Randomized controlled trials (RCTs) demonstrated efficacy and safety of endovascular treatment (ET) in anterior circulation large vessel occlusions (LVO). We aimed at investigating how stroke patients treated by thrombectomy in clinical practice and their outcome compare to cohorts and results of thrombectomy trials.In a prospective study, we consecutively included stroke patients treated by thrombectomy (2015–2017). Baseline characteristics, procedural and outcome data were analyzed. Outcome was assessed by modified Rankin Scale (mRS) at 90 days. Ordinal regression analysis was performed to identify predictors of outcome.Thrombectomy was applied in 264 patients . Median baseline National Institutes of Health Stroke Scale (NIHSS) was 16, 58.0% received concomitant intravenous thrombolysis, 62.1% were referred from external hospitals. Median Alberta Stroke Program Early CT Score (ASPECTS) was 7. Successful recanalization  was achieved in 72.0%. Symptomatic intracranial hemorrhage (sICH) occurred in 4.5%. Independent outcome (mRS 0–2) was achieved in 26.2%, poor outcome (mRS 5–6) in 49.2%. Only 33.5% met the stringent enrolment criteria of previous RCTs. Lower age, baseline NIHSS, pre-stroke mRS, higher ASPECTS, and successful recanalization were independent predictors of favourable outcome.The majority of stroke patients treated by ET in clinical practice would not have qualified for randomization in prior RCTs. Outcome in real-life patient cohorts is worse than in the highly selected cohorts from randomized trials, while rates of successful recanalization, sICH and outcome predictors are the same. Our findings support ET in broader patient populations than in the RCTs and may improve treatment decision in individual stroke patients with LVO in clinical practice. Several randomized controlled trials (RCT) demonstrated the beneficial effect of endovascular treatment (ET) in stroke patients with an anterior circulation large vessel occlusion (LVO) –5. TheseRecently, two RCTs, demonstrated efficacy of ET in patients with anterior circulation LVO beyond 6 h after symptom onset , 8, but Therefore, we studied procedural parameters and outcome of thrombectomy in a real-world prospective cohort of patients. We aimed to investigate how patients treated in clinical practice compare to patients from the RCTs. We further aimed to identify predictors of clinical outcome, and to compare outcome of the patients meeting ASA/AHA guideline criteria for thrombectomy to those not meeting these criteria.mothership), or transferred from external hospitals for ET (ship). The decision to treat was made interdisciplinary between a vascular neurologist and interventional neuroradiologist on a case-by-case basis guided by an institutional protocol. Factors taken into account for treatment decision included the elapsed time from symptom onset, stroke severity assessed by the NIHSS on admission, site of vessel occlusion, the ASPECT score, the pre-stroke disability, and extended imaging with CT-perfusion or MRI to identify potentially salvageable brain tissue, in particular, beyond 6 h of symptom onset or if the symptom onset time was uncertain. Patients received standard treatment including intravenous thrombolysis (IVT) prior to endovascular therapy if appropriate according to national and international guidelines.All consecutive patients (18 years and older) with acute ischemic stroke who were treated by mechanical thrombectomy between July 2015 and June 2017 in our center were prospectively enrolled. Patients were either referred directly to our hospital  score, concomitant anticoagulation, pre-existing vascular risk factors and information on stroke onset time.Additionally, the site of vessel occlusion – identified either by computed tomography (CT) or magnetic resonance imaging (MRI) - was recorded. The extent of early ischemic changes was assessed using the Alberta Stroke Program Early CT Score (ASPECTS) score on baseline imaging of all anterior circulation vessel occlusions. Recanalization result was defined using the modified Thrombolysis in Cerebral Infarction Score (mTICI). All patients received subsequent brain imaging within 24 h after intervention.This study was conducted in accordance with ethical principles for human studies and approved by the local ethics committee. Written informed consent was obtained from the patient or the proxy. Consent was waived when patients died before consent could be obtained or lacked the capacity to give consent and no proxy was available.Mechanical thrombectomy was performed by six interventional neuroradiologists on a 24/7 basis. Different devices were used according to the preference of the interventional neuroradiologist.Successful recanalization was defined as mTICI ≥2b. Symptomatic intracerebral hemorrhage (sICH) was defined as an occurrence of ICH combined with a deterioration of at least 4 points on NIHSS score or indication for surgery or death (according to the SITS-MOST criteria ). Time bPeriprocedural and post-treatment complications were documented until discharge. A follow up was performed after 90 days assessing the mRS by telephone interview with the patients, the next of kin or caregiver. The assessors were blinded to patients’ variables including the results of ET.2-tests or Fisher exact tests were used, as appropriate. Mann-Whitney-U tests were employed for continuous variables. Predictors of better outcome (lower mRS scores) were calculated by ordinal regression analyses on the mRS score at 90 days as the dependent variable. The variables sex, IVT, ship, successful recanalization (defined by mTICI 2b/3), anterior/posterior circulation, compliance with the ASA/AHA guidelines for thrombectomy in acute ischemic stroke patients , NIHSS of ≥6, ASPECTS of ≥6, treatment within 6 h after symptom onset, pre-treatment with IVT in eligible patients according to the guidelines from professional societies; ASA/AHA-group/non-ASA/AHA-group) and mRS before admission (categorized to 0–1 and 2–5) were considered as dichotomous independent variables. Side of vessel occlusion was considered as a multinomial variable  and age, baseline NIHSS, SOR and the ASPECT score as continuous independent variables.For between-group comparisons of categorical variables, χn = 243),An initial model containing the entire study sample with all independent variables (n = 200),the same independent variables plus the ASPECT score (n = 105). The variable ship was excluded from this subanalysis due to the high correlation between the variables ship and SOR.the same independent variables plus the ASPECT score and SOR .The resulting odds ratios (OR) with 95% confidence intervals (CI) and ship), while 100 patients (37.9%) presented directly to our center (mothership). Median age was 75 years (IQR 64–81), 49.6% were female. Seventy patients (26.5%) aged older than 80 years. The median baseline NIHSS score was 16, 153 patients (58%) received IVT before ET. Vessel occlusions of the anterior circulation were present in 221 (83.7%) cases, whereas 43 (16.3%) subjects suffered from LVO in the posterior circulation. In patients with anterior circulation stroke, the median ASPECTS was 7. The distribution of the pre-stroke mRS scores of the entire study population is displayed on Additional file A total number of 264 patients received mechanical thrombectomy within the study period. Baseline characteristics are displayed in Table ship  than by mothership .In patients with known time of symptom onset, median time of SOG was 240 min (IQR 180–310), median time of SOR was 293 min (IQR 227–370)  was achieved in 190 subjects (72%), 103 of them (39%) reached complete recanalization (defined by a TICI 3 score). In eleven cases (4.2%) of these the occluded artery was found to be already recanalized in angiography prior to any endovascular procedure. An overview of the devices used for thrombectomy is given in the Additional file b/3 was aPeriprocedural complications occurred in 19 patients (7.2%) and comprised vasospasm, dissections, bleedings, distal emboli. Within 24 h after intervention and during the entire hospital stay 117 patients 44.3%) suffered from complications which encompassed recurrence of stroke, groin haematoma/pseudoaneurysm, myocardial infarction, ICH, malignant media infarction, hemicraniectomy, severe pneumonia and others . Any ICH on follow-up imaging was found in 46 patients (17.4%), sICH occurred in 12 cases (4.5%) [see Additional file % suffereAt discharge, mortality rate was 16.7% (44 patients). In 17 patients (6.4%) death was attributable to the initial ischemic stroke syndrome . sICH precipitated death in eight patients (3.0%). In 16 cases (6.1%) reasons of death were related to initial stroke , in three patients (1.1%) causes of death were not related to stroke (e.g. cardiac decompensation etc.). In 20 (45.5%) of the patients, who died during the hospital stay, recanalization was futile.Follow-up information was available for 248 patients (94.0%). Independent outcome (mRS 0–2) was achieved in 65 cases (26.2%), while 122 patients (49.2%) suffered from a poor outcome (mRS 5–6). Mortality after 90 days was 29.8% , patients with available ASPECTS (p = 0.88), patients with available ASPECTS and information on time to recanalization (p = 0.95).An additional multivariable regression analysis including the interaction term (TICI2b/3 x ASA/AHA-group or non-ASA/AHA-group) showed no significant effect of the interaction term in the three models: entire study population (n = 6) or ASPECTS (n = 1).A total number of 257 patients were able to be classified either to the group of patients complying with the suggested guidelines of the ASA/AHA or to those who did not adhere to these criteria Table . In seveWhen applying the ASA/AHA guideline criteria for patient selection, only 91 patients (35.4%) met these criteria, while 166 patients (64.6%) did not comply with at least one of these criteria for thrombectomy: in 65 patients (39.1%) vessel occlusion occurred in the distal segment of the MCA (M2) or in the posterior circulation, 10 patients (6.0%) had a baseline NIHSS score < 6, 35 patients (21.1%) had an ASPECTS < 6, 37 patients (22.3%) suffered from pre-stroke disability with mRS > 1, 57 patients (34.3%) were treated beyond the 6 h threshold, and in 21 cases (12.7%) stroke occurred during sleep (wake up stroke).p < 0.001), had a lower median ASPECTS , showed a longer time of SOR , had a lower rate of good clinical outcome defined by mRS 0–2 , a higher rate of poor outcome assessed by mRS 5–6  and a higher median mRS score at 90 days  , a higher rate of patients with poor outcome (49.2%), and a lower rate of patients achieving a good outcome (26.2%), as compared to the “big five” RCTs of thrombectomy in acute ischemic stroke   and the First, with a median age of 75 years patients in our population were older than in the HERMES meta-analysis (median age 68 years) . One outSecond, patients treated by thrombectomy in our study had larger baseline ischemic stroke cores indicated by a median ASPECTS of 7 being lower than the median