The hydrophilicity and flammability of fiberboards have limited their real-life applications. In this study, a facile strategy for preparing the multifunctional coatings with superhydrophobicity and flame retardancy on medium density fiberboards (MDFs) has been investigated. The superhydrophobic and flame-retardant coating on the MDF surface was obtained by depositing polydimethylsiloxane (PDMS) and 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane (FDTS)-modified Mg/Al layered double hydroxide (LDH) particles step by step. The as-prepared coating exhibited superhydrophobic properties with a water contact angle (WCA) of ~155° and good self-cleaning properties. Furthermore, the limiting oxygen index (LOI) value of the superhydrophobic MDFs increased by 60.4% as compared to that of the pristine MDFs, showing improved flame retardancy. The peak heat release rate (PHRR) and total heat release (THR) of MDFs decreased after coating with PDMS@FDTS-Mg/Al LDH, suggesting that the superhydrophobic coating decreased the fire growth speed and risk of fire hazard of MDFs. This coating with multiple functions opens a new avenue for the protection and functionalization of MDFs. Fiberboards have been widely applied in various fields, for example in furniture and interior decoration materials. This is due to their abundance, low cost, and easy machinability ,2. In ge2 nanoparticles x+[Ap−x/p]x+ · mH2O, where M2+ and M3+ are a metallic bivalent cation and a metallic trivalent cation, respectively, and Ap− is an interlayer anion [3 and Mg(OH)2 [2/Al–LDH polypropylene composites with improved flame retardancy [−–modified LDH composite with enhanced flame-retardant properties [Furthermore, traditional flame-retardant methods of fiberboards involve mixing flame retardants with adhesives or fibers before hot pressing progress. At early stage, halogen-containing compounds are used as the most common flame retardants. However, the leakage of hazardous flame retardants would have a bad influence on the environment and human health . In ordeand clay ,22,23, aer anion ,25,26. A Mg(OH)2 ,28. Wangtardancy . Kuila foperties . Guo preoperties . HoweverHerein, we report a facile method to prepare superhydrophobic coating with flame retardancy on MDF via brushing coating of PDMS following by deposition of 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane (FDTS)-modified Mg/Al LDHs. In this coating system, Mg/Al LDH act as hierarchical roughness materials and FDTS functions as low surface energy film. PDMS serves as adhesive to anchor the modified Mg/Al LDH on the MDF surface. Additionally, due to the coated Mg/Al LDHs, the superhydrophobic coatings endow the MDF with improved flame retardancy. It is believed that the resulting multifunctional superhydrophobic coating can provide a new strategy for the protection and functionalization of MDFs.4Al2(OH)12CO3·3H2O) were purchased from the Usolf chemical technology company . Polydimethylsiloxane (PDMS) and its curing agent were supplied by Dow Corning Company .Wood fibers were provided by Zhejiang Great World Group . Chitosan was supplied by Macklin Biochemical Co., Ltd. . Glutaraldehyde (25 wt %) and absolute ethanol were provided by Sinopharm Chemical Reagent Co., Ltd. . 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane (FDTS) was purchased from Aladdin . Mg/Al layered double hydroxides (Mgw/v) was obtained by dissolving chitosan in an acetic acid solution of 1.5% (w/v) at room temperature for 2 h under stirring. Secondly, 20 wt %  glutaraldehyde was dropwise injected into the chitosan mixture with continuous agitation until the formation of chitosan hydrogel. After that, the wood fibers were mixed with chitosan hydrogel following a mass ratio of 5.0:100 (chitosan to wood fibers). Finally, the blended fibers were hot pressed at 180 °C, 4.5 MPa to form a board with a size of 200 mm × 200 mm × 8 mm. The target density of fiberboard was 0.83 g/cm3.Medium density fiberboards were prepared according to our previous studies . In a tyv/v) and dispersed by ultrasonic treatment for 30 min. After that, the above suspension was stirred for 24 h at room temperature. Then, the resultant suspension was centrifuged and washed several times with absolute ethanol. Finally, the FDTS-Mg/Al LDHs were obtained after dried at 60 °C.Firstly, 1.0 g of Mg/Al LDHs was added into 20 mL absolute ethanol containing 1% FDTS . The chemical group changes of Mg/Al LDH before and after modification were recorded by Fourier transform infrared spectroscopy  using the KBr pellet method. The crystalline structures of the pristine and modified Mg/Al LDH particles were observed by X-ray diffraction  with Cu Kα radiation at 40 kV and 30 mA, λ = 0.1540 nm. Surface morphology of MDF before and after coating treatment were observed using scanning electron microscopy . Prior to the observation, thin gold films were sputtered on all samples by using a low-conductivity sputtering coater. Surface chemical composition mapping images of MDF coated with PDMS@ FDTS-Mg/Al LDH were obtained by energy dispersive X-ray spectrometry (EDS). The water contact angles (WCAs) of MDF before and after coating treatment were measured using an OCA100 contact angle test system  at room temperature. A 4 μL water drop was used to measure the WCA and the average values of WCA were obtained by five measurements at different positions. Limiting oxygen index (LOI) values of uncoated and coated MDF with a size of 150 mm × 10 mm × 8 mm were tested using a JF-5 oxygen index instrument. Combustion parameters of MDFs (100 mm × 100 mm × 8 mm) before and after coating were tested via a cone calorimeter  using 50 kW m2– and –CF3 [XPS spectra was used to evaluate the surface chemical compositions of Mg/Al LDH before and after modification. and –CF3 . These i−1 ascribed to –OH stretching vibrations of bare Mg/Al LDH. After modified by FDTS, it can be clearly observed that the broader absorption peaks appeared at 3403 cm−1 in the spectra of modified Mg/Al LDH, indicating that the hydrogen bond interaction occurred between FDTS and Mg/Al LDH. When FDTS was added to ethanol, a hydrolysis reaction of FDTS occurred. Si–OH groups can be obtained due to the hydrolysis of the three Si–Cl groups of FDTS [−1 attributed to ν3 vibration of CO32− from the interlayer anion [−1, attributed to Mg–O and Al–O vibrations [−1 belonging to CF2 asymmetric stretching, FH2 symmetric stretching, and CF2 symmetric stretching in the spectra of modified Mg/Al LDH [In order to further investigate the modifications of Mg/Al LDH, FTIR spectra was carried out on Mg/Al LDH and FDTS modified Mg/Al LDH A. As sho of FDTS . Thus, ier anion . For botbrations ,36. Furtg/Al LDH ,38,39, w4Al2(OH)12CO3·3H2O) [XRD measurements were used to investigate the crystalline structure changes of Mg/Al LDH before and after modification. O3·3H2O) ,40,41. AThe superhyrophobicity is commonly considered to be due to the synergistic effects of micro- and nano- hierarchical structures and low surface energy. Therefore, it is necessary to investigate the surface morphologies changes of MDFs before and after coating treatment. In order to evaluate the wettability changes before and after coating treatment, the contact angle tests were carried out. As shown in Furthermore, dust or particle contamination on the PDMS@FDTS-Mg/Al LDH coating cannot be avoided in daily life. Hence, it was also important to investigate the self-cleaning properties of PDMS@FDTS-Mg/Al LDH coating. Fly ashes as contamination source were deliberately positioned on the coating a. The wa2 (MDF) to 224.9 kW/m2 (MDF coated with PDMS@FDTS-Mg/Al LDH). Moreover, the fire growth rate (FIGRA) decreased from 1.3 kW/m2 s (MDF) to 0.8 kW/m2 s (MDFs coated with PDMS@FDTS-Mg/Al LDH). As shown in As the most common parameters for investigating the difficulty of combustion, the LOI was used to evaluate the flame retardancy of MDFs coated with PDMS@FDTS-Mg/Al LDH. As shown in In order to investigate the combustion process of MDF in practical applications, the MDFs and MDFs coated with PDMS@FDTS-Mg/Al LDH (100 mm × 15 mm × 8 mm) were burned above an alcohol burner . As showIn summary, we have successfully fabricated superhydrophobic coatings on MDF with self-cleaning functionalities and flame retardancy. The PDMS@FDTS-Mg/Al LDH coating exhibited superhydrophobicity with a water contact angle of 155° and good household liquid repellency. The self-cleaning test showed that decontamination on the coating can be carried out by flowing water droplets, and muddy water can roll off readily. The LOI value increased from 24.0 (MDFs) to 38.5 (MDFs coated with PDMS@FDTS-Mg/Al LDH). The peak heat release rate and total heat release of MDFs coated with PDMS@FDTS-Mg/Al LDH reduced by 24.7% and 11.2% as compared to MDFs alone. The presented inorganic coating provided a new strategy for the protection and functionalization of MDFs.


