About 55% and 82% of stations in strata II and III, respectively, were dominated by silt-clay fractions (<63 μm mean diameter), coherent with TOM data. In addition, sediments in the deep area (>50 m) tend to be poorly sorted, very fine skewed, and platykurtic. Unlike data obtained 20 years ago which reported high content of silt-clay (58%), this study recorded a lower content (35%); therefore, changes in sediment load had been observed in southern SCS. Sediment distributions in deep sea influence the benthic community structure and thus play an important role in shaping the marine ecosystem. Several studies on sediment characteristics had been conducted in South China Sea (SCS), but only limited to coastal areas of regions within SCS territories. Therefore, this study was carried out to analyze the benthic sediment profile in an area beyond 12 nautical miles off the coast of Sarawak, southern SCS. Sediment samples were collected from 31 stations, comprising three depth ranges: (I) 20–50 m, (II) 50–100 m, and (III) 100–200 m. The total organic matter (TOM) contents were determined and subjected to dry and wet sieving methods for particle size analysis. TOM contents in the deep area (>50 m) were significantly higher ( Sedimen2 . The hab2 . The ana2 .It is presumable that the sedimentary environment will continue unchanged if the interaction remains stable unless some changes take place in any of the factors and within a period of time, which led to the alteration of sediment . SedimenAccording to Kao et al. , more thPreviously, there were a large number of literatures on sediment profile in the continental shelf of Sarawak. For example, Chong and Idrus  focused A three-month (16 August until 6 November 2015) sampling cruise was carried out in an area beyond 12 nautical miles off the coast of Sarawak. Sediment collection is one of the side activities from the main survey  of the cruise. The study area comprised sandy, muddy, and rocky bottoms and hard corals, besides consisting of sloping areas with a depth of more than 200 meters. Samples were collected from 31 selected stations  which coA is the initial dry weight  and B is the final weight .The total organic matter (TOM) analysis was conducted to examine the loss of weight on ignition and a method by Greiser and Faubel  was usedμm; coarse sand: 500 μm; medium sand: 250 μm; fine sand: 125 μm; very fine sand: 63 μm) and silt-clay fraction (particle size <63 μm). Udden-Wentworth's [Grain size analysis defines the sedimentary environment characteristic with a series of particle size distributions. The analysis was conducted to characterize the continuous distribution from small particles to many millimeters of sediment fractions. Dry sieving and wet sieving methods were used in the measurement of sand and silt-clay fraction, respectively, following the method by Holme and McIntyre . The mettworth's , 23 gradThe 3D graph of TOM by depth strata was plotted using OriginLab 9.0™ software. All data were subjected to one-way analysis of variance (ANOVA) and multiple-comparisons (Tukey's) test was used to examine significant differences among averages of mean value of stations in all contours. Correlation analysis was used to compare differences of mean between TOM with sediment fractions with significance level at 0.05 using Minitab 17™.In order to compare sedimentary environments with each other quantitatively, four parameters were used to describe the grain size distribution: (a) the particle average size, (b) the spread (sorting) of the sizes around the average, (c) the symmetry or preferential spread (skewness) to one side of the average, and (d) the degree of concentration of the grains relative to the average (kurtosis). These properties were determined by the logarithmic method of moments and descriptive terminology  by KrumbMapping technique to portray information into a map is the common and basic study on marine geographical information systems (GIS) works. Distributions mapped in this way provide a good visual relationship between sediment characteristics with sea depth and distance to coastline. Sediment data obtained from Sarawak waters were presented into a map using ArcGIS 10.1™ software. The information of total organic matter (TOM), sea depth, and sampling location (latitude and longitude) was compiled into visualized information into maps.a%, 37.19 ± 10.08b%, and 34.31 ± 5.08ab%, respectively. These results had shown that contour II had significantly higher organic matter compared to contour I whereas there is no difference of TOM value with contour III.As can be seen from μm, 500 μm, 250 μm, 125 μm, 63 μm, and <63 μm are referred to as very coarse sand (VCS), coarse sand (CS), medium sand (MS), fine sand (FS), very fine sand (VFS), and silt-clay (SC), respectively. Out of 9 stations in contour I, 4 stations recorded the highest percentage of FS fractions. Meanwhile, in contour II, out of 11 stations, 6 stations recorded the highest percentage of SC fractions. For contour III, out of 11 stations, 9 stations recorded a high percentage of SC fraction.In the present study, the distributions of particle size vary according to station . SedimenThese results had shown that FS was dominantly found in contour I, while SC was dominantly found in contour II and contour III, respectively. Thus, the average means were calculated to summarize sediment distributions of each depth contour. Statistical analysis was adapted to the sediment size frequency and sediment was described in terms of analysis of mean size, sorting, skewness, and kurtosis . The aveThe degree of asymmetry (skewness) of the sediment in Sarawak EEZ was dominated by fine sediment which is represented by a positive (+) value. Therefore, in contour I, there are 1, 7, and 1 stations that showed very fine skewed, fine skewed, and coarse skewed sediment distribution frequency, respectively. In contour II, there are 5, 3, 2, and 1 stations that showed very fine skewed, fine skewed, symmetrical, and very coarse skewed sediment distribution frequency, respectively. In contour III, there are 7, 3, and 1 stations that showed very fine skewed, fine skewed, and symmetrical sediment distribution frequency, respectively.The degree of peakedness (kurtosis) of the grains relative to the average was calculated. In contour I, 56%, 22%, and 22% of the stations showed platykurtic, mesokurtic, and leptokurtic characteristics, respectively. For contour II, 45%, 27%, and 27% of the stations showed platykurtic, mesokurtic, and leptokurtic characteristics, respectively. In contour III, 72%, 18%, and 9% of the stations showed platykurtic, mesokurtic, and leptokurtic characteristics, respectively. Lastly, as a summary, from all 31 stations sampled, 58%, 23%, and 19% of the stations showed platykurtic, mesokurtic, and leptokurtic characteristics, respectively.The fine fraction sediment forms a good bonding with the organic compound through strong cation exchange capacities for organic adsorption on the sediment surface , 25. Accr = 0.73), resulting in a high proportion of organic matter in sediments of the continental shelf of Sarawak. For example, in contour I, the highest silt-clay fractions were at station 48 which also recorded high organic content (53.5%). Apart from that, the high content of sand fractions showed a low amount of total organic content. As an example in contour I, the highest sand fractions were at station 74 which recorded a low amount of organic matter (22.2%). Thus, the results obtained showed a similar trend to studies that were conducted in the continental shelf of Southern California [Basically, it was suggested that the organic compounds are well composed in fine or silt-clay fraction. Similarly, in the present study, positive correlation was obtained between organic matter and silt-clay fraction  of sand than the data obtained from the last two decades of survey whereas only 42% of sand had been reported by Husain et al. . Howeverμm. In contrast, during this study, in contour I, 44% of the mean diameters ranged from 125 to 250 μm. Meanwhile, in contours II (55%) and III (82%), majority of the mean diameters of surface sediment are less than 63 μm. In addition, μm and this value is within medium to fine sand fraction where it is the best sorting value as reported by Inman and Chamberlain [A previous study conducted by Gorsline  showed tmberlain . A partimberlain . This phmberlain , environIn the present study, 78%, 54%, and 45% of stations in contours I, II, and III, respectively, demonstrated sediments that were moderately sorted. Meanwhile, 22%, 45%, and 54% of stations in contours I, II, and III, respectively, demonstrated sediments that were poorly sorted . SimilarAt a depth of 50–200 m in the present study, a high excess of fine sediment or very fine skewness was recorded. However, the study conducted by Husain et al.  reportedThis study provides useful information on the sediment profile in the continental shelf of Sarawak. High amounts of TOM were positively correlated with silt-clay fractions. For the sediment distribution, FS was dominantly deposited in contour I, while SC was dominantly deposited in contour II and contour III, respectively. The highest grain size distribution in the continental shelf of Sarawak is SC, followed by FS, VFS, MS, CS, and VCS. In addition, deeper sediments tend to be more poorly sorted, very fine skewed, and platykurtic compared to the shallow area. The increment of sand fractions in the surface sediment of the continental shelf of Sarawak should be seen as a serious problem. Therefore, there is a need for further research, in order to investigate the recent status of benthic sediment alteration for better management and actions by relevant agencies.


