Aims: To assess real-world effectiveness and safety of intravenous (IV) HX575, a biosimilar epoetin-α, in hemodialysis (HD) patients. Materials and methods: This prospective, observational, pharmacoepidemiological study of adult HD patients treated with IV HX575 for renal anemia for up to 24 months was conducted in 114 centers in 10 European countries. Of 2,086 enrolled subjects (safety sample), 2,023 had ≥ 1 follow-up visit (effectiveness sample). Results: Most (59.3%) patients were male, median age was 68 years. At enrollment, most (82.5%) had been treated with an erythropoiesis-stimulating agent, and 73.0% had adequate iron stores. At baseline, mean (± standard deviation) baseline hemoglobin (Hb) was 11.09 (± 1.14) g/dL and HX575 dose 106.5 (± 78.7) international units (IU)/kg/week; at month 24, Hb was 11.25 (± 1.19) g/dL and HX575 dose 113.0 (± 102.5) IU/kg/week. Variations in mean HX575 dose and Hb over the study were not statistically significant. As to safety, 140 patients (6.7%) experienced ≥ 1 adverse event; of these, 19 events  were related to HX575 treatment, 148  were reported as serious, including 12 events in 11 patients (0.5%) stated to be related. No cases of anti-epoetin antibodies or pure red cell aplasia were reported. Conclusions: MONITOR-CKD5 confirmed the real-world effectiveness and safety profile of IV biosimilar HX575. HD patients treated for up to 24 months showed stable dosing patterns and Hb outcomes. The safety profile of HX575 is likewise comparable to reference epoetin-α.  Rena® and Epoetin Alfa HEXAL® , was among the first biosimilars approved by the European Medicines Agency (EMA). The late-stage clinical development program for HX575 included a phase-III multi-center, double-blind, parallel-group randomized controlled equivalence trial (INJ-9) of 479 CKD5 patients being treated with reference epoetin-α  and randomized to either be converted to HX575 or continue with the reference medicine [HX575 , 9, 10, medicine , 7, 11. medicine . These smedicine . The MONITOR-CKD5 study  was a prThe methodology of the MONITOR-CKD5 study has been described elsewhere . Key eleMONITOR-CKD5 was designed as an international, prospective, longitudinal, observational, pharmacoepidemiological study of hemodialysis patients whose treating physicians prescribed IV HX575 per their best clinical judgment, but informed by local and regional clinical practice guidelines. Patients were recruited from 114 centers in 10 European countries as summarized in Supplemental Table S1. Eligible were male or female CKD5 adult (age ≥ 18) patients on hemodialysis with either original or grafted kidneys, diagnosed with renal anemia, and treated with commercially-available IV HX575. The safety sample consisted of all patients who received at least 1 dose of HX575. The evaluable sample was limited to patients for whom baseline and at least 1 follow-up visit with a valid Hb measurement were available, and the sample also excluded patients with major study protocol violations. The primary effectiveness outcome was Hb (g/dL) at baseline and follow-up visits. We also calculated at each follow-up visit the Hb change from baseline, as well as the erythropoietin resistance index (ERI), defined as the ratio of HX575 dose (IU/kg/week) to Hb (g/dL). Patients with ERI > 15 were considered hypo-responsive. They were further classified as chronic hypo-responsive if they had ERI > 15 for 4 or more consecutive months, or nonchronic hypo-responsive if there was no series of 4 consecutive months with ERI > 15. Two sources of safety data were used . First, site investigators recorded at visits 2 – 24 the occurrence of prespecified clinical events since the prior visit. Second, as MONITOR-CKD5 was a study in routine clinical practice, site investigators were instructed to report adverse events (AEs) using standard spontaneous reporting forms for all patients who received at least 1 dose of HX575. A total of 2,086 patients were enrolled and received at least 1 dose of HX575; this constituted the safety sample. Of these, 63 were removed because of either major protocol violations  or not having a minimum of 1 follow-up visit . This yiIn the evaluable sample, most patients (40.6%) were treated in Germany, followed by Romania (18.6%) and Italy (15.3%), accounting together for 74.5% of the study population . In terms of region, 74.7% of patients came from Western European and the remaining 25.3% from Central and Eastern European countries. The sample was predominantly male (59.3%). Median age was 68 years (range 20 – 93), and the sample was almost exclusively Caucasian . The most prevalent primary CKD etiologies included diabetic nephropathy (25.4%), chronic glomerulonephritis (20.4%), and renal vascular disease (16.4%). Median time on dialysis was 2.1 years (range 0 – 35). Most patients (83.4%) had vascular access through a fistula. Relevant major medical history and/or comorbidities included hypertension (81.0%), coronary disease (35.1%), type II diabetes mellitus (30.2%), peripheral vascular disease (19.8%), heart failure (17.1%) and, of note, neoplasia (7.3%). Of the 1,008 patients for whom data were available, 27.0% were iron deficient. The most common concomitant medications were antihypertensive (65.3%), phosphate-binding (61.1%), and antiplatelet (42.2%) agents. At enrollment, most patients (82.5%) had been treated with an ESA (including 56.8% with HX575) and therefore can be considered in the maintenance phase of renal anemia management; whereas the remaining 17.5% were ESA-naïve at baseline, and going through an anemia-correction phase first before transitioning to maintenance therapy. Mean (± standard deviation (SD)) Hb at enrollment was 11.09 (± 1.14), and 68.0% of patients had Hb values in the 10 – 12 g/dL target range. For most patients (75.5%), the lower limit of the individualized target Hb range prespecified by their treating physician was ≥ 10 g/dL but < 11 g/dL; whereas for 5.3% patients it was Hb < 10 g/dL, and Hb ≥ 11 g/dL for the remaining 19.2%. Also, for 74.4% of patients the prespecified individualized upper limit of the target, Hb range was ≥ 11.5 g/dL but < 12.5 g/dL; with 6.5% below and 19.1% exceeding that range. However, only 61.2% of patients met their individualized target range at visit 2 and only 59.4% at visit 24. Duration of treatment with HX575 was calculated as the difference in days from the last to the first available patient visit plus 1 day. Median HX575 treatment duration was 505 days or 1.4 years. Mean (± SD) HX575 dose rose nominally from 106.5 (± 78.7) international units (IU)/kg/week at enrollment to 113.0 (± 102.5) IU/kg/week at month 24, with a low of 106.1 (± 80.6) IU/kg/week at month 3 and a high of 117.7 (± 103.5) at month 20  Figure . Median Comparing baseline to visit 24, the proportion of patients receiving iron supplementation decreased by –5.5% (p = 0.017). There were statistically-significant increases in the proportions of patients prescribed vitamin B12 , vitamin C , vitamin D , and folate , but not vitamin E . At enrollment, 2.8% of patients had previously received a blood transfusion for renal anemia. Over the ensuing 23 months, transfusion rates varied between a low of 0.3% (visit 22) and a high of 1.2% (visit 3). Mean (± SD) Hb levels increased nominally from 11.09 (± 1.41) IU/kg/week at enrollment  to 11.25 (± 1.19) g/dL at month 24, with a high of 11.29 (± 1.18) at month 4  Figure . Median Mean (± SD) ERI scores rose nominally from 9.95 (± 7.78) at baseline to 10.55 (± 10.22) at visit 24, and median ERI declined nominally from 7.82 to 7.76, but these changes were not statistically significant (both p = n.s.) . Proportions of patients classified as hypo-responsive were 19.5% at baseline and 20.3% at 24 months (p = n.s.). Using ERI > 15 for ≥ 4 consecutive months as criterion, 133 patients (8.0%) were considered chronically hypo-responsive at some time during the study. At baseline, out of 1,008 patients for whom data were available, 73.0% had adequate iron stores, but 22.2% had functional and 4.8% had absolute iron deficiency. At month 24, for 131 patients with data available, these proportions were 69.5%, 26.0%, and 4.6%, respectively. These shifts in proportions from baseline to visit 24 were not statistically significant. Data were collected at every visit related to thromboembolic events (TEEs), non-TEE events, hospitalizations, and deaths that occurred since the previous visit. In the evaluable sample , 291 patients (14.4%) experienced a total of 319 TEEs . AEs were spontaneously reported on all patients receiving at least 1 dose of HX575 . Per these reports, 140 patients (6.7%) experienced 254 AEs, including 19 events in 16 patients (0.8%) stated to be related to HX575 treatment . Of all AEs, 148 occurring in 108 patients (5.2%) were reported as serious (SAE), including 12 events in 11 patients (0.5%) stated to be HX575-related. No patients were reported to have developed non-neutralizing or neutralizing anti-epoetin antibodies or pure red cell aplasia. The 24-month MONITOR-CKD5 study demonstrates that the efficacy of IV HX575, a biosimilar epoetin-α, in managing renal anemia in hemodialysis patients, established in the pivotal randomized controlled trial INJ-9  and valiImportantly, what our study adds is that this pattern of stable dosing and stable Hb effectiveness outcomes can be achieved sustainably for up to 24 months with HX575 under conditions of greater heterogeneity in patients, clinicians, and settings compared to the relative homogeneity that characterizes efficacy trials – and this in Western as well as in Central and Eastern European countries. Our effectiveness outcomes are also consistent with those of the hemodialysis patients in two international prospective observational studies of anemia-treatment patterns and outcomes with reference epoetin-α: the 5 Western European countries initially involved in the Dialysis Outcomes and Practice Patterns Study (EuroDOPPS), with 4,591 hemodialysis patients from 101 dialysis centers in 5 countries , and theRecombinant human erythropoietin was approved for reducing the need for RBC transfusions in anemic CKD patients. In the pivotal INJ-9 trial, on-study transfusion rates were 1.0% in the HX575-treated group in the 28-week randomized study; rising to 7.4% when including the 56-week open-extension study . In the The proportions of patients with adequate iron stores versus those with functional and absolute iron deficiency remained statistically similar over the 24-month period. There was, however, a significant decline from 66.5 to 61.0% in the proportion of patients receiving iron supplementation. The pivotal trial and Epo-PASS reports do not provide data on iron supplementation in conjunction with HX575 treatment , 12. LikWhile best practice guidelines advise to individualize ESA treatment and set a specific Hb target range for each patient, only ~ 60% of patients were within their target range over the course of MONITOR-CKD5. In fact, as Gesualdo et al.  reportedOn the safety side, 8.1% of patients experienced a shunt thrombosis, 17.1% were hospitalized, and 16.9% died over the 24-month period. No cases of anti-epoetin antibodies or pure red cell aplasia were reported, although one study reported a rate of 2% of patients developing non-neutralizing anti-epoetin antibodies . In addiWith the relative effectiveness and safety of HX575 established, policy, financing, and access to care issues can be addressed. In an era of payment constraints to dialysis providers, the use of biosimilar epoetin-α can generate significant savings. In the US, the introduction of bundled payments has led dialysis providers to revert back, at least in part, to red blood cell transfusions to manage renal anemia . While tThe MONITOR-CKD5 study has limitations. It was not a population-based study nor did it randomly select sites and, within sites, patients. Selection bias cannot be excluded. Germany, Italy, and Romania provided most patients, and this may have exerted extra weight in the analyses. All patients were treated with HX575, and there was no comparison group of patients treated with other ESAs . While limited to centers using HX575, efforts were made to enroll a variety of centers from each country with regards to patient volume, patient mix, and affiliation. In keeping with prevailing guidelines, setting target hemoglobin concentrations as well as dose and frequency of administration of HX575 was left to the discretion of the treating physician. Some rates reported here are visit-specific and influenced by the patients in the study at a given visit, a denominator that declined over time. The MONITOR-CKD5 study of hemodialysis patients underscores the real-world effectiveness of HX575, a biosimilar epoetin-α, in managing renal anemia. Patients treated for up to 24 months with HX575 showed Hb outcomes equivalent to reference epoetin-α under dosing patterns similar to the reference medicine. The majority of treated patients were maintained within guideline-recommended target Hb ranges. No unknown safety signals, including immunogenicity, were detected. The authors acknowledge the contributions of L. Gesualdo, co-chair of the study steering committee, who resigned after having been elected president of the Società Italiana di Nefrologia. The MONITOR-CKD5 study was funded by Hexal AG. The sponsor participated in the development of the protocol, the discussion of the results, and review of the manuscript for scientific content. GL, JM, DG, CC, FD, and FZ received compensation from Hexal AG for their participation in the work reported here. AK and NH are employees of Hexal AG. KM and IA are affiliated with Matrix45. By company policy, they cannot hold equity in sponsor organizations and cannot receive direct personal benefits, financial or other, from sponsor organizations. Matrix45 provides similar services to other biopharmaceutical companies without exclusivity constraints.


