexual dimorphism and metabolic rate in both their native and introduced ranges, DNA barcodes analyzed in this study and others are highly similar [Locustella luscinioides or Passer domesticus can distinguish them from other species, but are not useful for genetically diagnosing subspecies.In group III, the COI sequences for  similar . DNA barCettia cetti, although three recognized subspecies exist, the analyzed samples from Russia and Turkey had identical DNA barcodes. These samples may belong to Cettia cetti orientalis or Cettia cetti cetti, since these subspecies are observed both in Turkey and Russia. Another species in group III, Coracias garrulus has two subspecies in its range. Coracias garrulous garrulus was found in North Africa, Europe to Iran and southwest Siberia and Coracias garrulus semenowi was found in Iraq to west Xinjiang and south Kazakhstan. Both subspecies winter in South Africa but in distinct locations [For another species in the third group, ocations . For thiOriolus oriolus and Sylvia nisoria have two subspecies in their ranges. For Oriolus oriolous, one of the subspecies is observed in the eastern part of its range from west Siberia to the Indian subcontinent, whereas the other occurs in the western portion, from Europe to the Ural Mountains. All samples that were analyzed in this study were from the western subspecies, O. o. oriolus. Similarly, for Remiz pendulinus, all samples analyzed in this study belonged to R. p. pendulinus. As expected, all analyzed DNA barcodes were very similar to each other for these species, as they represent samples from only one subspecies for each species examined.Also in the third group were species for which all the barcodes in the BOLD most likely came from a single subspecies. Both Caprimulgus europaeus (six subspecies) and Sylvia curruca (two subspecies), two main divergent and geographically isolated haplotype groups were observed. First haplotype group was common in the western parts of the species’ range, whereas the second group was common in the east. Therefore, these species exhibit an allopatric population system. Mean intraspecific divergences were 1.7% and 3.2% for Caprimulgus europaeus and Sylvia curruca, respectively.All members of group IV had several genetically divergent subspecies with unique DNA barcodes. For both Erithacus rubecula, despite covering the ranges of different subspecies, there were only two haplotypes. The samples from Russia, Sweden, Turkey, and Norway were genetically different from the sample from Russia. The mean intraspecific divergence was 0.9%.In Parus major and Phoenicurus phoenicurus. Three and two main different haplotype groups were observed for Parus major and Phoenicurus phoenicurus, respectively. These different haplotypes had overlapping ranges. Mean intraspecific divergence was 1.7% for Parus major and 2.6% for Phoenicurus phoenicurus. The most common haplotype of Parus major was widely distributed, and observed in Sweden, Norway, Russia, Lithuania, and the study site (Aras). According to Kvist et al. [Phoenicurus phoenicurus, genetic lineages appear to be largely sympatric [A different pattern in group IV was observed for t et al. , this obympatric , since tThe first barcoding study of birds in Turkey showed that the bird community of Aras River wetlands, 1650 kilometers east of Bulgaria and 5 km west of Armenia, has mostly an eastern European-western Russian genetic signature, a result of its important location at the intersection of three major migratory flyways from western Palearctic to Middle East, India and Africa . Even thDNA barcoding can also help to identify patterns of migratory connectivity for populations breeding in unstudied areas. Many of the species that pass through the Aras banding station are on their way to remote breeding grounds in Russia and Siberia. Because breeding ecology information from this area is so limited, DNA samples from en route migrants can be used to establish patterns of genetic similarity. The degree of genetic similarity can then be used to infer distinct breeding populations and migratory connectivity.The gathering of the DNA barcoding data also made it possible to have a snapshot of the genetic diversity in the region before the possible building of the intended Tuzluca dam on the Aras River  that wouFor the first barcoding study on Turkey’s birds, we applied genetic barcoding to birds captured in the Aras River wetlands on the Kars-Iğdır border, and compared our findings to the previous barcoding studies at continental scales. Our results generally agreed with the DNA barcoding literature both in terms of the effectiveness of COI barcodes as an identification tool and the existence of a barcoding gap . SamplesS1 FigCoturnix coturnix b) Cuculus canorus c) Ficedula parva d) Oenanthe