The primary aim of this trial was to determine the recommended phase II dose (RP2D) of weekly paclitaxel (wP) administered in combination with oral metronomic cyclophosphamide (OMC).2) followed by an expansion cohort at RP2D. Dose-limiting toxicity (DLT) was defined over the first 28-day cycle as grade ≥ 3 non-hematological or grade 4 hematological toxicity (NCI-CTCAE v4.0) or any toxicity leading to a dose reduction.Patients ≥ 18 years of age with refractory metastatic cancers were eligible if no standard curative measures existed. Paclitaxel was administered IV weekly  in combination with a fixed dose of OMC (50 mg twice a day). A 3 + 3 design was used for dose escalation of wP , the maximal grade of drug-related adverse event was Gr1 in three patients, Gr2 in six patients, Gr3 in one patient and Gr4 in one patient (no AE in three patients). At RP2D, a partial response was observed in one patient with lung adenocarcinoma.In total, 28 pts.  were included, and 16/18 pts. enrolled in the dose-escalation phase were evaluable for DLT. DLT occurred in 0/3, 1/6 (neuropathy), 0/3 and 2/4 pts.  at doses of 40, 60, 70 and 75 mg/mThe combination of OMC and wP resulted in an acceptable safety profile, warranting further clinical evaluation.NCT01374620; date of registration: 16 June 2011.TRN: The online version of this article (10.1186/s12885-018-4678-x) contains supplementary material, which is available to authorized users. Metronomic chemotherapy refers to the frequent, typically daily, administration of cytotoxic drugs at doses that are significantly lower than the maximum-tolerated dose, with no prolonged drug-free breaks. Oral cyclophosphamide-based metronomic chemotherapy (OMC) is the most largely studied metronomic regimen, with greater than 30 retrospective studies and phase II trials reporting in vivo anti-angiogenic and immune-modulatory properties and significant clinical anti-tumor activity, which has been confirmed in heavily treated patients who have exhausted all effective treatments –3.The mode of action of paclitaxel involves the stabilization of microtubules through the inhibition of the depolymerization process , 5. ThisCompared with 3-week cycles, weekly administration of paclitaxel induces a clear increase in dose-intensity without significant enhancement of toxicity for fragile or heavily pretreated patients with ovarian , 9, lungWe hypothesize that metronomic cyclophosphamide and weekly paclitaxel combination is feasible combination. In this context, we performed a multi-center dose-finding phase I trial to determine the recommended phase II dose of weekly paclitaxel administered in combination with metronomic cyclophosphamide and to evaluate the safety and preliminary signs of activity of this combination.This was a 3 + 3 dose-escalation single-center study. The primary objective was to determine the recommended phase II dose of weekly paclitaxel administered in combination with a fixed dose of OMC.3, platelets ≥100,000/mm3, hemoglobin ≥9 g/L, albuminemia ≥36 g/L, lymphocytes count ≥700/mm3, bilirubin and AST/ALT ≤3 ULN or ≤ 5 ULN in case of liver metastasis, and creatinine clearance ≤60 mL/min), negative pregnancy test within 7 days, use of effective contraceptive measures, and absence of any psychological, familial, sociological or geographical condition potentially hampering compliance with the study protocol and follow-up schedule and before registration. Written informed consent must be provided according to ICG/GCP and national regulations. Exclusion criteria were as follows patients undergoing simultaneous therapy with other anticancer agents, prior treatment with paclitaxel, brain or leptomeningeal metastasis, patients not able to swallow and absorb the oral investigational agent, prior symptomatic neuropathy, uncontrolled infection and contraindication to metronomic cyclophosphamide .The main inclusion criteria were histology-proven malignancy, patients having exhausted all available standard of care, documented disease progression at study entry, target measurable according to RECIST 1.1, wash-out period of 28 days after the prior treatment, no persistent toxicity related to prior therapies, age between 18 and 65 years, WHO performance status ≤2 within 7 days prior to the study entry, correct biological parameters  were prescribed as clinically indicated by the treating physician. Oral metronomic cyclophosphamide was administered continuously at 50 mg twice a day. Paclitaxel was administered if all the following criteria were met: performance status ≤2, hemoglobin ≥9 g/L, granulocytes ≥1500/mm3, platelets ≥100,000/mm3, AST/ALT and bilirubin < 3 ULN and absence of dose-limiting toxicities (DLT).Eligible patients received weekly paclitaxel. Seven dose-levels were planned: 40 mg/mDLTs were weekly assessed during the first 28 days of treatment and included the following toxic events (NCI-CTCAE v4.0): prolonged (> 7 days) grade 4 neutropenia, febrile neutropenia with fever ≥38.5 °C, grade 4 thrombocytopenia, hemorrhage related to thrombocytopenia, hematological toxicity not allowing paclitaxel administration on Days 8 or 15, grade 3 or 4 non-hematological toxicity and oral metronomic interruption for at least 4 days.We planned an expansion cohort of 10 additional patients at the dose identified as the recommended phase II dose to better explore the tolerability and the activity of this combination.Other objectives were to describe the nature and severity of adverse events (NCI-CTCAE v4.0), assess the response after 2 cycles according to RECIST 1.1, estimate the progression-free and overall survival from the date of inclusion, and estimate the growth modulation index . We have described distribution of adverse events in the 1st cycle of treatment as well as the distribution of adverse events observed during the overall treatment.All estimates were provided with their 95% confidence intervals (95%CI). Progression-free and overall survival curves were estimated using the Kaplan-Meier method. Analyses were performed using Stata/SE (version 13.1) statistical software .ClinicalTrial.gov Register (NCT01374620). Written informed consent was obtained from each patient.This study was approved by the regional Ethics Committee  and the French Health Products Safety Agency . This study was registered in the n = 9, 32%), soft tissue sarcomas , head and neck carcinoma , other digestive carcinomas, liver cancer, lung cancer, , renal cell carcinoma, cervical cancer, bone sarcoma, testis cancer, ocular melanoma and unknown primary site . Twenty-seven patients (96%) had metastatic disease, mainly involving the lung , liver  or lymph nodes . At study entry, the performance status was PS = 0 in 19 patients (68%), PS = 1 in 8 patients (29%) and PS = 2 in 1 patient (4%). Previous treatments included surgery in 24 cases (86%), radiotherapy in 15 cases (54%) and previous systemic chemotherapy or targeted treatment in 27 cases (96%). The number of prior systemic treatment lines was 0 in 1 case (4%), one in 3 cases (11%), two in 2 cases (7%), and 3 or more in 22 cases (78%). Only one patient previously received cyclophosphamide (cyclophosphamide-vinorelbine for a para-testicular rhabdomyosarcoma), and no patient received prior paclitaxel.Twenty-eight patients were included between June 2011 and February 2013: 19 men (68%) and 9 women (32%). The median age was 54.5 years . The primary lesions were colorectal adenocarcinomas (2 of weekly paclitaxel), seven patients at dose-level 2 (60 mg/m2), 14 patients  at dose-level 3 (70 mg/m2) and four patients at dose-level 4 (75 mg/m2). All patients received at least one dose of paclitaxel.Three patients were enrolled at dose-level 1 (40 mg/m2).No DLTs were observed among the three patients enrolled at dose-level 1 (40 mg/m2), one was not assessable for DLT because he received the wrong dose (40 mg/m2); he was subsequently replaced by a fourth patient. This patient experienced DLT (Grade 3 neuropathy). Three additional patients were thus enrolled at the same dose-level; none of them experienced DLT.Among the three first patients enrolled at dose-level 2 (60 mg/m2). One of them was not assessable for DLT because he received only two injections of paclitaxel due to rapid disease progression with intestinal occlusion leading to death. A fourth patient was then enrolled. None of these patients experienced DLT.Three patients were enrolled at dose-level 3 (70 mg/m2). One of them experienced DLT: febrile neutropenia. Furthermore, this patient affected by cholangiocarcinoma died from disease progression immediately after the occurrence of DLT. A fourth patient was then enrolled; this patient also experienced a DLT .Three patients were enrolled at dose-level 4 (75 mg/m2).Consequently, the dose escalation was stopped, and the recommended phase II dose was defined as dose-level 3 . Considering the 13 patients treated at the recommended phase II dose and evaluable for DLT assessment, the probability of DLT is estimated at 8% (95%CI: 0.2 to 36%).n = 28), over the first cycle, the maximum grade of drug-related AE was Grade 1 in six patients, Grade 2 in 13 patients, Grade 3 in two patients and Grade 4 in 2 patients (no AE in 5 patients). At the recommended phase II dose (n = 14), the maximum grade of treatment-related AE was Grade 1 in three patients, Grade 2 in 6 patients, Grade 3 in 1 patient and Grade 4 in 1 patient (no AE in three patients).Figure Table The most frequent adverse events were hematological toxicities ; however, febrile neutropenia occurred in only two patients. Peripheral sensory/ motor neuropathy was reported in 12 patients (44%) during first cycle .Over the 1st cycle, the relative dose-intensity was > 75% for both drugs in 23/28 patients (82%). Two patients (7%) required transient treatment interruption classified as DLT. Treatment was definitively stopped for 2 other patients , and another patient received a reduced dose by error. Five patients definitively stopped the study treatment (at least one of the drugs) after 1 cycle, and 15 stopped after 2 cycles, whereas 8 patients received more than 2 cycles of the combination . The reasons for stopping the treatment were toxicity for 4 patients, progression for 21, patient’s choice for 1, physician’s decision for 1, and unknown for 1 patient.We have observed Grade 3 lymphopenia in 12 patients. The median duration of this grade 3 lymphopenia was 2,6 months . We have observed three infectious episodes in three patients: urinary tract infection, skin infection and febrile neutropenia.Table 2 when administered in combination with 50 mg OMC twice a day, (ii) DLTs were mainly hematological, (iii) this combination appeared well tolerated, and (iv) objective responses were noted in patients with heavily pretreated lung adenocarcinoma.The key-findings of this dose-finding phase I trial are (i) the recommended phase II dose of weekly paclitaxel is 70/mg/m2.The tolerance of the combination was mostly manageable without unexpected toxicity. The observed toxicity was as expected in terms of the nature and severity of these events. In this study, the addition of metronomic cyclophosphamide did not allow a dose escalation of weekly paclitaxel beyond 75 mg/m2 [2 [2 [The activity and safety of weekly paclitaxel as a single agent have been assessed in several phase II trials , 16–27. 2 , 21, 22,2 [2  or 100 m2 [2 [2  are rare2 [2 [2 , 22 to 32 [2 [2 . The med2 [2 [2 . The rep2 [2 [2 , 21, 22 2 [2 [2 , 20, 22.2 [2 [2 , 13, 24.The study had some limitations. The dose of metronomic cyclophosphamide (50 mg twice a day) could be discussed since some prior trials are based on 50–100 mg once a day. Five patients aged between 66 and 67 had been enrolled (inclusion criteria was up to 65), however regarding their very good shape, the study coordinator had provided waiver. We did not conduct any translational study to evaluate biomarkers associated with tumor response. At the time of this study, analysis of ALK, ROS and MET mutations were not part of the standard of care in lung adenocarcinoma. We do not know whether the two responding patients were affected by mutated lung adenocarcinoma. Furthermore, we did not enroll patients with ovarian cancer or bladder cancer (these patients have in most cases received weekly paclitaxel before to be considered for study entry).2 days 1, 8 and 15 of 28-day cycles. However, in the absence of randomization and an internal comparator, we cannot establish the therapeutic role of the addition of metronomic cyclophosphamide compared with weekly paclitaxel alone.To conclude, as previously reported , 16–27, Additional file 1Table S1. Adverse events (treatment related or not) reported over the entire treatment duration . Table S2. Characteristics and outcome of patient with lung cancer. (DOCX 31 kb)