To investigate the differences in the perioperative serum cortisol, C-reactive protein (CRP) and interleukin-6 (IL-6) levels between aged and middle-aged patients undergoing total hip arthroplasty (THA).Sixty patients (30 aged and 30 middle-aged) undergoing THA for osteoarthritis between August 2016 and January 2017 participated in this study. Blood samples were collected preoperatively and at 6 hours, 24 hours and 3 days after surgery to measure the cortisol, CRP and IL-6 concentrations. The clinical outcomes were assessed using the visual analogue scale (VAS) pain score and Harris hip score (HHS).P < 0.05). IL-6 at 6 and 24 hours after surgery, CRP at 3 days after surgery and the VAS score at 6 and 24 hours after surgery in the aged group were significantly higher than those in the middle-aged group (P < 0.05). In the aged group, weak correlations were found between the cortisol concentration 6 hours after THA and the IL-6 level 24 hours after THA  and between the IL-6 level 6 hours after THA and the VAS score 24 hours after THA .No significant differences were found between the two groups before the operation in the cortisol, IL-6 and CRP levels; the VAS score; or the HHS. Cortisol was significantly lower at 6 hours after surgery in the aged group than in the middle-aged group (Aged patients showed lower cortisol levels at 6 hours after surgery and higher IL-6 levels at 6 and 24 hours after surgery than middle-aged patients undergoing THA. Life expectancy has been prolonged considerably due to improved healthcare. The prevalence of osteoarthritis (OA) is almost 70% in women and 60% in men over the age of 65 years, and most people who suffer from OA display radiographic evidence and/or clinical symptoms , 2. TotaHowever, surgeries can trigger stress responses, including a series of immunological, metabolic and hormonal changes –10. As oAlthough several studies have compared cortisol levels between aged and middle-aged patients undergoing other operations, the conclusions have been inconsistent –19. To tPatients aged over 40 years and scheduled for primary unilateral uncemented THA for OA between August 2016 and January 2017 were screened in this prospective, non-randomized, observational study. The exclusion criteria were as follows: senile dementia, steroid use within the past 6 months, chronic malnutrition, smoking, alcoholism, drug abuse, rheumatoid arthritis, hypertension, diabetes mellitus and other systemic diseases that would influence the neuroendocrine and inflammatory responses. The administration of non-steroidal anti-inflammatory drugs (NSAIDs) was suspended four weeks before the operation. Of the 68 eligible patients, 7 patients refused to participate, and 1 patient withdrew from this study. Finally, 60 patients were included in the study. The patients were divided into an aged group (over 65 years) and a middle-aged group (40–65 years). This study was conducted in accordance with the Declaration of Helsinki and approved by the local Ethics Committee. All patients gave informed consent prior to their inclusion in the study.All surgeries were performed by one single experienced surgeon (B.S.). Generally, 1.5 g of cefuroxime was given within 30 min before the beginning of surgery, and general anaesthesia was used in all cases. The same operative technique was used. Through the posterolateral approach, the acetabular component was placed at 20° anteversion and 45° abduction, the femoral component was fixed at 15° anteversion, the capsular flap and the short external rotators were repaired in all cases, and wound drainage was indwelled in every patient.Preadmission counselling was given by qualified nurses, and the preliminary assessment was conducted by a doctor. Clear oral fluids were administered up to 3 h before the operation to minimize preoperative fasting. No sedative was used before surgery. One hundred milligrams of ropivacaine (100 mg: 10 ml) was diluted with 30 ml of saline and subcutaneously injected around the wound as a local infiltration analgesia.Forty milligrams of parecoxib was given by intramuscular injection at 6 h after surgery. A second dose of 40 mg of parecoxib was given at 8:00 a.m. on postoperative day (POD) 1, followed by oral celecoxib (200 mg) every 12 h. Ten milligrams of rivaroxaban was given orally 8 h after the operation and then administered at 24-h intervals on the subsequent days provided that no bleeding events occurred.At 8:00 a.m. on POD 1, wound drainage and the monitor were removed, and unrestricted food intake was permitted. Daily functional training, including walking and muscle power exercises, was guided and supervised by a physiotherapist.Venous blood samples were collected in the morning of the day before surgery from each patient as baseline. The primary outcome was the serum cortisol level. Blood samples to measure the cortisol, CRP and IL-6 levels were obtained at 6 h, 24 h and 3 days postoperatively. After removing cells and debris by centrifugation, the samples were stored at −80 °C prior to the analysis. All measurements were performed at the clinical key laboratory of West China Hospital, which was certificated by the College of American Pathologists (CAP). CRP was measured using the rate nephelometric assay on the IMMAGE®800 Access immunoassay system  and reported in milligrams per litre (mg/L). IL-6 and cortisol were measured with electrochemiluminescence immunoassays (ECLIs) on the modular analytics E170 module  and were presented in picograms per millilitre (pg/mL) and nanomole per litre (nmol/L), respectively.Visual analogue scale (VAS) pain scores (rest pain) were marked on the day of hospitalization and 6 h, 24 h, 2 days, and 3 days after surgery by a mediatinus . The Harris hip scores (HHS) were obtained at the time of hospital admission and 3 months postoperatively to evaluate the early recovery effect. Adverse events were recorded at each follow-up.t-test was adopted if the variances were homogenous; otherwise, a t-test was performed. For non-normally distributed and ranked data, a nonparametric test (Mann-Whitney rank-sum test) was conducted. For enumeration data, the Chi-square test was employed. Because some of our variables  were not normally distributed, analysis of variance (ANOVA) of repeated measurement data was not used. Pearson’s correlation coefficient and Spearman’s rank correlation coefficient were calculated for variables with normal and non-normal distributions, respectively, within each group to analyse their possible correlations. The analyses were performed using the SPSS statistical software . For all comparisons, a P value <0.05 was considered significant.Data were collected using Excel. Continuous variables are presented as the means and standard deviations (M ± SD). Dichotomous data are presented as numbers for each category. To compare data between two groups, the normality of all quantitative data was tested with the Shapiro-Wilk test. For normally distributed data, Levene’s test for equality of variances was used to test the homogeneity of variances of matching data from two groups, and the two independent samples The patient screening and processing steps are illustrated in Fig. The two groups were well matched for gender, height, weight, BMI, and operating time Table .Table 1DThe mean preoperative cortisol levels did not significantly differ between the two groups. Six hours after surgery, serum cortisol increased from 314.84 ± 89.65 nmol/L and 291.93 ± 111 nmol/L at baseline to 701.85 ± 156.32 nmol/L and 807.12 ± 161.34 nmol/L in the aged and middle-aged groups, respectively. Then, the cortisol concentrations dropped to 550.99 ± 210.73 nmol/L and 607.7 ± 214.31 nmol/L, respectively, at 24 h after the operation, which were still much higher than the baseline concentrations. The concentrations were almost the same at POD 3 (562.63 ± 142.7 nmol/L in the aged group and 609 ± 153.53 nmol/L in the middle-aged group) Table .Table 2CThe IL-6 concentration was 3.54 ± 1.55 pg/ml for the aged group and 3.96 ± 1.59 pg/ml for the middle-aged group before surgery, both of which were within the normal range. These concentrations increased to 64.14 ± 16.43 pg/ml and 52.81 ± 29.16 pg/ml, respectively, 6 h after surgery and continued to rise until they peaked at 24 h after surgery (165.35 ± 50.50 pg/ml and 114.53 ± 56.78 pg/ml). At POD 3, these concentrations plummeted to lower but still above-normal levels  Table .The CRP levels were barely changed 6 h after THA compared with the preoperative measurements in both groups and fell within the normal range. The CRP concentration soared to 90.47 ± 22.44 mg/L (aged) and 81.56 ± 21.09 mg/L (middle-aged) 24 h after surgery and peaked at 119.40 ± 32.06 mg/L (aged) and 100.27 ± 25.34 mg/L (middle-aged) 3 days after the operation Table .The aged group scored 3.47 ± 1.46 in the preoperative VAS evaluation, whereas the middle-aged group scored 3.33 ± 1.32. Six hours after the operation, both groups experienced aggravated pain and scored 5.47 ± 1.11 and 4.87 ± 1.07, respectively. However, the scores in both groups decreased swiftly, with the aged group scoring slightly higher than their preoperative evaluation score (3.73 ± 0.98) and the middle-aged group scoring slightly lower than their preoperative rating (3.20 ± 1.19) 24 h after surgery. Then, the VAS scores in both groups fell to lower levels than their preoperative counterparts. The aged group felt more severe pain both before and after surgery than the middle-aged group, although significant differences were only shown at 6 and 24 h postoperatively, which was in accordance with the higher IL-6 levels in the aged group Fig. .Fig. 3aThe middle-aged and aged groups scored 50.93 ± 6.40 and 48.67 ± 9.11, respectively, before THA and then 81.37 ± 3.94 and 80.67 ± 4.44, respectively, after THA. Both groups showed evident improvement in the hip function assessment after THA, but no significant differences between the two groups were shown before or after THA Fig. .r = −0.37, P = 0.04, Fig. r = 0.42, P = 0.02, Fig. r = 0.39, P = 0.04, Fig. The correlation analysis demonstrated only a possible weak correlation  and postoperative pain (VAS at 6 and 24 h after surgery). Further studies of perioperative glucocorticoid usage in aged patients should be conducted to demonstrate the benefits and possible side effects.