hispanica e) Emberiza citrinella f) Emberiza hortulana.The black triangles indicate localities with GPS coordinates, the red circles indicate countries for which GPS data were not available, and the blue squares indicate the study site (Aras River Research Station). Red and blue shaded areas indicate the general distribution areas for clades. a) (TIFF)Click here for additional data file.S2 FigLanius minor b) Acrocephalus palustris c) Saxicola rubetra d) Emberiza calandra.The black triangles indicate localities with GPS coordinates, the red circles indicate countries for which GPS data were not available, and the blue squares indicate the study site (Aras River Research Station) a) (PNG)Click here for additional data file.S3 FigAlcedo atthis b) Coracias garrulus c) Oriolus oriolus d) Galerida cristata e) Remiz pendulinus f) Cettia cetti g) Phylloscopus trochilus h) Locustella luscinioides i) Sylvia nisoria j) Muscicapa striata k) Luscinia svecica l) Turdus merula m) Sylvia atricapilla n) Passer domesticus o) Passer montanusRed, a) (DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Trachurus japonicus) using echo sounder technology. A linear model showed a high R2 value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions  are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors  are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel ( Surveillance of fish species composition and biomass provides useful information for management and conservation of marine and freshwater ecosystems. For example, a single survey of fish composition at a local habitat may suggest interactions among species  ,2, whileVarious sampling methods have been used for fish surveillance, depending on the objectives of studies and the field conditions; each has advantages and disadvantages. For example, echo sounder, which generates an acoustic pulse and detects echos from fish, allows one to survey large geographical areas in a short period of time, but it is inadequate for species identification and surveillance in environments with many obstacles such as coral reefs. In such environments, underwater visual censuses should be effective for fish counts and species identification, although data from these methods may contain bias resulting from fish and diver (observer) behavior –12. MoreDetection and quantification of species-specific environmental DNA (eDNA), DNA molecules originally shed from the bodies of organisms in the environment , might Trachurus japonicus) (-1) [In the present study, we assessed whether the distribution of a fish species in a sea could be monitored based on the spatial variation in the quantity of eDNA. To this end, we collected seawater samples over a wide area in Maizuru Bay, Sea of Japan, and quantified eDNA of the Japanese jack mackerel (ponicus) . This baus) (-1) , providius) (-1) –28. We cField research in the present study was approved by the harbormaster of Maizuru Bay (Permission Number 191 issued at 9 June 2014).2 , was ~8,000. Moreover, the underwater visual censuses indicated that jack mackerel usually exceeds 200 individuals/400m2 in June in Maizuru Bay while only four species reach 20–150 individuals/400m2 even in years that each species was abundant. In addition to those species, the other species are 20 or less individuals/400m2. Therefore, we can assume that detected signals by echo sounder in June in Maizuru Bay predominantly indicate jack mackerel. Only Japanese anchovy occasionally shows ~200 individuals/400m2. However, because the average body size of Japanese anchovy is ~2 cm in our research season, echo intensity of Japanese anchovy individual would be extremely small  [To examine the association between the estimated concentration of jack mackerel eDNA and the biomass estimation using echo sounder, water sampling and an acoustic survey were conducted on 18 June 2014 in west Maizuru Bay, which has a surface area of ca. 11 kmviduals) . TherefoThe echo sounder surveys started from the mouth of the bay (near St. 47) and moved southwestwardly to the end of the bay (near St. 1). Our survey cruises were conducted during the day, and it took 6 hours. The tracks of our ship are depicted in Seawater samples for eDNA analyses were collected both from the sea surface using buckets and from ~1.5 m above the sea bottom using van Dorn water samplers at 47 sites in west Maizuru Bay on 18 June 2014 . We collTotal eDNA was extracted from each filter using a DNeasy Blood and Tissue Kit  with a minor modification to adjust for eDNA extraction. Briefly, a sample filter was placed in the suspended part of a Salivette tube . Then, 440 μL solution composed of 40 μL Proteinase K and 400 μL AL Buffer was put on the filter and the tube was incubated at 56°C for 30 min. After incubation, the liquid held in the filter was