ASPECTS of 9 in the pooled analysis of the HERMES collaboration . Higher Moreover, 14% of our study sample had pre-existing disability reflected by a pre-treatment mRS score > 1. This, again, would have excluded patients from most thrombectomy trials. And again a higher mRS score representing pre-stroke disability was identified as an independent predictor of worse outcome in our analysis.Sixteen percent of the patients presented with a vessel occlusion located in the posterior circulation. LVO of the posterior circulation were excluded from the thrombectomy trials, thus it remains elusive to what extent this important subgroup of patients benefits from ET. While several studies already indicate that thrombectomy may be a safe and effective treatment modality in basilar artery occlusion (BAO) , 16, a sIt becomes evident, that a large proportion of patients treated by thrombectomy under routine conditions in a large neurovascular center does not comply with the narrow inclusion criteria of the randomized trials. Indeed, almost two third of our patients would not have met the suggested ASA/AHA-criteria for thrombectomy. A similarly high proportion of non-ASA/AHA-patients was included in the TREVO Stent-Retriever Acute Stroke (TRACK) registry further supporting the hypothesis that in real life only a small group of patients matches the ASA/AHA guidelines . SimilarWhen comparing the patient groups meeting the ASA/AHA-criteria with those failing to match these criteria, we observed better outcome in the ASA/AHA-group of patients with 38.1% showing an independent outcome (mRS 0–2) after 90 days as compared to only 20.9% in the non-ASA/AHA-group. This is very likely explained by the fact that the suggested ASA/AHA-criteria, in the same way as the inclusion criteria of the RCTs, select those patients with the highest likelihood of treatment benefit. This approach was previously referred to as “cherry picking” of patients of the large thrombectomy trials [Of note, in our entire patient cohort the recanalization rate (72.0%) as well as the rate of sICH 4.5%), as one of the most important safety outcome parameter, were within the range of the RCTs  –5, 10 an%, as oneNotably, we did not observe an interaction between successful recanalization and compliance with the ASA/AHA-group. Although interaction analyses might be underpowered, this finding suggests that the effect of achieving successful recanalization with regard to functional outcome is homogenous across both subgroups of patients (ASA/AHA eligible vs. non-eligible). Furthermore, regression analysis identified the same predictors of better outcome in both subgroups of patients, i.e. lower age, lower pre-stroke mRS, lower baseline NIHSS score, and successful recanalization (data not shown). In addition, compliance with the ASA/AHA-group of patients did not predict an independent outcome 90 days after stroke in our study population.We also performed a systematic analysis of death after thrombectomy and of severe complications that occurred during in-hospital stay. Beside cerebral causes of death, i.e. malignant brain infarction, severe non-cerebral complications, such as pneumonia, mesenteric infarctions with lactic acidosis, cardiac decompensation, or multi-organic failure, were causes of death. A reason for the high rate of severe complications might be rooted in the large proportion of elderly patients with pre-existing comorbidities in our study population.For clinical practice, these findings indicate, that although overall outcome may be worse in stroke patients not meeting the strict criteria of randomized trials or ASA/AHA guidelines, thrombectomy may as well be beneficial in these patients and less aggressive treatment may lead to a worse outcome. This is supported by results of the individual patient data meta-analysis by the HERMES collaboration that showed a benefit of ET in some of these subgroups , e.g. paThis study is limited by patients derived from a single center and lacking of a group of stroke patients with LVO not treated by ET. However, this study comprises a high number of prospectively enrolled stroke patients in clinical practice that were treated by ET in a center with a big catchment area. Hence, together with our findings available evidence strongly suggests that thrombectomy is feasible and safe in patients beyond those meeting the strict criteria of previous RCTs and ASA/AHA-recommendations, but the effectiveness needs to be confirmed in future RCTs.In a real-world setting the majority of stroke patients undergoing thrombectomy is treated outside the core inclusion criteria of the RCTs of stroke thrombectomy. Outcome of unselected patients treated in the “real world” is worse than in the highly selected patient cohorts of clinical trials owing to clinical characteristics that are associated with worse outcome . However, rates of successful recanalization, sICH, and parameters predicting better outcome after ET are the same in these patients. By this, our findings support the application of thrombectomy in broader patient populations not represented in the RCTs.Additional file 1. Distribution of the pre-stroke mRS score (n = 263). The distribution of the mRS scores before admission of the patients enrolled in this study is displayed on this table.Additional file 2. Different applied devices. An overview of the devices applied during intervention is given in this table.Additional file 3. Complications – periprocedural and during the hospital stay (n = 264). This table provides data on the complications which occurred during intervention and during hospital stay.


Saussurea costus and its mode of intervention in human cancer cells of breast, colon, and liver were investigated.  Apoptosis, a major form of programmed cell death, plays a vital role in regulating tissue development and maintenance of homeostasis in eukaryotes. Apoptosis can occur via a death receptor-dependent extrinsic or a mitochondrial-dependent intrinsic pathway and can be induced by various chemotherapeutic agents. In this study, the anticancer activity of S. costus leaves were extensively extracted in five solvents of different polarity. The cytotoxicity and anticancer effect of the extracted secondary metabolites were investigated against breast (MCF-7), liver (HepG2), and colon (HCT116) cancer cell lines using a Sulphorhodamine B (SRB) assay. Secondary metabolites extracted using hexane, methanol, ethyl acetate, and chloroform had the highest cytotoxicity and thus the greatest anticancer effect on all the cancer cell lines tested , while butanol was comparatively less active . Further investigation using DNA flow cytometry and fluorescent microscopy revealed that the extract arrested the cells in the G1 phase of cell cycle and induced apoptosis. Furthermore, the elevated expression level of proapoptotic proteins and decreased expression level of antiapoptotic proteins confirmed that the intrinsic  pathway was involved in mediating the apoptosis of cancer cells upon treatment with S. costus extract. These results altogether suggest that S. costus could be a potential anticancer agent.  In this study, the bioactives of S. costus extract is the potential source of the secondary metabolites that could be used as anticancer agent to treat diverse cancers of breast, colon, and liver. These results suggest that the  Cancer, abnormal growth of cells, also called malignancy, is the second leading cause of death globally and is responsible for an estimated 9.6 million deaths in 2018 [Hepatocellular carcinoma (HCC), the most common primary malignancy of the liver, is a leading cause of death in people with cirrhosis . ChronicS. costus to possibly isolate almost all bioactives, and then, these diverse solvent extracts were tested individually against diverse cancer cell lines.A variety of cytotoxic drugs reported to combat cancer usually are incompetent not only because of their efficacy but also because of their undesirable side effects. There is a need for novel anticancer compounds without adverse side effects. Plants are known to have a long history in cancer treatment  and mediSaussurea costus (Costaceae family), habitant of the subalpine region of Jammu and Kashmir, Himachal Pradesh, and Uttarakhand, is used to treat various ailments, viz., ulcer, headache, rheumatism, cough and cold, and throat infection, while in Korea it is frequently used in Korean traditional prescriptions for inflammatory diseases [S. lappa roots, has anticancer activity by inhibiting the proliferation of HL-60 human leukemia cells by induction of apoptosis through ROS-mediated mitochondrial permeability transition and cytochrome C release [1/S phase arrest and activating mitochondrial-mediated apoptotic pathways [S. costus, commonly known as kuth in Kashmiri, have not been investigated particularly in the cancer cell lines taken in this study.diseases . Its antdiseases –13 activdiseases , 15, antdiseases , 17, antdiseases , has gaidiseases  and tumodiseases . Costuno release . In anotpathways . All theApoptosis, a major form of programmed cell death, plays a vital role in regulating tissue development and maintenance of the homeostasis in eukaryotes –25. One S. costus could function as a chemotherapeutic agent in human cancers. We tested S. costus extracts for their effects in inhibiting cell proliferation of the cancer cells of breast, liver, and colon and characterized its underlying mechanisms of action. The demonstration of anticancer activity of the S. costus extracts against diverse cancer cell lines in this study indicates that the plant extract/its bioactives can be used in the treatment of diverse cancers. However, isolation of active compounds and their anticancer activity needs to be tested in vivo using animal models.In this study, we explored the possibility whether S. costus on the proliferation of different types of cancer cell lines, an SRB assay was performed. The viability (%) of the cancer cells was used as an indicator of the cell toxicity towards the treatment of cells with different concentrations of extract for 72 hrs. The results demonstrate that the viability of cells decreased in a dose-dependent manner , almost similar to that of a prominent anticancer drug, doxorubicin (IC50 < 1 μg/ml). The methanolic extract exhibited comparatively less cytotoxic effect  against all the cell lines tested  suggested that the major cellular populations of all tumor cell lines tested against all types of extracts were in the early apoptotic stage. However, a marked percentage of HCT116 cells were in the necrotic stage of apoptosis when treated individually with chloroform, ethyl acetate, and butanol.To further investigate the extract-induced inhibitory effect, cells treated with various extracts were analyzed under a fluorescent microscope for nuclear morphological changes (apoptosis or necrosis) after acridine orange/ethidium bromide staining. The major hallmarks of apoptotic cell death are DNA fragmentation and loss of membrane asymmetry. As shown in Figures S. costus extract was causing