Hypoxic hepatitis (HH) is a type of acute hepatic injury that is histologically characterized by centrilobular liver cell necrosis and that is caused by insufficient oxygen delivery to the hepatocytes. Typical for HH is the sudden and significant increase of aspartate aminotransferase (AST) in response to cardiac, circulatory or respiratory failure. The aim of this study is to investigate its epidemiology, causes, evolution and outcome.The screened population consisted of all adults admitted to the intensive care unit (ICU) at the Ghent University Hospital between January 1, 2007 and September 21, 2015. HH was defined as peak AST > 5 times the upper limit of normal (ULN) after exclusion of other causes of liver injury. Thirty-five variables were retrospectively collected and used in descriptive analysis, time series plots and Kaplan–Meier survival curves with multi-group log-rank tests.P = 0.007) among the causes, ranging from 33.3% in the hyperthermia subgroup to 52.9 and 56.2% in the septic shock and pulmonary embolism subgroups, respectively. The magnitude of AST increase was also significantly correlated (P < 0.001) with mortality: 33.2, 44.4 and 55.4% for peak AST 5–10× ULN, 10–20× ULN and > 20× ULN, respectively.HH was observed in 4.0% of the ICU admissions at our center. The study cohort comprised 1116 patients. Causes of HH were cardiac failure (49.1%), septic shock (29.8%), hypovolemic shock (9.4%), acute respiratory failure (6.4%), acute on chronic respiratory failure (3.3%), pulmonary embolism (1.4%) and hyperthermia (0.5%). The 28-day mortality associated with HH was 45.0%. Mortality rates differed significantly (This study surpasses by far the largest cohort of critically ill patients with HH. HH is more common than previously thought with an ICU incidence of 4.0%, and it is associated with a high all-cause mortality of 45.0% at 28 days. The main causes of HH are cardiac failure and septic shock, which include more than 3/4 of all episodes. Clinicians should search actively for any underlying hemodynamic or respiratory instability even in patients with moderately increased AST levels. Hypoxic hepatitis (HH), also referred to as “ischemic hepatitis” or “shock liver,” is a type of acute hepatic injury that is histologically characterized by centrilobular liver cell necrosis and that is caused by insufficient oxygen delivery to the hepatocytes . TypicalThe major causes of HH are septic shock, respiratory failure and cardiogenic shock , 2. PossHH is reflected by a typical pattern of liver enzyme alterations. It presents with a sudden and significant increase of AST, alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), reaching their peak levels around 24 h after ICU admission . InitialCurrently, only limited data from small retrospective studies are available, making HH an understudied disease. The largest cohort described to date was recently published and included 565 patients . The aimn = 29,874) who were admitted to the surgical, cardiac or medical ICU at the Ghent University Hospital between January 1, 2007 and September 21, 2015. HH was defined as a significant but transient increase in AST level above 5 times the upper limit of normal (ULN) after exclusion of other potential causes of liver injury. Different AST cutoff values for defining HH are used in the literature , supportive therapy  and need for dialysis), ICU and hospital length of stay, duration of the HH episode, and ICU and in-hospital mortality at 28 days. The peak of HH was defined as the point in time (T-ASTmax) when AST reached its peak value (ASTmax). In this study, the time origin was set to T-ASTmax (designated as time 0) and all recorded values are expressed in time relative to T-ASTmax for optimal comparison. Severity of illness scores were recorded over a time span of 24 h around T-ASTmax. Three categories for severity of HH were used: “5–10× ULN”, “10–20× ULN” and “> 20× ULN”. Seven underlying causes of HH were defined: cardiac failure, septic shock, hypovolemic shock, pulmonary embolism, acute respiratory failure, acute on chronic respiratory failure and hyperthermia.Routinely available biochemical parameters were recorded, including AST (U/L), ALT (U/L), LDH (U/L), bilirubin (mg/dL), alkaline phosphatase (AP) (U/L), gamma-glutamyl transpeptidase (U/L), lipase (U/L), international normalized ratio (INR), platelet count  and third (Q3) quartiles. For categorical variables, comparisons between groups are performed using the Pearson’s Chi square test for contingency. For continuous variables, a permutation test based on difference in medians between groups is used. A Tukey-like approach with permutation resampling is applied to adjust on 3.3.2 .During the study period, 29,874 adult patients with a male-to-female ratio of approximately 3:2 were admitted to the ICU at our center, of whom 4012 patients had peak AST levels exceeding 5 times the ULN. In 30.0% (1202/4012) of the cases, the elevation of AST was caused by HH, resulting in an overall ICU incidence of 4.0% . As only the first episode was eligible for analysis in patients with multiple episodes of HH during the study period, the final study cohort comprised 1116 patients.The all-cause mortality of patients admitted to the ICU at our center during the study period was 7.7% . Among these non-survivors, 19.8% (455/2302) had developed HH during their ICU stay. The all-cause mortality associated with HH was 45.0% (502/1116) at 28 days, of which 90.6% occurred during ICU stay. The survival curves by cause of HH are presented in Fig. In our study cohort, the median age was 66.0 (Q1–Q3 55.0–74.0) years with a male-to-female ratio of 3:2. The median SAPS-II score at T-ASTmax was 66.0 (Q1–Q3 43.0–81.0). 74.9% of patients required mechanical ventilation. Vasopressor and inotropic agents were used in 60.8 and 43.2%, respectively, and 9.5% were on dialysis at T-ASTmax. The causes of HH, in decreasing order of frequency, were cardiac failure (49.1%), septic shock (29.8%), hypovolemic shock (9.4%), acute respiratory failure (6.4%), acute on chronic respiratory failure (3.3%), pulmonary embolism (1.4%) and hyperthermia (0.5%). An episode of HH (length of time that AST levels are exceeding 5 times the ULN) had a median duration of 54.3 (Q1–Q3 26.4–94.2) h, during which the median recovery time (duration from peak AST to levels below 5 times the ULN) was 34.7 (Q1–Q3 14.6–67.2) h.P < 0.001 and median APS-II score 25.0 vs. 30.0, P = 0.005) as compared to non-survivors. They were less likely to have septic shock . Peak AST levels above 20 times ULN were less commonly seen in survivors . More characteristics, classified according to survival status, are presented in Table Survivors had significantly lower severity of illness scores  and a higher need for supportive therapy, such as inotropic agents , vasopressor agents , dialysis  and IABP . In addition, higher peak AST levels were associated with higher 28-day mortality rates . More characteristics, classified according to severity of HH and to cause of HH, are presented in Tables The three ASTmax subgroups (“5–10× ULN” vs. “10–20× ULN” vs. “> 20× ULN”) were also compared with each other. The causes of HH were equally represented within each subgroup. Patients with higher peak AST levels had higher severity of illness scores   U/L and 1180.6 (Q1–Q3 684.1–2770.8) U/L within a median time of 22.0 (Q1–Q3 9.7–43.0) and 21.4 (Q1–Q3 10.0–43.6) h after start of HH, respectively. In survivors, LDH returned to baseline (< 233 U/L) within a median of 5.1 (Q1–Q3 1.3–15.9) days after its peak. ALT declined more slowly, returning to baseline  within 8.6 (Q1–Q3 3.9–16.5) days after its peak. When considering all patients, including those with AST already exceeding 5 times the ULN at ICU admission, ALT and LDH peaked at T-ASTmax in the majority of patients  and to a lesser extent after T-ASTmax .The median relative increase of AST, ALT and LDH at T-ASTmax was 15.0, 8.7 and 4.7 times their ULN, respectively. LDH exceeded both AST and ALT at all time points in more than 80% of patients. The median AST/ALT ratio was 1.6 at the start of HH, increased to 1.8 at T-ASTmax and subsequently declined, reversing within a median of 32.5 h after T-ASTmax.In addition, Fig. The pattern of liver enzyme alterations described above was found in all ASTmax subgroups . The high number of early deaths in the pulmonary embolism, septic shock and hypovolemic shock subgroups mainly accounted for the early mortality associated with HH. 37.5, 24.3, 25.7% of these patients died within 24 h after T-ASTmax. The mortality rate in the pulmonary embolism subgroup exceeded at all times those of other subgroups and ultimately resulted in 56.2% mortality at 28 days. The septic shock and hypovolemic shock subgroups had a 28-day mortality of 52.9 and 43.8%, respectively. While the early mortality in the cardiac failure subgroup was less pronounced, yet still considerable as 14.8% of these patients died within 24 h, its 28-day mortality of 41.4% ranked fourth among all subgroups. The acute respiratory failure and acute on chronic respiratory failure subgroups had similar survival curves and outcome, with 39.4 and 37.8% mortality at 28 days. The lowest mortality rate of 33.3% at 28 days was observed in the hyperthermia subgroup. The survival curves by cause of HH are presented in Fig. Twenty-eight-day mortality rates differed significantly among the causes of HH (P < 0.001) with severity of HH, ranging from 33.2% in the “5–10× ULN” subgroup to 55.4% in the “> 20× ULN” subgroup. There was no significant difference (P = 0.363) in 28-day mortality between men (39.8%) and women (42.4%).The 28-day mortality also increased significantly  to define HH, while other studies have used higher cutoff values ranging from 400 to 3000 U/L . HH has Our study uncovered interesting results. Firstly, it indicated that HH may be more common than previously thought. In our study, an incidence of 4.0% was observed, which is higher than the pooled incidence of 2.5% from a meta-analysis of 1782 patients [Secondly, the typical liver test pattern that suggests diagnosis of HH was observed in all subgroups of our study cohort. HH was characterized by (1) a sudden and significant increase of aminotransferases with an AST/ALT ratio greater than 1, (2) a similar rise in LDH mostly exceeding both AST and ALT and (3) a steady decline of aminotransferases with reversal of the AST/ALT ratio approximately 1.5 days after T-ASTmax. In survivors, LDH normalized first, followed by AST and finally ALT . Bilirubin followed a similar pattern as the aminotransferases, but the magnitude of its increase was much less pronounced, with median levels never exceeding the ULN. The associated prolongation of INR and impairment of renal function as described by Raurich et al. [Finally, we observed a 28-day mortality of 45.0%, which is in line with the reported mortality of 49% from a meta-analysis of 1782 patients [n = 1116). It comprises twice as many patients as the second largest cohort study, which was recently published and included 565 patients [Our study has a number of strengths. To our knowledge, it is by far the largest cohort study of patients with HH (patients . Additiot with an ICU incidence of 4.0%, and it is associated with a high all-cause mortality of 45.0% at 28 days. The main causes of HH are cardiac failure and septic shock, which include more than 3/4 of all episodes. Clinicians should search actively for any underlying hemodynamic or respiratory instability even in patients with moderately increased AST levels.This study surpasses by far the largest cohort of critically ill patients with hypoxic hepatitis. HH is more common than previously though


Trichoderma reesei  QM6a is a model fungus for a broad spectrum of physiological phenomena, including plant cell wall degradation, industrial production of enzymes, light responses, conidiation, sexual development, polyketide biosynthesis, and plant–fungal interactions. The genomes of QM6a and its high enzyme-producing mutants have been sequenced by second-generation-sequencing methods and are publicly available from the Joint Genome Institute. While these genome sequences have offered useful information for genomic and transcriptomic studies, their limitations and especially their short read lengths make them poorly suited for some particular biological problems, including assembly, genome-wide determination of chromosome architecture, and genetic modification or engineering.T. reesei has evolved a robust repeat-induced point mutation (RIP) system to accumulate AT-rich sequences, with longer AT-rich blocks having more RIP mutations. The widespread distribution of AT-rich blocks correlates genome-wide partitions with gene clusters, explaining why clustering of genes has been reported to not influence gene expression in T. reesei.We integrated Pacific Biosciences and Illumina sequencing platforms for the highest-quality genome assembly yet achieved, revealing seven telomere-to-telomere chromosomes  with 1630 newly predicted genes and >1.5 Mb of new sequences. Most new sequences are located on AT-rich blocks, including 7 centromeres, 14 subtelomeres, and 2329 interspersed AT-rich blocks. The seven QM6a centromeres separately consist of 24 conserved repeats and 37 putative centromere-encoded genes. These findings open up a new perspective for future centromere and chromosome architecture studies. Next, we demonstrate that sexual crossing readily induced cytosine-to-thymine point mutations on both tandem and unlinked duplicated sequences. We also show by bioinformatic analysis that Compartmentation of ancestral gene clusters by AT-rich blocks might promote flexibilities that are evolutionarily advantageous in this fungus’ soil habitats and other natural environments. Our analyses, together with the complete genome sequence, provide a better blueprint for biotechnological and industrial applications.The online version of this article (doi:10.1186/s13068-017-0825-x) contains supplementary material, which is available to authorized users. Trichoderma is a fungal genus found in many different natural habitats including—among others—mushroom caps, rotting wood, and soil. Trichoderma reesei (syn. Hypocrea jecorina) is a widely used model organism for plant cell wall degradation and industrial enzyme production. The natural strain QM6a (ATCC13631) was first isolated from the Solomon Islands during the Second World War  [N. crassa survived RIPs due to either nucleolar sequestration [N. crassa ] , 59. HigpC+GpT)] , 61. We stration  or theirstration , respectblr1Δ, env1Δ  at 30 °C for 1.5 h. The protoplasts were harvested by centrifugation at 600g for 10 min at 4 °C, dissolved in 1.2 mL GuHCl solution  at 65 °C for 20 min and then mixed with 6.4 mL ice-cold ethanol to precipitate the genomic DNA. The pellet was dissolved in 10× TE with 0.6 mg/mL RNase H at 37 °C for 1 h and then in 0.4 mg/mL proteinase K at 65 °C for 1 h. The genomic DNA was purified with the phenol:chloroform:isoamyl alcohol (25:24:1) method and then recovered by standard precipitation with ethanol. Next, the quality of high molecular weight genomic DNA for Illumina MiSeq and PacBio sequencing was validated by PFGE. The genomic DNA was separated in a 1% agarose gel in 0.5× TBE buffer, using a CHEF DR II (Biorad) with 0.5× TBE running buffer, continuously refrigerated at 14 °C and 6 V/cm (current 110–125 mA) for 18 h. The Lambda DNA-Mono Cut Mix  was used as size marker. Visualization was performed after staining with ethidium bromide after the electrophoresis.QM6a was inoculated on petri-plates with malt extract agar (MEA) medium at 25 °C until full asexual sporulation was observed (~5 days). 