collected by centrifugation. To increase the yield of eDNA, 200 μL TE buffer was put on the filter and the liquid was again gathered by centrifugation. We added 200 μL AL buffer and 600 μL ethanol to the collected liquid, and transferred the mixture to a spin column. Subsequently, we followed the manufacturer’s instructions and total eDNA was eluted in 100 μL AE buffer. To check for cross-contamination during eDNA extraction procedures, we simultaneously extracted eDNA from an unused filter (extraction negative control). We first extracted eDNA from one of three filters from each of 94 sampling points (both surface and bottom sampling at 47 sites). The set of filters was defined as filter series 1. We then extracted eDNA from one of the other filters and the set of filters was defined as filter series 2. Finally, the remaining filters were DNA-extracted and defined as filter series 3.b (CytB) gene was performed using real-time TaqMan® PCR with a StepOne-Plus™ Real-Time PCR system . We amplified and quantified eDNA using primers and a probe specific to jack mackerel: forward primer, 5´-CAG ATA TCG CAA CCG CCT TT-3´; reverse primer, 5´-CCG ATG TGA AGG TAA ATG CAA A-3´; probe, 5´-FAM-TAT GCA CGC CAA CGG CGC CT-TAMRA-3´ . The primers specifically amplify a 127 bp fragment of the jack mackerel CytB gene. Each 20 μL TaqMan reaction contained 2 μL extracted eDNA solution, a final concentration of 900 nM forward and reverse primers and 125 nM TaqMan probe in 1 × PCR master mix (TaqMan gene expression master mix). Quantitative PCR (qPCR) was performed with the following conditions: 2 min at 50°C, 10 min at 95°C, and 55 cycles of 15 s at 95°C and 1 min at 60°C. For each eDNA sample , PCR was performed in triplicate. To quantify the number of jack mackerel CytB genes in each 2 μL eDNA solution sample, we simultaneously performed qPCR in triplicate using a dilution series of standards containing 3 × 101–3 × 104 copies of a commercially synthesized artificial DNA fragment that included the jack mackerel CytB sequence amplifiable with the above primer set. The artificial DNA fragments were pUC57 plasmids containing 327 bp of the partial CytB gene: the amplified 127 bp region and 100 bp upstream and downstream of the amplified region. Prior to qPCR analysis, the plasmids were digested with a restriction enzyme (EcoRI). In addition, a 2 μL pure water sample was analyzed simultaneously in triplicate as a negative control in the PCR (PCR negative control). In all the runs, R2 values of calibration curves were more than 0.992, the range of slopes were between –3.951 and –3.370, and the range of intercepts were between 39.055 and 42.264. Based on the calibration curve of each run and the Ct value of each sample, the copy number of CytB gene fragment was calculated. Finally, to confirm the species-specificity of those primers and the probe, we determined sequences of 47 samples that were randomly selected from amplified samples by Sanger sequencing. As a result of BLAST search using NCBI nucleotide database, all the sequenced fragments had a jack mackerel CytB gene sequence.To evaluate the amount of eDNA derived from jack mackerel, quantification of the copy of mitochondrial cytochrome Jack mackerel biomass was estimated using a calibrated quantitative echo sounder following a standard acoustic survey method . We useWe eliminated noise from the obtained echo intensity data using Echoview ver. 6.0 . According to a regression described in a previous study , we assua priori knowledge of the relevant spatial scale of fish biomass reflecting the spatial pattern of eDNA concentration inside the bay, we obtained 20 datasets of local echo intensity, which was determined as the cumulative echo signals within a defined volume of the water column, from the original echo intensity data  on cruise trajectories. To estimate the local echo intensity, we considered five levels of horizontal range (buffer area) and four levels of vertical range, which were used to define the water columns surrounding each sampling station. Horizontal ranges were within a 10, 30, 50, 150 and 250 m radius from each sampling station , and vertical ranges were within 2, 5, and 10 m from both the surface and bottom at each sampling station, as well as the entire vertical range of the sea . All possible combinations of these horizontal and vertical ranges yielded 20 datasets of local echo intensity for each sampling station: for example, a station might have a local echo intensity value of a 10 m buffer/2 m vertical water column, a 10 m buffer/5 m vertical water column, and so on. Within each water column, we obtained a series of sv values and the integrated sv between the vertical ranges , along the cruise trajectory. This integrated sv value is identical with area backscattering coefficient , which indicates echo strength per a water column with a cross-sectional area of 1 m2 . Then the average of integrated sv values within each water column was multiplied by the area of the water column, yielding the estimated cumulative echo signals within each column  in water sampled at St. 2, the nearest site to the fish market (unpublished data). In addition, we also considered (5) a binary  explanatory variable that indicates whether the local echo intensity was zero or not (represented as ‘no-echo’), because in some water columns the cumulative echo signal was zero-valued and hence we could not take the logarithm. Then we assigned echo = 0 and no-echo = 1 for these data for use in the analysis. Note that an explanatory variable, no-echo, was only included in the regression models that involved its counterpart variable, echo. As for echo, interactions between no-echo and other covariates were also considered. No serious collinearity was found in these explanatory variables (1–5), which was confirmed by calculating the variance inflation factor  local echo intensity (represented as ‘echo’ in the following formula), as our primary interest; (2) vertical position of water samples ; (3) filter series (‘filter’); and (4) a measure of the inverse of the distance of each sampling station from the fish market  as covariates. As mentioned above, there were 20 values of local echo intensity (echo) for each sampling site corresponding to the differences in the horizontal and vertical spatial scale of the water column. We assumed that the spatial scale of the local echo intensity was constant among sampling sites, whereas it might vary across the vertical sampling position. The three covariates appeared to affect the observed variation in eDNA concentrations. Filter series was considered because DNA concentration seems to decrease in later filter series; DNA extractions were conducted from series 1 to 3 at different times and filter series 2 and 3 would probably undergo freezing and thawing, causing reductions in eDNA concentration . Distancn factor . The resn factor  were excn factor .r = 0.87, 0.84 and 0.86 for filter series 1 vs. 2, 1 vs. 3 and 2 vs. 3, respectively; p < 0.0001 for all pairs). The quantity of jack mackerel eDNA varied among the 47 sampling sites  were selected for both surface and bottom water samples, whereas for the latter, water columns of 150 m in radius horizontally and 5 m vertically were selected for surface samples and water columns of 50 m in radius horizontally and 2 m vertically were selected for bottom samples .Statistically significant partial associations between the estimated eDNA concentration and echo intensity were observed ; Fig 5. Some other covariates were also significantly associated with eDNA concentration. We especially note the positive relationship between inv-dist, the distance index between the fish market and sampling sites, and eDNA concentration, which indicates that eDNA concentration tended to be high at sites that were located near the market . MoreoveUnlike surface samples, bottom samples frequently shows the lower bound of eDNA concentration . Analysis based on linear regression models sometimes underestimate an association between explanatory and dependent variables when data includes a high proportion of the lower bound value . To account for this, we also applied the Tobit regression model, which can accommodate censored dependent variables. As a result, we ensured that quantitatively similar results were obtained by fitting the Tobit regression model .2) is larger and the amount of flow should be greater than such previous research systems. Although water flow in the bay would homogenize eDNA, our results suggest that eDNA concentration indicates biomass of marine fish.We found significant partial associations between spatial variation in estimated eDNA concentration from jack mackerel and echo intensity. This suggests that the concentration of eDNA reflects the fish distribution and biomass across west Maizuru Bay . AlthougOn the other hand, the present study suggests that eDNA sources other than live individuals may confound distribution and biomass estimations using the eDNA method. For example, the concentration near the wholesale fish market St. 2) was much higher than at the other sampling stations and variation in eDNA concentration among sites were strongly associated with distance from the fish market , such th was muchThe relevant sizes of water columns selected by AIC were relatively small . Thus, our results might suggest limited vertical spread of eDNA.The vertical spread of eDNA might also be minor in west Maizuru Bay. The best vertical sizes differed for the total eDNA dataset compared to eDNA data without outliers ; the entire water column (from surface to bottom) was selected with the minimal AIC for all data, whereas small sizes (2 or 5 m) were selected for data without outliers . At St. To evaluate spatial variation in eDNA concentration, we used a quantitative analysis of eDNA instead of DNA metabarcoding analysis. Quantitative analysis has an advantage in that the obtained results, that is, eDNA concentrations, can be compared among studies. This advantage also allows comparisons of temporal variation in eDNA concentration, thereby facilitating the study of temporal trends or fluctuations in eDNA concentration e.g., ,44,45). ,45. 