decreased cell viability in all the cancer cell lines tested. For this purpose, we tested the hexane extract for having the least IC50 for all the four cancer cell lines. The level of Bcl-2 (antiapoptotic) and Bax (proapoptotic) expression in cancer cells was determined following treatment with the hexane extract of S. costus. Treatment of S. costus extract decreased the levels of Bcl-2 in all cancer cells, while the level of Bax expression is highly induced after 48 hours of treatment , the activation of the executioner caspase-3 and caspase-7 is regulated both by extrinsic (death ligand) and intrinsic  pathways. The BCL2 family of proteins, including both antiapoptotic (Bcl-2) and proapoptotic (Bax) regulators, is the hallmark of apoptosis regulation. We, therefore, addressed the potential mechanism by which reatment . It has aspase-3 .S. costus extracts have been investigated for their biological efficacy against cancer cells. The dried plant leaves were used for crude extract preparation using different solvents. We used several solvents  to extract all compounds of varying polarity. The four solvent extracts methanol, hexane, chloroform, and ethyl acetate showed strong cytotoxic activity against all the three cancerous cell lines tested (IC50 < 2.6 μg/ml). However, butanol was comparatively less effective . Cell cycle analysis showed that the extracts basically arrested all types of cancer cells in the G1 or G2/M phase of the cell cycle, though the percentage of cells showed a variation with respect to a particular cell line . For the fresh crude extract preparation, 100 g of fresh leaves was washed with distilled water and ground by a grinder with 500 ml of 80% aqueous ethanol. The leaves were then immersed in one liter of 80% aqueous ethanol, left for seven days at room temperature (18–24°C), and occasionally stirred. The ethanol extract was filtered using filter paper and concentrated to dryness under reduced pressure using a rotary evaporator at 37°C . The concentrated crude extract weight was 10 g, and the extraction yield was 10%. The crude extract was then reconstituted in 400 ml of distilled water and extracted with different solvents according to the polarity, viz., methanol, hexane, chloroform, ethyl acetate, and butanol , using liquid-liquid extraction method. After that, the solvent phase was separated and reevaporated using a rotary evaporator. The solvent was then left at room temperature (20–26°C) for complete evaporation for 3 days. 0.01 g of each crude extract was stored as a stock solution for bioactivity assays. The crude extracts were stored at -20°C for further studies.The dry leaves of μg/ml penicillin and heat-inactivated fetal bovine serum (10% v/v) in a humidified 5% (v/v) CO2 atmosphere at 37°C [The cells of the human hepatocellular carcinoma cell line (HepG2), colorectal adenocarcinoma cell line (HCT116), and breast adenocarcinoma cell line (MCF-7) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI-1640 supplemented with 100 μg/ml), and untreated cells were included as a control. Triplicate wells were incubated with different concentrations for 72 hours and subsequently fixed with TCA (10% w/v) for one hour at 4°C. After several washings, cells were stained by 0.4% (w/v) SRB solution for 10 min in a dark place. Excess stain was washed with 1% (v/v) glacial acetic acid. After drying overnight, the SRB-stained cells were dissolved in tris–HCl and the color intensity was measured in a microplate reader at 540 nm. The linear relation between the viability percentage of each tumor cell line and extract concentration was analyzed to get the IC50  using SigmaPlot 12.0 software [The cytotoxicity of different compounds was tested against human tumor cells using the Sulphorhodamine B assay (SRB). Healthy growing cells were cultured in a 96-well tissue culture plate (3000 cells/well) for 24 hours before treatment with the tested extracts to allow attachment of the cells to the plate. Cells were exposed to five different concentrations of each extract , and resuspended in 0.5 ml of PBS. 10 ml of 70% ice-cold ethanol was added gently while vortexing. Cells were kept at 4°C for one hour and stored at -20°C until analysis. Upon analysis, fixed cells were washed and resuspended in 1 ml of PBS containing 50 mg/ml RNase A and 10 mg/ml propidium iodide (PI). After 20 min incubation at 37°C, cells were analyzed for DNA contents by FACSVantage™ . For each sample, 10,000 events were acquired. Cell cycle distribution was calculated using CELLQuest software  [The cells were treated with the extract ICose, CA) .50 of the compound. Cells were harvested using trypsinization and washed twice with PBS. Ten microliters of the cells was transferred to a glass slide and mixed with 2 μl of AO/EB mixture (100 μg/ml of AO and 100 μg/ml of EB). The apoptotic, necrotic, and live cells were examined immediately under a fluorescence microscope [The cells were seeded in 6-well tissue culture plates and incubated for 48 hours with the ICcroscope .50) of hexane extract for 48 hours and lysed with cell lysis buffer. The total protein concentration was estimated by Coomassie plus protein assay kit , and 20–40 μg protein from the cell lysate was fractionated with SDS-PAGE followed by transfer to a nitrocellulose membrane. After probing with the respective primary antibodies, HRP-secondary antibodies were added, and the blots were developed using an enhanced chemiluminescence (ECL) detection kit. The membranes were stripped off the antibodies and incubated with the β-actin antibody at 1 : 5000 dilutions and redeveloped to detect the β-actin band. Bands specific to Bcl-2, Bax, and caspase-3 were quantified using ImageJ  and normalized to β-actin band intensity. All the experiments were performed in triplicates. For statistical analysis, two-tailed Student's t-test of GraphPad Prism 6.0  was used, and the P value of ≤0.05 was considered statistically significant. Caspase-3, Bax, Bcl-2, and β-actin antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA.The western blotting procedure was performed as described . In brie


Infectious meningitis is a serious disease and patient outcome relies on fast and reliable diagnostics. A syndromic panel testing approach like the FilmArray ME can accelerate diagnosis and therefore decrease the time to pathogen specific therapy. Yet, its clinical utility is controversial, mainly because of a remaining uncertainty in correct interpretation of results, limited data on its performance on clinical specimens and its relatively high costs. The aim of this study was to analyze clinical performance of the assay in a real life setting at a tertiary university hospital using a pragmatic and simple sample selection strategy to reduce the overall cost burden.N = 171 samples matched to our risk criteria and were subjected to FilmArray ME analysis. Those samples were also analyzed by reference methods: culture only (n = 45), PCR only (n = 20) or both methods (n = 106).Over a period of 18 months we received 4623 CSF samples . FilmArray ME analysis was restricted to CSF-samples with a high pretest probability of infectious meningitis, e.g. positive Gram-stain, samples in which leukocytes and/or bacteria were evident or urgent suspicion of infection was communicated by clinicians. 56/171 (32.75%) were FilmArray ME positive. Bacterial pathogens were detected in 30/56 (53.57%), viral pathogens were detected in 27/56 (48.21%) and yeast DNA was detected in 1/56 (1.79%) of positive samples. Double detection occurred in 2/56 samples. In 52/56 (92.86%) FilmArray ME positive samples, results could be confirmed by the reference assays .The FilmArray ME assay is a fast and reliable diagnostic tool for the management of infectious meningitis and can easily be implemented in routine diagnostic workflows. However, correlation of test results and underlying clinical symptoms requires experienced users and the awareness of potentially false negative or false positive results. Moreover, considering the need for antimicrobial susceptibility testing, the use of molecular tests as a stand-alone diagnostic cannot be recommended. Infectious meningitis is a serious threat with high morbidity and mortality rates –4. In baN. meningitides, a worldwide leading meningitis pathogen, and L. monocytogenes detected in none of the samples [The Biofire FilmArray ME Panel provides for a simultaneous multiplex testing of a comprehensive panel of 14 pathogens using a minimal amount of CSF (e.g. 200 μl). This sample-to-answer system was FDA-cleared in 2015. However, in the multicenter evaluation study, reported detection rates for bacterial pathogens typically found in infectious meningitis cases were surprisingly low. Most notably,  samples . Meanwhi samples –13 with  samples . Aside fThe study was conducted over a period of approximately 18 months (September 2015 through February 2017) in our laboratory. CSF samples demonstrating abnormality in Gram-stain (e.g. leucocytes and/or bacteria visible) and CSF samples of patients with urgent suspicion of infection as communicated by clinicians were selected for additional FilmArray ME analysis .Bacillus subtilis inhibition-assay for preliminary assessment of presence of antibiotics [2, one drop of CSF sediment was inoculated in thioglycollate medium (thioglycollate broth) and incubated at 37 °C. All Plates and broth are inspected at least once daily for bacterial growth. Species identification relied on MALDI-TOF technique combined with classical biochemical identification methods.CSF samples were centrifuged for 10 min at 3000 rpm at room temperature. Supernatant was used for ibiotics . With onLDTs), specific primers and probes are designed based on sequences and assays published elsewhere [www.sepsitest-blast.de/en/index.html) and the BLAST tool of NCBI (www.ncbi.nlm.nih.gov/blast).Nucleic acid extraction (RNA and DNA) was performed on a QIAsymphony SP/AS instrument with the QIAsymphony DSP Virus/ Pathogen Mini kit using 200 μl of native CSF sample. During extraction process, universal PCR inhibition control (DNA/RNA) was added. Elution volume was 60 μl. Quantitative real-time-PCR reaction was set-up using 5–10 μl of eluate, reverse-transcription, amplification and analysis were performed on a LightCycler 480 instrument. The quantitative real-time PCRs used in CSF diagnostic are laboratory developed tests . Hydration solution was injected into the FilmArray ME pouch to rehydrate the freeze-dried reagents, sample mixture was thereafter injected. The pouch was transferred to the instrument and run was initiated. Nucleic acid purification, reverse transcription, multiplex PCR and melting curve analysis are performed automatically, with a runtime of about 1 h. Test reports are generated by instrument software.n = 4623 CSF samples , including samples sent for both molecular diagnostic and bacterial culture as well as samples sent only for either purpose. In detail, bacterial culture was performed in n = 2682 samples, molecular diagnostic was performed in n = 1891 samples and n = 50 samples were sent for serology purpose. The overall positivity rate was 5.97% with positive results in 137/2682 samples analyzed by culture (5.11%) and 139/1891 positive results in samples analyzed by molecular methods (7.35%). More details are given in supplementary Tables Over a period of 18 months we received a total of n = 171 samples matched to our risk criteria and were subjected to FilmArray ME analysis. Of those, n = 117 samples had abnormality in gram-stain  and n = 54 samples were selected as suspicion of infectious meningitis was communicated by clinicians. Of note, in 24/54(44.44%) of samples selected upon clinical request, gram stain and primary bacterial culture was not performed as suspicion for viral infection was communicated. 