2 × 10Illumina MiSeq sequencing was carried out at the DNA sequencing facility in the Institute of Molecular Biology, Academia Sinica  in May 2016 (sequencing was coordinated by the author SYT). A shotgun paired-end library (average size of 550 bp) was prepared using the Illumina TruSeq DNA nano Sample Prep Kit. The Illumina library (including PCR amplification and quantification) was prepared automatically by the NeoPrep Library Prep System, and then sequenced on the MiSeq platform  with the MiSeq Control software version 2.5.1 and Sequencing Analysis Viewer version 1.8.20. Sequencing data were sent to Illumina BaseSpace automatic analysis during running. For report version 2.2.9 with MiSeq 600 cycler V3 chemistry, 96.16% of clusters passed the filter and 78.32% of bases qualified higher than Q30 at 2 × 300 n.®Cell V3 (PN 100-171-800), and MagBead (PN 100-133-600), loading at 200 pM on the plate. Data were collected with Stage Start and 180 min movies. Seven SMRT®Cell cells were used to generate 263,312 reads and 3,397,762,180 bases.For PacBio continuous long-read sequencing, high-quality genomic DNA was submitted to the Ramaciotti Centre for Genomics . Sequencing was coordinated by Carolina Correa and Tonia Russel. High molecular weight DNA was sheared with g-TUBE (Covaris PN 520079), aiming at DNA fragments of about 20 kb. The library was constructed with a 20 kb size-selected protocol using DNA Template Prep Kit 2.0 (PN 001-540-726), purified, and further selected for long insert size with a 0.35× AMPure (AMPure PB PN 100-265-900) bead size selection. The library was sequenced on a PacBio RSII device using the reagents DNA/Polymerase Binding Kit P4 (PN 100-236-500), DNA Sequencing Reagent 2.0 PN (100-216-400), SMRThttp://www.pacb.com/products-and-services/analytical-software/smrt-analysis/). The assembly was performed with HGAP (3.0 protocol) with the following parameters: (1) PreAssembler Filter v1 ; (2) PreAssembler v2 ; (3) AssembleUnitig v1 ; (4) BLASR v1 mapping of reads for genome polishing with Quiver . The final assembly contained nine unitigs  with a total 34,993,035 bp and approximately 87× genome coverage. N50 was 5,311,312 bp and max was 6,835,650 bp. The per-base quality value (QV) is higher than 45 and a little less than 50 . A BLASTN search revealed that the circular mitochondrial genome  was completely identical in nucleotide sequence with the QM9414 mitochondrial genome (Accession: NC_003388.1) [The PacBio data were assembled with the SMRT Analysis Software v2.3.0 (03388.1) . The shohttp://www.usadellab.org/cms/?page=trimmomatic) to trim and remove reads (33.15%) that fell below a quality score threshold of 30 (Q30) and were shorter than 30 bp. Next, CLC Genomics Workbench 7.5 (http://www.clcbio.com/blog/clc-genomics-workbench-7-5/) was used to map the qualified reads to three different QM6a genomic drafts: (1) QM6a-v2.0 (http://genome.jgi.doe.gov/Trire2/Trire2.home.html) plus the complete mitochondrial genome [The two runs of Illumina MiSeq with 300-bp paired-end reads were combined into one. The forward and reverse data were 6.8 Gb. Reads were preprocessed with Trimmomatic V0.32  thel genome  plus thel genome ; and (3)reads 33.5% that fhttp://www.bioinformatics.nl/cgi-bin/emboss/revseq) was used to reverse and complement the nucleotide sequences of ChII, ChIV, and ChVI, respectively, so that all seven chromosomes had a shorter (left) arm at 5′ termini and a longer (right) arm at 3′ termini. The final sequences were first compared to the original version using BLAST searches , from the largest to the smallest. Artemis was used to determine the percentage of G+C in 500 bp non-overlapping windows. The centromere of each chromosome was their longest interspersed AT-rich block. The EMBOSS revseq software tool  genome annotation pipeline [T. reesei protein sequences from NCBI were used for ab initio gene predictions. Secondly, the Augustus v3.0.3 gene prediction software (http://augustus.gobics.de/) [Neurospora spp. and Fusarium spp. were used for Augustus training to impose constraints on the predicted gene structure, including splice sites, translation initiation sites, and stop codons. Thirdly, we isolated QM6a poly(A) RNA and performed a MiSeq RNA sequencing experiment. The resulting mRNA reads  were applied to Trinity  for de novo transcriptome assembly. The expressed sequence tag (EST) results predicted by “Maker” were used to identify new genes. Finally, we integrated all the ESTs and protein sequences from the two publicly available databases  as well as all the ESTs assembled from two published transcriptome datasets [The MAKER v2.31.8 (pipeline  was applics.de/)  was alsodatasets , 25.http://genome.jgi.doe.gov/Trire2/Trire2.home.html)(TreeseiV2_FilteredModelsv2.0.transcripts.fastaTreeseiV2_FilteredModelsv2.0.proteins.fastahttp://genome.jgi.doe.gov/TrireRUTC30_1/TrireRUTC30_1.home.html)GSE53629 (SRR1057947: QM9414 Cellulose replication 1SRR1057948: QM9414 Cellulose replication 2SRR1057949: QM9414 Cellulose replication 3SRR1057950: QM9414 Sophorose replication 1SRR1057951: QM9414 Sophorose replication 2SRR1057952: QM9414 Sophorose replication 3SRR1057953: QM9414 Glucose replication 1SRR1057954: QM9414 Glucose replication 2SRR1057955: QM9414 Glucose replication 3http://cole-trapnell-lab.github.io/cufflinks/) were downloaded for transcriptome assembly and differential expression analysis, including bowtie2-build (bowtie v2.2.3), tophat2 (v2.0.13), and cufflinks (v2.2.1). For read alignment, the parameters of tophat2 were as follows: (1) min-intron-length: 20 bp; (2) max-intron-length: 5000 bp (to reflect introns and splicing elements of five diverse fungi); and (3) no transcript GTF (Gene Transfer Format) file was provided to guide assembly. There were ~95% trimmed paired reads that could be aligned to the QM6a complete genome. After each QM9414 RNA-seq raw dataset was assembled into transcripts, the cuffmerge program was used to merge all GTF files into one. The gffread program was used to extract transcript sequences.The Illumina-HiSeq.sra files were converted into fastq using the fastq-dump program of the SRA Toolkit. Trimmomatic v0.32  was usedhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44648 )SRR764963: QM6a Glucose 48 h replication 1SRR764964: QM6a Glucose 48 h replication 2SRR764965: QM6a Glucose 48 h replication 3SRR764966: QM6a Straw 24 h replication 1SRR764967: QM6a Straw 24 h replication 2SRR764968: QM6a Straw 24 h replication 3SRR764969: QM6a Straw + Glucose 5 h replication 1SRR764970: QM6a Straw + Glucose 5 h replication 2GSE44648 , tophat2, and cufflinks were used. All parameters were as for the QM9414 dataset, except the colorspace option was used for the bowtie1 program. There were ~33% reads that could be aligned to the QM6a complete genome.T. reesei protein sequences from NCBI > Neurospora spp. and Fusarium spp. (Augustus v3.0.3) > de novo assembly QM6a-RNA (Trinity v2.0.6) > QM6a v2.0 + RutC-30 v1.0 + QM6a-RNA (SOLiD) + QM9414-RNA (Illumina HiSeq 2000).Next, we performed gene filtering to finalize all predicted protein sequences, the filtering order was all  Trichoderma research community [We also manually integrated almost all the annotation results reported by the ommunity , 24, 25.ommunity .N. crassa database (Broad Institute), F. fujikuroi (JGI), Sordaria macrospora (NCBI), S. cerevisiae (SGD), and S. pombe (PomBase and NCBI). The e-values of all BLAST results were <1.0 × 10−5. The Retrieve/ID mapping tool (UniProt) was used to map gene ID (UniProt sprot v2.0 and NCBI BLAST result) and to determine gene ontology (GO)  database, Universal Protein Resource (UniProt) Prot v2.0, S. cerevisiae, A. nidulans, and N. crassa are 52–1002, 27–1903, and 46–1740 bp, respectively [6, whereas reads per kilobase (RPK) = (read counts/transcription length)  and QM9414 (Illumina MiSeq) RNA reads with the following parameters: (1) min-intron-length: 20 bp; (2) max-intron-length: 5000 bp (because it was reported previously that the ranges of intron length in ectively ); and  with default parameters. RepeatMasker (version 4.0.6) and the Repbase Library (http://www.repeatmasker.org) were used to scan 12 different fungal genomes for interspersed repeats and low complexity DNA sequences. The output of the program is a detailed annotation of the repeats that are present in the query sequence, as well as a modified version of the query sequence in which all the annotated repeats have been masked (default: replaced by Ns). To obtain high-confidence data, we first analyzed the genome sequences of S. cerevisiae because the number and location of five different Ty elements in all its 16 yeast chromosomes had been reported previously [Novel repeat elements were identified by RepeatModeler-1.0.4  was downloaded [The latest RIPCAL program . Table A2. Error corrections of the seven PacBio unitigs using the Illimina-MiSeq reads. Table A3. Characteristics and assembly of the seven QM6a chromosomes. Table A4. Mapping of all trimmed paired-end Illumina MiSeq reads to three QM6a genome drafts. Table A5. The MiSeq reads mapped to the QM6a-v2.0 draft genome. Table A6. Sixteen false predicted genes in QM6a-v2.0. Table A7. The QM6a-v2.0 draft genome compared to the complete QM6a genome sequence. Table A8. The HiC draft genome compared to the complete QM6a genome sequence. Table A9. PCR primers. Table A10. The complete QM6a genome sequence versus the RUT-C30-v1.0 draft genome. Table A11. Gene Ontology of some newly-predicted genes. Table A12. Sequencing quality of 18 different fungal genomes. Table A13. RIP indices of various sequences in QM6a and Neurospora crassa.Table A14. Repeat-induced C-to-T mutations observed in the hph alleles in all F1 progeny. Table A15.Saccharomyces cerevisiae Ty elements by chromosome. Table A16. Transposable elements in 12 well-assembled fungal genomes. Table A17. Repetitive sequences in different chromosomal regions. Table A18. Repetitive sequences in seven QM6a chromosomes. Table A19. Partitioning of four gene clusters by the AT-rich islands. The TPM values in glucose (48 hrs), in straw (24 hrs) and in straw (24hrs) then glucose (5 hrs) are shown [in straw  hrs and Additional file 2: Table B1. Genome annotation of the complete QM6a genomes, including AT-rich blocks, predicted genes, repetitive features, gene ontology (GO), gene names in T. reesei, Saccharomycetes cerevisiae and Neurospora crassa, as well as gene identity numbers in QM6a-v2.0 and RUT-C30-v1.0. (Columns A-H) All the genes that were not annotated in QM6a-v.20 and Rut-C30-v1.0 are highlighted with yellow background, whereas those only annotated in Rut-C30-v1.0 are highlighted with yellow-green background. (Columns O-AE) Comparative transcriptomic analysis of all QM6a genes in different carbon sources using two published transcriptome datasets: (1) The SOLiD sequencing reads from QM9a grown first in glucose for 48 hours, then in straw for 24 hours and finally with the addition of glucose for 5 hours [Table B2. Annotation of the complete QM6a genome. All the annotated QM6a genes are compared to those in QM6a-v2.0, Rut-C30-v1.0, all Trichoderma reesei proteins from NCBI, the QM6a-HiC annotation results by Druzhinina et al. [Fusarium fujikuroi (JGI), Sordaria macrospora (NCBI), Saccharomyces cerevisiae (SGD) and Schizosaccharomyces pombe (PomBase and NCBI). The e-values of all BLASTP results were < 1.0 x 10-5. Table B3. TPM values of all new QM6a genes revealed by two publicly-available transcriptome datasets [Table B4. Locations of 62 previously identified CAZyme and SSCP gene clusters [ 5 hours . The TPMa et al. , the anndatasets . Table Bclusters  in 42 chAdditional file 3: Table C1. Straw-induced QM6a-v2.0 genes identified by Ries et al. [Table C2. Straw-induced QM6a-v2.0 genes not identified by Ries et al. [Table C3. New QM6a genes that are highly induced (≥ 20-fold) by straw and their TPM values. Table C4. New QM6a genes that are significantly induced (5-20-fold) by straw and their TPM values. Table C4. Two new QM6a genes that are highly induced by cellulose and their TPM values. Table C5. Nine new genes having higher TPM values in QM6a than in QM9414. Table C6. Centromere-encoded genes and their TPMs in QM9414.s et al.  and theis et al.  and theiAdditional file 4. Sequence alignments of the 24 centromeric repeats.Additional file 5. Nucleotide sequences of the hgh alleles from all strains listed in Figure 7.