Uremia is likely a risk factor for deep neck infection (DNI). However, only a few relevant cases have been reported, and evidence sufficient to support this hypothesis is lacking. The aim of the study is to investigate the effects of end-stage renal disease (ESRD) on DNI.We used the database of the Registry for Catastrophic Illness Patients (RFCIP), a subset of the National Health Insurance Research Database (NHIRD) in Taiwan, to conduct a retrospective follow-up study. Between 1997 and 2013, a total of 157,340 patients in Taiwan with ESRD who received dialysis were registered in the RFCIP, whom were matched with a database consisting of 1,000,000 randomly selected patients who represented the national population, to conduct the follow-up study for investigating the incidence of DNI in the ESRD and control cohorts.p < 0.001). Kaplan–Meier analysis indicated that the ESRD group had a significantly higher cumulative incidence of DNI (p < 0.001). According to Cox regression analysis, the hazard ratio of ESRD for DNI was 2.23 (p < 0.001). The therapeutic methods (non-surgery and surgery), performance of tracheostomy, duration of hospitalization did not differ significantly between the two groups, except more ESRD-DNI patients were admitted to intensive care units. The mortality rate of patients with DNI in the ESRD group was significantly higher than that in the control group . Furthermore, the Kaplan–Meier analysis demonstrated a poorer survival outcome in the ESRD group (p = 0.029). However, the individual survival outcomes following non-surgical and surgical therapies in the ESRD group did not differ significantly (p = 0.31).In the ESRD group, 280 DNIs were identified with an incidence rate of 43 per 100,000 person-years. In the comparison group, 194 DNIs were identified with an incidence rate of 20 per 100,000 person-years. The incidence rate ratio was 2.16 (ESRD is a predisposing factor for DNI, increasing its risk by twofold. In the patients with ESRD, DNI was not associated with higher rates of surgical debridement, tracheostomy, and mediastinal complications or longer hospital stays; however, it was associated with poorer survival outcomes, regardless of the therapeutic method. Deep neck infection (DNI) is a common disease and usually presents at emergency departments, which usually requires intensive care and aggressive treatment. The wide availability of antibiotics, improvement of diagnostic technology, and the concept of early surgical debridement had significantly reduced the morbidity and mortality of DNI , 2. HoweWang 2003) analyzed 196 DNI cases retrospectively and considered uremia a risk factor for complicated DNI . In Huan analyzedESRD is widely prevalent, particularly in Taiwan, which has the highest global incidence rate for renal replacement therapy –22. PatiTo our knowledge, no studies investigating the effects of ESRD on DNI have been published. Hence, we used the National Health Insurance Research Database (NHIRD) in Taiwan to conduct a retrospective cohort study to investigate these effects, particularly concerning the incidence rate and risk of DNI (primary endpoints), as well as its interventions, complications, and survival outcomes (secondary endpoints).In March 1995, the Taiwan government started the National Health Insurance Program and accordingly enacted an insurance policy, which considerably increased the national insurance coverage from 60% to 92%. By 2016, the coverage reached 99.6% . In 2000We conducted the study in accordance with the guidelines of the Declaration of Helsinki. This study was exempted from obtaining informed consent from the participants, because the database was nationwide and the data were deidentified. All information of the insurants was unidentifiable, and this study did not violate their rights or adversely affect their welfare. The research was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB number: 201601249B1).In the Taiwan health insurance system, the category of catastrophic illness patients (CIP) has been instituted for a particular group who benefit from the treatment of relevant catastrophic illnesses. The verification of the eligibility for CIP certification requires strict investigation. For instance, a patient who has relevant diagnosis codes for ESRD and has received renal replacement therapy for at least 3 months can apply for CIP certification. Patients with CIP certification for ESRD pay concessional fees for ESRD-associated therapy, including dialysis. Insurants with CIP certification are categorized in the Registry for Catastrophic Illness Patients (RFCIP) associated with the NHIRD.Using the RFCIP, we defined a study cohort consisting of patients with ESRD who received dialysis from 1 January 1997 to 31 December 2013 in Taiwan. We used the following ESRD-associated ICD-9 codes, which was defined for RFCIP : 585 , LHID2005, and LHID2010. For example, LHID2000 consists of 1,000,000 insurants in 2000 who were randomly statistically selected  from all insurants in Taiwan. No statistically significant differences exist in age, sex, or health care costs between the LHID2000 sample group and all enrollees, according to a National Health Research Institutes report , and LHIA DNI was defined as a severe infection in the deep neck that required hospitalization for intensive care. Therefore, while determining the incidence of DNI in the study and control cohorts, we searched for diagnoses during hospitalization that included the following ICD-9 codes: 528.3 , 478.22 , 478.24 , and 682.11 (cellulitis and abscess of neck) [For each ESRD case, one control without ESRD 1:1) with matching gender, age, urbanization level, income level, DM and hypertension was randomly selected from the LHID2000 database to form a control group. The index date of the study group was the date of registry in the RFCIP for patients with ESRD, and an index date matching that of patients with ESRD was created for the control group. DNI diagnosed before the index date was excluded. A total of 127,283 controls matching the study patients were included. DNI diagnosis codes were used in the two groups to determine the incidence rate of DNI with a follow-up to the end of 2013. The follow-up period was from the index date to the diagnosis of DNI, and some patients were censored because of death , hypertension (ICD-9-CM codes: 401–405), liver cirrhosis , systemic autoimmune disease , chronic obstructive pulmonary disease (COPD) , coronary artery disease (CAD) (ICD-9-CM codes: 410–414), and cerebrovascular accident (CVA) (ICD-9-CM codes: 430–438) , 31, 32.We assessed the therapeutic methods used for treating patients with DNIs in the two groups. The treatment methods were divided into three subgroups: antibiotics alone, antibiotics with abscess aspiration or drainage, and surgical intervention. The intervention, including abscess aspiration or drainage and surgical debridement, was identified using the claims records during each hospitalization for DNI treatment. If the patients received surgical intervention, they were included in the surgery subgroup, irrespective of the performance of abscess aspiration or drainage. If the patients received abscess aspiration or drainage without surgery, they were included in the aspiration subgroup. If the patients received only antibiotic treatment during hospitalization, they were included in the antibiotic subgroup.For evaluating prognosis, we analyzed the duration of hospitalization, care in intensive care units (ICUs), performance of tracheostomy, and mediastinal complications, which were defined according to the receipt of mediastinal surgery during hospitalization or the following ICD-9-CM codes: 510, 513 and 519.2. The mortality and mediastinitis-related mortality were also investigated in both cohorts. Mortality was defined as death occurring during DNI treatment. The mediastinitis-related mortality was defined as death during DNI treatment accompanied by a diagnosis of mediastinitis.p < 0.05.The sociodemographics and incidence rate of DNI were compared between the ESRD and control groups using the Pearson’s chi-squared test. The Fisher exact test was used to compare the rates of tracheostomy, mediastinitis, and mortality. The duration of hospitalization was compared using the Student’s t test. The results of Kaplan–Meier analysis revealed the cumulative incidence of DNI in both groups. Furthermore, the analysis was used to investigate the survival outcomes. The risk of DNI for the study group was estimated using a Cox proportional hazard model. All the analyses were performed using SAS software, version 9.4 , and the level of statistical significance was set at p < 0.001).Table p < 0.001)  , and proportion of the three therapeutic methods in the ESRD group resembled those in the control group. Nearly three-quarters of patients with DNI were treated using antibiotics (ESRD: 74.3% and control: 75.3%), and one-quarter of the patients received surgical intervention (ESRD: 24.3% and control: 21.7%). Abscess aspiration for DNI treatment accounted for a small portion in both groups (ESRD: 1.4% and control: 3.1%). The performance of tracheostomy  and duration of hospitalization  did not differ significantly between the two groups; however, more patients in the ESRD group received care in ICUs . In total, seven and two patients developed mediastinal complications in the ESRD and control groups, respectively , and approximately half of those DNI cases with mediastinal complication result in mortality . The duration of hospitalization ranged from 1 to 89 days. In the 3 months after DNI, the patients with ESRD had a significantly higher mortality rate than did the controls . Kaplan–Meier analysis was used to investigate the survival outcomes, and log-rank tests revealed significantly poorer results in the ESRD cohort during the 3 months after hospitalization for treatment of DNI (p = 0.029) , whereas conservative treatment constituted the remainder. These results are inconsistent with those of previous studies. This inconsistency is likely due to previous studies being conducted in medical centers or tertiary hospitals, which receive and treat more severe DNI cases , 10–13. p = 0.032) (Table p = 0.029)   in the ESRD group, and log-rank tests revealed no significant difference in the two methods (31) Fig. . This rep = 0.249), and the rates of mediastinitis development in both cohorts were relatively consistent with the preceding data. Furthermore, in the aforementioned studies, the mortality rate in patients with DNI and mediastinitis ranged from 40% to 41% [p = 0.515), and the ratio was in accordance with the preceding data. Thus, our findings indicate that ESRD in patients with DNI is not associated with an increased risk of mediastinal complications; nevertheless, these complications entail a high risk of mortality.In previous case-series-based studies, the rate of mediastinitis developing after DNI was reported to be 1.7%–2.7% , 18, 19,Our nationwide study had some limitations. We used ICD-9-CM codes to define patients with DNIs and identified the associated interventions, complications, and mortality in both cohorts to evaluate the prognostic outcomes; however, we could not obtain information concerning the extent of disease, pathogens, laboratory tests, and imaging findings, which is usually used for evaluating the disease severity. The effects of these omitted factors on ESRD and DNI require further investigation. Furthermore, we investigated the 3-month mortality as an alternative to the DNI-specific mortality because the definite cause of death could not be identified using the database; consequently, further research is necessary.This nationwide population-based study was the first to investigate the epidemiological data of DNI development in patients with ESRD and the relevant prognosis. It corroborated that ESRD is a predisposing factor for DNI. DNI in patients with ESRD is not associated with higher rates of surgical intervention, tracheostomy, mediastinitis, and mediastinitis-related mortality or a longer duration of hospitalization relative to those of patients without ESRD; however, it would lead to a poorer survival outcome. Such outcomes are not related to the therapeutic intervention, and their cause requires further research.