44,42) in approximately 6 hours. This implies that a snapshot of localized fish resources over a large area can be revealed using the eDNA method combined with a grid survey such as we conducted in the present study. Several studies on stream organisms that conducted surveillance over large areas successfully found rare or threatened species using the eDNA method [Our findings suggest that the eDNA method will allow for quick surveillance in seas and large lakes. We collected water samples at 47 stations in west Maizuru Bay  within a water column multiplied by water area. (a) Buffer area is defined as a circle with a radius of assigned distance from a sampling station. Buffer area is usually identical with water area of a column although when there were land areas within the buffer area, actual water area (green) is a circle area without the overlapping land area (dark gray). (b) Green areas indicate the vertical range of a water column. We obtained sv values that reflect fish biomass in a cube 1 m on a side using quantitative echo sounder. The integrated sv is the sum of sv values beneath each data point in assigned vertical range.Local echo intensity was determined as the mean of s(EPS)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableCoefficient of linear regression models (a) and Tobit regression models (b) that are selected by AIC.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Neuromuscular respiratory failure is a rare complication of systemic immunoglobulin light chain amyloidosis. We describe a case of a 70-year-old Caucasian man with multiple myeloma who presented with worsening dyspnea. The patient was diagnosed with and treated for congestive heart failure but continued to suffer from hypercapnic respiratory insufficiency. He had restrictive physiology on pulmonary function tests and abnormal phrenic nerve conduction studies, consistent with neuromuscular respiratory failure. The diagnosis of systemic immunoglobulin light chain amyloidosis was made based on the clinical context and a cardiac biopsy. Despite treatment attempts, the patient passed away in the intensive care unit from hypercapnic respiratory failure. Autopsy revealed dense diaphragmatic amyloid deposits without phrenic nerve infiltration or demyelination or lung parenchymal involvement. Only 5 cases of neuromuscular respiratory failure due to amyloid infiltration of the diaphragm have been described. All cases, including this, were characterized by rapid progression and high mortality. Therefore, diaphragmatic amyloidosis should be on the differential for progressive neuromuscular respiratory failure in patients with multiple myeloma or any other monoclonal gammopathy. Given its poor prognosis, early recognition of this condition is essential in order to address goals of care and encourage pursuit of palliative measures. A 70-year-old Caucasian man with a previously diagnosed, IgG-kappa multiple myeloma presented with a 3-month history of worsening dyspnea, dysphagia, and weight loss. The diagnosis of multiple myeloma was established one year prior to presentation with a serum kappa free light chain (K-FLC) level of 685 mg/dL and a lambda FLC level of 0.13 mg/dL (kappa/lambda (K/L) ratio of 5269). The patient had no lytic bone lesions or liver or heart involvement and had a normocellular bone marrow with 70% plasma cells. He was never noted to have macroglossia or albuminuria. His past medical history was otherwise notable for hypertension, type II diabetes mellitus, atrial fibrillation, and chronic kidney disease with Bence Jones proteinuria (K-FLC proteinuria of 691 mg/dL). Upon initial evaluation, the patient was hypoxic to 78% in room air. He appeared tachypneic, with an increased work of breathing and accessory muscle use. Auscultation revealed overall reduced breath sounds, bibasilar crackles, and an irregular cardiac rhythm with a holosystolic grade III/VI murmur loudest at the cardiac apex.2 of 60, and a pO2 of 60 mmHg. His labs otherwise revealed an bicarbonate of 33 mmol/L, a creatinine of 2.55 mg/dL (increased from a baseline of 1.2 mg/dL), a hemoglobin of 10.3 g/dL, and a white blood cell count of 7.4 × 103 per μL. The patient also had a B-type natriuretic