6/30 samples of that subset had positive gram stain (20.00%), whereas 25/30 samples had negative gram stain (83.33%). Overall, gram stain was performed in 147/171 samples. Leucocytes were seen in 122/147 (82.99%) samples, bacteria in 15/147 (10.20%) samples and yeast in 1/147 (0.68%) of those samples.Overall, n = 45), molecular analysis only (n = 20) or both methods (n = 106). 56/171 (32.75%) were FilmArray ME positive. Bacterial pathogens were detected in 30/56 (53.57%) and viral pathogens were detected in 27/56 (48.21%) of positive samples. Additionally, Cryptococcus neoformans was detected in 1/56 samples (1.79%). Double detection occurred in two samples. An overview of pathogens detected is given in Fig. Samples analyzed by FilmArray ME were also tested by reference methods: culture only (only n = 0 or bothn = 116), positive Film Array ME results were obtained in 44/116 samples . Organisms have been seen in 16/117 samples selected for positive gram stain (bacteria in 15/117 samples (12.82%), yeast in 1/117 samples . Within the subset of samples selected upon request by clinicians, (n = 54), positive Film Array ME results were obtained in 44/54 samples .Within the subset of samples selected for leucocytes seen in gram stain . Residual material of the two samples positive for E. coli K1 and S. agalactiae was not sufficient for additional 16S-rRNA amplification, as proper DNA extraction for this method requires a high input volume (e.g. 1000 μl of CNS liquid). No specific real-time PCR assay for detection of E.coli K1 was applied. One sample that was FilmArray positive for HSV-1 could not be confirmed by two independent molecular detection methods. Neither our laboratory developed real-time PCR assay, nor a commercially available real-time PCR assay yielded a positive result . In four samples that were positive for S. pneumoniae and Parechovirus by specific in-house PCRs, respectively (NPV =98.26%). In two samples, FilmArray analysis detected two pathogens . Presence of VZV (1.4 × 104 copies/ml) and H. influenzae was confirmed by specific real-time PCRs, but conventional assays were unable to detect HHV-6, and these results were thus regarded as false-positive. In 10 samples, pathogens not found by the FilmArray ME panel were detected by culture  or PCR .Two samples (1%) revealed negative FilmArray results, but were positive for n = 1560, n = 969, n = 705 and n = 253 CSF samples analyzed by Film Array ME, respectively). Figure Results were compared to the multicenter evaluation study  and threp < 0.0001). In addition, we could increase the amount of bacterial pathogens to 53% of the specimens, again this is more than in other published studies that observed positive ranges for bacterial pathogens between 15 and 37% [p < 0.0001).We here report on our real-life experience of the implementation of the FilmArray ME panel into a diagnostic laboratory in a German university hospital setting. We implemented a simple sample selection strategy in our workflow: FilmArray ME analysis was restricted to CSF-samples with a high pretest probability of infectious  meningitis. Decision was made in our laboratory based on gram stain and/or urgent suspicion of infectious meningitis communicated by clinicians, e.g. neither the clinical decision for lumbar puncture was affected, nor was explicit ordering of the assay permissible. Using this selection criteria we selected 171/4623 CSF specimens for additional syndromic molecular testing. Overall we observed positive results in 56/171 32%), which is higher than in other published studies , 34–38 a%, which  and 37% . Indeed,S. pneumoniae (17/30) was the most frequent bacterial pathogen detected by the FilmArray ME assay. Additionally, in 10/30 specimens rapid detection of N. meningitides (n = 5), H. influenzae (n = 2) or L. monocytogenes (n = 3) was important for optimal clinical management including rapid implementation of post exposure treatments in contact persons (antibiotic prophylaxis and/or vaccination).In concordance with other studies , 34–38, S. pneumoniae and Parechovirus were detected by specific in-house PCRs. Although it might be crucial to detect a bacterial or viral meningitis in the individual case, our data confirm the high negative predictive value of the multiplex PCR assay that has been emphasized by others [Analytically we observed good concordance (52/56 positive results could be confirmed) between the FilmArray ME panel results and our comparator assays. Only in two of the samples the FilmArray ME panel yielded a discordant negative result compared to the local reference methods. Namely, in each of one sample y others .E. coli and S. pneumoniae. One sample positive for S. agalactiae in FilmArray E panel showed no inhibition of growth in B. subtilis inhibition assay. Notably, culture negative yet PCR positive infectious meningitis cases caused by S. agalactiae have been described previously, underlining the importance of molecular diagnostic for optimization of patient management [S.agalactiae was indeed false positive and the communication of the test result might have led to a dispensable antibiotic administration.In four samples, potentially false positive results of FilmArray ME panel occurred, including three samples tested positive for bacterial pathogens by multiplex PCR that remained culture negative. Among the reasons for failure of primary culture is antibiotic administration prior to lumbar puncture . In the nagement –42. OtheHSV-1. Beside the probable useless administration of Acyclovir following a false positive HSV-1 result, one should also consider the questionable relevance of Herpesviridae detection in CSF in general. As these viruses feature life-long latency after primary infection, subclinical reactivation with replication can be seen in conjunction with different underlying clinical conditions [Herpesviridae detection in multiplex PCR testing might lead to delayed diagnosis of the actual underlying disease, as described by Gomez et al. for a patient suffering from tuberculous meningitis [In our study, one sample remained disconcordant for nditions , 44. Thuningitis .HHV-6 is included in the FilmArray ME panel. This virus exhibits not only latency and also has the potential of chromosomal integration. Therefore, careful interpretation of HHV-6 detection in FilmArray ME panel is mandatory. The clinical diagnosis should generally not be made by molecular detection of HHV-6 in CSF alone [HHV-6 detection in our study remains unclear in its consequences for patient management and outcome.The herpesvirus SF alone , 46. As Bacillus sp., CoNS, S. aureus, and K. pneumoniae grew on culture media, whereas BK virus and Streptococcus spp. were detected by PCR. Albeit the questionable clinical relevance of Bacillus sp., Streptococcus spp. and CoNS in a CSF sample, detection of S. aureus, K. pneumoniae and BK-Virus may account for serious infections. This underlines, that reasonable and effective CNS diagnostic should combine classical cultural and molecular methods, rather than focus on one test system alone.An obvious limitation of any syndromic molecular panel testing approach is the limited number of pathogens included into the panel. Even though the FilmArray ME panel includes a broad range of pathogens, overreliance on negative results might be crucial especially in geographical regions with unusual etiologic agents of meningitis this might yield to ineffective testing . In our It has been proposed recently that implementation of the FilmArray ME Panel in routine diagnostic may help for cost saving in direct antimicrobial utilization and might decrease diagnostic costs even with uncontrolled routine availability of the assay , 48.However, both Naccache et al. and Radmard et al. reported massive overutilization of the test, when no eligibility criteria was implemented , 33. In Eichinger et al. reported their findings of implementation of the FilmArray ME panel as a POCT in children with suspected meningitis in a children hospital . They hiOur implementation approach is independent from approval by infectious disease specialists and it does not involve any ordering modifications. The latter opportunities have been proposed for diagnostic stewardship programs , howeverWe report our real-life experience of the implementation of the FilmArray ME panel into our laboratory workflow, no sample randomization was done. Clinical specimens were tested prospectively, yet our selection strategy might exhibit a selection bias. Notably, sample selection strategy was mainly based on gram-stain abnormalities ensuring on-spot decision making, but not representing the ideal method for leucocyte detection. Furthermore, we did not assess all samples by all comparator assays, in particular for samples that were not selected for FilmArray ME analysis, diagnostic was performed according to orders and no additional assays were performed. Thus, we cannot rule out, that pathogens have been overlooked in some samples. Moreover, no clinical data was analyzed, hence effects on patient outcome or antibiotic utilization in our study remain unclear. Nevertheless we believe that our data gain new insights in how syndromic panel testing may be implemented into laboratory routine and therefore may help to identify the ideal approach to ensure its clinical utility.The FilmArray ME is a useful tool for fast and reliable diagnostic of infectious meningitis in a real life diagnostic setting and can be easily implemented in routine diagnostic workflows. Beyond that, our strategy of risk-assessment driven selection of samples might help to avoid unnecessary testing if bacterial pathogens are suspected. However, correlation of test results and underlying clinical symptoms requires experienced users and the awareness of potentially false negative or false positive results. Moreover, considering the need for antimicrobial susceptibility testing, the use of molecular tests as stand-alone diagnostic cannot be recommended.Additional file 1: Table S1. Overview of analytical methods performed and detection rates in n = 4623 CSF samples received.Additional file 2: Table S2. Overview of pathogens detected by different methods. Numbers of pathogens detected by routine diagnostic procedures, numbers detected by Film Array ME Panel as well as confirmatory results are given.