S-transferase (GST) activities were assessed following exposure. The toxicities of BFRs tested can be ranked by LC50 as tetrabromobisphenol A (TBBPA) < 4,4′-isopropylidenebis[2-ethanol] (TBBPA-OHEE) < Pentabromochlorocyclohexane (PBCH) < 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) < hexabromocyclododecane (HBCD) < hexabromobenzene (HBB) < Tetrabromophthalic anhydride (PHT4). No adverse effect was observed in di(2-ethylhexyl) tetrabromophthalate (TBPH) or dibromoneopentyl glycol (DBNPG)-treated embryos. The rate of spontaneous movement was decreased in a concentration-dependent manner following exposure to four of the nine compounds. GST activity was elevated following treatment with PBCH, TBBPA, HBCD, and HBB. The results indicate that exposure to several BFRs may activate an antioxidant response and alter behavior during early development. Some of the BFRs, such as TBBPA and TBBPA-OHEE, induced adverse effects at concentrations lower than chemicals that are currently banned. These results suggest that zebrafish are sensitive to exposure to BFRs and can be used as a comparative screening model, as well as to determine alterations in behavior following exposure and probe mechanisms of action.Brominated flame retardants (BFRs) are used to reduce the flammability of plastics, textiles, and electronics. BFRs vary in their chemical properties and structures, and it is expected that these differences alter their biological interactions and toxicity. Zebrafish were used as the model organism for assessing the toxicity of nine structurally-diverse BFRs. In addition to monitoring for overt toxicity, the rate of spontaneous movement, and acetylcholinesterase and glutathione- Brominated flame retardants (BFRs) are used in a wide variety of consumer products, including electronics, textiles, insulation, and carpets to raise the ignition temperature of materials . There aGreat variety in chemical structure of brominated flame retardants exists, resulting in a wide range of physicochemical properties. Since most flame retardants are not covalently bound to the products they are designed to protect, the potential for volatilization or leaching from products over time is significant. As a result, BFRs have been reported in biological samples, including breastmilk and cord blood, and house dust and sediments worldwide ,8,9,10. Of the BFRs, tetrabromobisphenol A (TBBPA) is produced in the highest volume, particularly with the ban of PBDEs and hexabromocyclododecane (HBCD) under Europe’s REACH program . TBBPA and HBCD are the most commonly used BFRs in the US, though many more are used in mixtures and detected as environmental contaminants. For instance, Firemaster 550 and Firemaster BZ-54 are predominantly comprised of two chemicals: 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) and di(2-ethylhexyl) tetrabromophthalate (TBPH). TBB and TBPH were detected in 50% of the air samples over the Great Lakes between 2008 and 2010 . Paired Danio rerio) during early stages of development as a model organism for comparison of adverse effects of nine selected BFRs on development and behavior. Zebrafish offer the robustness of a whole-organism model with the high-throughput capability comparable to cell culture. Further, use of zebrafish as a model organism that can be quickly and easily manipulated for toxicological and pharmacological testing, and development is rapidly expanding [The purpose of the present study was to evaluate the toxicity of a variety of common-use used brominated flame retardants. PBDEs and polychlorinated biphenyls (PCBs) have been shown to disrupt hormone homeostasis and neurological development ,14, leadxpanding . Finallyxpanding .S-transferase (GST) and acetylcholinesterase (AChE) enzyme activities to probe potential mechanisms of action. AChE activity is a commonly used ecotoxicology assay for probing the neuro-muscular junction; GST activity was assessed as an indicator of oxidative stress.We hypothesized that the nine chemicals selected as a representation of BFRs with varying physicochemical properties, usage, and production would exert variable effects on embryo viability. Zebrafish embryos were exposed to the selected BFRs at varying concentrations, and mortality, developmental malformations, and behavioral alterations were assessed. In addition, we investigated glutathione-Artemia salina twice daily. The fish were spawned in tanks, and embryos were collected daily within two hours of spawning and maintained in Petri dishes until use. All rearing and exposure protocols were approved by the Institutional Animal Care and Use Committee.Adult tropical 5D zebrafish were maintained at 28 °C on a 16:8 light:dark photoperiod in a recirculating water system. Deionized water with 0.6% sea salt was used to maintain the conductivity at 500 μS/cm and a pH of 7.0. The fish were fed All BFRs used in this study were purchased from AccuStandard  in neat form, dissolved in DMSO , and stored in the dark at room temperature. The full names, abbreviations, and properties are listed in Exposure solutions were prepared by a two-fold serial dilution in culture water and 0.5% DMSO to achieve treatment concentrations of 1.25, 2.5, 5.0, 10, and 20 ppm BFR. Control embryos were treated with a 0.5% DMSO solvent vehicle. Nominal concentrations are reported in this study. The embryos were collected at 2 h post-fertilization (hpf) and dechorionated using a protease at 6 hpf. The chorion was removed to ensure that it did not selectively inhibit uptake of BFRs. Five embryos were placed in each well of a 48-well plate containing 0.75 mL of exposure solution in each well at 6 hpf and remained in a static exposure until 168 hpf. Exposures were held in a temperature-controlled incubator at 28 °C on a 14:10 light cycle. Embryos were evaluated daily for morphological malformations, embryo viability, and mortality. For all studies, 12 replicates per concentration per chemical were used. A Motic SMZ-161 light stereoscope  at 20 × magnification was used to view the embryos to determine development and observe physical malformations. Embryos used for enzyme activity were exposed under the same conditions until 120 hpf. At the end of the experiments, embryos were euthanized with tricaine methanesulfonate .Zebrafish embryos exhibit spontaneous movement (coiling and flexing) during the 17–30 hpf developmental window [50 values. If mortality was not observed (up to 10 ppm), the embryos were exposed to concentrations of 10 ppm. Six wells of embryos per sample were pooled, euthanized, and frozen (N = 4). Protein was homogenized in 100 μL of 100 mM phosphate buffered saline (PBS) with a motorized micropestle in a microcentrifuge tube. The homogenate was vortexed and centrifuged for 10 min at 13,000 × g. The supernatant was isolated in a clean microcentrifuge tube. AcSCh was added to extracts for a 3 mM final concentration. The PBS/DTNB solution was pipetted into 96-well plate for each sample, and 10 μL of protein extract were added to the wells. The absorbance was measured at 405 nm after 2 and 10 min of the addition of protein to the wells. AChE activity was calculated using the following equation: OD10−OD2/ODCAL−ODH2O; where OD is the optical density of the sample at 10 or 2 min. A calibrator was used to normalize between runs (ODCAL) and the optical density of the PBS/DTNB solution was subtracted (ODH2O). AChE activity was normalized to protein concentration, which was determined using a NanoDrop 1000 Spectrophotometer .Acetylcholinesterase activity was determined at 120 hpf following exposure to the selected BFRs. Activity was determined following a modified Ellman Protocol . The conN = 4 biological replicates. Protein was extracted in 150 μL cold homogenization buffer  at pH 7.4 with a motorized pestle in a microcentrifuge tube. The homogenate was centrifuged for 10 min at 13,000× g, and the supernatant was reserved. Lysate was added to 60 mM CDNB and 15 mM GSH in 100 mM sodium phosphate buffer (pH = 6.6). GST enzyme activity was determined using a Shimadzu UV Spectrophotometer , and the rate of CDNG-GSH conjugate formation was monitored at 340 nm. The activity rate was normalized to protein concentration, which was determined using a NanoDrop 1000 Spectrophotometer.GST activity following exposure to BFRs was assessed in whole larval lysates using CDNB as a model substrate. The method used was an adaptation of the Habig et al. protocol . BFR expp < 0.05, N = 12). Rates of spontaneous movement were analyzed for statistically significant differences using one-way analysis of variance (ANOVA), with Tukey’s post-hoc test (N = 12). AChE and GST activities were analyzed for statistical significance using one-way ANOVA, with an N = 4 for each study.Fisher’s exact test was used to determine the statistical significance of differences in the rates of mortality and malformations  activity was evaluated in this study as an indicator of oxidative stress. Although no prior data suggest that GSTs catalyze BFR compounds, GST’s function, in general, is to catalyze the deoxification and elimination of electrophiles within the cell. As such, their expression is controlled by activation of an antioxidant responsive element (ARE) in the 5’ promoter region; therefore, elevated GST enzymatic activity often indicates induction of oxidative stress. Previous studies have shown that GST activity in zebrafish does not increase following PBDE exposure [gstpi) was down-regulated following exposure to 6-OH-BDE47 [Glutathione-exposure . FurtherOH-BDE47 . GST actOH-BDE47 ; howeverTBBPA is one of the mostly commonly used flame retardants , yet it Derivatives of TBBPA, such as TBBPA-OHEE, have also been found in bird, fish, and mollusk samples ,28. In tIn our study, HBCD exposure induced curved body malformations, in line with previous reports ,32. The Although it was one of the most toxic BFRs identified in the current work PBCH is, historically, the least studied. PBCH has not been extensively used as a flame retardant, nor has its toxicity been evaluated in other model systems, to our knowledge. In addition to inducing mortality in the low ppm range, exposure to 2.5 ppm PBCH induced the greatest increase in GST activity. Further characterization of PBCH-induced toxicity may be warranted if production and use increase.HBB is a flame retardant primarily used in Japan and China and, consequently, it is not often identified in European samples ,35. ReceIn the embryonic zebrafish model, PHT4 and DBNPG did not elicit adverse effects. With a LC50 of 18 ppm, PHT4 is considerably less toxic in our model than most of the BFRs and PBDEs tested, suggesting that the human health risk associated with PHT4 exposure is comparatively low. It is heavily used as a flame retardant in urethane foam, but little to no information was previously available concerning its potential to induce toxicity . DBNPG dExposure to the two components of the FM-550 mixture, TBPH and TBB, elicited very different responses. TBB was moderately toxic with an LC50 value comparable to most PBDEs, while TBPH did not elicit an adverse effect at any concentration tested. TBPH and TBB are often in a mixture of a 4:1 ratio by weight, and thus TBPH is found in greater abundance in the environment . In factOne of the benefits of using zebrafish during early development as a model organism, is the ability to evaluate alterations in behavior. Exposure to several BFRs in the current study resulted in a decrease in spontaneous movement prior to mortality. This endpoint has been demonstrated to be very sensitive and precedes mortality and the onset of malformation. In the case of the BFRs tested in this study, alterations in spontaneous movement indicated there would be a reduced rate of survival. However, further investigation into acetylcholinesterase activity as a potential target, did not result in inhibition. This assay was conducted at 120 hpf rather than 24 hpf. Noyes et al. had similar findings for TBBPA induced hypoactivity at 24 hpf, as well as hypoactivity at 120 hpf . There dThis study compares the toxicity of nine BFRs and builds upon our previous research on PBDEs. The findings of this study suggest that the use and production of TBBPA should be reexamined, and that exposure to TBBPA may pose a risk to human and environmental health that is greater than that of PBDEs. Several of the chemicals tested, such as TBPH and DBNPG, did not induce observable adverse effects, which is encouraging given the high production rates. Future studies are needed to further assess mechanisms of action of TBBPA and HBCD. While increased GST activity is a possible indicator of oxidative stress, further testing will be required to definitively show these chemicals are causing oxidative stress in zebrafish embryos. Additionally, the mechanisms underlying alterations in early spontaneous movement should be explored. Finally, this study highlights the need for caution and comparative studies when substitute chemicals are chosen following the phase out of current-use chemicals. This concept extends to the organophosphate flame retardants and will be addressed in future studies.