H exon. CSR depends on non-coding (CSRnc) transcription of donor Iμ and acceptor IH exons, located 5′ upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iμ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called “immune privileged” tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response  in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJ Affinity maturation and class switch recombination (CSR) are tightly regulated, molecular processes that occur in a specialized microenvironment within secondary lymphoid organs known as the germinal center (GC). Upon T-dependent antigen stimulation, IgM+ naive B cells re-localize into the B cell follicle, undergo clonal expansion within the GC. The GC reaction is an iterative process of mutation-selection that leads to the generation of antigen-specific high affinity, class-switched memory B cells and long-lived antibody secreting plasma cells  (n = 18). Written informed consent was obtained from each volunteer in each blood sample draw. All procedures in human subjects were performed after Institutional Review Board approval from the National Institute of Public Health (CI: 971/82-6684). Plasma and PBMCs were isolated according to the manufacturer's instructions, aliquoted and stored at −80°C and liquid N2, respectively. Total RNA was extracted from PBMCs with TRIzol and stored at −80°C until used.Pre-immune (day 0), day 7, 15, 30, and 180 post-vaccination peripheral blood samples (18 mL) were obtained by venipuncture in 2 × 8 mL Vacutainer® CPT™ tubes from healthy volunteers vaccinated with Hepatitis B and/or Tetanus toxoid/Diphteria .Cryopreserved PBMCs were thawed at 37°C and resuspended in RPMI 10% FBS, washed with PBS 1X and fixed with 1% paraformaldehyde for 20 min at room temperature. After washed with FACS solution , cells were incubated for 30 min at 4°C with the following antibody cocktail: anti-CD3 PerCP/Cy5.5 , anti-CD19 FITC , anti-CD20 PE Cy7 , anti-CD27 APC , and anti-CD38 PE . Flow cytometry analysis was performed in a FACS Aria II . Doublets and CD3+ events were gated out. Plasmablasts were defined as CD36 B cells were seeded per well on 6-well plate incubated in RPMI medium supplemented with 10% FBS, streptomycin and penicillin at 37°C with 5% CO2. Two activation conditions were stablished at different time points , 1 μg/ml anti-human CD40 (G28.5), 5 μg/ml CpG ODN 2006 (Invivogen) and 25 ng/ml recombinant IL-21 (eBiosciences). For T-independent activation, 5 μg/ml CpG ODN 2006 (Invivogen), 0.05% S. aureus Cowan , 5 ng/ml Pokeweed Mitogen (Sigma).PBMCs were isolated by Ficoll-Paque™ density gradient from blood bank buffy coats. B cells were enriched through negative selection using B cell Isolation Kit II . 1 × 10IGHM, IGHG1, IGHG3, and AID gene was performed using specific primers and TaqMan probes(IDT). The primers and probes used to quantify the CSRnc transcripts are detailed in Table HPRT with PrimeTime® Predesigned qPCR Assays was performed as the reference gene. The fold difference was calculated using 2ΔΔCT, using resting enriched B cells as calibrator and non-B cells as negative control. HPRT was used as normalizer for every condition.Total RNA from PBMCs was extracted using TRIzol (Invitrogen). The integrity of the RNA was measured with Agilent RNA 6000 Nano. SuperScript™ III One-Step RT-PCR(Invitrogen) was used for reverse transcription and amplification. Quantitative PCR of CSRnc transcripts for H are upstream of the corresponding switch region (SH), thus we first mapped S regions based on the frequency distribution of the AGCT motif in 500 bp bins along the whole IGH locus   that can be programmatically accessed through the R programming language  are not mapped as such in the current version of the human genome GRCh38). I8. IH arelanguage . To map s Viewer . CSRnc rrecount2 was used to extract read counts from each of the nine CH constant region coding genes , as well as, from the corresponding CSRnc I exon coordinates as a GRanges object (2-transformed CH (coding) and CSRnc RPKM average per sample was used as an approximation of abundance of transcription. For CSRnc transcription, log2 RPKM per sample average adopted a quasi-normal distribution with a mean of 2.65 log2 RPKM (SD ± 3.79), which corresponds to 6.29 RPKM. As an initial exploration to which tissues and in which diseases CSRnc transcription takes place, we used the log2 RPKM average as a cut-off to define “high” expression (>2.65 log2 RPKM) or “low” expression (< 2.65 log2 RPKM). The mean CH transcription average log2 RPKM was 7.82 (SD ± 5.16). Given the difference between coding and CSRnc transcription, and to address the relative expression between coding and CSRnc transcription for each Ig loci, coding and CSRnc log2RPKM values for each Ig gene were standardized by transformation to Z-scores.s object . The logAlthough all RNA-seq samples in TCGA and GTEx follow a homogeneous ontology categorization, metadata associated to SRA projects is widely heterogeneous and commonly insufficient. To obtain a more homogenous categorization of nearly half of our dataset, we used disease  annotati0 is that there is no difference in the probability distribution between Z clusters and tissue. A 2 × 2 contingency table was built for each tissue between the number of samples belonging to a given Z cluster and the remaining samples not belonging to that cluster. A two-sided Fisher's test was performed with the R function fisher test . P-value adjustment with the Benjamini-Hochberg method was performed to correct for multiple testing using the R function p. adjust . A False Discovery Rate (FDR) < 0.01 was considered as a significant enrichment.To define CSRnc transcription profile variation in healthy tissue (GTEx dataset), we performed tissue sample enrichment analysis according to Z-cluster using a two sided Fisher's Exact Test. The Hk-means clustering using the Cluster software 3.0 (https://sourceforge.net/projects/jtreeview/).To define CSRnc transcription profiles, Z-scores for each I-exon were subjected to ware 3.0 . Ten clulimma R package v3.34  and was included for further analysis  that were selected for downstream analysis  at ENSEMBL  region within the s Figure . This remyelomas , 24. Fors Figure . Iγ2 ove ENSEMBL . The genprojects , 36, indprojects . Overallprojects ; howeverin vitro for 3 and 6 days. We obtained whole blood total RNA and quantified Iμ, Iγ3, and Iγ1 CSRnc and AID transcripts by qRT-PCR . Transcription of AID showed the same pattern as CSRnc transcripts  or at day 14 , however at day 14 was higher than at day 7 (p = 0.01)  nor at day 14 (mean 1.02 ± 0.9. p = 0.57)  and increased at day 14 . As expected, Iγ3 transcription at day 14 was higher than at day 7 (p = 0.0002)  Figure . Iγ1 tra) Figure . Contras) Figure . No chan) Figure .p = 0.011), and returned to pre-vaccine levels 14 days post-vaccination, as previously described . However, plasmablast and Iμ transcription correlated at day 7 . Interestingly, plasmablast fold-change at day 7 negatively correlated with Iγ3 transcription . No changes in Iμ, Iγ1, and Iγ3 transcription was detected in response to influenza vaccination  in any of the 10 IH exons in a substantial fraction of the whole dataset  (Table H log2-transformed RPKM average (2.65) for each non-zero RPKM sample  was used to define an expression cutoff. “High” CSRnc transcription was defined as a mean log2 RPKM of 2.65 or higher. Only 29.8% of RNA-seq samples  were above this expression cutoff. “Low” CSRnc transcription was defined as a mean log2 RPKM < 2.65 . The CSRnc transcription levels varied according to IH. Higher transcription (log2 RPKM), as well as, a more widespread transcription (proportion of samples) was found for Iμ in all datasets  obtained with 5 Figure . Table 3s Figure , supportH relative transcription profile. To do so, the entire non-zero dataset including GTEx, TCGA, and SRA Z-scores was clustered in 10 groups using k-means clustering . Clusters Z0, Z2, and Z8 were characterized by low (mean log2 RPKM < 2.65 or Z score < 0) CSRnc transcription in all IH classes. Clusters Z4, Z6, and Z7 were characterized by “high” expression of Iμ only (mean log2RPKM > 2.65). Cluster Z3 showed “high” expression in Iμ and Iα2. Cluster Z1 showed high expression in Iμ, Iγ1, and Iγ4. Finally, clusters Z5 and Z9 showed “high” expression in all IH's  such as terminal ileum, transverse colon, stomach, lung, and esophageal mucosa  and sigmoid colon (mean 2.1 ± 3.2). Interestingly, salivary gland expression was among the tissues with highest transcription, and non-mucosal tissues such as thyroid and pituitary gland showed high average IH transcription. Another notable difference in average log2 RPKM was between visceral adipose tissue  and subcutaneous adipose tissue (mean 1.9 ± 1.5). Conversely, samples of tissues such as brain, skeletal and cardiac muscle, skin, as well as, chronic myelogenous leukemia cell line K562 and transformed dermal fibroblast cell lines showed the lowest CSRnc transcription levels (mean log2 RPKM < 2.65)  Figure .H by tissue type. The transcription pattern of each IH in whole blood was significantly different (p < 2.2e−16)   Figure . Iμ tran Figures , in whicα1.1 and Iα1.2 transcription however, some differences were noted. Iα1.1 transcription was higher in terminal ileum and transverse colon than in blood (p < 0.001). In contrast, Iα1.2 transcription was higher in spleen and salivary gland than in blood (p < 2e−16). Iα1 transcription was similar to Iα1.2 transcription, but also was higher in stomach, esophageal mucosa than in blood (p < 1.1e−05)  Figure . Thus, Iγ4 and Iε. Transcription for both was higher in spleen, terminal ileum and EBV-transformed B-cells than in blood (p < 0.01). However, Iγ4 transcription was higher in thyroid, visceral adipose tissue (omentum), testis, than in blood (p < 2e−16). Similarly, Iε transcription was higher in in thyroid than in blood (p < 0.00003)  Figure .H relative expression (Z-score) revealed that terminal ileum, spleen, transverse colon, whole blood and salivary gland share a similar expression pattern and are highly enriched (FDR < 0.001) in clusters Z5 and Z9   (FDR < 0.001), whereas others such as breast, vagina, liver were enriched in cluster Z3 and Z6 (Iμ only). Testis, thyroid, pituitary, and omentum, characterized by the highest Iγ4 transcription, were enriched in cluster Z1  (FDR < 0.001). Finally, the remaining tissues such as subcutaneous adipose