peptide level of 572 pg/mL and serial cardiac troponin I levels of 0.22, 0.22, and 0.21 ng/mL drawn 6 to 8 hours apart. His K-FLC level was 1379 mg/dL with L-FLC of 0.65 mg/dL (K/L-FLC ratio of 2121).The patient's arterial blood gas revealed a pH of 7.31, a pCOA chest radiograph showed mild pulmonary vascular congestion with small bilateral pleural effusions. His echocardiogram was notable for elevated pulmonary artery systolic pressure of 64 mmHg, biatrial dilatation with normal sized ventricles, and a preserved ejection fraction. A ventilation-perfusion scan was negative for acute or chronic pulmonary embolus.Subsequently, the patient was admitted to the cardiac stepdown unit for inotropic diuresis and noninvasive positive pressure ventilation (NIPPV). After diuresis, a right heart catheterization (RHC) revealed PA pressure of 34/9/18 mmHg (systolic/diastolic/mean) and a pulmonary capillary wedge pressure of 10 mmHg.Despite effective diuresis, the patient went into a worsening hypercapnic respiratory failure with almost continuous dependence on NIPPV. His pulmonary function tests revealed restrictive physiology with FEV1 of 61%, FVC of 58%, and FEV/FVC of 104%. A subsequent nerve conduction study was suggestive of bilateral phrenic axonal neuropathy.With his worsening dysphagia and respiratory muscle weakness, the course was further complicated by aspiration pneumonitis. A barium swallow esophagram showed narrowing of the gastroesophageal junction and tertiary contractions in the esophagus. An upper endoscopy was unrevealing and biopsy samples were negative for amyloidosis, confirmed by lack of Congo red staining (not shown). A fat pad biopsy was also negative for amyloid.Due to deteriorating cardiopulmonary and renal status, the patient was transferred to our cardiac intensive care unit. A repeat RHC was performed and a right ventricular biopsy was diagnostic of cardiac amyloidosis. The patient subsequently received 3 doses of bortezomib and dexamethasone but passed away soon after from the multiorgan system failure, approximately 5 weeks after admission.Autopsy revealed IgG-kappa amyloid deposits in the left and right ventricles , the diaThe differential diagnosis of hypercapnic respiratory failure is extremely broad. It is ultimately the result of inadequate ventilation, which leads to an increase of the partial pressure of carbon dioxide in the blood. This subsequently leads to acidemia and concurrent hyperkalemia, both of which promote arrhythmias, muscle weakness, and CNS depression leading to death. This failure can be caused by disease in any organ system that is essential to the initiation and propagation of breathing including the airways, alveoli, central nervous system, peripheral nervous system, respiratory muscles including diaphragm, and chest wall. We propose that in this case the failure was caused by a mechanical defect in diaphragmatic function secondary to light chain amyloid infiltration of the diaphragm. We demonstrate sparing of the phrenic nerve and lung parenchyma on pathology, further supporting the hypothesis that diaphragmatic infiltration is the primary cause of respiratory failure in this reported case.This rapidly progressive hypercapnic respiratory failure secondary to infiltration of the diaphragm by amyloid is only the fifth such case described in the literature. We have conducted an extensive search using National Library of Medicine database, looking for the words “amyloid”, “amyloidosis”, and “diaphragm”. We were able to find only 4 other relevant cases in the literature. The first published article describes a 56-year-old black man with multiple myeloma . AutopsyTo our knowledge, there is only one case report that proposes an alternative theory of respiratory failure driven by a peripheral nerve infiltration with amyloid . This coBoth our case and that by Berk et al. show abnormal nerve conduction studies. However, in the absence of pathology to suggest nervous system involvement or demyelination, it is very unlikely for phrenic nerve neuropathy to be the cause of respiratory failure in such patients. Therefore, nerve conduction studies should not be considered diagnostic. This becomes important when measures such as diaphragmatic pacing are considered, as it may be of benefit when the nervous system is involved but unlikely to benefit a diaphragm infiltrated by amyloid.This case report highlights a rare but a rapidly fatal complication of systemic amyloidosis. The reported cases over the past 30 years show a 100% association between amyloid infiltration of the diaphragm and respiratory failure. In 4 out of the 5 cases of diaphragmatic amyloidosis reviewed, the diagnosis of respiratory failure carried an 80% mortality rate within 1 month. Only in 1 case did the patient survive hospitalization [