Salvia miltiorrhiza injection (SMI) is a classical traditional Chinese medicine, which plays an active role in the treatment of many diseases such as promoting blood circulation, removing blood stasis, reducing inflammatory reaction, and improving acute lung injury (ALI). Previous studies have shown that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in the pathophysiological process of ALI. However, the relationship between SMI and MMPs/TIMPs remains unclear. In this study, Wistar rats were randomly divided into control group (NC), Salvia miltiorrhiza group (SM), lipopolysaccharide group (LPS), and Salvia miltiorrhiza treatment group (Tsm). The four groups were subdivided into four time points , and specimens were collected after animal sacrifice at each time point. Serum TNF-α and IL-6 levels were detected by ELISA. The degree of lung injury was determined by lung tissue hematoxylin-eosin staining, lung wet/dry weight (W/D) ratio, and lung permeability index. The changes in lung MMPs/TIMPs protein and mRNA were detected by Western blot and real-time quantitative PCR. The results showed that rats injected with LPS experience acute lung injury, and the ratio of MMPs/TIMPs in lung tissues increased gradually with time. In the Tsm group, the ratio of MMPs/TIMPs decreased gradually, and likewise, the balance was gradually restored, while indicators related to lung injury were gradually declined. These data suggest that SMI alleviates LPS-induced acute lung injury; this protective effect may be related to regulation of the balance of MMPs/TIMPs ratio. Acute lung injury (ALI) is a common critical illness in clinic that can manifest as severe hypoxia and dyspnea, has a relatively high mortality rate, and is difficult to treat , 2. The The extracellular matrix (ECM) is the main structural part of the alveolar-capillary membrane, which not only plays a mechanical-supporting and mechanical-connecting role between cells but is also important in maintaining normal tissue structure and function , 10. StuSalvia miltiorrhiza injection (SMI), a Chinese Materia Medica standardized product extracted from the root of red-rooted Salviae Miltiorrhizae , which is officially recorded in Pharmacopoeia of the People's Republic of China (version 2010) and plays an active role in the treatment of many diseases such as promoting blood circulation, removing blood stasis, reducing inflammatory reaction, and improving coagulation [gulation .Previous studies have shown that SMI has a protective effect on ALI , 15, butSalvia miltiorrhiza injection (SMI) was purchased from Chiatai Qingchunbao Pharmaceutical Co., Ltd. . It is a classical traditional Chinese medicine standardized aqueous product extracted from the root of red-rooted Salvia . SMI is an injectable solution through modern formulation and preparation process; the main ingredient is tanshinone, which is officially recorded in Pharmacopoeia of the People's Republic of China (version 2010) and plays an active role in the treatment of many diseases [diseases .Specific pathogen-free (SPF) male Wistar rats  were purchased from Charles River Experimental Animal Co., Ltd.  and raised in the Xiamen University Laboratory Animal Center (License number: SYXK (Min) 2018-0010). The animals were housed one week before the start of experiment under standard laboratory conditions at a stable temperature (22 ± 2 °C), humidity (50 ± 10%), and a 12/12 h light/dark cycle with food and water ad libitum , 16. AllSalvia miltiorrhiza (SM) group, lipopolysaccharide  group, and Salvia miltiorrhiza treatment group (LPS + SM: Tsm group). In the LPS group, an experimental model of ALI was established by intravenous injection of 5 mg/kg LPS [Salvia miltiorrhiza injection. The Tsm group was given 50 mg/kg Salvia miltiorrhiza injection one hour after 5 mg/kg LPS injection. The NC group was injected with 2 mL/kg normal saline as a control. The four groups were further divided into 2, 6, 12, and 24 hours subgroups with six rats at each time point. The rats were anesthetized via intraperitoneal injection with 2.25% pentobarbital sodium  [In total, 96 Wistar rats were randomly divided into a normal control (NC) group, g/kg LPS . The SM , China) , and bloα  and IL-6  levels were measured according to the ELISA procedures as described by the manufacturers, in order to investigate the inflammatory reaction in rats.The rats were anesthetized, followed by collecting 3 mL of blood from the abdominal aorta. After centrifuging the blood samples at 3,500 g for 10 min, the supernatants were collected. TNF-μm thickness [Small pieces of upper left lung tissue were fixed in 4% formaldehyde for 24 h, followed by conventional paraffin embedding and tissue sectioning at 5 hickness . The tisThe wet/dry weight ratio is an index of pulmonary edema. The inferior lobe of the left lung was excised and the wet weight was recorded before being placed in an incubator at 60°C for 72 h to obtain the dry weight . The lunDetection of sample protein was performed by bicinchoninic acid (BCA) protein quantitative kit . The absorbance was measured at 562 nm wavelength with a multifunction microplate reader, and a protein standard curve was drawn. A tracheoalveolar lavage was performed 3 times using 1 ml phosphate buffered saline (PBS) per lavage on the upper right bronchi and the bronchoalveolar lavage fluid (BALF) was collected (90% recovery). The protein contents of BALF and plasma were calculated according to the absorbance values. To determine the extent of the lung injury, the pulmonary permeability index (PPI) was calculated using the following equation: PPI = BALF protein content/plasma protein content .β-actin was used as an internal control for quantitative real-time PCR  detection of MMPs and TIMPs. cDNA synthesis kit was purchased from Tiangen Biotech Co., Ltd. . The forward and reverse primer sequences  for MMP2, MMP9, TIMP1, TIMP2, and β-actin are shown in –ΔΔCt method [Approximately 50 mg of right middle lobe lung tissue was used for total RNA extraction  using the TRIzol  method . After at method .μg total protein to 1/3 volume of 4 × SDS-PAGE  sample buffer, boiling the protein sample for 10 min, and separating the proteins by SDS-PAGE. After transferring the separated proteins onto PVDF membrane , the protein blots were prepared according to the conventional procedures of Western blot analysis [β-actin absorbance. Related antibodies and companies required in Western blot analysis are shown in Approximately 50 mg lower right lung tissue samples were collected from the different time point groups of rats, followed by washing, homogenization, and lysing . After canalysis . After ds). The independent sample t-test was used for comparison between groups. One-way ANOVA was used for comparison within groups. Pearson's correlation analysis was used for the relevant trend variables. p < 0.05 was considered statistically significant.All statistical analyses were performed with SPSS 21.0 software . Each measurement is presented as mean ± SD , a Chinese Materia Medica standardized product extracted from the root of red-rooted Salviae Miltiorrhizae , is an injectable solution through modern formulation and preparation process. The main ingredient is tanshinone, which plays an active role in the treatment of many diseases such as promoting blood circulation, removing blood stasis, reducing inflammatory reaction, and improving coagulation, as well as other effects [ effects , 47.α and IL-6 in the Tsm group to significantly decrease, along with a reduced inflammatory reaction. Additionally, the lung W/D ratio and lung permeability index were decreased significantly, suggesting that Salvia miltiorrhiza reduced pulmonary edema. The pathological analysis showed that the degree of lung injury was gradually reduced over time, suggesting that SMI reduced lung injury. At the same time, we found that the expression of MMP2 and MMP9 in the Tsm group was gradually downregulated over time. The ratio of MMP2/TIMP2 and MMP9/TIMP1 decreased gradually in the Tsm group until the ratio gradually regained balance. This suggested that the protective effect of SMI on ALI might be mediated by rebalancing of MMPs/TIMPs ratio.In this study, SMI one hour after LPS injection caused serum TNF-In summary, our data suggest that SMI can alleviate acute lung injury caused by LPS injection by restoring the balance of MMPs/TMPs ratio. This presents a new avenue of research to pursue clinical applications of this potential treatment.