Lacinutrix venerupis has recently been considered a potential fish pathogen. Here, we report the complete genome sequence of L. venerupis DOK2-8, which possesses several virulence-related genes. This strain may be potentially virulent to other marine organisms, and its genomic information will provide important insights into the biodiversity of the genus Lacinutrix. Lacinutrix, which is a member of the family Flavobacteriaceae (Proteobacteria: Gammaproteobacteria) was created in 2005 and comprises 10 valid marine species, including L. algicola, L. cladophorae, L. copepodicola, L. gracilariae, L. himadriensis, L. iliipiscaria, L. jangbogonensis, L. mariniflava, L. undariae, and L. venerupis  are currently available in the GenBank database; however, the genome of L. venerupis has not yet been sequenced. Since 2015, we have screened several indigenous bacterial strains with advantageous characteristics for biotechnological applications. Here, we present the first complete genome sequence of an L. venerupis strain isolated from the Republic of Korea.The genus ine fish . Four drL. venerupis Cmf 20.8T (HG970752); hence, it was finally classified as L. venerupis DOK2-8. The isolate showed strong extracellular proteolytic activity, as determined with the method of Amoozegar et al.  version 3.0 and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/books/NBK174280).The DOK2-8 strain was isolated from marine sediment collected from Dokdo, East Sea, Republic of Korea (37°14′25.9″N 131°52′02.6″E), using the standard dilution plating technique on marine agar 2216 (Difco) followed by incubation at 28°C. The 16S rRNA of the isolate (GenBank accession number MG493235) showed >99.9% similarity to Lacinutrix species strains were assessed using the orthologous average nucleotide identity algorithm , and the lowest (75.5%) was with L. himadriensis (LIQI00000000).The final assembled circular chromosome of DOK2-8 comprised 3,192,399 bp, with a 30.6% G+C content (92.5% coding region percentage), and encoded 2,857 genes, 2,811 coding sequences, 6 rRNAs , 36 tRNAs, and 4 noncoding RNAs. Overall genome similarities among DOK2-8 and the other four htpB chaperonin, which were homologous to those of other Gram-negative species, were detected. Moreover, three putative hemolysin genes were detected in the DOK2-8 genome. These results indicate that L. venerupis DOK2-8 may have potential virulence to other marine organisms, similar to other species in the family Flavobacteriaceae  as KACC 19202. The 16S rRNA and complete genome (chromosome) of L. venerupis DOK2-8 have been deposited at DDBJ/ENA/GenBank under the accession numbers MG493235 and CP019352, respectively.


Trichoderma are fungicolous on fungi and have been extensively studied and commercialized as biocontrol agents. Multigene analyses coupled with morphology, resulted in the discovery of T. hypoxylon sp. nov., which was isolated from surface of the stroma of Hypoxylon anthochroum. The new taxon produces Trichoderma- to Verticillium-like conidiophores and hyaline conidia. Phylogenetic analyses based on combined ITS, TEF1-α and RPB2 sequence data indicated that T. hypoxylon is a well-distinguished species with strong bootstrap support in the polysporum group. Chemical assessment of this species reveals a richness of secondary metabolites with trichothecenes and epipolythiodiketopiperazines as the major compounds. The fungicolous life style of T. hypoxylon and the production of abundant metabolites are indicative of the important ecological roles of this species in nature.Some species of  Trichoderma was based on morphology. Most species in this genus are usually fast growing, produce highly branched conidiophores with cylindrical to nearly subglobose phialides and ellipsoidal to globose conidia123556789Trichoderma59Trichoderma species complex and reveal taxonomic deversity2678910Traditionally the taxonomy of species of Trichoderma species are most frequently found in vegetable matter, decaying wood and soil, plant rhizosphere, as well as on other fungi56Trichoderma species comprise diversity and abundant genes associated with secondary metabolites productivity15141516171819202115TrichodermaTrichoderma arundinaceumFungi associated with other fungi as saprobes, commensals or parasites are termed fungicolous fungi13Trichoderma were obtained from stroma of Hypoxylon anthochroum. They are described, illustrated and named as a new species Trichoderma hypoxylon. Its phylogenetic positions were also explored, which inferred from sequence analyses of the combined internal transcribed spacer (ITS), partial RNA polymerase II subunit (RPB2) and translation elongation factor 1 alpha (TEF1-α) exon genes. Detailed comparisons were made between the new taxa and their related fungi. Considering T. hypoxylon is a new fungicolous species and its special life style, we reasoned that it is valued to study the chemical profiles to get the linkage to the biological roles.In the search of novel bioactive compounds, we carried out resource and diversity investigation of fungicolous fungi in China and Thailand. Two isolates of T. hypoxylon are less than 97% similar to other Trichoderma species. Based on phylogenetic analysis of single gene of ITS, RPB2 and TEF1-α, T. hypoxylon formed a clade with Trichoderma taxi and Trichoderma rubi. The position of this clade showed closed relationship with section Hypoceanum, Polysporum, Psychrophila and other species in Trichoderma , maximum likelihood (ML) and maximum parsimony (MP) trees generated shared the same topology. In MP analyses, 1642 (57.8%) characters are constant, 216 (7.6%) characters are parsimony-uninformative, and 981 (34.6%) characters are parsimony informative. In MP analyses, 1642 characters were constant, 981 were parsimony-informative, and 216 variable characters were parsimony-uninformative. Maximum likelihood tree was Presented . They also clustered in 7 recognized subclades and a new subclade including T. hypoxylon, Brevicompactum (MLBP/BIPP = 94%/1.00), Deliquescens (MPBP/BIPP = 100%/1.00), Hypoceanum (MPBP/MLBP/BIPP = 100%/99%/1.00), Polysporum (BIPP = 1.00), Psychrophila (MPBP/MLBP/BIPP = 98%/96%/1.00), and green spore sections (MPBP/MLBP/BIPP = 100%/100%/1.00), whereas two strain of Trichoderma atroviride Bissett positioned inside Polysporum section. The tree topology is basically congruent with previous reportsBased on the analyses, 60 strains of T. hypoxylon , and T. taxi and T. rubi formed a clade independent from other Trichoderma species with strong support (MPBP/MLBP/BIPP = 100%/100%/1.00). However, T. hypoxylon isolates were distinguished from T. axi and T. rubi with high support (MPBP/MLBP/BIPP = 100%/100%/1.00).The new species Trichoderma hypoxylon Jing Z. Sun, Xing Z. Liu & K.D. Hyde, sp. nov.Index Fungorum number: IF552046, Facesoffungi number: FoF: 02075, Type:—HMAS 246918Hypoxylon anthochroum. Sexual morph Undetermined. Asexual morph forming Acremonium- to Verticillium-like conidiophores and hyaline conidia. Phialides lageniform to cylindrical, hyaline, 4.5–12 × 3–3.5 μm . Colony radius on CMD after 72 h at 20 °C. 41.2–46.1 mm; 25 °C. 37.4–38.3 mm; 30 °C, 8.32–9.12 mm; and 35 °C 0 mm (n = 5). Colony radius on SNA after 72 h at 20 °C, 24.9–26.8 mm; 25 °C, 27.1–29.2 mm; 30 °C, 10.4–12.1 mm, and 35 °C, 0 mm (n = 5).Etymology:—hypoxylon refers to the genus of host fungus Hypoxylon anthochroum.Distribution:—Chiang Rai, ThailandHost:—Hypoxylon anthochroum, a saprobic fungus on dead wood.Material examination: Thailand, Chiang Rai Province, isolated from the stroma of Hypoxylon anthochroum, 5 May, 2014. MFLU16–1263 , HMAS 246918  ex-type living culture, MFUCC 15–0683, CGMCC 3.17906, CGMCC 3.17907.Notes: The asexual morph of Trichoderma hypoxylon produces Acremonium- to Verticillium-like conidiophores and hyaline conidia. Phylogenetically T. hypoxylon is related to T. taxi and T. rubi and together formed a new independent clade distinguished other Trichoderma species  are also longer than T. taxi (2.4–3.1 × 2.0–2.5 μm)T. rubi (2.3–3.3 × 2.0–2.7 μm)T. hypoxylon did not produce pigment on medium, whereas T. rubi produced brownish pigment and yellow crystals species . HoweverT. hypoxylon cultivated on PDA medium were carried out to assess the production of secondary metabolites. Four major peaks with high yields were obtained by HPLC chromatography , aspergillazine A (2), aspergillazine C (3) and harzianum B (4)  . The assTrichoderma species was mainly based on the morphology6Trichoderma are accepted based on phylogenetic analysis5Trichoderma remain to be discovered and described5T. hypoxylon, phylogenetic tree containing all species of Trichoderma species was constructed and the putative position of T. hypoxylon was shown 15Spicaria elegansT. viridesT. viridesFusarium, Myrothecium and Trichoderma in order Hypocreales15Hypocrea sp.  strain F000527 yield of 18.13 mg/LT. hypoxylon with yield of 50 mg/L. The high amount of trichothecenes in T. hypoxylon, indicating that it has antagonistic potential against fungal hosts.cterized . TrichodTrichoderma strains were isolated from the host by single spore isolation as detailed in Chomnunti et al.et al.Samples were collected on 5 May 2014 in Chiang Mai Province, Thailand. et al.Methods and morphology were described basically following counterparts by Jaklitsch & VoglmayrApproximately 50 mg of fungal material from each culture was placed in 600 μl of 2% CTAB buffer and ground with a plastic pestle. Genomic DNA was extracted using a modified CTAB extraction protocolTrichoderma species was conducted seperately with N. berolinensis and N. eustromatica as outgroup. Then, single and combined genes analyses of ITS, TEF1-α and RPB2 sequence data of 60 phylogenetic closed Trichoderma species in Brevicompactum, Deliquescens, Hypoceanum, Longibrachiatum, Polysporum, Psychrophila and green spore sections were carried out. N. berolinensis and N. eustromatica were arranged as outgroup taxa.The ITS and TEF1-α data sets used sequences of isolates CGMCC 3.17906, CGMCC 3.17907 and reference sequences were downloaded from GenBank . The ITSMaximum parsimony (MP) analysis was conducted by PAUP 4.0b10Maximum-likelihood (ML) analysis was performed in RAxMLBayesian Inference (BI) analysis was performed with MrBayes 3.1.2All trees were viewed in TreeView 1.6.61H-NMR spectra were recorded on a Bruker Avance-500 spectrometer using TMS as internal standard, and chemical shifts were recorded as δ values. ESI-MS utilized on an Agilent Accurate-Mass-QTOF LC/MS 6520 instrument. HPLC analysis was performed on a Waters HPLC system  using an ODS column  with a flow rate of 1 mL/min.2Cl2: Acetone (V:V = 1:1) elution to obtain fifteen fractions 1–15. The fractions 3–7 (200 mg) was separated by semi-preparative RP-HPLC  to afford 4 . The fraction 11 (35 mg) was separated by semi-preparative RP-HPLC  to afford 1 , 2 , and 3 .The fungal strain was cultured on 20 slants of potato dextrose agar at 25 °C for seven days. The fermented PDA substrate was extracted repeatedly with ethyl acetate by exhaustive maceration (4 × 200 mL), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (330 mg). The residue was fractionated by Sephadex LH-20 CC using CH1H NMR  δH: 2.72 ; 2.28 ; 4.48 ; 5.60 ; 5.55 ; 4.24 ; 4.15 ; 8.58 ; 7.41 ; 7.12 ; 3.96 ; 3.91 ; 9.40 ; Positive ESIMS: m/z 433.1 [M+H]+.White powder (MeOH)251H NMR  δH: 3.14 ; 2.42 ; 4.16 ; 5.95 ; 6.07 ; 4.33 ; 4.23 ; 6.90 ; 7.09 ; 6.69 ; 3.90 ; 3.83 ; 9.38 ; 9.90 ; Positive ESIMS: m/z 449.1 [M + H]+.Yellow powder (MeOH)261H NMR  δH: 2.96 ; 2.40 ; 4.31 ; 5.42 ; 5.49 ; 4.16 ; 3.77 ; 6.85 ; 7.11 ; 6.69 ; 3.90 ; 3.84 ; Positive ESIMS: m/z 451.1 [M + H]+.Yellow powder (MeOH)1H NMR  δH: 3.84 ; 2.57 ; 2.03 ; 5.65 ; 1.92‒1.96 ; 1.40‒1.43 ; 1.97‒1.99 ; 5.41 ; 3.63 ; 3.13 ; 2.83 ; 0.72 ; 0.95 ; 1.71 ; 6.03 ; 7.32 ; 6.61 ; 6.64 ; 7.32 ; 6.07 ; Positive ESIMS: m/z 401.2 [M + H]+.White powder (MeOH)How to cite this article: Sun, J. et al. A new species of Trichoderma hypoxylon harbours abundant secondary metabolites. Sci. Rep.6, 37369; doi: 10.1038/srep37369 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Background:Callicarpa macrophylla (Varbenaceae) is a medicinal shrub and is traditionally used in India, China, and South Asia. Methods: The plant material was collected from lower Himalayan region of Uttarakhand in India. The essential oils from three different aerial parts were analyzed by GC-MS. Antioxidant activity, phenolic assay, and various pharmacological activities were determined by using existing methods which are generally practiced widely. Results: Over 51, 53, and 40 compounds were identified in C. macrophylla leaves essential oil (CMLEO), C. macrophylla pre mature seeds and fruits essential oil (CMEO-I) and C.macrophylla mature seeds and fruits essential oil (CMEO-II), respectively. These oils differ in relative contents of major compounds viz; β-selinene , phyllocladene , caryophelline oxide , 9E-epi-caryophelline , longipinocarvone , and 1,8-cineole . The oils exhibited good in vitro antioxidant activity. The maximum activity was found in CMEO-II with IC50 values 7.37 ± 0.11, 11.49 ± 0.87, 14.59 ± 0.18, 15.66 ± 0.03, and 17.49 ± 0.13 µL/mL. The essential oils showed qualitative and quantitative diversity in the makeup of essential oils constituents. The oils were found to exhibit anti-inflammatory, analgesic, and antipyretic activity on swiss albino mice compared to the standard drugs, viz; ibuprofen, paracetamol and indomethacin. Conclusion: It is inferred from the study that the plant parts can be used scientifically in traditional systems as folk herbal medicine. Furthermore, we have generated a database for future reference and judicious exploitation of these oils from their natural setting. C. macrophylla growing wildly in Uttarakhand Himalaya of India was investigated for chemical composition and pharmacological activity of its essential oils. This plant belonging to family Varbenaceae is an erect, stellate-pubescent, perennial shrub, with stems terete and leaves in sub or unequal pairs on 0.3–1.5 cm long petioles, elliptic-dentate with gland- tipped teeth, glabrescent or so above, 10–20 (–25) × 4–6 (–8) cm while flowers in axillary, 1–3 cm long, peduncled, cymose, corymbose panicles , followed by 2.5 mL of 10% Cl3CCOOH after incubation at 50 °C for 20 min. The content was centrifuged for 10 min at 650 rpm. 5 mL of the supernatant was mixed with 5 mL of distilled water and 1 mL of 0.1% FeCl3. The optical density (OD) of the solutions was measured at 700 nm using UV-spectrophotometer . The same procedure was followed for controls and standards. BHT, catechin, and gallic acid were used as standard antioxidants. The reducing power was evaluated by the procedures reported earlier . In brie2+ chelating ability of the antioxidant measured calorimetrically at 562 nm using ferrous ion-ferrozine as a reference complex [2·4H2O and 0.2 mL of 5mM ferrozine were added to 5–25 μL of essential oils followed by methanol to make up the volume to 5 mL. The solutions were homogenized and allowed to react for 10 min. OD was measured at 562 nm. The chelating activity on Fe2+ of EOs were compared with that of EDTA (0.01 mM) and citric acid (0.025 M). The following equation was used to calculate the percent of chelating activity.t = absorbance of the sample, A0 = absorbance of the control at 562 nm).The procedure is based on the Fe complex . 0.1 mL 3OH. The solutions were placed in the dark for 30 min. OD of the samples, standards, and control were read at 517 nm. BHT, catechin, and gallic acid were used as standards. The percent DPPH radical scavenging activity was calculated by the following equation.t = absorbance of the sample, A0 = absorbance of the control at 517 nm).The scavenging effect on the DPPH radical was evaluated by following the developed and reported procedures . 5, 10, t = absorbance of sample, A0 = absorbance of control at 546 nm).Nitric oxide (NO) obtained from sodium nitroprusside (SNP) was measured by the Griess reagent with the composition of 1% sulfanilamide, 0.1% naphthylethylenediamine dichloride, and 2.0 mL orthophosphoric acid. The scavengers of NO competed with oxygen, leading to reduced production of NO. 2 mL of SNP (10 mM) in phosphate buffer saline (PBS) with pH 7.4 was homogenized with different concentrations (5–25 µL/mL) of EOs dissolved in acetone. The content was incubated at 25 °C for about 150 min. OD of the pink color developed was read at 546 nm. Ascorbic acid was taken as standard . NO scavt = absorbance of sample, A0 = absorbance of control at 560 nm).In brief 1 mL nitroblueterazolium (156 mM), 1 mL nicotinamide adenine dinucleotide (468 mM) and 0.1 mL of phenanzinemethosulphate solution (PMS) in 0.1 M of phosphate buffer solution (pH 7.4) was mixed to various concentrations (5–25 μL) of EOs followed by incubation at 25 °C for 5 min. The OD was measured at 560 nm against blank containing all reagents except PMS. Ascorbic acid was taken as standard . The % s4.7H20 (1 mM), 90 μL aq. 1, 10 phenanthrolein monohydrate (1 mM), 2.4 μL (0.2 M) phosphate buffer (pH 7.8) and 150 μL of H2O2 (0.17 mM) was added in 1.5 mL of EOs (5–25 µL). OD was measured at 560 nm for samples control and standards (ascorbic acid). The % OH scavenging activity was calculated as:t = absorbance of sample, A0 = absorbance of the control at 560 nm). The hydroxyl (OH) scavenging activities of EOs were evaluated by using the developed protocols . BrieflyPermission from the institutional ethical committee  was taken prior to execute the experiments. Swiss albino mice were procured from the Lab Animal Division of the Central Drug Research Institute, Lucknow, U.P. in India. The animals were randomly divided into eleven groups with six mice in each and were kept under standard laboratory conditions. Three concentrations  of EOs were given orally with the dose level of 10 mL/kg body weight. All the concentrations of EOs were separately triturated by addition of small amount of tween-20 and saline water to make the final volume of 10 mL. Ibuprofen, indomethacin, and paracetamol were used as standard drugs and saline water as control.The anti-inflammatory activity of EOs were evaluated according to the protocols reported . BrieflyThis activity of the EOs were studied in HCHO induced arthritis. Briefly, 0.1 mL HCHO (1%) solution was injected in the right hind paw of the mice . The EOsIn this activity, the animals were treated with glacial acetic acid intraperitoneally to induce pain sensation . After 1(Sacchromyces cerevisiae) (10 mg/kg b. wt.) as per the reported protocol [To evaluate antipyretic activity pyrexia was induced in mice by subcutaneous injection of 20% brewer’s yeast protocol . The micIn order to evaluate the toxic effect of essential oils, different doses  at 10 mL/kg body weight were given to experimental animals. For toxic effect of drug under experiment, the behavioral changes were recorded for 24 h and the numbers of deaths if any were recorded up to 48 h.p < 0.05 were considered to be statistically significant.The experimental data generated were represented as mean ± S.E and the results were analyzed using one way analysis of variance. The value of Over 40–53 compounds were identified in CMLEO, CMEO-I, and CMEO-II which contributed to 96.55%, 94.56%, and 95.35% of the total oil, respectively. The constituents identified in CMLEO, were β-selinene (37.51%), phyllocladene (9.76%), caryophelline oxide (7.34%), caryophelline-9-epi (E) (6.23%), longipinocarvone (4.96%), β-caryophelline (3.26%), juniper camphor (3.13%), vulgarone (2.92%), 1, 8-cineole (2.23%), α–muurolol (1.76%), and sphathulenol (1.06%) . In CMEOβ-Salinene has been found as a major compound in all the EOs. The oils also showed diversity both in qualitative and quantitative makeup of essential oils. CMEO-I contained longicamphnylone (3.08%), 4-camphenylbutan-2-one (0.80%) cedren-13-ol (0.52%), nopinone (0.50%), hexanoic acid (0.47%), terpinen-4-ol (0.43%), bornyl acetate (0.42%), and leden oxide-I (0.40%) which were absent in other oils. Muurolene-14-oxy-α (2.50%), α-muurolol (1.76%), β-copaen-4-α-ol (1.03%), globulol (0.42%), β-oplopenone (0.33%) etc were found only in CMLEO, while 5-octen-2-one (0.53%), β-ocimene (0.31%), α-pinene (0.40%), and khusinol (0.10%) were identified only in CMEO-II . The essential oils showed good reducing activity as the function of concentration compared to standard antioxidants . CMEO-II showed the highest reducing property of the extracts .2+ chelating with maximum chelating values of 63.82% recorded for CMEO-II, at 25 µL/mL of dose level compared to standard antioxidants.As shown in The EO showed good to moderate DPPH radical scavenging activity. CMEO-II was a better DPPH radical scavenger than CMLEO and CMEO-I, compared to standard antioxidant viz. BHT, catechin, and gallic acid . DPPH scCMEO-II, CMEO-I, and CMLEO exhibited good superoxide scavenging activity with maximum at higher concentration. CMLEO-II revealed highest scavenging activity (56.98 ± 0.630%) followed by CMLEO (56.49 ± 0.412%) and CMEO-I (53.13 ± 0.724%), respectively, as compared to ascorbic acid (60.76 ± 0.313%) .The EOs were good NO radical scavengers but moderate in comparison to the standard, ascorbic acid. CMEO-II was the strongest NO scavenger among the oils and scavenged 43.51 ± 0.329–70.68 ± 0.115% NO radical from 5 µL to 25 µL .The oils exhibited OH radical scavenging in a dose-dependent manner similar to DPPH, NO, and superoxide methods. Maximum scavenging activity was observed at the concentration level of 25 μL in ascorbic acid (68.48 ± 0.708%) followed by CMEO-II (64.4 ± 0.900%), CMLEO (61.22 ± 0.340%), and CMEO-I (57.03 ± 0.520%), respectively .50 values for scavenging and chelating activity of the EOs indicated their efficiency against oxidative stress as well as free radicals. CMEO-II showed the minimum IC50 value in all the activity tests among the oils and the highest antioxidant activity, but was moderate in comparison to the standards , followed by citric acid (IC50 = 9.42 ± 0.95 µg/mL), CMEO-II (IC50 =11.49 ± 0.87 µL/mL), CMEO-I (IC50 = 13.42 ± 0.17 µL/mL), and CMLEO (IC50 = 14.38 ± 0.27 µL/mL). The DPPH radical is a highly reactive species and used for evaluation of free radical scavenging activity of antioxidants. It accepts an electron and a hydrogen to become a stable diamagnetic molecule [50 = 7.95 ± 0.11 µg/mL) > catechin (IC50 = 8.18 ± 0.11 µg/mL) > BHT (8.55 ± 0.10 µg/mL) > CMEO-II (IC50 =15.66 ± 0.03 µL/mL) > CMLEO (IC50 =18.35 ± 0.18 µL/mL) > CMEO-I (IC50 = 20.19 ± 0.11 µL/mL). It has been reported earlier that the plant-derived products mitigate the harmful effects produced by nitric oxide radicals in human body [50 value 7.37 ± 0.11 µL/mL, which was comparable to ascorbic acid , followed by CMLEO (2.3 mm3 on day 1 and 2.22 mm3 on day 10) and CMEO-I (2.26 mm3 on day 1 and 2.24 mm3 on day 10) at 20% dose level. The inhibition property was observed in a dose-dependent manner  was observed in paracetamol-treated group up to 24 h. The uniform fall in temperature up to 24 h was observed by increasing the dose . It has C. macrophylla can be beneficial to treat inflammations, pain, and fever. These activities might be due to presence of a complex mixture of terpenoids.Infection in the body is a cause of fever or other diseased states like tissue damage, inflammation, and graft rejection etc. . RegulatC. macrophylla showed activities in various degrees against inflammation, pain, and fever. According to the research, higher levels of prostaglandin, particularly PGE2, produce inflammation, pain, and fever because of cyclooxygenase activation [The essential oil of tivation . As a retivation ,35.C. macrophylla neither cause any changes in behavior nor any death of mice under experimentation during the period of observation. It was thus considered that the EOs were practically non-toxic.Essential oils from parts of C. macrophylla showed a qualitative and quantitative make-up of constituents. The oils also showed different pharmacological activities. From the results, it could be inferred that the essential oils might be used as drugs for treatment of inflammation, fever, and pain in traditional systems of medicine with scientific knowledge. Clinically, this herb can be a good source of herbal medicine for the treatment of diseases indigenously. The study will also help to generate a database of species which can be exploited scientifically and judiciously in the future by local people and so that ecological balance is maintained. The EOs from