CTNNB1 (β-catenin) gene is well known for its crucial role in cell adhesion and the Wnt-signaling pathway. Previous studies have shown that gain-of-function mutations in the CTNNB1 gene contribute to the occurrence and development of a variety of carcinomas in humans. Recently, de novo, heterozygous, loss-of-function mutations of the CTNNB1 gene were found that partially explain intellectual disability in some patients. Other major clinical symptoms in these patients included microcephaly, abnormal facial features, motor delays, speech impairments, and deformities of the hands and feet. In addition, approximately 75% of these patients had mild visual defects, such as astigmatism, hyperopia, or strabismusThe A 15-month-old Chinese boy, presenting with retinal detachment, lens and vitreous opacities, hypertonia of the extremities, mild thumb adduction, microcephaly, and developmental delay, is described. Targeted gene sequencing using an ophthalmic gene panel was performed to test for familial exudative vitreoretinopathy; however, the pathogenic gene was not found.Genomic DNA analysis was performed to search for causing mutations.CTNNB1 gene . Sanger sequencing of the patient and his parent confirmed this mutation and demonstrated it to be de novo. To the best of our knowledge, this is the first case report of a loss-of-function mutation of the CTNNB1 gene in an Asian population.Whole-exome sequencing revealed a novel nonsense variation in exon 11 of the CTNNB1 gene. Our finding expands the mutant spectrum of CTNNB1 gene and adds new understanding of the phenotype.Severe ophthalmic phenotype has not well been connected with loss of functional mutation of  In addition to its role in the cell–cell adhesion, β-catenin serves as a coactivator in the Wnt-signaling pathway, which plays essential roles in a multitude of developmental and homeostatic processes.–3 The CTNNB1 protein has dual functions due to its unique structure, which contains 12 central armadillo repeats motif flanked by the C-terminal transactivation domain beginning with a Helix-C motif and an N-terminus that mediates degradation of the protein. It has been demonstrated that conditional loss- and gain-of-function mutations in β-catenin result in various phenotypes in distinct organs in mice, and somatic gain-of-function mutations in β-catenin are highly associated with tumorigenesis and tumor metastasis in humans.,6 More recently, loss of function mutations in CTNNB1 were found to be associated with human disease in a cohort of 765 patients with intellectual disabilities (IDs). Subsequently, several single case reports, as well as a study of 16 patients with CTNNB1 de novo mutations, further confirmed that the CTNNB1 gene is a pathogenic gene associated with ID and contributes to several other phenotypes, including microcephaly, abnormal facial features, hand and foot deformities, motor delays, speech delays, truncal hypotonia, peripheral hypertonia, behavioral abnormalities, and visual defects.–11 The present study describes a de novo mutation of the CTNNB1 gene in a 15-month-old Chinese boy who presented with retinal detachment, lens and vitreous opacities, microcephaly, and developmental delay.The 2The study protocol was approved by the ethics committee of Shanghai Children's Medical Center, and informed consent was obtained from the family of each patient.3The patient is a 15-month-old male, born from nonconsanguineous and healthy Chinese parents, without family history of birth defects, developmental delay, and/or IDs. He was delivered by cesarean section at 40-week gestation, had a birth weight of 3.6 kg, and was the first child of the couple. Neonatal clinical examination at the local hospital did not reveal any abnormalities.At 3 months of age, he underwent an ophthalmologic examination after his parents noted that his eyes did not react to light. Results of an ultrasound of the eyes showed a retinal detachment and lens and vitreous opacities in both eyes. Intraocular pressures were as follows: oculus dexter: 15.3 mm Hg; oculus sinister: 14.7 mm Hg. Falciform retinal folds and fundus hemorrhages were also reported. Because familial exudative vitreoretinopathy was initially suspected, targeted gene sequencing using an ophthalmic gene panel was performed; however, the pathogenic gene was not found. Fundus fluorescein angiography in both eyes was normal. Ultrasound examination of the eyes at 4 and 7 months revealed persistent retinal detachments in both eyes.At 9 months of age, the patient was 73.0-cm tall, weighed 8.0 kg, had a head circumference of 42.0 cm (<−2 standard deviation [SD]), but could not roll or sit by himself. He was also noted to have mild thumb adduction and hypertonia of the extremities.At the age of 15 months, the patient was taken to the Medical Genetics Department at Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine  to obtain a definitive diagnosis. Physical examination showed that he was 80.3-cm tall, weighed 10.0 kg, and head circumference of 44.0 cm (<−2SD). He had hypertonia, low-set ears, and high palate arch. He still had no reaction to light and appeared to have a complete lack of vision. He could raise his head but still could not sit or walk without support. No language development was found. Magnetic resonance imaging of the patient's brain, electroencephalogram, abdominal ultrasound, and hearing examination revealed no abnormalities. His genetic/metabolic workup, including karyotype, liver function tests, alkaline phosphatase, glucose, lactate, electrolytes, and urinalysis were all normal. Base calling and sequence read quality assessment were performed using Illumina HCS 2.2.58 software  with the new versions of HiSeq control software and Real Time Analysis. The reads were mapped to the reference human genome  using NextGENe software . WES generated a total of 34,350 variants. The Ingenuity Variant Analysis  was used to analyze the candidate variants. The following variants were initially excluded: the common variants with the minor allele frequency greater than 1% in the control databases, including 1000 Genomes Project, the National Heart, Lung and Blood Institute Exome Sequencing Project, and the Exome Aggregation Consortium; the benign variants, including synonymous, harmless missense predicted by PolyPhen-2  and SIFT software, and those predicted to have no impact on splicing by MaxEntScan . Subsequently, clinical symptoms of developmental delay, ophthalmic diseases, and microcephaly subsequently served as filtering indexes to analyze the candidate variants. Finally, a novel heterozygous nonsense variation  in exon 11 of the CTNNB1 gene was identified in the affected individual. Sanger sequencing was used to examine the proband and parents in order to further confirm the WES results and to assess whether the identified variation was present in either of the parents. The Sanger sequencing results confirmed the presence of the CTNNB1 gene variant in the patient and showed that the parents were normal , indicating that the variant was de novo.At this point of the investigation, we concluded a rare inherited syndrome of the patient. The patient was screened for causal variants using whole-exome sequencing (WES) as previously described by our own lab.4CTNNB1 gene , which was demonstrated to be de novo via Sanger sequencing. The mutant of c.1627C>T was classified as pathogenic (PVS1+PS2+PM2), based on the American College of Medical Genetics and Genomics/the Association for Molecular Pathology variant-interpretation guidelines.The present case involves a 15-month-old Chinese boy with a complex phenotype, which included retinal detachment, lens and vitreous opacities, hypertonia of the extremities, mild thumb adduction, microcephaly, and developmental delay. WES of the patient revealed a novel heterozygous nonsense mutation in exon 11 of the CTNNB1 gene is located at 3p22.1 (chr3:41240942–41281939) and contains 15 exons, which encodes a protein consisting of 781 amino acids . CTNNB1 belongs to the armadillo family of proteins that are involved in genetic programs that control embryonic development and adult homeostasis and play an important role in the Wnt-signaling pathway and cell adhesion. The p.Thr551Met mutation, identified in an autism spectrum disorders (ASD) cohort study, was the first de novo mutation in the CTNNB1 gene that was found to be connected to ID. However, the pathogenicity of this mutation was not well recognized due to a lack of functional studies and few cases with supporting evidence. Subsequently, 3 de novo loss of function mutants were found in ID patients demonstrating that germline mutations of the CTNNB1 gene could lead to Mendelian inherited disease, which had a phenotype included in the Online Mendelian Inheritance in Man (OMIM) database (OMIM# 615075). To date, 20 loss-of-function mutations of the CTNNB1 gene have been reported in 21 patients,–11 including 9 nonsense, 7 frameshift, 2 splice, and 2 whole-gene deletions. The mutants are distributed in exons 3, 4, 5, 6, 9, 10, 12, 13, and introns 7 and 10 . Two missense mutations (p.Thr551Met in exon 11 and p.Leu388Pro in exon 8) were not listed in the table due to a lack of evidence for loss of function.The (Table ). Although the child was too young to perform the related tests, his parents realized that his intelligence was below the level of his peers. Another patient, with a mutation in exon 11 of CTNNB1 (p.Thr551Met), presented with ASD, moderate ID, though no further clinical data were provided. Consequently, a meaningful comparison of clinical feature could not be conducted. The most significantly different feature is that approximately 75% of patients with CTNNB1 loss of function mutations have mild visual defects, mostly characterized by astigmatism, hyperopia, or strabismus, while the patient in this study suffered from severe ophthalmic disease resulting in complete loss of vision. Wnt/β-catenin signaling is essential in vertebrate eye development and conditional loss-of-function mutations of β-catenin in mice can lead to abnormal lens morphology and retinal anomalies,15; however, the severe eye disease observed in the patient in this study was rarely described in humans with CTNNB1 loss of function mutations. Moreover, approximately 5% of patients with a molecular diagnosis have multiple molecular diagnoses, which involve more than 1 clinical diagnosis, and cause more serious phenotypic symptoms. Taking this into account, the WES data were reanalyzed with only the ophthalmic disease phenotype in order to determine whether other pathogenic genes were responsible for or aggravated the eye symptoms in the patient. In total, 11 heterozygous variations were found. Of these, 10 were recessively inherited genes , and 1 was a missense mutation in the KMT2D gene (p.Arg3707Gln), which was present in his mother, though she lacked similar clinical features, indicating that the mutation was benign. Therefore, it appears that the CTNNB1 gene mutant was responsible for the clinical features of the patient in the present study. CTNNB1 should be added to the growing ophthalmic disease list when designing an ophthalmic gene panel.Most of the phenotypic characteristics of the patient in the present study were consistent with other reported cases in the literature Table . AlthougCTNNB1 gene (p.Gln558X). The patient had a severe ocular phenotype, which had not been well recognized. Different analyses of the WES data suggested that no other pathogenic gene contributed to the phenotype. This study expands the mutant spectrum of the CTNNB1 gene and enhances the understanding of this phenotype. To the best of our knowledge, this is the first case report of a loss-of-function mutation of the CTNNB1 gene in Asia.In conclusion, the present study reported on a Chinese patient with a novel de novo heterozygous nonsense mutation in