Praia tianmunica is 15,556 bp long. It contains 37 genes and a control region of 505 bp in length. Gene rearrangement is present in the mitogenome of P. tianmunica. The maximum-likelihood and Bayesian phylogenetic analyses showed that the genus Praia was closely related to genus Labriocimbex.The complete mitochondrial genome of  Praia Wankowicz, 1880 is a small Palearctic genus of Cimbicidae with four species that have been reported  + Asicimbex. A recent cladistic analysis of Cimbicidae based on morphological characters showed that Praia is a basal lineage of Cimbicinae  were collected in Laodian, Xitianmushan, Lin’an, Zhejiang, China  in April 2018. Genomic DNA was prepared in 150 bp paired-end libraries, tagged, and analyzed with the high-throughput Illumina Hiseq 4000 platform. A total of 16,724,850 raw reads was obtained. DNA sequences were assembled using MitoZ  using Leptocimbex yaniae as a reference (coverage = 4595). First, the intergenic region between trnH and nad4 generated by MitoZ was proved to be an error by reassembly using nad4 as the reference in Geneious. Then, we used trnM-trnQ (135 bp), trnY-trnC-trnI (215 bp), and a randomly selected sequence from the middle of the control region (150 bp) as reference sequences to examine the longest interval region further. By consistently obtaining similar coverage of the assembly contigs, we were able to confirm the 505 bp non-coding region between trnQ and trnY. Annotations were generated in the MITOS web server . The gene arrangement of P. tianmunica was consistent with that of Labriocimbex sinicus, Trichiosoma anthracinum, and T. vitellina. Four overlapping regions and 17 intergenic regions were scattered throughout the genome.The complete mitogenome of atp8, nad2, and nad4L were excluded) of 44 Symphytan species and two Apocritan species. The Bayesian inference with Phylobayes under the MtArt-CAT model .Phylogenetic analysis was based on ten unsaturated amino acids (n et al. . All rel


Exposure to total body irradiation (TBI) causes dose- and tissue-specific lethality. However, there are few effective and nontoxic radiation countermeasures for the radiation injury. In the current study, mice were pretreated with a traditional antimicrobial agent, FZD, before TBI; the protective effects of FZD on radiation injury were evaluated by using parameters such as the spleen index and thymus index, immunohistochemical staining of intestinal tissue, and frequency of micronuclei in polychromatophilic erythrocytes of bone marrow. The intestinal epithelial cell line IEC-6 was used to investigate the underlying mechanisms. Our results indicated that FZD administration significantly improved the survival of lethal dose-irradiated mice, decreased the number of micronuclei, upregulated the number of leukocytes and immune organ indices, and restored intestinal integrity in mice after TBI. TUNEL and western blot showed that FZD protected intestinal tissue by downregulating radiation-induced apoptosis and autophagy. Meanwhile, FZD protected IEC-6 cells from radiation-induced cell death by inhibiting apoptosis and autophagy. To sum up, FZD protected against radiation-induced cell death both in vitro and in vivo through antiapoptosis and antiautophagy mechanisms. With the rapid development of nuclear technology and the extensive application of nuclear medicine, ionizing radiation (IR) is increasing the health risk, and a series of pathological changes might occur and consequently cause tissue damage and even lead to death. Exposure to high-dose radiation induces acute radiation syndrome (ARS), manifested by DNA damage, cell death, organ and tissue damage, and so on . ExposurIntestinal injury is a very common complication of radiotherapy. When the digestive system is severely damaged, acute gastrointestinal syndrome (AGS) is the predominant cause of death within the first week after IR exposure , and AGSAlthough research on a radioprotector is increasingly extensive, there are still problems to be solved. When AGS occurs, the routine treatment is aimed at reducing apoptosis of the gastrointestinal mucosal epithelial cell and restoring gastrointestinal function . ChemicaFurazolidone, a traditional antimicrobial agent, has been extensively used to treat humans , 8. FZD Trypan blue solution (prod. no. T8154), phosphatase inhibitor cocktails 2 and 3 , and furazolidone (prod. no. V900742) were from Sigma-Aldrich . A protease inhibitor cocktail (ref. no. 11836 153 001) was from Roche Diagnostics . Primary antibodies anti-Atg5 (#2630), anti-beclin-1 (#3738), cleaved caspase-3 (#9661), Bcl-2 (#3498S), PARP (#46D11), H2AX (#2595S), LC3I/II (#4180S), BAX (#2772), and P53 (#9282) were purchased from Cell Signaling Technology , and anti-actin was purchased from Sigma-Aldrich (prod. no. A3853). Secondary antibodies goat anti-rabbit IgG- (H+L) HRP conjugate (cat. no. 170-6515) and goat anti-mouse IgG- (H+L) HRP conjugate (cat. no. 170-6516) were obtained from Bio-Rad Laboratories .2,37°C incubator.The intestinal epithelial cell line IEC-6 was obtained from the Beijing Cell Bank of the Chinese Academy of Sciences and periodically tested for the absence of mycoplasma. The cell line was cultured in Dulbecco's modified Eagle's medium mixture medium (Invitrogen Inc.) supplemented with 5% fetal bovine serum (Invitrogen Inc.), 0.01 mg/ml insulin (Absin), and 1% penicillin/streptomycin  in a humidified 5% COMale ICR mice with 20~24 g were purchased from the Center of Experimental Animals of Jilin University . All mice were randomly divided into different groups. A normal diet and water could be provided for one week prior to the study allowed for acclimatization. The environment temperature was maintained at 25°C ± 2°C and a 12 h light/dark cycle. Animal treatments complied with a protocol approved by the Jilin University Institutional Animal Care and Use Committee.An X-ray generator  was utilized to deliver radiation at a dose rate of 3 Gy/min. The distance between the source and the target was 70 cm.Firstly, mice were exposed to 2 Gy, 4 Gy, 8 Gy, and 12 Gy disposable TBI, respectively, and the median lethal dose was tested. Secondly, furazolidone was a synthetic organic compound that belongs to nitrofurans that were initially derived from furfural. It exhibited antibacterial and antihelmintic properties in humans and animals. The mice were randomly divided into 4 groups: TBI+saline, TBI+60 mg/kg FZD, TBI+100 mg/kg FZD, and TBI+140 mg/kg FZD. The FZD was given by oral gavage 30 minutes before a sublethal TBI (4 Gy) and once daily for 4 days. Mice were finally euthanized on the 15th day after TBI.At 15 days postirradiation, peripheral blood from the orbital sinus was collected and the number of white blood cells was analyzed. Mice were sacrificed by cervical dislocation, and the spleen and thymus were removed and weighed. The organ index was calculated according to the following formula: organ index = organ weight (g)/body weight (g). The chest cavity of mice was opened to remove the sternum, and then, the bone marrow of the sternum was squeezed with hemostatic forceps. Put a drop of bovine serum on a clean slide, mixed well and pushed the slide to dry. The bone marrow was fixed with methanol and stained with Giemsa. The number of micronuclei (MN) in 1000 polychromatic erythrocytes was counted by microscopy, and the rate of MN (‰) in bone marrow polychromatic erythrocytes was calculated.The intestine was removed and fixed in the formalin. Tissue was embedded in paraffin and cut into cross sections per segment in each mouse. The slides were stained with hematoxylin/eosin (H&E) for histopathology analysis by light microscopy. Gastrointestinal (GI) damage was diagnosed when the small intestines displayed denuded mucosa with no villus and no apparent crypts, respectively. Intestinal cell apoptosis was assessed in intestinal sections with TUNEL stain.Intestinal protein extracts were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes using the Protean Mini Cell (Bio-Rad). After completion of the transfer, membranes were blocked with 5% nonfat milk in TBS/0.1% Tween 20 for 120 min. Incubation with the primary antibody (as indicated) was conducted overnight at 4°C. Incubation with a peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1 : 2000) was performed for 120 min at room temperature. Finally, chemiluminescent analysis was performed.3 cells/well) and pretreated with FZD  followed by 8 Gy X radiation. The OD values were recorded after 2 h of CCK-8 incubation at 450 nm using a plate reader. The proliferation rate of cells was calculated by the following formula: cell viability =  × 100%.Cell Counting Kit-8  was used according to the manufacturer's protocol. Cells were seeded in 96-well plates (3.5 × 10Quantification of apoptotic cells was performed according to the Annexin-V/PI Apoptosis Detection Kit manufacturer's instructions (BD 556547). Analyses were performed by using a flow cytometer . Annexin-V-positive and PI-negative cells were regarded as apoptotic cells.4/ml with 3 parallel wells in each group. After various treatments, cells were stained with a MitoSOX Red probe at a final concentration of 10 μM at 37°C in the dark for 15 min. Then, cells were washed with PBS three times, and the activity of mtROS was analyzed using a flow cytometer .Mitochondrial ROS (mtROS) level in IEC-6 cells was detected by the MitoSOX Red (Invitrogen M36008) assay according to the manufacturer's instructions. The cells were seeded in 6-well plates with a density of 2.5 × 10μl propidium iodide  in the presence of RNase A  in PBS for at least 30 minutes and then subjected to flow cytometric analysis .The cell cycle was performed by propidium iodide (PI) staining and analyzed using flow cytometry. The cells were pretreated with DMSO (SolarBio) and FZD  for 2 h followed by radiation. After 72 h, cells were collected by trypsinization, washed with PBS, and fixed in 75% ethanol at 4°C for at least 24 hours. Cells were washed twice in PBS, and nuclear DNA was stained with 250 t-test (nonparametric) and analysis of variance (ANOVA) for data mean comparisons. Fisher's test was used for evaluating the survival of mice. Differences were considered significant at p < 0.05. Results are presented as means ± standard deviations.Statistical analysis was performed using GraphPad Prism 5 software with the unpaired The median lethal dose of TBI in mice was estimated by overall survival after different doses of irradiation. 