tissue, arteries, brain, skeletal, and cardiac muscle, skin, transformed fibroblasts and K562 cell lines, were enriched in Z0, Z2, and Z8  (FDR < 0.001)  (FDR < 0.001).We tested if a particular CSRnc expression profile was associated to a particular tissue. Consistently with the regression analysis, de-convolution of CSRnc transcription according to I) Figure . Some ti) Figure , and in H coding transcript log2RPKM as a proxy of the amount of B cells in the tissue. In general, CH transcription was 10–100-fold higher than CSRnc transcription. To correlate CSRnc transcription with corresponding CH transcription, Z-scores were used. As expected for each class, CSRnc and CH Z-scores where significantly correlated (p < 1.0e−16), suggesting that the higher numbers of B cells or plasma cells in a given tissue, higher CSRnc transcription. However, we have noticed that for most classes, a fraction of samples deviates from the expected orthogonal correlation, indicating higher CSRnc transcription relative to the CH transcript. This is particularly notable for Iμ, but also, Iγ1, Iγ4, and Iα2 , lymphoid-primed multipotent progenitors , common lymphoid progenitors , thymic CD34+CD7− CD1a− CD4−CD8− (Thy1) precursors and fully B cell-committed progenitors  expressed Iμ  in kidney, brain and muscle.We analyzed CSRnc expression in fetal tissue by using project SRP055513 data, which reported an extensive RNA-seq analysis in twenty fetal tissues during gestational weeks 9–22 . Remarkak Figure . Higher H average log2 RPKM distribution) across 33 cancer types  and acute myeloid leukemia (AML) were the highest. In general, CSRnc transcription in neoplastic tissue mimicked its non-neoplastic counterparts, being high in lung, stomach, and testicular germ cell carcinomas. Conversely, CSRnc transcription was low in glial cell and skin cancers  Tumors where average CSRnc transcription is lower than in healthy tissue, such as in DLBCL, prostate, thyroid, liver, and colon cancer  Tumors where average CSRnc transcription was higher than its healthy tissue counterpart, such as breast, rectum, testicular germ cell, pancreas carcinomas, ovarian cystadenoma, and skin melanoma  Tumors with no difference in CSRnc transcription between healthy and neoplastic counterpart, such as stomach and esophagus  and neoplastic tissue allowed the identification of three distinct patterns Figure : (1) Tum Figures . (2) Tuma Figure . (3) Tums Figure . Interess Figure . In adres Figure .H transcription   Table S. However66  Tablen Figure .recount2  located in the IGHM-IGHD intergenic region was identified, which overlaps with a previously described repeat Σμ region, downstream of an atypical switch region (σδ) involved in non-classical CSR to IgD in mice (+ EBV-transformed cell lines and human tonsillar B cells undergo μ-δ CSR by non-homologous recombination using σδ as acceptor switch region (δ (Σμ) can be actively transcribed , suggesting that CSRnc transcription may not be the result of plasmablast mobilization to peripheral blood. Moreover, we observed increased Iγ1 and Iα1 transcription in natural H7N9 infection, but not upon influenza vaccination , and its transcription is required for CSR regardless of the acceptor class , but not subcutaneous adipose tissue. Visceral adipose lymphoid B cells are immunologically active cells implicated in adipose tissue homeostasis that may play important pro-inflammatory roles associated with metabolic syndrome and obesity , testis (Z1 and Z2), terminal ileum (Z5 and Z9), and whole blood  indicate CSRnc transcriptional heterogeneity in tissue donors involved in the GTEx project. This could result from different tissue microenvironments , as well as, genetics, which may modify CSRnc transcription patterns and possibly CSR itself. A recent study identified four SNP's in the human IgH locus presumably involved in CSR that affect immunoglobulin levels  The anti-tumor response is largely mediated by the presentation of tumor neo-antigens to T cells and Treg balance. However, antibody-mediated anti-tumor activity can be achieved by antibody—dependent cytotoxicity or other mechanisms (2) The presence of ectopic (or tertiary) lymphoid structures (ELS) (3) The tumor microenvironment, including certain cytokines may modify CSR patterns in infiltrating and ELS B cells, regardless the antibody effector function. (4) AID activity is a known contributor to off-target mutagenesis and genomic instability in B cell malignancies  Thees (ELS) , 62 and es (ELS) , breast es (ELS) , and coles (ELS) . (3) Thegnancies . Aberrangnancies .H expression in certain tumors analyzed in the TCGA project resembles their non-neoplastic counterpart; however, some cancer types have significantly less CSRnc transcription, whereas others show the opposite. Most of the evidence we have gathered so far indicate that the origin of IH transcription is in the tumor infiltrating and ELS B cells, rather than the tumor cells per se .This study was conceived and designed by HK-M and JM-B. The experimental procedures were performed by H-KM, MO-M, HV-T, and JT-S. Flow cytometry acquisition and analysis was conducted by HK-M and LCB. RNA-seq data bioinformatics and statistical analysis were done by HK-M, LC-T, and JM-B, and supervised by AJ. HK-M and JM-B drafted the manuscript. LCB, LC-T, and AJ critically reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake. Tricholoma matsutake is an ectomycorrhizal agaricomycete predominantly associated with pines and oaks. It is a commercially valuable edible mushroom [mushroom  with gremushroom , which imushroom .T. matsutake consumers has steadily expanded market demands for this mushroom. On account of the continued deterioration of ecological systems and the environment and excessive picking, the natural productivity of this gourmet mushroom has gradually declined. To date, no artificial cultivation method has yet been developed for T. matsutake due to the lack of information concerning precise soil requirements and cues for sporophore formation [T. matsutake and its products are in a short supply in the market, and, as such, its economic value has risen sharply. To address demands for the mushroom and reap higher profits, merchants frequently pass off counterfeit or adulterated T. matsutake products as genuine items, for example, sliced dried Agaricus blazei [T. matsutake is an urgent necessity.The growing number of ormation . Therefos blazei . EstabliT. matsutake are generally divided into two categories, namely, morphological [T. matsutake, and the detection result is easily influenced by variety, habitat, and processing method [Traditional methods of identifying ological  and physological , and eacological . Morpholg method ,10. ThusRapid developments in modern biotechnology have enabled the wide use of molecular biological methods for authentication due to their accuracy, convenience, and speed. PCR-based methods, in particular, have been broadly adopted by many laboratories and fields ,12. CompvicK for Staphylococcus aureus [L21 [CruA [PEP [HMG-I/Y [FatA [BnAccg8 [lectin [β-actin [hsp [Cotton-ppi-PPF [ACP1 [Sad1 [SAH7 [hmga [10kDa zein [Ivr1 [zSSIIb [Adhl [gos9 [RBE4 [SPS [T. matsutake have yet to be established or reported.Identification of species-specific diagnostic marker, which requires species specificity, a consistent and low copy number, and low heterogeneity in the same species ,14, has s aureus , Ribosomeus [L21  for gene21 [CruA , PEP [18[HMG-I/Y , FatA [2/Y [FatA , and BnA [lectin ,22, β-ac[β-actin , and hsptin [hsp  for soyb-ppi-PPF , ACP1 [2PF [ACP1 , Sad1 [2P1 [Sad1 , and SAHd1 [SAH7  for cottH7 [hmga ,29,30, 1kDa zein ,32,33, Iin [Ivr1 ,30,34,35 [zSSIIb ,37, and hl [gos9 , RBE4 [3s9 [RBE4 , and SPSBE4 [SPS  for riceBE4 [SPS . HoweverT. matsutake fruiting bodies is crucial for downstream molecular experiments. At present, the cetyltrimethyl ammonium bromide (CTAB) method, which is especially suitable for plant DNA extraction, is commonly used to extract mushroom DNA [T. matsutake fruiting bodies is difficult to acquire using this method because the mushroom contains thick cytoderms or capsules [T. matsutake fruiting bodies is an important endeavor.Extracting high-quality DNA from room DNA . Howevercapsules  and is rPol gene, which encodes RNase H and integrase, was selected as a candidate species-specific diagnostic DNA marker for T. matsutake through sequence alignment and BLAST. The gene was verified to be a valid species-specific diagnostic marker given its features of appearance as a single copy in genomic DNA, good species specificity, and real-time quantitative detection limit as low as 32 pg. A suitable method for extracting the DNA of T. matsutake, i.e., high-concentration salt precipitation method, was also determined. The benefits of the present study are multifold: it provides a convenient and accurate approach for detecting T. matsutake and its related processed products, it aids efforts to improve food safety and detect food adulteration, and it develops an effective technique for detecting adulterations in the wild-mushroom market.In this work, the T. matsutake fruiting bodies from Yunnan was obtained using the CTAB, SDS-CTAB, high-concentration salt precipitation, and kit methods. The results of 1% agarose gel for genomic DNA  showed no homology with other genes from different varieties of the mushroom.Among the genes selected, the Pol gene was a retroelement from T. matsutake, which related to RNase H and integrase of retrotransposons [T. matsutake worldwide, this finding suggested that retroelements associate with ectomycorrhizal basidiomycetes and might be useful as genetic markers for identification, phylogenetic analysis, and mutagenesis of this fungal group [Pol gene, it just confirmed the statement above. Hence, we chose the Pol gene as the candidate species-specific diagnostic marker of T. matsutake.It has been reported that retrotransposons have been incorporated into the genome of their hosts and inherited to the host progenies since the earliest establishment of their parasitism. In a previous study, this nsposons . The reval group . After tT. matsutake species  and three T. matsutake strains  was subjected to 18s rDNA amplification  with a slope of −3.081 was determined between the DNA quantities and Ct values , the quality of genomic DNA was further analyzed on 1% agarose gel with ethidium bromide (0.1 μg /mL), and run with 1 × TAE buffer. Finally, the DNA solutions were stored at −20 °C.EcoR I (15 U/μL) , 3 μg genomic DNA, and added ultrapure water to 20 μL, and put the tube in a water bath (37 °C) for 2 h, after that added 2 μL 10× loading Buffer  to stop the reaction. The digestion products was further analyzed on 1% agarose gel with ethidium bromide (0.1 μg /mL), and run with 1× TAE buffer.The DNA digestion was performed in a 20 μL reaction system containing 2 μL of 10× H Buffer, 1 μL T. matsutake fruiting bodies obtained from Yunnan and Jilin was performed using Hind III and EcoR I, respectively, according to the manufacturer’s instructions (TaKaRa) to ascertain the copy number of the Pol gene in the same species. The cleaved DNA was separated by 0.8% garose gel in 1×TAE at a constant voltage of 20 V overnight. Thereafter, the DNA fragments were transferred to a nylon membrane  from the 0.8% agarose gel. A 775 bp DNA fragment of the Pol gene was amplified with Southern blot-F/R and used as the hybridized probe, which was labeled with DIG-dUTP . Pre-hybridization was carried out at 42 °C for 2 h, after which the pre-hybridization solution was poured off. The probe was denatured into single-strand DNA for 10 min at 100 °C and then cooled for 5 min. Exactly 4 µL of the denatured probe and 8 mL of the hybridization solution were added to a hybridization bag, and hybridization was performed at 42 °C overnight. Finally, the nylon membrane was washed twice with 3× SSC/0.5% SDS, and autoradiography was performed for 2–3 days.Complete enzyme cleavage of DNA from T. matsutake were sought in Genbank, and several genes were preliminarily selected as detection targets according to their detailed gene information. Afterword, BLAST analysis was performed to determine the homology of the targets. The gene that with the lowest homology compared with all the DNA sequences in Genbank was selected as the candidate.Genes belonging to All primers and TaqMan fluorescent dye-labeled probes were designed using ABI Prism Primer Express version 3.0 software  and synthesized by Sangon Co. Ltd. .Pol gene was assessed through qualitative PCR with the primer pol-F/R and through quantitative PCR with the primer pol-F/R, as well as the probe pol-P. Southern blot-F/R was used for Southern blot assay. The detailed sequences of these primers and probe are listed in The universal primer 18S rDNA-F/R was used to evaluate DNA quality. The Pol gene amplification, the procedures applied for 18S rDNA amplification were followed, but annealing was optimized at 56 °C, 58 °C, 60 °C, 62 °C, and 64 °C separately. After comparison, the annealing temperature 60 °C was chosen. Qualitative PCR was performed using an ABI SimpliAmp thermal cycler (Applied Biosystems) in a 25 µL reaction system containing 2.5 μL of 10×buffer, 0.2 mM dNTP, 0.4 μM of each primer, 2.5 units of Taq DNA Polymerase (TaKaRa Biotechnology Co. Ltd.), 100 ng DNA template, and 16.3 µL of ultrapure water. The 18S rDNA amplification was carried out to test DNA quality via the following program: 5 min at 95 °C; 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C; and 10 min at 72 °C. During pol-F/R separately by Sangon  company.The PCR amplified products were analyzed on 2% agarose gel with ethidium bromide (0.1 μg/mL), and run with 1× TAE buffer. The PCR products, that conducted the homology analysis, were sequenced using Pol gene was also conducted in an ABI StepOne Plus Real-Time System  using the TaqMan probe methods. TaqMan probe-based qPCR was performed using a 20 µL reaction system containing 1×TaqMan Gene Expression Master Mix , 200 nM primers, 200 nM probe, and 50 ng of DNA. Each sample was quantified twice for each biological replicate.Real-time quantitative PCR of the T. matsutake was diluted seven times from 200 ng/µL to 12.8 pg/μL  over a gradient using the non-T. matsutake DNA, and a series of PCRs with 1 µL DNA sample were conducted to determine the detection limit of qualitative PCR, and the products were analyzed using 2% agarose.Genomic DNA from T. matsutake DNA, and with 1 µL DNA sample, the detection limit of Taqman quantitative PCR was determined.To evaluate the sensitivity of Taqman-based quantitative PCR, genomic DNA was serially diluted six times to final concentrations ranging from 100 ng/μL to 32 pg/μL  using the non-T. matsutake fruiting bodies was obtained following the high-concentration salt precipitation method, and the Pol gene was selected and validated as an ideal species-specific diagnostic marker for the PCR-based detection of T. matsutake sources after assay of its species specificity, copy number, high homology in different varieties, and sensitivity. The detection limit of Taqman-based quantitative PCR analysis was 32 pg, which means this method could be used to detect processed T. matsutake products containing low amounts of the target genomic DNA.In conclusion, this work demonstrated that High-quality DNA from


To evaluate the outcome of patients treated with stereotactic ablative body radiotherapy (SABR) with curative intent for stage I non-small cell lung cancer (NSCLC) with regard to local, regional and distant tumor control, disease-free survival (DFS), overall survival (OS) and toxicity.Data of 300 patients treated with SABR for NSCLC cancer for the period of November 2007 to June 2016 were retrospectively analyzed. Of which, 189 patients had single primary lung lesion and were included in the study. The prescribed dose for the tumor was 48 Gy, given in 12 Gy × 4 fractions for all patients. In 2010, an improved protocol was established in advanced technology for the planning CT, dose calculation and imaging. Cumulative incidence function (CIF) of local, regional, distant or any recurrences were computed using competing risk analysis with death as a competing event. Survivals (DFS and OS) were estimated using the Kaplan-Meier method and Cox proportional regression was used for comparisons. Toxicities were graded according to the common terminology criteria for adverse events version 4.0 (CTCAE v.4).N = 80). At 1, 2 and 4 years, the cumulative incidence function (CIF) of local relapses were 8% [4–13%], 15% [10–21%] and 18% [12–25%], the CIF of regional relapses were 4% [2–8%], 10% [6–16%] and 12% [8–19%], the CIF of distant relapses were 9% [5–14%], 15% [11–22%] and 20% [15–28%] and the CIF of any relapses were 14% [10–20%], 28% [22–36%], 34% [27–43%], respectively. After 1, 2 and 4 years, the OS rates were 83% [95% CI: 78–89%] (N = 128), 65% [95% CI: 57–73%] (N = 78) and 37% [95% CI: 29–47%] (N = 53), respectively. The median survival time was 37 months. The DFS after 1, 2 and 4 years reached 75% [95% CI: 68–81%] (N = 114), 49% [95% CI: 42–58%] (N = 60) and 31% [95% CI: 24–41%] (N = 41), respectively. No grade 4 or 5 toxicity was observed.Diagnosis was histologically confirmed in 42% of the patients (We observed a long-term local control and survival after SABR for peripheral stage I NSCLC in this large series of patients with the expected low toxicity. The standard treatment for stage I non-small cell lung cancer (NSCLC) is surgery, with excellent local control and survival outcome , 2. HoweWe retrospectively evaluated the local, regional and distant tumor control, as well as the disease-free survival (DFS), OS and toxicity in our unselected patient population that was treated over the last 10 years. In recent years, there have been advances in the radiotherapy techniques, delivery for SABR and treatment methods in our department, however, the 12 Gy × 4 fractions prescription has remained unaltered.Data of 300 patients with stage I NSCLC   treated 18F-FDG-PET uptake and morphological abnormalities suggestive of malignancy  (N = 78) and 37% [29–47%] (N = 53), respectively. The median survival time was 37 months. DFS rates were 75% [95% CI: 68–81%] (N = 114), 49% [95% CI: 42–58%] (N = 60) and 31% [95% CI: 24–41%] (N = 41) after 1,2 and 4 years, respectively , 18 with regional relapses (10%), 29 with distant relapses (15%) and 51 with at least one relapse (27%).At 1, 2 and 4 years, the cumulative incidence function (CIF) of local relapses were 8% [4–13%], 15% [10–21%] and 18% [12–25%], the CIF of regional relapses were 4% [2–8%], 10% [6–16%] and 12% [8–19%], the CIF of distant relapses were 9% [5–14%], 15% [11–22%] and 20% [15–28%] and the CIF of any relapses were 14% [10–20%], 28% [22–36%], 34% [27–43%], respectively  and a worse DFS . Treatment period and previous cancer history were not found to be significantly associated with OS nor DFS.Stage T1b (vs T1a) was associated with a worse OS (univariate HR (uHR): 1.49, multivariate HR (mHR): 1.66, 95% confidence interval (CI): [1.06–2.59], p = 0.07).Treatment period was associated with a decrease of incidence of any recurrences (univariate sHR (usHR): 0.42, multivariate sHR (msHR): 0.49 [0.29–0.90], p = 0.02). The association between treatment period and local relapses was slight but inconclusive . Stage T1b was not associated with incidence of local, regional or distant relapses.Previous cancer history was associated with an increase of distant relapses , chest wall pain , dyspnea , radiation pneumonitis , dermatitis , cough , rib fractures , and esophagitis . No grade 4 or 5 toxicity was observed.For all patients, grade 1 to 3 toxicities were: fatigue  of local relapses, regional relapses and distant relapses were 15 and 18%, 10 and 12%, 15 and 20%, respectively. The CIF of any relapses at 2 and 4 years were 28 and 34%, respectively. Our results in terms of clinical outcomes are comparatively similar to recently published data. In a recent review of 72 SABR studies, Chi et al. reported a 5-year OS rate of 41.3% after SABR .Regarding toxicities, our data are also coherent with the recent review of Chi et al. with a low incidence of grade 3 radiation pneumonitis and the overall incidence of rib fractures. The incidence of grade 3 radiation pneumonitis was 2% compared to 3.4% in Chi et al. and the incidence of rib fractures for our study against Chi et al. was 2% vs. 3.2%.The main limitation of our study is its retrospective nature, although all the patients are treated at a single radiation center after referrals from several hospitals.. Therefore, it is likely that some local relapses were actually radiation-induced fibrosis.A second limitation is the absence of a central review of follow-up imaging. However, most follow-up images were reviewed in the local multidisciplinary thoracic oncology board of the patient’s hospital, including a radiologist and/or a nuclear physician. All suspected lesions were counted as local relapses, which can be seen as another limitation with an under-estimated local control. Histological confirmation was not a prerequisite for classifying lesions as recurrence. Differentiation between benign radiographic changes and local recurrence after SABR remains a challenge as many patients developed radiation-induced fibrosis in the treated lung region , 19. ThePatients treated without histological confirmation had to meet the following criteria: an increase in tumor size over time, a high FDG-PET uptake and morphological abnormalities suggestive of malignancy. When all these criteria are fulfilled and there were no signs of infection, the probability of benign diagnosis is less than 4% , 15. TheAs compared to the recent international Advisory Committee on Radiation Oncology Practice (ACROP) recommendations, our methods especially since 2010, are in tandem in terms of the radiation preparation, prescription methods, delivery, and technologies . One difThe substantial technological advancements during the recruitment of patients can be viewed as another methodological weakness, although they represent real-life practice. The patients were treated with a nominally constant dose prescription. However, considering the fact that the dose was first prescribed at the isocenter using a pencil beam algorithm, and later at the isodose encompassing the PTV using an AAA algorithm, proved a significant dose escalation by at least 25%. The studied outcome parameters were not clearly improved by the new technologies or with the derived prescription method, but we remark a slight association between the treatment period and the incidence of any recurrences. However, the decrease in relapses is concomitant with an increase of deaths. The positive impact of the treatment period must therefore be further demonstrated. The limitations of this non-randomized comparison highlights the difficulties in radiation oncology to assess the potential clinical improvement of new technologies, as illustrated by Chi et al. when trying to compare SABR with hypo-fractionated particle beam therapy . TypicalWe observed a long-term local control and survival after SABR for peripheral stage I NSCLC in this large series of patients with the expected low toxicity.