This study was aimed at a serial evaluation and comparison of the prognostic values of Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE) II scores for neurologic outcomes in comatose, out-of-hospital cardiac arrest (OHCA) survivors, treated with targeted temperature management (TTM).We analysed a prospective cohort of comatose OHCA patients, with TTM, admitted to an emergency intensive care unit (ICU), between January 2010 and December 2015. SOFA and APACHE II scores were calculated initially, and then at day 1, 2, 3, 5, and 7 after ICU admission. Primary and secondary outcomes were the 28-day neurologic outcome and the 28-day mortality, respectively. Prognostic value of the SOFA and APACHE II scores was analysed using the receiver operating characteristic curve.Of the 143 selected patients, 62 survived and 34 had good neurologic outcomes at day 28. There was no significant difference in the SOFA and extracerebral SOFA scores between the good and poor neurologic outcome groups. However, the APACHE II scores were significantly higher in the good outcome group; they displayed good discriminatory power in predicting poor outcomes, unlike the SOFA scores. The APACHE II score at day 3 had the highest prognostic value for predicting poor neurologic outcomes with an area under the cure of 0.793, and with a cut-off value of 20, the APACHE II score predicted poor neurologic outcomes with a sensitivity of 43.75%, a specificity of 94.12%, a positive predictive value of 94.59%, and a negative predictive value of 41.56%.Identifying APACHE II score might assist as one piece of multimodal prognostic approach for the assessment of neurologic outcomes in OHCA survivors treated with TTM. Out-of-hospital cardiac arrest (OHCA) is a major public health concern, with a global average incidence of 55 adult OHCAs of a presumed cardiac cause, per 100,000 person-years , 3. FollAlthough brain injury accounts for most of these deaths, whole-body ischaemia-reperfusion injury causes the activation of immunologic and coagulation pathways, leading to multiple organ failure . This tyAlthough the Acute Physiology and Chronic Health Evaluation (APACHE) II score , one of This single-centre retrospective, observational, registry-based study was performed at the emergency intensive care unit (ICU) of a university-affiliated teaching hospital in Korea. Data were extracted from the OHCA registry, which prospectively collected data of consecutive patients with OHCA, between January 2010 and December 2015. All OHCA survivors in our institution are entered into OHCA registry. The institutional review board of the University of Ulsan College of Medicine reviewed the study protocol and approved the study . Informed consent was waived due to the retrospective nature of this study. The cohort included successfully resuscitated patients above the age of 18 years, who experienced non-traumatic OHCA with subsequent cardiopulmonary resuscitation, in whom the return of spontaneous circulation (ROSC) was achieved, who had neurologic impairments immediately after ROSC (defined as a patient’s inability to follow commands), and who were treated with therapeutic hypothermia , 13.All eligible patients were admitted to the emergency ICU. Post-resuscitation care, including coronary reperfusion or TTM, in accordance with the then-current advanced cardiac life support guidelines, was provided to them. TTM was performed using Arctic Sun Energy Transfer Pads , with the aim of achieving a body temperature of 33–36 °C. The target temperature was maintained for 24 hours and then patients were rewarmed at a rate of 0.25°C/h. During TTM, the temperature was monitored using an oesophageal temperature probe. Propofol and opioids (morphine or remifentanil) were used for sedation and analgesia. Neuromuscular blockades were administered to control shivering, if the need arose. All the patients received standard intensive care, according to the institutional ICU protocols.Data on the following variables were obtained from the registry: age, sex, pre-existing illnesses, presence of a witness on collapse, first monitored rhythm, aetiology of cardiac arrest, collapse-to-ROSC interval, initial core temperature, pre-induction time, induction time, rewarming time, vital status, mortality , and Cerebral Performance Category (CPC) score at 28 days. In addition, we calculated the SOFA and APACHE II scores during the first 7 days after the ROSC. The SOFA score has a range of 0–4 points for each of the 6 organ systems  . The APAU test for continuous variables and the Chi-square test or Fisher’s exact test for categorical variables. To estimate the effect of both day and group on the scores, we performed the linear mixed model that accounted for data clustering and dependency. After confirming the group-by-time interaction effect, the scores were compared the group effects within time points. All reported P-values are two sided, and P-value of <0.05 was considered statistically significant. The prognostic value of the SOFA and APACHE II scores, to predict poor neurologic outcomes, was analysed using the receiver operating characteristic curve with the area under the cure (AUC). The optimal cut-off value of the scores was determined using Youden’s index. Data manipulation and statistical analyses were conducted using SAS® version 9.4  and IBM SPSS of Windows, version 21.0 .Continuous and categorical variables are represented as median with interquartile range (IQR), and number (%), respectively. Comparisons between patients were performed using the Mann–Whitney During the study period, a total of 143 patients with OHCA were admitted to the emergency ICU for post-resuscitation care. Among them, 62 patients (43.4%) survived till the 28-day mark, and 34 patients (23.8%) had good neurologic outcomes at day 28. The demographic and clinical characteristics of the patients are summarized in Although the SOFA and extracerebral SOFA scores of the survivors were significantly lower than those of the non-survivors, for all the study time points, except for day 1 in the ICU , there wThe performance of the SOFA, extracerebral SOFA, and APACHE II scores in predicting 28-day mortality and poor neurologic outcomes is presented in In this study, we conducted the serial evaluation of SOFA and APACHE II scores in comatose OHCA patients treated with TTM and evaluated the predictive power of both scoring systems for 28-day mortality and poor neurologic outcomes. Despite significant differences in the SOFA scores between survivors and non-survivors, this score was found to be an ineffective tool for the discrimination of poor neurologic outcomes. Compared with the SOFA score, the APACHE II score was found to be a good predictor of poor neurologic outcomes as well as mortality; the performance of APACHE II scores in predicting poor neurologic outcomes was the best on day 3 in the ICU, with an AUC of 0.793. Although a substantial number of patients  were excluded from the analysis due to death on day 3 in the ICU, APACHE II scores, at a cut-off value of 20, predicted poor neurologic outcomes with a sensitivity of 43.75%, a specificity of 94.12%, a positive predictive value of 94.59%, and a negative predictive value of 41.56%.The SOFA scores displayed an acceptable discriminatory power for survivors, at admission, with an AUC value of 0.634, which was consistent with that of previously conducted studies , 15, 16.The APACHE II scores displayed a good predictive power for poor neurologic outcomes, across all time points and demonstrated the highest predictive value  on day 3 in the ICU. The superiority of the APACHE II score when compared to the SOFA score could be attributed mainly to the measurements of the patients’ previous health status  and demographic status (age) . Only onThere are several limitations in our study. First, this was a single-center retrospective study based on a prospective cardiac arrest registry and, therefore, limitations pertaining to data gathering and analysis are inevitable. Second, despite following the standardized treatment protocol for post cardiac arrest, including the performance of TTM, the results cannot be generalized. Third, the results might be biased with regard to mortality and consequently, drop-outs in the early phase. However, we also performed the linear mixed model to estimate the effect of both day and group on the scores and compared the group effects within time points to minimize the confounding.Neurologic prognostication is an important clinical issue in the management of post-cardiac arrest patients, and is still under investigation. In this study, we found that APACHE II score after day 3 showed acceptable levels of discriminatory power, in terms of discriminating the good neurologic outcome group from the poor neurologic outcome group, as well as survivors from non-survivors. APACHE II scores calculated on day 3 in the ICU might be used as one piece of multimodal prognostic approach for predicting neurologic outcomes in post cardiac arrest patients treated with TTM following an OHCA. Further multicenter studies would be warranted to validate our results.S1 TableaCatecholamine doses are given as μg/kg/min for at least 1 hour. PaO2, partial pressure of oxygen; FiO2, fraction of inspired oxygen; MAP, mean arterial pressure.(DOCX)Click here for additional data file.S2 Table2, alveolar-arterial oxygen gradient; FiO2, fraction of inspired oxygen; PaO2, partial pressure of oxygen; ABGs, arterial blood gases.MAP, mean arterial pressure; A-aDO(DOCX)Click here for additional data file.S3 TableValues are presented as median with interquartile range or number (percent). SOFA, Sequential Organ Failure Assessment; APACHE II, Acute Physiology and Chronic Health Evaluation II.(DOCX)Click here for additional data file.S4 TableValues are presented as median with interquartile range. SOFA, Sequential Organ Failure Assessment; APACHE II, Acute Physiology and Chronic Health Evaluation II; ICU, Intensive Care Unit.(DOCX)Click here for additional data file.S5 TableSOFA, Sequential Organ Failure Assessment; APACHE II, Acute Physiology and Chronic Health Evaluation II; ICU, Intensive Care Unit; SE, standard error; CI, confidence interval.(DOCX)Click here for additional data file.