We isolated an influenza strain named A/Swine/Fujian/F1/2010 (H1N2) from a pig suspected to be infected with swine flu. The results of electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis suggest that it was a reassortant virus of swine (H1N1 subtype), human (H3N2 subtype), and avian influenza viruses. To further study the genetic evolution of A/Swine/Fujian/F1/2010 (H1N2), we cloned its whole genome fragments using RT-PCR and performed phylogenetic analysis on the eight genes. As a result, the nucleotide sequences of HA, NA, PB1, PA, PB2, NP, M, and NS gene are similar to those of A/Swine/Shanghai/1/2007(H1N2) with identity of 98.9%, 98.9%, 99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. Similar to A/Swine/Shanghai/1/2007(H1N2), we inferred that the HA, NP, M, and NS gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were derived from classical swine influenza H3N2 subtype, NA and PB1 were derived from human swine influenza H3N2 subtype, and PB2 and PA genes were derived from avian influenza virus. This further validates the role of swine as a “mixer” for influenza viruses. Swine influenza is an acute and highly contagious flu caused by swine influenza virus (SIV), which infects human and pigs. Its clinical symptoms in swine include high fever, decreased appetite, lethargy, sneezing, coughing, and difficulty in breath . In viruAt present, there are three subtypes of swine influenza in the world, namely, H1N1, H3N2, and H1N2, which can be further divided into classic swine H1N1, avian H1N1, human-like H3N2, reassortant H3N2, and reassortant H1N2 , 11. In From 2009 to 2013, our laboratory carried out epidemiological study of swine flu in Fujian Province. An influenza strain was isolated from 13 pigs suspected to be infected. We then performed electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis to infer its biological characteristics and evolutionary history. As a result, we confirmed that the virus was a “swine-human-avian” reassortant virus.Materials and Reagents. The lungs and lymph nodes used in the experiment were collected from a large-scale swine farm suspected to be infected with swine influenza in Fujian Province, China. EDS-76 and ND were positive sera provided by Fujian Institute of Animal Husbandry and Veterinary Medicine; the standard antigens and its antisera of H1, H3, H5, H7, and H9 subtypes and antisera of N1, N2, and N9 subtypes were purchased from Harbin Veterinary Research Institute; RNA extraction kit Trizol, plasmid extraction kit, LATaq DNA polymerase, RNasin Inhibition, M-MLV, dNTP, BamHI, HindIII, and PMD18-T vector were purchased from Dalian Bao Biological Company; Escherichia coli engineering strain DH5a was preserved by our laboratory.Test Animals. BALB/c mice were collected from Fujian Medical Laboratory Animal Center; 5-week-old piglets were purchased from a large-scale pig farm in Fuzhou, which were confirmed to be negative in serological antibody examination; SPF eggs were purchased from Fuzhou Dabei Agricultural Biotechnology Co. and incubated to 9 to 11 days of age.Acquisition and Processing of Disease Materials. The lung and lymph node tissues of pigs suspected to be infected with swine influenza were cut into pieces and ground into homogenate under sterile conditions. Hank's solution was added to it to make 10% suspension solution, which was then undergone frozen and thawed for three times, centrifuged at 12000 rpm for 10 min. The supernatant was filtered and sterilized, and placed at −70°C freezer for further use.Virus Isolation and Culture. The supernatant was inoculated into allantoic cavity of 9 to 11-day-old SPF chicken embryos with 0.1 mL/embryo, and each sample was inoculated into 4 embryos. The inoculated embryos were incubated in an incubator at 37°C and a relative humidity of 55%. The eggs were candled twice a day to inspect the vitality of chicken embryos for 6 days. The embryos died within 24 hours were abandoned, whereas those died between 24 and 96 hours were stored overnight at 4°C. Allantoic fluid was collected and the hemagglutination activity was detected. If the hemagglutination result is positive, we preserved chicken embryo allantoic fluid; otherwise the allantoic fluid were blindly passed for three generations and discarded if the result was still negative.Swine Influenza RT-PCR. A pair of primers was designed according to the M gene conserved region of swine flu registered in GenBank, i.e., SIV-F: 5′-CAAGACCAATCCTGTCACCTC-3′, and SIV-R: 5′-AAGACGATCAAGAATCCAC AA-3′. It was then amplified into a fragment of 684 bp by synthetic company Dalian Biological Engineering Technology. The viral RNA was extracted by conventional methods. The 10.0 μL RT reaction system consists of DEPC water (1.25 μL), 5 × AMV Buffer (2.0 μL), 25 mg MgCl2 (1.0 μL), dNTP mixture (1.0 μL), RNase inhibitor (0.25 μL) of AMV (0.5 μL), random primer (1.0 μL), and RNA sample template (3.0 μl). The RT reaction conditions were 30°C for 10 min, 42°C for 1 h, and 99°C for 5 min. The 25.0 μL PCR reaction system consists of Nuclease-Free Water (10.0 μL), GoTaq Green Master Mix (10.0 μL), upstream primer (0.5 μL), downstream primer (0.5 μL), and RT reaction product (4.0 μL). The PCR reaction conditions were 95°C for 5 min, 94°C for 1 min, 54°C for 1 min, 72°C for 1 min with 35 cycles, and then 72°C for 10 min and 4°C for preservation. After the reaction, the PCR products were electrophoresed on 1% agarose gel, and the positive product was sequenced by Bioengineering (Shanghai) Co., Ltd.Electron Microscopy. After allantoic fluid was centrifuged by differential and density gradient method, the morphology and size of the virus were observed by transmission electron microscopy.Agar Diffusion Test. The isolated chicken embryo allantoic fluid was purified, which can be used as antigen after adding SDS. After that, it was under agar diffusion test with standard serum. The results were collected at 24 h, 48 h, and 72 h, respectively.The HA subtype of swine influenza virus was identified by comparing the isolated virus with the standard positive sera of swine influenza by hemagglutination assay (HA) and hemagglutination inhibition assay (HI). Neuraminidase inhibition assay (NI) was used to identify NA subtypes.Hemagglutination Assay (HA). We added 50 μl of virus antigen in the first well of each row and used a pipette to mix well; we then added 50 μl of the mixed solution to the second well, diluted consecutively until the 11th well. The 12th hole without antigen was taken as a control. After that, we added 50 μl 1% pigeon erythrocyte suspension to each well, oscillated using microoscillator for 1 min and recorded the results after 20 ~ 30 min quiescence in room temperature.Hemagglutination Inhibition Assay (HI). According to the hemagglutination test, we made 8-unit and 4-unit antigen by adding physiological saline. We then added 50 μl 8-unit antigen into the first well and 50 μl 4-unit antigen into the 2nd, 3rd,…, and 11th well and 50 μl normal saline to the 12th well as negative control. After that, we took 50 μl standard positive serum to the first well, mixed them even, and took 50 μl to the second well. The process was repeated until the 11th well. After 15 min at room temperature, we added 50 μl 1% pigeon erythrocyte suspension, oscillated using microoscillator for 1 min and recorded the results after 20 min quiescence in room temperature. The hemagglutination inhibition titer of the serum was the maximum dilution of sera with 50% inhibition of erythrocyte agglutination. Hemagglutination inhibition titer ≥ 4log3 serum was determined as positive.Neuraminidase Inhibition Assay (NI). The allantoic fluid of the virus was tested against the standard antiserum of three NA subtypes.50 was calculated by Karber's method.The virus liquid of each dilution was inoculated into 9-day-old SPF chicken embryos with 4 pieces/group and 0.1 mL/piece. The embryos were then sealed with wax and cultured at 37°C for 7 days. The embryos which died within 24 hours were abandoned and allantoic fluid from embryos which died after 24 hours were collected for hemagglutination assay and the EIDVirus allantoic fluid was centrifuged at 3000 rpm for 15 min, and the supernatant was distributed into several tubes for further use. We then added SDS , chloroform , ether , hydrochloric acid , 1% potassium permanganate (for 5 min), formaldehyde , and 75% ethanol (for 5 min) and treated them with water at 56°C for 0,5, 10,15, 20, 30, 60, 90, 120, and 180 mins overnight. The treated virus was inoculated into 9-day-old SPF chicken embryo with 0.1 mL/embryo. Allantoic fluid or the embryo died in 24 ~ 96 h were collected for HA assay.BALB/c Mice Infection Assay. The virus with the dose 2 × 105EID50 was inoculated intranasally into ten 6-week-old BALB/c mice and physiological saline was inoculated into 10 mice as controls. One BALB/c mouse was dissected on the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days after inoculation. The lungs were harvested for virus isolation. The mental and diet status of the mice were also observed for 2 weeks.Infection Assay on Piglets. Twelve 5-week-old piglets, which were tested negative for swine influenza virus, were divided into experiment and control group evenly. Piglets in experiment were inoculated intranasally with 2 × 106EID50 dose viruses and those in control group were inoculated with normal saline. The body temperature of all piglets was measured every day after inoculation, and the diet and mental status of piglets were observed continuously for 28 days.Primer Synthesis. According to the H1N2 subtype of swine influenza virus in GenBank, 12 pairs of primers containing 8 gene fragments were designed by oligo 6.0 software. The primers were available upon request.Virus RNA Extraction and RT-PCR Reaction. Total RNA was extracted from the viral RNA extraction kit Trizol. The RT reaction conditions were DEPC water (1.25 μL), MgCl2 (1.0 μL), 5 × MMLV Buffer (2.0 μL), dNTP Mixture (1.2 μL), MMLV (0.5 μL), RNase Inhibition (0.25 μL), Uni-12 primer (1.0 μL), RNA (3.0 μL); the reaction conditions were 30°C for 10 min, 42°C for 60 min, and 99°C for 5 min and preserved at 4°C. The PCR reaction conditions were 10× PCR Buffer (2.0 μL), dNTP Mixture (1.2 μL), DNA polymerase (0.2 μL), Up Primer (0.4 μL), Down Primer (0.4 μL), and template (2.0 μL) and filled to 20 μL by adding H2O. The reaction conditions were as follows: predenaturation at 95°C for 5 min, denaturation at 94°C for 1 min, annealing at 53.5-61.0°C for 1 min, extension at 72°C for 1 min, and extension at 72°C for 10 min. The PCR products were identified by 1.0% agarose gel electrophoresisGene Cloning and PCR Identification. The PCR product was ligated into PMD18-T vector and transferred into DH5α strain. The recombinant plasmid was identified by 1.0% agarose gel electrophoresis, and the positive plasmid was sent to Bioengineering (Shanghai) Co., Ltd., for sequencing.http://www.megasoftware.net/). The phylogenetic trees were generated using the Neighbor-Joining method with 1000 bootstrap replicates.The sequences were put into GenBank for homology search using BLAST, and the sequences of subtypes human influenza H3N2, swine flu H3N2, classical swine flu H1N1, and representative swine flu H1N2 were downloaded and analyzed by DNAStar software. The sequence alignments were done by MUSCLE software in MEGA6 . Finally, the EIDWe first performed BALB/c mouse infection assay. The mice in the experiment group showed symptoms of mental exhaustion, coarse coat, and dyspnea symptoms 3 days after inoculation, but no mice died. The control group was normal. Influenza virus was isolated from the lungs of mice from 1 to 5 days after inoculation, and no virus could be isolated after 7 days after inoculation.We then infected virus with 6-weed-old piglets. Similar to mice, piglets in experiment group developed symptoms of high body temperature, loss of appetite, cough, and runny nose after 4 days of inoculation, and the symptoms were relieved after 7 days until all the piglets were recovery. No piglets died in the process. In contrast, piglets in control group were normal.The PB2, PB1, PA, HA, NP, NA, M, and NS genes of A/Swine/Fujian/F1/2010 (H1N2) strain were amplified by RT-PCR, respectively. After sequencing, we obtained 2341 bp, 2302 bp, 2190 bp, 1709 bp, 1543 bp, 1443 bp, 991 bp, and 882 bp gene fragments , which whttps://blast.ncbi.nlm.nih.gov/Blast.cgi), DNAStar (https://www.dnastar.com/), and MEGA6 using Neighbor-Joining method with 1000 bootstrap replicates. We used K80 model to estimate the distances between nucleotides. Specifically, the eight gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were compared with human influenza, avian influenza, and swine influenza virus deposited in NCBI flu database.We then performed nucleotide homology and genetic phylogenetic analysis using BLAST  with identity 98.9%, 98.9%,99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. According to the phylogenetic tree, the HA, NA, PB1, PA, PB2, NP, and M gene fragments of A/Swine/Fujian/F1/2010 (H1N2) is genetically close to those of A/Swine/Shanghai/1/2007(H1N2), while its NS fragment is close to A/Swine/Guangxi/13/2006 (H1N2). We illustrated the phylogenetic trees of HA, NA, PB1, PA, and PB2 in Figures 4The analysis of 8 gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain showed that the HA, NP, M, and NS gene fragments were originated from classical pig influenza H3N2 subtype, NA and PB1 were derived from human swine influenza H3N2 subtype, and PB2 and PA gene were from avian influenza virus, as shown in Swine as a host of influenza virus can simultaneously infect different types and subtypes of influenza viruses. Therefore, most scholars believe that swine plays an important role in influenza pandemic as a “mixer” of virus reassortment . When piCurrently, there are a lot of reports about swine influenza virus reassortment all over the world. For example, the H3N2 reassortant virus is isolated from North Carolina in 1998, whose HA, NA, and PB1 fragments are derived from human influenza H3N2 subtype virus; NP, NS, M, PB2, and PA were derived from classical swine influenza H1N1 viruses; and PB2 and PA were originated from H9N2 avian influenza viruses. In 2007, Yao et al. reported a two-source H3N2 reassortant swine influenza virus . The M a50 of the isolated strains was 10−4.6/0.1 mL, it could infect mice and piglets without killing them, of which the isolated strains were less pathogenic.In this study, a virus was isolated from the lymph nodes and lungs of pigs suspected to be infected with swine flu viruses. Our results showed that the virus had strong pathogenicity to chick embryo and could kill the chicken embryo in 48 hours. The allantoic fluid had hemagglutination activity. It is confirmed to be a H1N2 subtype virus by HA, HI, and NA assays, electron microscopy, and whole genome sequencing and thus named A/swine/Fujian/F1/2010 (H1N2). Based on the role of pigs as intermediate hosts in the SIV interspecies transmission chain and the complexity of influenza ecology in China, it is of great importance to understand the prevalence of influenza viruses circulating in swine herds. Although EIDBased on the whole genome sequencing analyses, A/Swine/Fujian/F1/2010 (H1N2) was a “swine-human-avian” reassortant virus, highly similar to A/Swine/shanghai/1/2007 (H1N2), another reassortant virus isolated in Shanghai, China. We inferred that the HA, NP, M, and NS genes were derived from the swine classical H1N1 influenza virus. The NA and PB1 genes were derived from human H3N2 subtype influenza viruses and PA and PB2 genes were from the avian influenza virus. The genetic characteristics of swine influenza virus isolation show that swine does play an important role as a “mixer” during the spread of influenza virus. Studies on the molecular and genetic evolution of swine flu are of great importance for timely detection of swine flu. The surveillance of the variation of swine influenza is critical for prevention and control of flu outbreaks.


Aspergillus caespitosus, Aureobasidium sp. RBSS-303 and Aspergillus flavus HF5 as confirmed using gene sequencing of ITS regions of the ribosomal DNA (rDNA). The obtained equilibrium data for Pb(II) biosorption of A. caespitosus fitted better to Langmuir isotherm with maximum sorption capacity of 351.0 mg/g and A. sp. RBSS-303 and A. flavus HF5 showed good fit to Freundlich isotherm with maximum sorption capacity of 271.5 and 346.3 mg/g respectively. The values of thermodynamic factors ascertained the nature of adsorption process is endothermic with A. caespitosus and A. flavus HF5 but exothermic with A. sp. RBSS-303. The experimental data for Pb(II) biosorption fits very well to pseudo second order kinetic model. With HCl the maximum 85.5, 75.3, 73.7% recovery of Pb(II) was obtained from A. caespitosus, A. sp. RBSS-303 and A. flavus HF5, respectively. The observed percentage loss in sorption capacity of Pb(II) was 3.9% by A. flavus HF5, 12.2% by A. caespitosus and 26.6% by A. sp. RBSS-303 after five cyclic studies of sorption and desorption. Results from the study confirmed the efficiency order of A. caespitosus > A. flavus HF5 > A. sp. RBSS-303 to remove and recover Pb(II) from aqueous solution. Finally, the fungal biosorbents can be used as soil conditioning agent after compositing into valuables fungal protein.The present investigation aimed to study and compare the efficiency of non-viable fungal isolates to remove divalent lead (Pb(II)) from aqueous streams. The selected fungal isolates showed identity with  A massive amount of toxic contaminants is discharged into water bodies and has become a severe hazard to environment with accumulation of non-biodegradable toxins in food chain . Among d3 or below) of metal ions. Therefore, the exploration of new most favorable technology to switch the conventional methods is the need to get rid of noxious waste to the lower point of requisite by regulation  were screened to recover the accumulated Pb(II) and to regenerate the exhausted biosorbents. Pb(II) loaded biomass (0.025 g) was washed with distilled water and mixed in 30 mL of 0.01 M desorbents at 180 rpm and 28 ± 2 °C. Regenerated biomass was washed, filtered and finally dried at 60 °C to calculate the loss in weight. The regenerated biomass was used for next sorption experiment and this sorption desorption cyclic studies were carried out for five times. The value of eluted Pb(II) per gram of biomass (qdes) was calculated from desorbed Pb(II) concentration (Cdes) as follows:3) volume of solution. The percentage desorption of Pb(II) was calculated asDifferent desorbents . Different desorbents were screened after 1 h contact time with Pb(II) loaded biosorbents during desorption studies. The initial pH of used desorbents (0.01 M) varied from 2.6 (hydrochloric acid) to 12.0 (sodium hydroxide). Plot of desorbents pH against desorption efficiency  efficiently removed the Cd(II) loaded to the biomass. The elution efficiency decreased with increase in pH value 7.2 (H2O), little elution was observed. Elution efficiency again reached to the values 68.6, 49.6 and 59.1% at pH 11.3 (sodium carbonate) and dropped to 17.0, 2.4 and 14.8% at pH 12.0 (NaOH) for A. caespitosus, A. sp. RBSS-303 and A. flavus HF5 respectively.To study the pragmatic approach of biosorption function in treatment of industrial effluents, recoveries of adsorbed metal ions along with adsorbents reuse are very important . Howeverncy Fig.  gave a ly et al.  suggeste3 of Pb(II) solution having initial concentration of 100 mg/dm3 for 6 h. The elution studies were carried out at optimized pulp density  after 2 h of incubation at 28 ± 2 °C and 180 rpm. The values of sorption capacity decreased from 174.2 to 152.9 and from 79.9 to 58.8 mg/g with A. caespitosus and A. sp. RBSS-303 and non-significant change (from 160.7 to 154.4 mg/g) was observed with A. flavus HF5 in going from first to fifth cyclic  loaded biomass was generated after incubating 0.5 g biosorbent/dm3)2 solution at pH 4.5 were shown in Fig. Sacha inchi shell biomass (SISB) by SEM that revealed the enhancement in weight percent of Pb(II) (15.72%) and Cu(II) (6.33%) ions on the surface of SISB after biosorption. Similarly Siddiquee et al. [Scanning electron microscopy is a useful technique to study the morphology of biosorbent and its modification after sorbate interactions. SEM–EDX analysis of Pb(II) biosorption by dried powdered biomass after 6 h of contact time with 100 mg/L Pb from aqueous solution. The results endorsed that the sorption performance was strongly affected by culture age and physical state of fungal biomass, temperature, initial Pb(II) concentration and sorbent dosage. The kinetic studies showed the good fit of adsorption equilibrium data to pseudo-second order model. The high correlation coefficient values (0.99) of equilibrium experimental data proved the favorable biosorption process. Elution studies were carried out to regenerate the biomass using 0.01 N HCl with 85.5, 75.3 and 73.7% Pb(II) elution from A. caespitosus; A. sp. RBSS-303 and A. flavus isolate HF5 respectively. Finally, sorption/desorption cyclic studies indicated that A. caespitosus, A. sp. RBSS-303 and A. flavus HF5 could be used as cost effective, easily cultivatable future cleaning up microorganisms for removal and recovery of Pb(II) from polluted water stream.These studies demonstrated the comparative feasibility and selectivity of