Lignin is the second most abundant natural polymer on earth. Industries using lignocellulosic biomass as feedstock generate a considerable amount of lignin as a byproduct with minimal usage. For a sustainable biorefinery, the lignin must be utilized in improved ways. Lignin is recalcitrant to degradation due to the complex and heterogeneous structure. The depolymerization of lignin and its conversion into specific product stream are the major challenges associated with lignin valorization. The blend of oligomeric, dimeric and monomeric lignin-derived compounds (LDCs) generated during depolymerization can be utilized by microbes for production of bioproducts.Burkholderia sp. strain ISTR5 (R5), a proteobacteria belonging to class betaproteobacteria, order Burkholderiales and family Burkholderiaceae, was isolated and characterized for the degradation of LDCs. R5 strain was cultured on 12 LDCs in mineral salt medium (MSM) supplemented with individual compounds such as syringic acid, p-coumaric acid, ferulic acid, vanillin, vanillic acid, guaiacol, 4-hydroxybenzoic acid, gallic acid, benzoic acid, syringaldehyde, veratryl alcohol and catechol. R5 was able to grow and utilize all the selected LDCs. The degradation of selected LDCs was monitored by bacterial growth, total organic carbon (TOC) removal and UV–Vis absorption spectra in scan mode. TOC reduction shown in the sample contains syringic acid 80.7%, ferulic acid 84.1%, p-coumaric acid 85.9% and benzoic acid 83.2%. In UV–Vis absorption spectral scan, most of the lignin-associated peaks were found at or near 280 nm wavelength in the obtained absorption spectra. Enzyme assay for the ligninolytic enzymes was also performed, and it was observed that lignin peroxidase and laccase were predominantly expressed. Furthermore, the GC–MS analysis of LDCs was performed to identify the degradation intermediates from these compounds. The genomic analysis showed the robustness of this strain and identified various candidate genes responsible for the degradation of aromatic or lignin derivatives, detoxification mechanism, oxidative stress response and fatty acid synthesis. The presence of peroxidases (13%), laccases (4%), monooxygenases (23%), dioxygenase (44%), NADPH: quinone oxidoreductases (16%) and many other related enzymes supported the degradation of LDCs.In the present study, a novel bacterium p-coumaric acid. Some other intermediates like catechol, acetovanillone, syringaldehyde and 3,4-dihydroxybenzaldehyde from the recognized bacterial metabolic pathways existed during growth on the LDCs. The ortho- and meta cleavage pathway enzymes, such as the catechol-1,2-dioxygenase, protocatechuate 3,4-dioxygenase, catechol-2,3-dioxygenase and toluene-2,3-dioxygenase, were observed in the genome. In addition to the common aromatic degradation pathways, presence of the epoxyqueuosine reductase, 1,2-epoxyphenylacetyl-CoA isomerase in the genome advocates that this strain may follow the epoxy Coenzyme A thioester pathway for degradation.Numerous pathway intermediates were observed during experiment. Vanillin was found during growth on syringic acid, ferulic acid and  Depleting fossil fuel reserves and climate change have compelled us to switch towards renewable alternatives for the production of fuels and chemicals. Lignocellulosic biomass is a renewable, easily available and low-cost alternative, which is being projected to replace Petro-based products. The lignocellulose biomass components include cellulose 40–50%), hemicellulose 20–30%) and lignin 10–25%) [0–25% [1,0–30% and0–50%, heLignin is a complex aromatic heteropolymer recalcitrant to degradation. The monomers present in lignin are linked by β-ether 45–55%), biphenyl (10–25%) and other types of linkages in varying amounts depending on the type and source of biomass –55%, bip. The noxPseudomonas, Bacillus, Pandoraea and Burkholderia, etc. have generally been reported to develop these types of modifications. Few strains of fungi Humicola grisea var thermoidea, Trichoderma viride, Talaromyces bassochlamydoides, etc. and bacteria Bacillus sp. LP1, Pandoraea sp. ISTKB, etc. were reported for the utilization of lignin and LDCs containing different functional groups [Sphingobacterium sp., Pseudomonas sp., Acinetobacter sp. [Alcaligens sp. 3K [Streptomyces sp. [Ralstonia, Sphingomonas, Bacillus, Klebsiella, Ochrobactrum tritici [Microbes while utilizing the recalcitrant compounds  as the sole source of carbon and energy undergo various types of stress. Due to adverse metabolic, respiratory pressure on the microbes, they adapt to cope with these types of stress for survival. They adjust to the harsh environment by modification in their extracellular secretory protein system and with the help of porin, peroxiredoxins, etc. Only a few species of the bacteria such as l groups , 9, 10. l groups . But thel groups . In natucter sp. , 12. Ades sp. 3K . Nishimuyces sp. . Several tritici , 16.Burkholderia, a proteobacteria belonging to class betaproteobacteria, order Burkholderiales and family Burkholderiaceae, is widely known for the biodegradation of diverse types of substrates and other biotechnological applications [Burkholderia glumae BGR1 for the production of p-hydroxybenzoic acid from p-coumaric acid [Burkholderia sp. efficiently utilized indole, propiconazole, di-(2-ethylhexyl) phthalate (DEHP) [Burkholderia genus is limited, thus limiting its application in lignin valorization.ications –21. Jungric acid . The Bure (DEHP) , 23. Usee (DEHP) –26. The p-coumaric acid, p-hydroxybenzoic acid, vanillin, vanillic acid, veratryl alcohol, guaiacol, catechol, gallic acid), which makes R5 a model strain for aromatic compound degradation studies. This strain was studied for the degradation of different LDCs containing different types of functional groups like aldehyde, carboxylic, methyl, methoxy and hydroxyl groups, etc. Enzyme assays (Lignin peroxidase (Lip), Manganese peroxidase (MnP) and laccase), total organic carbon, UV–Vis absorption spectral scan on spectrophotometer (200–800 nm wavelength) and GC–MS analysis were done to confirm the degradation of LDCs. The genomic analysis supported and highlighted the robust metabolic machinery of this strain. The findings will enhance the understanding of the role of Burkholderia genus in LDC utilization and its prospective application as a biofunneling bacteria for an economical biorefinery.There are limited reports available regarding the utilization of various LDCs by beta-proteobacteria. In the present study, a novel isolate R5 was able to metabolize a number of different aromatic compounds  were streaked on separate plates and were further screened for their potential for LDC degradation as sole carbon source. Although the degradation pattern was monitored for 168 h, the highest degradation was observed at 120 h for all the isolates. The bacterial strain R5 showed growth on all the LDCs used, followed by R8 with growth on 8, R7 and R6 on 6, R9 on 4 LDCs , syringaldehyde (RT 13.5) and isovanillin (RT 9.3). Essential enzymes which are responsible for conversion of syringic acid into vanillin include phenylacetate-CoA oxygenase reductase, Enoyl-CoA aldolase and methyltransferases. . 120 h sp-Coumaric acid representing coumaryl monomer of lignin structure is mostly found in the grasses [p-coumaric acid using p-hydroxyphenylacetate 3-hydroxylase, reductase component. Following the oxidation, reduction reaction to the final products after 120 h sample mainly comprised guaiacol (RT 13.6) and malonic acid (RT 21.4). From the intermediates obtained, it is evident that the degradation pattern of p-coumaric acid is similar to the β-ketoadipate pathway. grasses . 3,4-dihFerulic acid represents the coniferyl monomer of the lignin structure. Degradation profile indicated that the main metabolite present after 72 h of incubation was vanillin (RT 8.3), acetovanillone (RT 9.8) and in trace amounts, vanillic acidand catechol; similar results were reported by . After 1Benzoic acid is a representative of benzoyl monomer of the lignin structure; its degradation occurred through the β-ketoadipate pathway. After 72 h of reaction, peaks of intermediates like 3, 4, 5-trihydroxyacetophenone (RT 9.8), 1,2-benzenedicarboxylic acid (RT 29.4) were found in the spectra. 120-h sample contains peaks of maleic acid (RT 34.5), Oxalic acid RT 11.9) and muconate (RT 13.5). The product profiling of the benzoic acid degradation signals towards the degradation via beta-ketoadipate pathway; similar pathway was also hypothesized by other researchers .9 and mu.The common intermediate obtained in the degradation of all the LDCs was vanillin. This shows the importance of biological funneling, i.e. bacteria can effectively degrade the various LDCs and bring at a common product. This funneling of the compounds paves the way for lignin valorization study.R5 proved to be efficient for the degradation of LDCs. The genome size of this strain is found to be 8.16 Mb with GC content of 68.11, and other general genome features are given in Table . KEGG-KADifferent electron donors, electron acceptors and other pertinent genes attributed to respiration are present in the genome. Surplus quinone oxidoreductases, soluble cytochromes, formate dehydrogenases and additional related electron transporters illustrate the significance and support in the metabolism of recalcitrant compounds , glutathione transferase, catalase, etc. . These tBurkholderia genus is known for its aerobic mode degradation of aromatic hydrocarbons . Numerous genes related to respiration, stress response and aromatic metabolism are present in the genome of strain R5. Transporters, receptors, metal ion binding and other membrane proteins found in the genome showed the presence of active transportation and metabolism machinery for the utilization of substrates.2O2 and organic hydroperoxides produced