40 mice were randomly averagely divided into 4 groups and, respectively, exposed to 2 Gy, 4 Gy, 8 Gy, and 12 Gy disposable TBI. The median lethal dose of TBI was 6 Gy Figures . To idenTo investigate whether FZD protects mice from TBI-induced hematopoietic injury, the peripheral blood cell counts were measured at 15 d after 4 Gy TBI. Compared with saline-treated mice, the numbers of WBCs exhibited a clear increase in FZD-administrated mice . To deteIn order to detect the target tissue which FZD protected against radiation damage, the liver, spleen, kidney, and lung tissues were taken for hematoxylin-eosin staining. Compared with the control group, liver, spleen, kidney, and lung tissues failed to change after TBI . Since TSince the villi of the intestine were significantly shortened following radiation treatment, FZD preferred to enrich in small intestine. We further detected whether FZD has a radiation protection effect on the intestine tissue. Previous studies have shown that exposure to IR provokes DNA damage and triggers various forms of cell deaths , 5. ThenTo further investigate the protective effect of FZD, we then designed in vitro experiments and an intestinal epithelial cell line, IEC-6, was used to determine the underlying mechanisms. After 12 ng/ml FZD treatment, cell survival significantly increased compared with the DMSO group by the CCK-8 assay . Also, aIonizing radiation can cause DNA damage in cells and then cause a series of complex reactions, including cell cycle arrest, DNA repair, and apoptosis. To further investigate whether FZD had an effect on radiation-induced cell cycle arrest in IEC-6 cells, IEC6 cells were divided into four groups: DMSO, FZD (12 ng/ml), IR+DMSO, and IR+FZD (12 ng/ml). The cells in the G0/G1 phase markedly decreased, and the cells in the S phase and G2/M phase accumulated in the IR group. In the IR+FZD group, the cells in the G1/G0 phase were significantly increased and the cells in the S phase and G2/M phase were decreased, indicating that FZD induced G0/G1 cell cycle arrest and delayed the cell cycle process, which is beneficial to damage repair. Also, FZD inhibited the S phase delay, which is beneficial to decreasing the occurrence of polyploid giant cells caused by S phase uncoupling and reducing the risk of cell transformation Figures .Because radiation can directly or indirectly increase ROS levels in cells, ROS-induced apoptosis is one of the main mechanisms of radiation. FZD had a protective effect against the radiation-induced cell death, and the mechanism may be related to reducing ROS release and inhibiting the ROS/caspase-3 signaling pathway. To determine whether FZD had the protective effect through ROS release and apoptosis, the ROS level and apoptosis were detected. As shown in Figures It is well known that a higher dose of TBI could cause ARS in which three syndromes were included: hematopoietic bone marrow syndrome, gastrointestinal syndrome, and brain syndrome , 18. So Since the primary pathological changes in bone marrow syndrome are the suppression of bone marrow hematopoietic functions, including the decrease in peripheral blood WBCs , we analSince the intestine is one of the most radiosensitive organs, intestinal injury is a very common complication of radiotherapy . AGS staPrevious studies have shown that exposure to IR provokes DNA damage and triggers various forms of cell deaths . Our resIn vitro experiments were then used to verify the underlying mechanisms. The data showed that FZD inhibited IEC-6 death caused by irradiation. FZD maintained most of cells in the G0/G1 phase and delayed the cell cycle process, which is beneficial to damage repair. Also, FZD inhibited the S phase delay, which is beneficial to decreasing the occurrence of polyploid giant cells caused by S phase uncoupling and reducing the risk of cell transformation. Our results showed that FZD inhibited radiation-induced ROS generation, apoptosis, and autophagy. In addition, FZD treatment reduced the protein expression of MAP LC3II/LC3I, beclin-1, and ATG5 following radiation treatment, suggesting that FZD could also inhibit radiation-induced autophagy. However, the molecular mechanism of how FZD protects cells from autophagy and apoptosis remains to be further studied.In summary, FZD treatment can improve the survival of mice treated with lethal dose radiation by alleviating TBI-induced injury and inhibiting autophagy and apoptosis. FZD is a clinically used drug with few side effects and might be used as an efficacious medical radiation countermeasure. However, the detailed radiation protection activity, clinical application, molecular mechanism, and drug target of FZD need to be further studied.


Taiwania circumdata (Coleoptera: Chrysomelidae: Cassidinae), an important insect pest on sweetpotato and water spinach in Southern China. This mitogenome was 13,546 bp long and encoded 13 protein-coding genes (PCGs), 19 transfer RNA genes (tRNAs) and 2 ribosomal RNA unit genes. The T. circumdata mitogenome with an A + T content of 77.9% presented a positive AT-skew (0.126) and a negative GC-skew (−0.160). Eleven PCGs started with a typical ATN codon, whereas the remaining two PCGs used TTG (nad1) and AAT (cox1) as the initial codon. All the 19 tRNAs had a typical secondary cloverleaf structure, except for trnS1 (AGN) which lacked the dihydrouridine arm. Phylogenetic analyses using Bayesian inference and maximum likelihood methods based on the concatenated nucleotide sequences of 13 PCGs recovered a phylogeny of Bruchinae+  + (Criocerinae + Cassidinae)). In Cassidinae, T. circumdata and Laccoptera ruginosa formed a clade, which was sister to three Cassida species.In this study we sequenced and annotated the nearly complete mitochondrial genome (mitogenome) of  Taiwania circumdata. This species is a serious insect pest on sweetpotato and water spinach in southern China. Adult specimens were collected from Nanchang City, Jiangxi Province, China, in August 2016. Samples have been deposited in College of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China. The total genomic DNA was extracted from a single specimen using a DNeasy Tissue Kit (Qiagen). The T. circumdata mitogenome was amplified with a set of universal and specific primers, and sequenced in both directions.Cassidinae is one of the most species-rich groups in Chrysomelidae, with approximately 6000 described species Chaboo . To bettT. circumdata, with 13,546 bp long (GenBank accession number MF946562). The region that we failed to sequence in T. circumdata was located between rrnS and nad2, and generally contained three transfer RNA genes  and a putative control region. This mitogenome encoded 13 protein-coding genes (PCGs), 19 tRNAs, two ribosomal RNA unit genes (rrnL and rrnS). The order and orientation of the mitochondrial genes was identical to the inferred ancestral arrangement of insects  and a negative GC-skew (−0.160), as found in most insect mitogenomes. The rrnL was 1265 bp long with an A + T content of 82.1%. Among the 13 PCGs, the lowest A + T content was 70.3% in cox1, while the highest was 85.6% in atp8. Eleven PCGs started with a typical ATN codon: five  with ATT and six  with ATG. The remaining two PCGs started with AAT (cox1) and TTG (nad1). Four PCGs terminated with TAA or TAG, whereas the remaining nine terminated with an incomplete stop codon TA or T which could be corrected via post-transcriptional polyadenylation. All of the 19 tRNAs had a typical cloverleaf secondary structure, except for trnS1 (AGN) which lacked the dihydrouridine arm.The nucleotide composition of the Anoplophora glabripennis from the family Cerambycidae was used as an outgroup. Phylogenetic analyses were performed with Bayesian inference in MrBayes 3.2.3 (Ronquist et al. T. circumdata and Laccoptera ruginosa formed a clade, as sister to three Cassida species.The concatenated nucleotide sequences of 13 PCGs in fourteen leaf beetles, representing five subfamilies, were used in phylogenetic analyses.