Objective: The present study aimed at assessing the sociodemographic and psychiatric characteristics of 40 to 65 year- old suicide attempters, who were referred to an emergency department within 4 hours of the attempt.Method: A total of 93 suicide attempters (Mean age=46.59 years) who were referred to an emergency department, were assessed in this study. Patients completed questionnaires covering sociodemographic data, personality traits, mood, and impulsivity. Psychiatric status of the patients was rated by experts.Results: Experts rated 85 (92.4%) of the suicide attempters as having a psychiatric disorder. Based on self-ratings and compared to normative data, 42 (46.6%) patients were psychopathologically ill. It was found that suicide attempts were not related to impulsive personality traits, mood disorders, sociodemographic patterns, or gender .Conclusion: The pattern of results suggests that further unknown factors were involved in pushing people to attempt suicide. As regards to the rate of suicide in Iran, recent estimations reported a prevalence of 5.3/100 000, with a higher rate in males (7 100 000) than in females (3.6 100 000) . Menon et al.  and PompGender differences were found as a sociodemographic characteristic of suicide and suicide attempters. Males are involved in more completed suicides than females, but it was found that the latter make more attempts, with a ratio of 3:1 , 5, 9-11Last, the present study expands upon previous studies in Iran , 12, 13 The following three hypotheses were formulated: First, following Arsenault-Lapierre et al.  and MilnProcedurePatients who were referred to the emergency department of a general hospital in Hamadan (Iran) following a suicide attempt within the previous 4 hours were approached. All patients were informed about the aim of the study, and the voluntary basis of participation. Furthermore, all participants were assured about the anonymity of the data, and written informed consent was requested. Then, a psychiatrist or clinical psychologist conducted a structured psychiatric interview (see below), and patients completed a self-rating questionnaire on psychopathology (see below). The ethical committee of Hamadan University of Medical Sciences  approved the study; furthermore, this study was in accordance with the Declaration of Helsinki.SampleA total of 93 patients  females) took part in the study. Inclusion criteria were as follows: 1. suicide attempt within the last 4 hours; 2. age 40 to 65 years; 3. signing written informed consent; 4. willing and able to submit to a clinical interview; and 5. completing a self-rating questionnaire on psychopathology (see below). Exclusion criteria were as follow: 1. status of continuous suicidality; 2. status of psychosis. A total of 156 patients were approached, of whom 45 (28.8%) did not meet the inclusion and exclusion criteria, 8 (5.1%) refused participation, and 10 (6.41%) withdrew after the beginning of the study. Thus, 59.6 % (N= 93) completed the study. Gender ratio of non-participants was approximately 1:1.Clinical AssessmentFirst, sociodemographic data were assessed , and the method employed in the suicide attempt was determined . Next, to diagnose patients’ psychiatric disorders according to the DMS-IV-TR , a cliniAs mentioned, to assess possible psychopathological traits from the patients’ view, patients were asked to complete the 175-item Millon Multiaxial Inventory-III ), a selfStatistical Analysis2-tests was performed to calculate possible associations between gender and sociodemographic variables. Next, a series of t-tests was performed to calculate gender differences in psychopathology (as self-rated). With performing further X2-tests, we calculated the associations between gender and psychiatric diagnoses (as rated by experts). First, a series of XThe level of significance was set at alpha ≤.05 and statistical analysis was performed with SPSS® 22.0  for Apple Mac®.Sociodemographic VariablesWith respect to gender differences, compared to males, more female patients reported to be married, rather than single, and they also reported not being in paid employment, and if employed, they did not hold a management position.Psychopathology (self-rating and experts’ ratings)Incidence of psychiatric disorder as rated by expertsIncidence of psychiatric characteristics as self-rated by patientsGender RatioWe observed a female/male ratio of 1:1.58. The key findings of the present study were that among middle- aged suicide attempters, who were referred to an emergency room, 46.6% (self-rating) to 91.4% (experts’ ratings) were psychopathologically ill, while no other patterns, whether sociodemographic or illness-related, were observed. The present findings add to the current literature in suggesting that beyond psychopathology, further unknown factors seem to be involved in suicidal behavior. Three hypotheses were formulated.Our first hypothesis was that psychiatric diagnoses would be in the range 67% to 87%. In the present sample, the rate of psychiatric diagnosis was 91.4%. Whereas this incidence is descriptively higher, the general direction is that suicide attempts are related to psychiatric disorders. The present pattern of results is, therefore, in accordance with numerous previous studies , 9, 10. With the second hypothesis, we expected higher rates of personality characteristics related to impulsivity  and mood disorders, but this was not confirmed. However, no specific psychiatrically predominant characteristic could be observed . TherefoOur third hypothesis was that self-ratings for psychopathological features would be in percentiles above the cut-offs for psychopathology see ; howeverAgain, the data were at odds with previous results such as those of Milner et al . MoreoveThe novel findings of this study should be balanced against the following limitations. First, the quality of the data did not allow us to identify specific life circumstances or triggers. Specifically, impulsive behavior and critical life events increase the risk of suicide attempts. Critical life events are, for instance, continually increasing or repetitive psychosocial difficulties such as loss of a loved person, or chronic somatic illnesses. Second, we asked about the civil status  as a proxy for social interaction and commitment, although civil status does not reflect the quantity and quality of a person’s social relationships. In this respect, low levels of interpersonal trust was the third leading risk factor for suicide attempts, after major depressive disorders and previous suicide attempts . This obIn addition, not all suicide attempters were referred to an emergency department, and not all were referred to the emergency department of the general hospital at which this study took place. Thus, the sample of suicide attempters may be biased and not representative of suicide attempters as a whole.Suicide attempters aged 40 to 65 years who were referred to an emergency department were psychopathologically ill, both from patients and from experts’ view. Suicide and attempted suicide should be a topic of routine counseling of patients with psychiatric disorders to reduce the risk of suicide.


Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by swelling and painful joints, eventually leading to joint destruction. There is still a lack of effective therapy to treat RA. The Juanbi pill is a Chinese medicine that has been widely used to treat active RA in China for hundreds of years, relieving pain and protecting the affected joints from malformation. However, there is no solid evidence to show the effect of the Juanbi pill on the management of active RA.We will conduct a multicenter, randomized, double-blind, placebo-controlled clinical trial to determine whether the traditional Chinese medicine Juanbi pill could relieve joint pain in RA and protect the joints. A total of 120 patients with active RA will be enrolled and treated with the Juanbi pill or a placebo for 3 months. The primary outcome measures are as follows: rate of in the American College of Rheumatology (ACR)50, change in the 28-joint Disease Activity Score (DAS28) from baseline at beginning of therapy to 3 months, and a change in the van der Heijde modified Sharp score measured from baseline to 12 months. The secondary outcome measures are as follows: rate of change in ACR20, ACR70, Health Assessment Questionnaire-Disability Index (HAQ-DI), and change in score in the Patient Assessment of Arthritis Pain, Patient Global Assessment of Arthritis, and the Athens Insomnia Scale (AIS) from baseline to 2-week, 1-month, 2-month, 3-month, 6-month, and 12-month follow up. In addition, the rate of change (score) in the ACR50 and DAS28 from the baseline to 2-week, 1-month, 2-month, 6-month, and 12-month follow up are also the secondary outcome measures.Although the Juanbi pill has been used in China for many years to treat RA, there is a lack of consensus about its effectiveness. This trial will provide convincing evidence about the effect of Juanbi pill on active RA.NCT02885597. Registered on 30 August 2016.ClinicalTrials.gov, The online version of this article (10.1186/s13063-018-2555-1) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by pain, swelling, synovial inflammation, joint damage, and bone destruction, resulting in severe disability and increased mortality rate . The preBi syndrome, and Juan Bi (means getting rid of Bi syndrome in Chinese) decoction from the book Yi Xue Xin Wu from the Qing Dynasty was specially designed to treat Bi syndrome [Cinnamomum cassia Presl, Gentiana macrophylla Pall., Angelica sinensis (Oliv.) Diels, Ligusticum chuanxiong Hort., Glycyrrhiza uralensis Fisch., Caulis Piperis Kadsurae, Morus alba L, Olibanum, and Radix Aucklandiae, and has been widely used to treat arthritis in China [Herbal medicines, which are complementary and alternative medicines (CAM), have been used for centuries in China to treat different illnesses , 6. In tsyndrome . The Juain China –10. Our Juanbi decoction combined with methotrexate (MTX) has been widely used in China for treatment of RA, and case report studies show that Juanbi decoction can help MTX attenuate joint pain and swelling –13. HoweThis study is a multicenter, randomized, double-blinded, placebo-controlled clinical trial with two parallel arms Fig. . The aimParticipants will be recruited from three hospitals  by poster and hospital official website advertisements. The planned recruitment period is 12 months. Informed consent will be obtained from all participants before randomization.Adults (aged ≥ 18 y) with RASatisfy American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for RA, 2010 Onset of symptoms within 12 months before enrollmentActive disease at the time of enrollment as indicated by 28-joint Disease Activity Score (DAS28) greater than 3.2 , and no Paid or unpaid employment but measurable work Participants who fulfill the following criteria are eligible:RA combined with other autoimmune disease, such as adjuvant arthritis, lupus arthritis, or osteoarthritisRA combined with abnormal liver and kidney functionWomen who are pregnant, planning pregnancy, or breast-feedingSevere chronic or acute disease interfering with attendance for therapyAlcohol or substance abuseUnable to understand or sign an informed consent formThe exclusion criteria are as follows:All patients will be randomized into the Juanbi group or the placebo group. Patients in the Juanbi group will receive