into water and alcohols, respectively. Detoxification of peroxides by alkyl hydroperoxide reductase plays a vital role in cell protection from oxidative stress [The genomic profile showed the presence of various genes encoding enzymes, which are essential for the scavenging of harmful chemical species in the culture media. Alkyl hydroperoxide reductase is responsible for the reduction of He stress . Apart fe stress . Thus, t2−, which is converted into O2 or H2O2 by Superoxide dismutase (SOD); this enzyme helps to cope up with the oxidative stress. Different types of SODs were found in the genome and have been recently shown to be responsible for demethylation of lignin [p-coumaric acid and other substrate’s degradation were hypothesized to proceed through demethylation and methyl shift reaction. Methoxy functional group-containing compounds like syringaldehyde, vanillin, and guaiacol were degraded by strain R5 due to the presence of Rieske type Fe–S cluster in demethylases enzyme [2O2 generated by them for dynamic participation in LDCs degradation [During the degradation process of aromatics by an oxidoreductase, free radicals are generated. One of them is superoxide Of lignin . Kumar ef lignin . In the s enzyme . In addis enzyme . Benzoats enzyme , 50. NADs enzyme . Oxidaseradation .Bacillus ligniniphilus L1 [Lip, MnP and laccase enzyme activity shown by the strain R5 on different LDCs showed the presence of peroxidase and laccase in the genome. Catalase/peroxidase is a potential catalase enzyme which performs the function of peroxidase and is responsible for the degradation of aromatic hydrocarbons. Several peroxidase and multi-copper oxidases were observed in the genome, reiterating its robustness for the degradation of LDCs. In addition to Lip, MnP, and laccase enzymes, other key enzymes for lignin degradation such as enoyl CoA hydratase, acetyl-CoA hydratase, dehydrogenases, and peroxidases are also present in the genome of strain R5 which is similar to hilus L1 .Pseudomonas putida, Escherichia coli and Pandoraea sp. [Genomic profile of R5 shows the existence of different pathways; one of them is Epoxy–CoA facilitated degradation of aromatic hydrocarbons. Epoxy CoA-thioester pathway genes were found in the genome of the bacteria. The aromatic compounds are degraded by the formation of epoxide bond by enzymes Acyl CoA reductase and 1,2-phenylacetyl CoA epoxidase. The enoyl CoA hydratase and Epoxide hydrolase are responsible for the degradation of intermediate and formation of diol derivative, respectively. Further metabolism to final products takes place using NAD-dependent succinate semialdehyde dehydrogenase . This tyraea sp. . The genraea sp. . Presencraea sp. .The analysis and level of existence of such a diverse gene set have helped us identify key lignin or aromatic biodegradation enzymes which will also allow for the valorization of lignocellulosic biomass.Burkholderia sp. based on the 16S rDNA sequencing analysis, possesses most of the key upper funneling pathways required for the transformation of LDCs. In addition to dominating enzymes such as Laccase and peroxidase, it has genomic machinery that plays an important role in the protection and survival of the cells against the harsh environment and oxidative stress. Along with the beta-ketoadipate pathway, this strain also has machinery for the Epoxy CoA-thioester pathway for the degradation of substrates. Conclusively, the study analyzed the core genes present in the genome of R5 for LDC utilization and represented the metabolic pathways followed for the degradation of LDCs. The intermediate metabolic compounds formed during degradation of syringic, ferulic, and p-coumaric acid indicated vanillin mediated degradation of the compounds. Biofunneling of complex aromatic compounds generated from lignin overcomes the heterogeneity and provides opportunity for bioconversion.We have isolated and characterized a bacterial strain from the soil, capable of utilizing various types of LDCs. Based on experiments performed and data analysis, we infer the remarkable inherent capabilities of the strain R5 for the utilization of 12 LDCs. Strain R5 identified as 9H10O5), p-coumaric acid (C9H8O3), vanillic acid (C8H8O4), ferulic acid (C10H10O4), vanillin (C8H8O3), guaiacol (C7H8O2), 4-hydroxybenzoic acid (C7H6O3), gallic acid (C7H6O5), benzoic acid (C7H6O2), syringaldehyde (C9H10O4), veratryl alcohol (C9H12O3) and catechol, (C6H6O2) in the present study were procured from the Sigma Aldrich (United States), Hi-Media , Merck and Tokyo Chemical Industry co. Ltd. (Japan) unless and otherwise stated.All the chemicals used, syringic acid 3Fe; 0.05Ca(NO3)2·4H2O; 0.085NaNO3 and 1 g/L Kraft lignin at 30 °C; rpm 180, pH 8 [p-coumaric acid, vanillic acid, ferulic acid, vanillin, guaiacol, 4-hydroxybenzoic acid, gallic acid, benzoic acid, syringaldehyde, veratryl alcohol and catechol to obtain a final OD of 0.1, to isolate and screen the potential strain. Conical flasks containing the stipulated reaction mixture were incubated at 180 rpm at 30 °C and pH8 was set using 0.1 N HCl and 0.1 M NaOH. For tracking the degradation trend of these LDCs, 2 mL sample was taken out daily for bacterial growth for 168 h.Soil samples were collected from the Jawaharlal Nehru University, New Delhi  forest soil where the degradation of wood was taking place. 1 g soil was mixed with 50 mL autoclaved distilled water in a sterile falcon tube and vortexed for proper mixing; 1 mL solution taken from it and transferred into 250 mL conical flask containing 50 mL 1 g/L Kraft lignin-MSM and cycloheximide (50 mg/L) was used to inhibit any fungal growth. The composition of Mineral Salt Media (MSM) used (g/L) was 7.8Na80, pH 8 . MSM wasThe potential LDC degrading strain was identified using 16S rDNA sequence analysis. Bacteria were grown in 10 mL LB broth and the cell pellet harvested during exponential phase by centrifuging at 12,000 rpm for 5 min. Then, DNeasy Ultraclean microbial kit was used to extract the genomic DNA, as per manufacturer’s protocol. Amplification of 16S rDNA was carried out using primers P0 5′ (GAGAGTTTGATCCTGGCTCAG) 3′ and P6 5′ (CTACGGCTACCTTGTTACGA) 3′. Obtained 16S rDNA was gel purified using gel purification kit (Qiagen) and then sent for sequencing to Central Instrumentation Facility, University of Delhi South Campus. The Strain was identified after blast search of sequence in NCBI database and then phylogenetic tree was constructed using neighbor-joining method .Samples were withdrawn daily at a fixed time and absorbance was recorded followed by spectral scan using Varian Carry 100 Bio UV–Vis Spectrophotometer. Preliminary analysis of the degradation of LDCs was done using Varian Carry 100 Bio UV–Vis Spectrophotometer in the spectral scan mode . In a pr−1 cm−1 [2O2 (0.1 mM) [−1 cm−1 [2+ oxidize to Mn3+ and formed complex with malonate which gives absorbance at 270 nm, ε270 = 11,590 M−1 cm−1 [4 1 mM , 0.1 mL enzyme, 0.2 mL H2O2 0.1 mM (0.5 mM in water). Enzyme activity is generally expressed in U/mL, defined as the volume of enzyme required to oxidize 1 μmol of the substrate per minute at 25 °C [For spectrophotometric enzyme assay, the samples were centrifuged at 8000 rpm for 15 min and the supernatant was kept at 4 °C for Lignin peroxidase (Lip), laccase and Manganese peroxidase (MnP) enzyme assays. Laccase enzyme activity was analyzed by using ABTS as substrate. The reaction mixture comprises ABTS (0.5 mM) as substrate, enzyme, and sodium tartarate buffer  incubated for 10 min and then absorbance reading was determined at 420 nm at a time interval of 30 s for 3 min, ε420 = 36,000 M−1 cm−1 . LiP ass(0.1 mM) . LiP act[−1 cm−1 . The manN,O-bis(trimethylsilyl)trifluoroacetamide) were added for derivatization [For GC–MS analysis, 50 mL sample withdrawn was centrifuged at 8000 rpm for 10 min and then acidified to pH 2–3. Then extraction was done using an equal volume of ethyl acetate, shaken for 30 min for proper mixing so that migration of organic compounds takes place in ethyl acetate. Thereafter, this solution was kept in separating funnel, and an organic layer containing LDCs was taken and dried to 2 mL; then 50 µL Pyridine and 100 µL BSTFA (tization . This sotization .http://www.genome.jp/kaas-bin/kaasmain)” [Bacteria were inoculated in LB-broth and allowed to grow at 180 rpm, 30 °C for 14–16 h. Culture was harvested during log phase, centrifuged at 8000 rpm, at 4°C and supernatant was discarded. Bacterial pellet was used for DNA isolation for whole genome sequencing. Genomic DNA of the strain was isolated by using DNeasy Ultraclean microbial kit, Qiagen (Germany). The Illumina MiSeq platform was used for sequencing of R5 genome, and raw data processing, selection of quality reads, assembly, generation of scaffolds, and gene prediction were performed as described earlier . Arrangeasmain)” . Islandvasmain)” , 64.Additional file 1: Figure S1. Genes engaged in respiratory mechanism of Burkholderia sp. ISTR5. Figure S2. Transcriptional regulators involved in the regulation of various functions in Burkholderia sp. ISTR5. Figure S3. Transporters responsible for the movement of substrates and ions in Burkholderia sp. ISTR5. Figure S4. Oxidoreductases responsible aromatic compounds degradation found in Burkholderia sp. ISTR5. Figure S5. Representation of various stress regulation proteins found in Burkholderia sp. ISTR5. Table S1. KEGG Pathway classification of R5. Table S2. Representation of monooxygenases present in Burkholderia sp. ISTR5. Table S3. Different dioxygenases found in Burkholderia sp. ISTR5. Table S4. Peroxidases found in genome of Burkholderia sp. ISTR5. Table S5. Peripheral pathway for aromatic compounds degradation in Burkholderia sp. ISTR5. Table S6. Metabolism of central aromatic hydrocarbons in Burkholderia sp. ISTR5. Table S7. Genes responsible for overcoming stress conditions.