Little is known about the etiology of childhood diarrhea in the United Arab Emirates (UAE) especially after the introduction of rotavirus vaccines. This study aimed to identify gastrointestinal pathogens in children with diarrhea (cases) and the carriage rate of these pathogens in asymptomatic children (controls).Stool samples were collected from 203 cases and 73 controls who presented to two major hospitals in Al Ain city, UAE. Samples were analyzed with Allplex™ Gastrointestinal Full Panel Assay for common entero-pathogens. The association between diarrhea and the isolated pathogens was calculated in a multivariate logistic regression model. The adjusted attributable fractions (aAFs) were calculated for all pathogens significantly associated with cases.P < 0.001). Rotavirus, norovirus GII and adenovirus were significantly more prevalent in cases. Their aAFs with 95% ci are 0.95  for rotavirus, 0.86  for norovirus GII and 0.84  for adenovirus. None of the 13 bacteria tested for were more commonly found in the cases than in controls. Cryptosporidium spp. were more significantly detected in cases than in controls. Co-infections occurred in 27.9% of the children. Viruses and parasites were significantly more likely to occur together only in the cases.At least one pathogen was identified in 87 samples (42.8%) from cases and 17 (23.3%) from controls (Multiplex PCR revealed high positivity rates in both cases and controls which demand a cautious interpretation. Rotavirus remains the main childhood diarrhea pathogen in UAE. Effective strategies are needed to better control rotavirus and other causative pathogens.The online version contains supplementary material available at 10.1186/s12879-020-05693-1. Escherichia coli, Salmonella spp., Vibrio cholerae, Clostridium difficile, noroviruses and adenoviruses [.Infectious diarrhea is a main reason for children’s visits to clinics and emergency rooms. Globally, it is the fifth leading cause of death among children younger than 5 years particularly in lower-income countries . While toviruses .Although it has been shown that, in the United Arab Emirates (UAE), 87% of parents seek medical care for their children when they develop gastroenteritis with 10% of these cases requiring hospitalization with an average length of stay of 2.6 days, little is known of the microbiology of gastroenteritis in this country . A hospiUsual laboratory investigations for gastroenteritis focus on salmonella, shigella and viruses such as rotavirus. Other possible microorganisms go therefore undetected. Traditional diagnostic tests such as culture, immunoassay and microscopic examination are time-consuming, require special laboratory setup and often lack sensitivity. Culturing most viruses that cause diarrhea is difficult. Other techniques for virus identification such as electron microscopy and immunoassay demand special expertise which is often lacking in many clinical diagnostic laboratories , 6.Multiplex polymerase chain reaction (PCR) based testing has been recently added to the list of microbiological diagnostic tools for several infectious diseases. It allows rapid and simultaneous amplification of several targets with good sensitivity and specificity , 7. AlthThe study was conducted at two major government-run referral hospitals  in Al Ain, a large inland city in the Eastern Region of the Emirate of Abu Dhabi, UAE with a population estimates of 766,936 people .Between December 2017 and April 2019, after obtaining parental informed consent, stool specimens were collected from children less than 5 years of age who presented to the two hospitals with diarrhea, defined as at least three loose stools within the previous 24 h, defined as “cases”. During the same period, stool specimens were collected from children attending the same two hospitals without diarrheal disease in the last 30 days, defined as “controls”. Only one stool sample was collected from each participating child. The exclusion criteria for cases included current antibiotic treatment, chronic conditions , long-term immunosuppressive therapy and severe or life- threatening co-morbidity. The exclusion criteria for controls included acute infection  and having a sibling recruited as a case or control. We also obtained data on demographics, clinical characteristics and exposures  through a brief survey of parents at time of enrollment.E. coli, EAEC (aggR), Enteropathogenic E. coli, EPEC (eaeA), Escherichia coli O157 (E. coli O157), Enterotoxigenic E. coli, ETEC (lt/st), hypervirulent Clostridium difficile and Enterohemorrhagic E. coli, Shiga toxin-producing E. coli, STEC (stx1/2). The second bacterial panel detects Aeromonas spp., Campylobacter spp., Clostridium difficile toxin B, Salmonella spp., Shigella spp./ Enteroinvasive E. coli (EIEC), Vibrio spp. and Yersinia enterocolitica. The parasitic panel detects Blastocystis hominis, Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica and Giardia lamblia. The PCR reaction was performed under the following cycling conditions: 20 min at 50 °C for one cycle; 15 min at 95 °C for one cycle; 10 s at 95 °C, 1 min at 60 °C and 30 s at 72 °C for 45 cycles; 10 s at 95 °C, 44 times. Seegene Viewer Software  was used for detection and data analysis. Samples were classified as pathogen positive at a cycle threshold value of < 40 as per manufacturer’s instructions [Following collection, fecal specimens were transported on a daily basis to the principal investigator’s research laboratory at the UAE University and stored at − 80 °C until further analysis. An automated nucleic acid extraction instrument  was used for the extraction of both RNA and DNA from the stool samples. A weighed aliquot of stool sample was suspended in 1-ml of ASL buffer  and incubated for 10 min at room temperature. After clarification by high-speed centrifugation, the sample was loaded into the Nimbus equipment for nucleic acid extraction. After nucleic acid extraction, PCR reactions were set up and run on the CFX96 Real Time Detection System . For that purpose, we used a multiplex one-step real-time PCR platform, the Allplex™ Gastrointestinal Full Panel Assay  as per manufactures’ instructions , 12. ThiThe descriptive results were expressed as number of participants and percentages. Proportions were compared with the Chi squared or Fisher exact test for small values. As the continuous variables did not follow a normal distribution (as confirmed by the Shapiro-Wilk test), they were expressed as median values and interquartile range  and were compared with the non-parametric Kruskal Wallis test.P values. When there were zero participants in both cases and controls for an individual pathogen, the model could not be computed and thus no results could be reported.The association between diarrhea and the isolated pathogens was calculated in a multivariate penalized maximum likelihood logistic regression model (Firth’s method) to take into account results with zero participants. The model was corrected for age as a confounder, and the results were expressed as adjusted odds ratios (aOR) with 95% confidence intervals (ci) and their respective P value. The results of the unmatched and age-matched analyses were compared.As the number of controls was significantly less than the cases, although we used a penalized maximum likelihood logistic regression model to adjust for age and gender between cases and controls, we still decided to validate the obtained results by executing the same analyses in a post-hoc frequency age-matching case-control analysis. The cases and controls were age-matched within a range of 6 months and were analyzed in a conditional logistic regression model. The association between diarrhea and the isolated pathogens was calculated in an exact logistic regression model (Cox and Snell method) to take into account results with zero participants. The results were expressed as adjusted odds ratios (aOR) with 95% ci and their respective Adjusting for potential confounders in the above model, we also calculated, for each pathogen significantly associated with diarrhea in the logistic model, the adjusted attributable fraction (aAF), which is the proportional reduction in diarrhea that would occur if exposure to the respective pathogen was eliminated. aAF (with 95% ci) was calculated from the aOR, with the Woolf approximation for small samples, using the equation:P value < 0.05 defined statistical significance.Co-infection amongst the three groups of pathogens was analysed in a logistic model. We calculated their respective odds ratio (OR), with 95% ci and also analyzed the existence of any interaction amongst them, i.e. if the presence of parasites, for example, increased the likelihood of infection with bacteria or viruses, and so on. For all calculations, the statistical software package STATA version 15 was used  and a two-tailed P = 0.01) who had a median age of 11 months (IQR: 2–26). History of sick contact was provided in 30.6% of cases in comparison to approximately 5% in controls  (Table A positive PCR result for at least one pathogen was identified more often in 87 samples 42.8%) from cases than in 17 23.3%) from controls (1) Table . Overall.8% from .3% from 1) Table . Of note1) Table . Further1) Table .Table 2P values > 0.05) in the logistic regression analysis (Table P = 0.07).Co-infections occurred in 27.9% of the children, with parasites being present with either viruses or bacteria in 23 children (8.3%). Although amongst all participants, there were no interactions among the three groups of pathogens , this study reveals that in the UAE, rotavirus remains the main pathogen detected in children younger than 5 years of age who present with diarrhea despite the introduction of rotavirus vaccine into the country several years earlier. These results are in agreement with findings from the Global Rotavirus Surveillance Network that included 16 countries in the Americas and Africa regions . In contNorovirus GII was the second prevalent pathogen among the studied children with aAF of 0.86. While the role of norovirus in childhood gastroenteritis is well established in many parts of the world, limited data exist about the contribution of norovirus to the burden of diarrheal disease in the UAE and neighboring countries. A systematic review of studies from 15 out of the 24 countries of the Middle East and North Africa region revealed norovirus infection rates between 0.82 and 36.84% with GII.4 being the most predominant genotype detected . Given rAdenoviruses were the third leading cause of diarrhea in our cohort with aAF of 0.84. The clinical course of adenovirus gastroenteritis is often mild and indistinguishable from other viral gastroenteritis. Of note, the Allplex assay is only able to detect enteric adenoviruses belonging to species F (types 40 and 41). Other diarrhea-causing adenovirus species such as A, C and D will be missed when using the Allplex assay –24. ThisE. coli including EPEC, ETEC and EAEC were frequently detected in our studied children. EPEC and EAEC are of debatable diarrheal causation. EPEC is subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC) strains based on the presence of EPEC adherence factor plasmid [Salmonella, Shigella, Campylobacter, Yersinia, C. difficile, and STEC in people with diarrhea accompanied by fever, bloody or mucoid stools, severe abdominal cramping or tenderness, or signs of sepsis. The IDSA guidelines emphasize that results obtained by culture-independent diagnostic testing, including molecular assays such as multiplex PCR tests, should be cautiously interpreted within the clinical context as these assays may not necessarily detect viable organisms [Although none of the 13 bacteria tested for using the Allplex assay were more significantly prevalent in the cases than in controls, diarrheagenic  plasmid . Recent  plasmid . Further plasmid . The 201rganisms .Cryptosporidium spp. were found significantly more often in cases than in controls in the unmatched analysis, but not in the age-matched multivariate analysis. Cryptosporidium spp. are major cause of gastroenteritis with variable presentations ranging from asymptomatic shedding, self-limiting watery non-bloody diarrhea to a prolonged life-threatening disease in the immunocompromised individuals . The uneP < 0.05), with rotavirus and norovirus GII being the most common co-infection in symptomatic children and seemingly aggravating the severity of diarrhea [Co-infections were found in 27.9% of the studied children. In particular, viruses and parasites were significantly more likely to occur together, but only in the cases. This confirms a previously reported case-control study conducted in southwest China showing that co-infections with two enteric pathogens was higher in cases than in controls  for pathogens that were significant in a multivariable logistic regression model. Table S3. Prevalence of enteric pathogens in of 276 enrolled children (203 cases and 73 controls) by age group. Figure S1. Monthly detections of the most prevalent pathogens among 276 enrolled children. Figure S2. Number of participants with co-infections with gastrointestinal pathogens in 276 enrolled children.