both the Juanbi pill  and MTX (5 mg per week) for 3 months, whereas patients in the other group will receive a Juanbi pill placebo  and the same dose of MTX as the Juanbi group. The Juanbi pill and the placebo are to be dissolved in 200 mL of hot water and taken orally twice a day for 3 months. The Juanbi pill will be manufactured, packaged, and labeled by Jiangyin Tianjiang Pharmaceutical Co., Ltd., based on good manufacturing practice standard. The crude herbs, including Rhizoma Et Radix Notopterygii (Qiang Huo) 18 kg, Radix Angelicae Pubescentis (Du Huo) 18 kg, Radix Gentianae Macrophyllae (Qin Jiao) 18 kg, Caulis Piperis Kadsurae (Hai Feng Teng) 36 kg, Ramulus Mori (Sang Zhi) 54 kg, Radix Angelicae Sinensis (Dang Gui) 54 kg, Rhizoma Chuanxiong (Chuan Xiong) 12.6 kg, Olibanum (Ru Xiang) 25.2 kg, Radix Aucklandiae (Mu Xiang) 25.2 kg, Radix Glycyrrhizae (Gan Cao) 9 kg, and Ramulus Cinnamomi (Gui Zhi) 9 kg, were supplied in one batch from Haozhou Hongyu Chinese Herbal Medicine Co., Ltd. or Anhui Huizhongzhou Chinese Herbal Medicine Co., Ltd. The test results of all herbs meet the standard. All herbs were stored in a specialized cool and dry place. The Juanbi pill is made as follows: (1) extraction: all herbs listed are put in a ceramic container and 1000 L of distilled water is added to macerate the herbs for 1 h. Then, the mixture is boiled at 100 °C for 1 h for the first extraction, and repeated twice to obtain three extractions in total. Note that the third extraction should contain only 500 L of distilled water and have only 30 min boiling time; (2) concentration: the three extractions are mixed and the mixture is concentrated at 60 °C (660 mmHg). The mixture is sprayed dry to produce an extract powder, which is smashed and screened through mesh size 80. The pills are packed in quantities of 4 g per bag and stored in a clean room at approximately 20 °C and 50% humidity. The Juanbi pill placebo contains 10% Juanbi pill and 90% bitterant, lactose edible essence, pigment , and starch, and has a similar shape, smell, color, and taste to the actual Juanbi pill. The Juanbi pill and the placebo all met the quality inspection standards.The duration of the intervention is 3 months, and follow up is 9 months. Study visits will take place at baseline and at 2, 4, 8, 12, 24, and 52 weeks. Every patient will be asked to visit within 3 days of the given time point.Randomization will be conducted by a credible pharmaceutical manufacturer , which will provide both the Juanbi pill and the placebo. A random number list will be generated using Excel  to randomize participants either to the Juanbi pill group or the placebo group in a 1:1 ratio. When a participant is recruited, the investigator will provide the pharmaceutical manufacturer with a number, and the pharmaceutical manufacturer will randomly post the Juanbi pill or the placebo to the participant according to the random number list. The number provided and the corresponding number list will be recorded and will be kept in a locked cabinet at the pharmaceutical company.The pharmaceutical company staff will not participate in the trials, and the investigator, physicians, nurses, outcome measurement operative, statisticians, and the participants will be blinded to the group information until the end of the trial, when all statistical analyses are finished.The primary outcome measure will be as follows: (1) the progression of ACR 50 after 1, 2, and 3 months of treatment compared to baseline; (2) the change in DAS28 before and after 1, 2, and 3 months of treatment; and (3) the difference between the two groups in the van der Heijde modified Sharp score before and after 12 months of follow up.The ACR50 is a scale to measure change in RA symptoms , 16. TheAlthough compared to ACR50 and ACR70, ACR20 has been accepted as the efficacy benchmark in RA clinical trials and has greater discriminant capacity to distinguish patients on active treatment from placebo control , 17, 18,The DAS28 is widely used as an indicator of RA disease activity and response to treatment, and clinical trials have used DAS28 to assess treatment effect , 21. TheThe 28 joints are 10 proximal interphalangeal joints and 10 proximal interphalangeal joints of the hands and wrists, elbows, knees, and shoulders bilaterally. GH is patient general health measured by VAS (0–100 mm); “0” is the best and “100” is the worst , 22–24.erosion and the joint narrowing in the 10 metacarpophalangeal joints, 8 proximal interphalangeal joints, 10 metatarsophalangeal joints, and 2 interphalangeal joints of the big toes. In addition, erosion is also assessed in the two interphalangeal joints of the thumbs, first metacarpal bone, radius, ulnar bones, trapezium and trapezoid, navicular bones, and right and left lunate bones bilaterally, whereas joint narrowing is also assessed in the third, fourth, and fifth carpometacarpal joints, multiangular-navicular joints, capitate-navicular-lunate joints, and right and left radiocarpal joints bilaterally. The maximum erosion score in the hand is 5, and in the foot it is 10, according to the degree of erosion. Thus, the maximum number of erosions in the hand is 160, and in the feet it is 120. The grade of joint space narrowing is as follows:1 = focal or doubtful2 = general, less than 50% of the original joint space3 = general, more than 50% of the original joint space or subluxation4 = no joint space remaining, luxation, or ankylosisThe van der Heijde modified Sharp score is the radiographic assessment of the Therefore, the maximum score for joint space narrowing is 120 in the hand and 48 in the feet. The van der Heijde modified Sharp score is the sum of the erosion and joint space narrowing score , 25.The ACR50 and DAS28 will be estimated at baseline and after 1, 2, and 3 months of treatment, and at the 6-month and 12-month follow up. The van der Heijde modified Sharp score will be measured only at baseline and after 12 months.The secondary outcome will be to compare the rate of change in the ACR20/70, HAQ-DI, Patient Assessment of Arthritis Pain, Patient Global Assessment of Arthritis, and AIS from baseline to 2 weeks, 1 month, 2 months, 3 months, and at the 6-month and 12-month follow up. The rate of change in the ACR50 and DAS28 from t baseline to 2 weeks, 1 month, 2 months, and 6-month and 12-month follow up are also secondary outcome measures, and the change in score on the 36-item Short-Form Health Survey Questionnaire (SF-36) from baseline to 1 month, 2 months, 6 months, and 12 months is also calculated.The HAQ-DI is a subscale of the ACR20/50/70. It is a completely patient-reported outcome, bridged between biochemical and physical measurements, and widely used in RA clinical trials to assess the disease activity and the patient’s disability . The HAQPatients with RA often have sleep disturbance, and the more severe the disease, the more severe the sleep problems , 28. TheThe SF-36 consists of 36 items and measures eight dimensions, which are physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning, and mental health . The SF-Pharmacopoeia of the People’s Republic of China (2015 version). Our preliminary experiment from December 2015 to May 2016 into the use of the Juanbi pill combined with MTX versus MTX alone did not show any side effects during the 3-month treatment period or in the 3-month follow-up period. We still need to perform a series of measures including subjective description and laboratory tests  to assess the safety of the Juanbi pill during the entire trial.The Juanbi pill has been used for hundreds of years in China, and the herbs in the Juanbi pill are safe according to the recommended amount in At each visit, patients will be asked whether there are any adverse effects during the study period. In addition, we will perform laboratory testing of the participants’ blood, urine, feces, and kidney and liver function. We are ready to provide an appropriate treatment to the participant immediately if an adverse event is reported. Emergency services will be provided in the case of serious adverse events, and we will be prepared to report the event to the Institutional Review Board within 24 h from the time of recognition.Study recruitment started in December 2016, and is expected to end in December 2017. The final follow up of all participants will end on 31 December 2018. The overview of the participant process is shown in Fig. We calculated the sample size according to our primary study. We conducted a preliminary experiment from December 2015 to May 2016 into the efficacy of the Juanbi pill combined with MTX versus MTX alone, and we found that patients taking the Juanbi pill combined with MTX achieved 84.8% ACR50, whereas patients in the MTX group achieved 55.5% ACR50 response. According to the formula of the rate in completely random design:1 and n2 are the number of participants in the Juanbi and placebo groups, respectively, and uα/2=1.96 when type 1 error is 0.05, uβ = 1.282 when type II error is 0.1 in two-sided tests: p1 and p2 [where n1 and p2 . It was p value < 0.05 will be defined as a statistically significant result. Means and standard deviations will be used to describe the continuous variables, such as demographic and clinical outcome variables, whereas percentages will be used for categorical variables such as the rate. Continuous variables following the normal distribution will be analyzed by Student’s t test; otherwise, non-parametric tests will be used to compare group differences.Efficacy and safety analyses will be conducted according to the intention-to-treat principle. The method of last observation carried forward will be applied in analysis of missing values. All statistical analyses will be performed using Statistical Packages of Social Sciences software (SPSS) (version 21.0). A This is a 12-month clinical trial in which participants are required to take the research medication for 3 months with follow-up visits at 6 and 12 months, attending seven assessment visits in all. Participants will receive disease activity assessment seven times , and six safety assessments . Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine is responsible for quality control.All the pills, including the Juanbi pill and the placebo, are provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. In addition, the pharmaceutical company will conduct the randomization of participants and allocation to the three centers. Before we began the clinical trial, we carried out unified training to make sure the multicenter physicians, nurses, and outcome measurement operative involved in the trials fully understood the process of the entire trial. When the clinical trial started we set investigators to supervise the three centers every month, making sure: (1) every center has recruited the planned number of participants; (2) all the participants recruited fully meet the inclusion criteria and do not meet the exclusion criteria; and (3) all the participants have fully followed the clinical trial process, and the case report form (CRF) has been filled in as required in the recruitment and follow up. Meanwhile, we call every participant to verify the case report form and data collection are recorded in an electronic CRF monthly during the course of the clinical trial.Chinese herbs have been widely used worldwide as CAM. A recent survey conducted in San Francisco showed that patients frequently used Chinese herbal products in conjunction with Western prescription medicines, which would be documented in the medical record . AlthougBefore we conducted this trial, we closely searched PubMed, EMBASE, the Cochrane Library, CINAHL, ISI web of knowledge, CNKI (including China Doctor/Master Dissertation Full Text Database and China Proceedings Conference Full Text Database), Vip Journal Integration Platform (VJIP), Wan Fang Data, Chinese BioMedical (CBM) databases (Sinomed), and clinical trials from the inception to December 2015, and we found no definite evidence about the effectiveness and safety of the Juanbi pill in the treatment of active RA. Therefore, we decided to conduct a multicenter, randomized, controlled clinical trial to closely study the effectiveness and safety. We will use the ACR50, DAS28, and van der Heijde modified Sharp score to estimate disease activity and damage in the affected joints; moreover, we will calculate the patients’ life quality using scales such as the HAQ-DI, AIS, and SF-36 as secondary outcome measures. We will assess the safety of the Juanbi pill in two categories: patients’ self-reporting of quality of life and symptoms, and laboratory examinations such as blood, urine, feces, and kidney and liver function tests, to obtain a full assessment. Due to lack of evidence about the effectiveness of the Juanbi pill, we will apply the pill combined with MTX to treat RA to ensure the compliance of participants and meet ethical considerations, since MTX is a first-line treatment among disease-modifying anti-rheumatic drugs, and can achieve 53.8% DAS28 remission , 30.To the best of our knowledge, this is the first well-designed, randomized, controlled trial investigating the efficacy of the Juanbi pill combined with MTX for the treatment of active RA. This study is built on our preliminary open experiment with a small sample, as well as hundreds of years’ use of the Juanbi pill in China for the treatment of RA.This study is designed to answer whether the Juanbi pill combined with MTX is preferred in the treatment of active RA, compared with MTX alone. If this trial succeeds, it will provide the patients and physicians an option of combining the Juanbi pill with MTX to obtain better disease remission. Moreover, this trial will provide data about the effect of the Juanbi pill on joint pain and function, quality of life, and safety. The results will aid clinical decision-making for the management of active RA and will provide useful information that can be incorporated into future guidelines.Recruitment started in December 2016 and is expected to finish in December 2018.Additional file 1:SPIRIT 2013 checklist: Recommended items to address in a clinical trial protocol and related documents. (PDF 284 kb)


Pediculus humanus capitis, is subdivided into several significantly divergent mitochondrial haplogroups, each with particular geographical distributions. Historically, they are among the oldest human parasites, representing an excellent marker for tracking older events in human evolutionary history. In this study, ancient DNA analysis using real-time polymerase chain reaction (qPCR), combined with conventional PCR, was applied to the remains of twenty-four ancient head lice and their eggs from the Roman period which were recovered from Israel. The lice and eggs were found in three combs, one of which was recovered from archaeological excavations in the Hatzeva area of the Judean desert, and two of which found in Moa, in the Arava region, close to the Dead Sea. Results show that the head lice remains dating approximately to 2,000 years old have a cytb haplogroup A, which is worldwide in distribution, and haplogroup B, which has thus far only been found in contemporary lice from America, Europe, Australia and, most recently, Africa. More specifically, this haplogroup B has a B36 haplotype, the most common among B haplogroups, and has been present in America for at least 4,000 years. The present findings confirm that clade B lice existed, at least in the Middle East, prior to contacts between Native Americans and Europeans. These results support a Middle Eastern origin for clade B followed by its introduction into the New World with the early peoples. Lastly, the presence of Acinetobacter baumannii DNA was demonstrated by qPCR and sequencing in four head lice remains belonging to clade A.The human head louse,  Pediculus humanus, is an obligatory haematophagous parasite that thrived exclusively on human blood for at least 5–7 million years ago , , 1], D   and E [2 et al.) , all tencytb gene (20/24) were analyzed and combined with all known modern and ancient haplotypes generated in our dataset.For the phylogenetic analysis we used the maximum likelihood method (ML)  and a meWithin clade A (12/20), seven sequences  of these ancient head lice belonged to the widespread modern haplotype A5. This haplotype is the most prevalent worldwide  and is present in all continents. It occupies a central position in haplogroup A, as shown in the MJ network, and is also found in pre-Columbian Peruvian and Chilean mummies from the New World. The five remaining clade A sequences, four from comb B and one from comb C, yielded two novel haplotypes which were found to be unique, provisionally named here as A55 and A56, respectively. A thorough inspection of the haplotype network revealed that the A55 haplotype deviated from haplotype A5 by one mutation step while the A56 haplotype derived on haplotype A5 by two mutations steps. Nevertheless, the mutations that we found in both haplotypes A55 and A56 are synonymous, i.e. resulting in no amino acid changes, compared to the A5 haplotype. The sequences of these two novel haplotypes were deposited in GenBank under accession number: KX232678-KX232679.Within the clade B ancient head lice, all eight sequences  belonged to the modern haplotype B36, which is the most common within the B-haplogroup . This haplotype occupied a central position in the MJ network, and is found in Africa, Europe and America, as well as in pre-Columbian Chilean mummies.rpoB gene of Acinetobacter was positive for four samples (4/24) of ancient lice DNA tested (34<Ct<36) from comb B. The qPCR products generated were directly sequenced. The four obtained sequences of the partial rpoB gene (182-bp) were 100% identical to one another and were identified as A. baumannii on the basis of a BLAST search. These sequences had 180 of 182 base positions in common (98.9% identity) with a reference strain of A. baumannii (GenBank accession number CP012952) and 181 of 182 base positions in common (99.4% similarity) with A. baumannii isolated from modern human head lice collected from elementary school children in Thailand (GenBank accession number KP161047). However, these ancient sequences have one mutation (position 81) that has not previously been described in modern A. baumannii  [The mtDNA analysis in this study revealed that the head lice remains from approximately 2,000 years ago have  Africa) , 19, 20. Africa) . Interes Africa) .Accordingly, the present findings show that clade B lice existed in the Mediterranean region long before the discovery of America, meaning that clade B lice did not originate from America as was previously thought but probably existed, at least in the Middle East, prior to contact between Native Americans and Europeans. These results are consistent with the hypothesis suggesting that this clade could originate from Asia, which is reported to have populated the Americas , 18.The remaining samples had haplogroup A and yielded three haplotypes, in which two haplotypes  were unique to the ancient head lice examined in this study, while haplotype A5, which is common worldwide, including in ancient head lice from Chilean and Peruvian mummies of the New World , 26, 31.Homo, similarly to those known to date from 2.3 to 0.03 million years ago (MYA) [Homo sapiens), showing signs of a recent demographic expansion out of Africa about 100,000 years ago, first to Eurasia and subsequently to Europe, Asia and the New World [Homo neanderthalensis, who expanded in Europe and Asia, and only became associated with modern humans during the period of overlap as the result of a recent host switch [Homo sapiens between Africa and the rest of the world [in situ for thousands of years until the second contact with European colonists in the 16th century [Prior research has suggested that the known lice clades evolved on different lineages of go (MYA) , 20 and,ew World . Haplogrt switch . Thus, che world , this haB. quintana in 4,000-year-old teeth [et al. [B. quintana and that soldiers had evidence of infection with either R. prowazekii or B. quintana, concluding that louse-borne infectious diseases affected nearly one-third of Napoleon’s soldiers buried in Vilnius, and indicating that these diseases might have been a major factor in the French retreat from Russia.Aside from their role as biological markers used to infer human evolutionary history, lice remains can provide information relating to past human sanitary conditions and diseases, because these lice are vectors of bacterial pathogens. Yet, genetic traces from pathogens can be identified in archaeological remains and ancient DNA has successfully been used to identify ld teeth  and was ld teeth . Raoult  [et al.  showed tA. baumannii DNA in ancient human head lice remains belonging to clade A. Specifically, the rpoB sequence that was found has not been reported previously.This study reveals for the first time the presence of A. baumannii was first isolated from the body lice of homeless people in Marseille (France) as well as from diverse countries worldwide [A. baumannii DNA was also found in head lice collected from elementary school children in Paris belonging to clade A [et al. showed that A. baumannii could be detected in clade A and C head lice collected from elementary school children in Thailand [In recent years, orldwide . A. baum clade A  and dete clade A . In 2015Thailand .A. baumannii was reported to occur in temporary head lice in several occasions, the clinical significance of this finding is unknown [A. baumannii was shown to cause nosocomial infections and severe community-acquired infections such as pneumonia, bacteremia, endocarditis, and meningitis, due to its increasing resistance to a wide range of antibacterial agents [Although  unknown , 39. Morl agents , 41.A. baumannii in Roman-era head lice remains belonging to clade A.The present work confirms that clade B lice existed, at least in the Middle East, prior to contacts between Native Americans and Europeans. Our results cannot support the previous hypothesis that clade B has an American origin and was imported from America to the Old World after its discovery by Christopher Columbus. We also identified the presence of nosocomial pathogen, Further study of the lice remains would be necessary to shed more light on the patterns of human migration worldwide, their lice and the flow of louse-borne pathogens at different times in history.S1 Fig1, 2 and 3 showed qPCR amplification targeted a 88-bp DNA fragment of cytb gene (24/24 positive with Ct varied between 32 to 38); 4 showed qPCR amplification targeted a 100-bp DNA fragment of 12S gene (22/24 positive with Ct varied between 32 to 38).(PPTX)Click here for additional data file.S2 Fig(PPTX)Click here for additional data file.S1 TableHaplotypes highlighted in blue are the newly identified haplotypes from sequences available in GenBank.(XLSX)Click here for additional data file.