Camellia oleifera) is a unique edible-oil tree in China, and anthracnose occurs in wherever it is cultivated, causing great economic losses each year. We have previously identified the Ascomycete fungus Colletotrichum fructicola as the major pathogen of anthracnose in Ca.oleifera. The purpose of this study was to characterize the biological function of Snf1 protein, a key component of the AMPK (AMP-activated protein kinase) pathway, for the molecular pathogenic-mechanisms of C. fructicola.Tea-oil tree (Saccharomyces cerevisiae Snf1. Targeted CfSNF1 gene deletion revealed that CfSnf1 is involved in the utilization of specific carbon sources, conidiation, and stress responses. We further found that the ΔCfSnf1 mutant was not pathogenic to Ca.oleifera, resulting from its defect in appressorium formation. In addition, we provided evidence showing crosstalk between the AMPK and the cAMP/PKA pathways for the first time in filamentous fungi.We characterized CfSnf1 as the homolog of C. fructicola and, therefore, a potential fungicide target for anthracnose control.This study indicate that CfSnf1 is a critical factor in the development and pathogenicity of  Camellia oleifera) is a commercial shrub native to China and has been widely cultivated throughout southern China for more than 2000 years with abundant edible oil in its seeds [2 and produces 250,000 tons of edible oil each year, however, it is still unable to meet the consumer demand for tea oil. One of the major limiting factors is the serious diseases occurred in Tea-oil tree.Tea-oil tree  and the γ subunit Snf4 [Magnaporthe oryzae, Verticillium dahliae, and Cochliobolus carbonum [C. fructicola. Here, we identified the protein kinase CfSnf1 from C. fructicola and characterized its functions.AMPK pathway is also called Snf1 (sucrose non-fermenting 1) pathway in nit Snf4 . The sernit Snf4 . In highnit Snf4 . The actnit Snf4 . Besidesnit Snf4 , 23–25. carbonum –28. DespS. cerevisiae Snf1 sequence as the trace, we acquired its single homolog in C. fructicola genome database by a BLAST_P search. Then, we submitted its sequence to NCBI database (https://www.ncbi.nlm.nih.gov/) (GenBank accession number MN094751) and named it CfSnf1. CfSnf1 was predicted to encode 741 amino acids and phylogenetic analysis revealed that CfSnf1 shows sequence conservation among other fungi Snf1 proteins; CfSnf1 shows higher amino acid sequence homology with that of C. gloeosporioides (96% identify and 96% similarity) and lesser homology with S. cerevisiae Snf1 (still 43% identify and 56% similarity)  suggested that CfSnf1 contains a S_TKc  domain, a UBA (ubiquitin-associated) domain, an Adenylate Sensor domain, and three low complexity regions  . Putative transformants were screened on hygromycin media, then verified by PCR amplification, and we thus obtained two deletion mutants ΔCfSnf1#3 and ΔCfSnf1#5 . Since these two mutants showed the same biological phenotypes, we selected ΔCfSnf1#3 for further analysis. To complement the mutant strain, the genomic DNA sequence of CfSNF1 containing a 1.5-kb promoter was retransformed to the ΔCfSnf1#3 mutant and restored all of the mutant defects.To characterize the functions of CfSnf1, a V. dahliae and M. oryzae [CfSnf1 mutant, and complemented strain ΔCfSnf1/CfSNF1 were cultured in the plates of PDA (Potato Dextrose Agar), MM , and MM supplemented with 1% Glycerol or 1% Pectin as sole carbon source for 4 days. All the strains showed similar growth levels on PDA and MM, while ΔCfSnf1 mutant showed significantly reduced growth rates to WT and ΔCfSnf1/CfSNF1 on MM supplemented with Glycerol or Pectin  tag to the C-terminus of CfSnf1 and introduced it into the Δdia Fig. . This reCfSnf1 mutant, and ΔCfSnf1/CfSNF1 on PDA plates supplemented with osmotic stress  for 4 days. We found that ΔCfSnf1 mutant showed significant higher inhibition rates than that of WT and ΔCfSnf1/CfSNF1 in all three osmotic stresses .For normal growth and infection, fungi must undergo many types of stresses in nature, such as osmotic stress, oxidative stress and cell wall integrity stress etc. Studies in yeast concluded that ScSnf1 plays critical roles in response to various environmental stresses , 31, 32.ses Fig. a and b. CfSnf1 mutant caused no lesions on both unwounded and wounded tea-oil tree leaves and its non-penetration on cellophane membranes, we wondered whether the mutant could not produce any functional appressorium. To further elucidate the mechanisms of the abolished pathogenicity in ΔCfSnf1 mutant, the conidia of WT, ΔCfSnf1 and ΔCfSnf1/CfSNF1 were inoculated onto hydrophobic artificial surfaces to induce the appressorium formation. At 4 h post inoculation (hpi), all the strains exhibited more than 80% conidial germination rate and showed no significant difference among them. However, the germ tubes of ΔCfSnf1 were elongated abnormally and no appressoria were developed at 24 hpi, in contrast with the normal germ tubes and more than 80% appressorium formation rate in WT and ΔCfSnf1/CfSNF1  plates at 28 °C in the darkness, unless indicated otherwise. The strains were cultured in liquid PDA medium in darkness shaking at 28 °C for 2 days and collected for the extraction of genomic DNA.C. fructicola, C. gloeosporioides, V. dahliae, Neurospora crassa, M. oryzae, C. carbonum, and S. cerevisiae were acquired from the NCBI database (https://www.ncbi.nlm.nih.gov/). The phylogenetic tree was constructed by MEGA 5.05 programs using neighbor-joining method with 1000 bootstrap replicates. The domain of CfSnf1 was predicted by the SMART website (http://smart.embl-heidelberg.de/).The Snf1 proteins of CfSNF1 targeted gene deletion was performed by one-step replacement strategy [CfSNF1 gene and 1.4-kb HPH gene were amplified using primes . After sequencing, the fused-pYF11 plasmids were transformed into the protoplasts of ΔCfSnf1 mutant for complementation.strategy . Two abo3, 0.52 g KCl, 0.152 g MgSO4·7H2O, 1.52 g KH2PO4, 0.01 g VB1, 1 ml 1000 × trace elements, 10 g Glucose, and 15 g agar in 1 L ddH2O), and MM with the Glucose was substituted by 1% Glycerol or 1% Pectin. After 4 days incubation, the colony diameters were measured and statistically analyzed.The strains were cultured on PDA, MM (6 g NaNO2O2). After 4 days incubation, the colony diameters were measured and the inhibition rates were statistically analyzed.The strains were cultured on PDA and PDA added with different stresses, including osmotic stress , cell wall integrity stress (0.01% SDS and 400 μg/ml CR), and oxidative stress  for 4 days, then filtered with three layers of lens paper and the conidia were collected and statistically analyzed. For appressorium formation, the collected conidia were resuspended to a concentration of 1 × 10For pathogenicity assay, the mycelial plugs of the strains were inoculated onto the leaf margin of the detached Tea-oil tree leaves. The inoculated leaves were kept in a humidity plate with a 12 h light and 12 h dark cycle for 4 days and then photographed. The penetration assay on cellophane membranes was performed as previously described , 40 withCfSnf1 mutant. The fluorescence of the hyphae and conidia was observed under microscope.The green fluorescent protein (GFP) tag was fused to the C-terminus of CfSnf1 and transformed into the ΔAdditional files 1: Figure S1. Generation of the CfSNF1 gene deletion mutants in C. fructicola. (A) Strategy for the construction of gene replacement fragment. ~ 1.0-kb left fragment (LF) and right fragment (RF) flanking the CfSNF1 gene and 1.4-kb HPH gene from pCX62 vector were amplified, then the LF and RF were ligated to the flanks of HPH, respectively, by overlap PCR. The PCR products were further cloned into the pMD19-T vector and obtained the vector of pMD-CfSNF1-KO. After sequencing, the 3.4-kb fragments, which contain the LF, RF and HPH, were amplified for gene replacement. (B) Schematic illustration for deletion strategy of CfSNF1 gene. (C) Validation of the gene deletion mutants by PCR amplified with primers 1 (NBF/NBR) and primers 2 (BWF/HPHR). M: marker; +: positive control; −: negative control; #3 and #5: mutants.Additional files 2: Figure S2. CfSnf1 is not involved in the tolerance of cell wall stress and oxidative stress. (A) The WT, ΔCfSnf1 and ΔCfSnf1/CfSNF1 strains were cultured onto PDA plates with different cell wall stresses (CR and SDS) and oxidative stress (H2O2) at 28 °C for 4 days and photographed. (B) Statistical analysis of inhibition rates of the strains to cell wall stresses and oxidative stress. Error bars represent SD of three replicates.Additional files 3: Table S1. Primers used in this study.


Begonia ×semperflorens), geranium (Pelargonium ×horturum), petunia (Petunia ×hybrida), and snapdragon (Antirrhinum majus) at 20°C under six sole-source LED lighting treatments with a photosynthetic photon flux density (PPFD) of 160 μmol∙m–2∙s–1 using B (peak = 447 nm), green , R (peak = 660 nm), and/or mint W  LEDs that emitted 15% B, 59% G, and 26% R plus 6 μmol∙m−2∙s−1 of far-red radiation. The lighting treatments (with percentage from each LED in subscript) were MW100, MW75R25, MW45R55, MW25R75, B15R85, and B20G40R40. At the transplant stage, total leaf area, and fresh and dry weight were similar among treatments in all species. Surprisingly, when petunia seedlings were grown longer (beyond the transplant stage) under sole-source lighting treatments, the primary stem elongated and had flower buds earlier under MW100 and MW75R25 compared to under B15R85. The color rendering index of MW75R25 and MW45R55 were 72, and 77, respectively, which was higher than those of other treatments, which were ≤64. While photosynthetic photon efficacy of B15R85 (2.25 μmol∙J–1) was higher than the W light treatments (1.51−2.13 μmol∙J–1), the dry weight gain per unit electric energy consumption (in g∙kWh–1) of B15R85 was similar to those of MW25R75, MW45R55, and MW75R25 in three species. We conclude that compared to B+R radiation, W radiation had generally similar effects on seedling growth at the same PPFD with similar electric energy consumption, and improved the visual color quality of sole-source lighting.Arrays of blue  and red  light-emitting diodes (LEDs) used for plant growth applications make visual assessment of plants difficult compared to a broad  spectrum. Although W LEDs are sometimes used in horticultural lighting fixtures, little research has been published using them for sole-source lighting. We grew seedlings of begonia ( The plug trays were rotated daily inside each LED module to mitigate any positional effects. For each radiation treatment, the percentage of each waveband and R: far red  was calculated using 100-nm wavebands; the phytochrome photostationary state (PSS) was estimated as described by Sager et al.  that provided pairwise comparisons between treatments using Tukey’s honestly significant test at 15R85 was negative, while MW100 had a CRI value of 64 and substituting MW with R LEDs by 25% or 55% increased the CRI to 77 or 72, respectively (20G40R40 (CRI = 56). The CRI values for all of our W light treatments were lower than the recommended CRI value ≥80, but were much higher than a typical B+R spectrum used in horticulture.CRI evaluates the accuracy of light sources to render human color perception of objects compared to a reference light source  –26. The ectively . However75R25 (4116 K) and MW45R55 (3018 K) can be categorized into neutral W and warm W, respectively. In addition, the CCT of MW45R55 was within the recommended CCT ranges (2700−4000K) for a natural color perception [15R85 (14766 K) and B20G40R40 (9842 K) were outside of the range of W light.The color appearance of light emitted by a light source can be described with CCT . CCT is rception . The CCT100, MW75R25 and MW25R75 were 26−33% taller than those grown under B15R85. Similarly, when petunia seedlings were grown longer (beyond the transplant stage) under the sole-source lighting treatments, the primary stem elongated and had flower buds earlier under MW100 and MW75R25 compared to under B15R85  and thus had a lower R:FR (8:1−17:1) value than other treatments (100 and MW75R25 could be influenced by a lower R:FR.Plants absorb most PAR (400−700 nm), especially B and R radiation, but they transmit and reflect most FR radiation. Therefore, under a plant canopy, the R:FR is decreased. Plants detect a reduction in the R:FR through phytochrome photoreceptors, which trigger shade-avoidance responses including promotion of extension growth –32. For eatments . Thus, t100 and MW75R25 was 59% and 45%, respectively while B15R85 had 0% of G radiation . With few significant spectral effects of lighting treatments on leaf expansion and net assimilation, plant dry weight was similar among radiation treatments in all species , is considered as the appropriate metric for electrical efficiency of light sources for plant growth [15R85 had the highest PPE value of 2.25 μmol∙J–1  as the MW100. We also calculated dry weight gain per unit electric energy consumption, or DWE (in g∙kWh–1). While B15R85 had a higher PPE (in μmol∙J–1) than the W light treatments, the DWE of B15R85 was similar with those of MW25R75, MW45R55, and MW75R25 in begonia, petunia, and geranium , and particularly, some mixtures of MW and R LEDs showed higher visual quality and optimum color quality for W light. These results suggest that W radiation can be used for sole-source lighting to produce young plants with similar growth attributes and electric energy consumption while improving human vision.